Original Articles Polish Journal of Veterinary Sciences Vol

22nd Meeting of the European Society of Veterinary Pathology 6th Meeting of the European Society of Veterinary Clinical Pathology European College of Veterinary Clinical Pathology

Olsztyn, 15-18 September 2004

Short original articles Polish Journal of Veterinary Sciences Vol. 7, No. 3 (2004), Supplement

Contents

1. Aupperle H., Garbade J., Schneider K., Steiger K., Dhein S., Schoon H.-A. Morphological and immunohistochemical characterization of alterations induced by doxorubicin and the chronological course of recovering of various tissues in rabbits ...... 5 2. Bakuła T., Apoznański J., Obremski K., Wiese I., Gajęcki M. Histopathological pattern of alimentary tract of pigs fed with fish and rape oil supplemented feed ...... 9 3. Bossowska A., Gonkowski S., Wojtkiewicz J., Skobowiat C., Kaleczyc J., Całka J., Majewski M. Nitric oxide synthase (NOS) in dorsal root ganglia neurons associated with the porcine IMG: occurrence and reaction to mechanical injury ...... 13 4. Bossowska A., Józefowicz A., Gonkowski S., Wojtkiewicz J., Kaleczyc J., Pidsudko Z., Majewski M. Proliferative enteropathy (PE)-induced changes in the expression and co-incidence patterns of SP and/or CGRP in DRG neurons supplying the porcine descending colon ...... 17 5. Bossowska A., Józefowicz A., Wojtkiewicz J., Gonkowski S., Kaleczyc J., Pidsudko Z., Majewski M. Influence of proliferative enteropathy on expression pattern of somatostatin (SOM) and galanin (GAL) in extrinsic primary afferent neurons supplying the porcine descending colon ...... 21 6. Bownik A., Siwicki A.K., Prevost G. In vitro effects of leukocidin LukE/LukD on the rabbit immunocompetent cells ...... 25 7. Całka J., Wolf G., Kaleczyc J., Wąsowicz K. Increased expression of NADPH-d/nitric oxide synthase after facial axotomy; ultrastructural and light microscopic analysis ...... 29 8. Dang X., Tian S., Tian D., Huang Z. The pathological investigation and analysis of the chicks cadmium chloride intoxication ...... 33 9. Dzienis A., Majewski M., Wojtkiewicz J., Jana B. Changes in density of sympathetic nerve terminals and steroidogenic activity of porcine ovaries after dexamethasone-induced polycystic ovarian syndrome ...... 37 10. Franke-Radowiecka A., Całka J., Wąsowicz K., Podlasz P., Kaleczyc J. The study on the NADPHd-positive innervation of the porcine mammary gland ...... 41 11. Gonkowski S., Wojtkiewicz J., Bossowska A., Kaleczyc J., Całka J., Majewski M. Proliferative enteropathy-induced de novo synthesis of NPY in intramural ganglia neurons of the porcine descending colon ...... 45 12. Gonkowski S., Wojtkiewicz J., Bossowska A., Kaleczyc J., Sienkiewicz W., Majewski M. Co-incidence pattern of SP and CGRP in neural structures of the porcine descending colon affected by proliferative enteropathy ...... 49 13. Gonkowski S., Wojtkiewicz J., Bossowska A., Kaleczyc J., Sienkiewicz W., Majewski M. Proliferative enteropathy (PE)-induced changes in the number of vasoactive intestinal polypeptide-immunoreactive (VIP-IR) neural elements in the porcine descending colon ...... 53 14. Janeczek M., Janeczek W., Sender-Janeczek A., Chrószcz A., Czerski A., Henklewski R. Clinical evaluation of fillings made with glass-ionomer and composite materials in sandwich technique in canine molar teeth ...... 57 15. Jedlińska-Krakowska M., Jakubowski K., Kowalski A. The level of selected antioxidative indices and testosterone in the blood of rats injected with vitamin E and selenium under an increased ozone concentration in the air ...... 61 16. Katkiewicz M., Osińska B. Lafora’s bodies in borzoi dog. Case report ...... 65 17. Kowalski I.M., Szarek J., Babińska I., Lipińska J., Fabczak J. Pathomorphological characteristic of supraspinal muscles in rabbits after short-term electrostimulation 69 18. Majewski M., Wojtkiewicz J., Gonkowski S., Bossowska A., Pidsudko Z., Kaleczyc J. NOS-, VIP- or GAL-immunoreactivity disappear from proliferative enteropathy-affected inferior mesenteric ganglion (IMG) neurons supplying porcine descending colon ...... 73 19. Majewski M., Wojtkiewicz J., Gonkowski S., Bossowska A., Sienkiewicz W., Kaleczyc J. Proliferative enteropathy (PE)-induced changes in the expression pattern of Dβ H, NPY and/or SOM in porcine IMG neurons supplying the descending colon ...... 77 20. Małaczewska J., Rotkiewicz Z., Siwicki A.K. Effect of Methisoprinol, KLP-602 and NDV infection on the selected biochemical indices of the allantoic fluid of chicken embryos ...... 81 21. Mikulska-Skupień E., Szweda W., Procajło Z., Bigoszewski M. Levels of C-reactive protein and interferon gamma in pigs vaccinated with deleted vaccine against Aujeszky’s disease after experimental infection with virulent Herpesvirus suis type 1 ...... 85 22. Osińska B., Katkiewicz M. Vimentin – the marker in dog kidney injury ...... 89 23. Pavlidou E. T-zone lymphoma in a dog. Preliminary morpho-immunohistochemical study ...... 93 24. Pidsudko Z., Majewski M. The distribution and chemical coding of porcine urinary bladder trigone-projecting neurons located in prevertebral ganglia other than IMG ...... 97 25. Rogala E., Skopińska-Różewska E., Wojtasik E., Chorostowska-Wynimko J., Sommer E., Filewska M., Siwicki A.K. Caffeic acid feeding of pregnant mice influences the prenatal development of their offspring ...... 101 26. Rymuszka A., Siwicki A.K. In vitro influence of synthetic pyrethroid – cypermethrin on phagocytes of heterothermic and homeothermic animals ...... 105 27. Saurek D., Katkiewicz M. The influence of dexamethason on the ovarian and uterine structure and ER, PR and alfa-inhibin expression in pseudopregnant rabbits ...... 109 28. Sienkiewicz W., Kaleczyc J., Łakomy M. Somatostatin-immunoreactive nerve structures in the ileum and large intestine of pigs undergoing dysentery 115 29. Sierosławska A., Siwicki A.K. In vitro immunosuppressive effects of oxytetracycline on phagocytic cells and lymphocytes isolated from rainbow trout (Oncorhynchus mykiss) and carp (Cyprinus carpio L.) head kidney ...... 119 30. Siwicki A.K., Morand M., Kazuń B., Trapkowska S. Immunopathogenesis of herpesviruses: influence of channel catfish herpesvirus (CCHV) on macrophage and lymphocyte activity – in vitro comparative study ...... 123 31. Siwicki A.K., Terech-Majewska E., Szarek J., Trapkowska S., Kazuń B. Pathogenesis of Birnaviridae – influence of infectious pancreatic necrosis virus (IPNV) on cell-mediated immunity, total Ig level and lysozyme activity in Salmonid ...... 127 32. Skopiński P., Skopińska-Różewska E., Kamiński A., Dziedzic-Gocławska A., Sommer E., Chorostowska-Wynimko J., Cendrowska I., Chrystowska D., Sadło J., Siwicki A.K. Chocolate feeding of pregnant mice resulted in epigallocatechin-related embryonic angiogenesis suppression and bone mineralization disorder ...... 131 33. Szarek J., Koncicki A., Zduńczyk Z., Jankowski J., Andrzejewska A., Babińska I., Lipińska J. Ultrastructural pattern of the muscle fibres of turkeys fed a diet containing oxidized fat and infected with the

O78K80H9 pathogenic serotype of Escherichia coli ...... 135 34. Szarek J., Kowalski I.M., Andrzejewska A., Lipińska J., Babińska I., Felsmann M.Z. Ultrastructural characteristics of supraspinal muscles in rabbits after short-term electrostimulation ...... 139 35. Szarek J., Lorek M.O., Lipińska J., Gugołek A., Babińska I., Truszczyńska M., Felsmann M.Z. Pathomorphological pattern of the liver and kidney in polar fox fed a diet supplemented with a new generation of feed ...... 143 36. Szarek J., Siwicki A.K., Andrzejewska A., Babińska I., Lipińska J., Truszczyńska M., Terech-Majewska E. Pathomorphological pattern of the kidney in European sheatfish (Silurus glanis) after iridovirus infection and lysozyme dimer application in a bath ...... 147 37. Witkowski L., Sobczak-Filipiak M., Kaba J., Rzewuska M., Kita J. Rhodococcus equi infection in foals – clinical and pathomorphological changes ...... 151 38. Wojtkiewicz J., Gonkowski S., Bossowska A., Kaleczyc J., Całka J., Majewski M. CGRP-immunoreactive structures in the porcine superior cervical ganglion (SCG) – decentralization/deafferentation-induced changes ...... 155 39. Wojtkiewicz J., Gonkowski S., Bossowska A., Kaleczyc J., Majewski M. Proliferative enteropathy (PE)-induced changes in the distribution pattern of vesicular acetylcholine transporter-immunoreactive (VAChT-IR) nerve terminals in the porcine inferior mesenteric ganglion (IMG). . 159 40. Zmysłowska I., Guziur J., Szarek J., Krause J. Potential pathogens in intensive rearing of Wels catfish (Silurus glanis L.) ...... 163 41. Zmysłowska I., Harnisz M., Guziur J., Szarek J. Antibiotic-resistant bacteria isolated from fish and from water during intensive fattening ...... 167 42. Żmudzka M., Malicka E., Sobczak-Filipiak M., Lechowski R. Telomerase activity in squamous cell carcinoma in dogs ...... 171 Polish Journal of Veterinary Sciences Vol. 7, No. 3 (2004), Supplement, 5-7

Short original article Morphological and immunohistochemical characterization of alterations induced by doxorubicin and the chronological course of recovering of various tissues in rabbits

H. Aupperle, J. Garbade1, K. Schneider1, K. Steiger, S. Dhein1, H.-A. Schoon

Institute of Veterinary Pathology, Faculty of Veterinary Medicine, University of Leipzig, An den Tierkliniken St. 33, 04103 Leipzig, Germany 1 Department of Cardiac Surgery, Heart Center, University of Leipzig, Strumpel St. 19, 04289 Leipzig, Germany

Abstract

Alterations caused by doxorubicin (DOX) in rabbits and their course of recovering was characterized morphologically and immunohistologically. After the 2nd DOX dose lymphatic organs were markedly hypocellular, testes and skin were atrophic, and myocardial degeneration occurred. Two weeks after the last DOX dose hematopoetic organs appeared to be hyperplastic. After six weeks hematopoetic tissues and testes were normal, whereas the skin was still atrophic. Myocardial fibrosis, as well as renal cysts and fibrosis developed. In conclusion, using doxorubicin the different reaction patterns of the various organs should consider forensic aspects (i.e. potentia generandi) and carefull interpretation of experimental results.

Key words: doxorubicin, toxicity, histopathology, immunohistochemistry, rabbit

Introduction Materials and Methods

Doxorubicin (adriamycin), an anthracycline anti- Thirty four 3 months old, male white New Zealand biotic, is used in chemotherapy of various neoplasia. rabbits were treated intravenously with 3 mg/kg  The side-effect cardiotoxicity (Suzuki et al. 1997) is doxorubicin hydrochloride (Ribodoxo -L 50) once used as an experimental model in different species. a week for six weeks. All procedures were done in However, other organs (i.e. hematopoetic tissues, accordance with the “Principles of laboratory animal skin, kidneys, intestine) are affected by anthracyclines, care” (1985). Nine rabbits died during the treatment period after the 1st (n=1), 2nd (n=1), 3rd (n=3) or too (Van Vleet et al. 1979, Maral and Jouanne 1981). 2 days after the 6th (n=4) DOX dose. Seven animals The aim of this study was the morphological and im- died two weeks after the last (6th) DOX dose. Twelve munohistological characterization of the alterations rabbits were anesthetized (isoflourane) and eutha- caused by doxorubicin (DOX) in rabbits and the chro- nized by explantation of the heart six weeks after the nological course of recovering (regeneration or repar- last DOX administration (day 84). Six rabbits served ation respectively) after treatment in various organs. as untreated controls.

Correspondence to: H. Aupperle, tel.: 0341-9738277, fax: 0341-9738299, e-mail: [email protected] 6 Aupperle H. et al.

Specimens of the heart, bone marrow, spleen, tive cells increased markedly after the end of treat- intestinal lymph nodes, skin, testes and all major or- ment. gans were fixed in 4% formalin, embedded in para- Heart. In rabbits, which died during treatment hy- plast and stained with hematoxylin and eosin, and drothorax (n=7), hydropericard (n=6) as well as as- Picro-Sirius-Red stained slides were additionally cites (n=6) were observed. The cardiac ventricles of examined by polarized light (Gru¨ninger 1996). Im- DOX treated animals were markedly dilated. After munohistochemistry was performed to detect PCNA the 2nd DOX dose histopathologically signs of a dif- (proliferating cellular nuclear antigen), CD79 (B-lym- fuse mild eosinophilic degeneration, loss of cross stri- phocytes), MAC 387 (macrophages). Results were ation and/or vacuolar degeneration occurred. Later, evaluated semiquantitavely. metabolic active fibroblasts building collagen fibres appeared and few lymphocytes and macrophages were scattered in the interstitium. Six weeks after the last application mild diffuse myocardial fibrosis but only Results single inflammatory cells were observed. Examination of Picro-Sirius-Red stained slides in polarized light Skin. After two weeks most of the rabbits develop- revealed mainly yellow lightening collagen fibres ed alopecia at the neck and in two cases perinasal and which can be interpreted as collagen I according to periocular, too. The skin at the neck showed his- Gru¨ninger (1996). topathologically moderate orthoceratotic hyper- Intestine. Moderate diarrhea accompanied by keratosis and severe epidermal and adnexal atrophy. a moderate mucosal atrophy and mild lympho-plas- The proliferation marker PCNA was detected only in macellular enteritis were the main findings in four single epidermal and adnexal cells. After the end of animals which died during the treatment period. treatment increasing numbers of adnexal cells but Kidneys. No gross lesions of the kidneys were only few epidermal cells expressed PCNA. Six weeks seen. However, after the 3rd DOX dose multiple mild- after the last DOX dose only in 5 of 12 animals the ly dilated tubules appeared in the inner cortex. Six epidermis appeared to be normal compared to the weeks after the last treatment numerous tubular cysts, controls. Seven rabbits still showed skin atrophy with lined by atrophic epithelial cells and surrounded by mild adnexal PCNA expression. mild (n=4) to severe (n=8) interstitial fibrosis had Testes. After 2 applications of DOX the testes developed. In polarized light Picro-Sirius-Red stained showed signs of increasing atrophy resulting in tubuli slides showed that fibrotic areas mainly consisted of seminferi mainly consisting of mildly vacuolized Ser- collagen I fibres. toli cells and only single remaining PCNA expressing Lungs, liver, brain, skeletal muscles, bones spermatogonia. Testes were still atrophic 2 weeks were normal in all treated and control animals. after the last DOX dose, but after four more weeks in 8 of 12 rabbits normal active spermotogenesis was observed. Three rabbits showed signs of beginning tes- ticular regeneration, while the testes of one animal Discussion were still completely inactive and atrophic. Spleen, intestinal lymph nodes. After 2nd DOX Cardiomyopathia induced by anthracyclines is administration spleen and lymph nodes showed a well described side effect which is often used in marked depletion of lymphoid cells. B-lymphocyte experimental studies. However, the effects on other follicles (CD79 +) were small and the number of organs are described sparsely (Van Vleet and Ferrans MAC 387 positive cells (macrophages) and the prolif- 1980, Suzuki et al. 1997) but may be responsible for eration activity (PCNA) were diminished. Two weeks the death of numerous animals during the experi- after the last DOX dose lymphatic follicles were mod- ments because of dermatitis, diarrhea, hemorrhagic erately hyperplastic and the number of MAC 387 diathesis and opportunistic infections. In our study positive cells increased mildly. Four weeks later bone marrow, spleen and lymph nodes showed spleen and lymph nodes appeared to be normal in all marked hypocellularity after the 2nd DOX dose corre- treated animals compared to the controls. sponding to Van Vleet et al. (1979), but already two Bone marrow. Already in the rabbit which died weeks after the last DOX dose the immune system after the first DOX dose severe hypoplasia of the appeared to be very active again, especially B-lym- bone marrow had developed. Three rabbits showed phocytes and macrophages were proliferating. Tes- signs of hemorrhagic diathesis (bleedings in the skin, ticular atrophy resulted from DOX by the induction gastrointestinal tract, retrobulbar hematoma) after of apoptosis of germinal cells (Shinoda et al. 1999) the 2nd DOX dose. Two weeks after the last DOX and a decreased proliferation activity (PCNA express- dose hematopoesis was active again and bone marrow ion). We showed that regeneration of the testes was appeared to be mildly hypercellular compared to the possible, but it took about 6 weeks. In contrast, corre- findings 4 weeks later. The number of MAC 387 posi- sponding to the findings of Maral und Jouanne Morphological and immunohistochemical ... 7

(1981), the skin was still atrophic six weeks after the References last DOX dose in most cases. The proliferation activity (PCNA expression) reappeared first in the adnexal Fajardo LF, Eltringham JR, Stewart JR, Klauber MR (1980) epithelia but was retarded in the basal layer of the Adriamycin nephrotoxicity. Lab Invest 43: 242-253. epidermis. Alterations in the heart and kidneys were Gru¨ninger BU (1996) Zur Pathogenese von Angiopathien in accordance to those described in the literature im Endometrium der Stute – Morphologisch-funktionelle ¨ (Fajardo et al. 1980, Suzuki et al. 1997). In contrast to Untersuchungen. Diss., Leipzig, Univ. Veterinarmed. Fakul. regenerative tissue the reparative processes leading to Maral RJ, Jouanne M (1981) Toxicology of daunorubicin in fibrosis were finished in full extend after six weeks. In animals and man. Cancer Treat Reports 65 (Suppl 4): both organs predominantly collagen fibres type I were 9-18. produced. Shinoda K, Mitsumori K, Yasuhara K, Uneyama C, Onodera In conclusion, using doxorubicin the markedly dif- H, Hirose M, Uehara M (1999) Doxorubicin induces ferent reaction patterns and regeneration time of the male germ cell apoptosis in rats. Arch Toxicol 73: various organs should be considered. These results 274-281. may be important in the view of forensic aspects (i.e. Suzuki T, Minamide S, Iwasaki T, Yamamoto H, Kanda H(1997) Cardiotoxicity of a new anthracyline derivate potentia generandi), in further therapy consequences (SM-5887) following intravenous administration to rab- (i.e. cardiac or renal insufficiency) and in the interpre- bits: Comparative study with doxorubicin. Invest New tation of experimental results. It seems likely, that Drugs 15: 219-225. DOX may influence pathogenetic pathways by the Van Fleet JF, Ferrans VJ (1980) Clinical and pathologic modified expression of growth factors and inflamma- features of chronic adriamycin toxicosis in rabbits. Am tory reactions during immune suppression and follow- J Vet Res 41: 1462-1469. ing reactivation. Van Vleet J, Greenwood L, Ferrans VJ (1979) Pathologic features of adriamycin toxicosis in young pigs: non- skeletal lesions. Am J Vet Res 40: 1537-1552. 8 Aupperle H. et al. Polish Journal of Veterinary Sciences Vol. 7, No. 3 (2004), Supplement, 9-12

Short original article Histopathological pattern of alimentary tract of pigs fed with fish and rape oil supplemented feed

T. Bakuła, J. Apoznański, K. Obremski, I. Wiese1, M. Gajęcki

Division of Veterinary Prevention and Feed Hygiene, Department of Veterinary Health Protection, Faculty of Veterinary Medicine, University of Warmia and Mazury in Olsztyn, Oczapowskiego St. 13, 10-717 Olsztyn, Poland 1 Commercial-Service Firm “Doktor”, Lotyń, Poland

Abstract

The effect of 3 and 6% of fish oil and 3% of rape oil addition to feed on histopathological pattern of the porcine alimentary tract was studied. The lessions were observed in all the animals. The most desirable alimentary tract patern was observed in the pigs nourished with feed containing 3% of fish oil, and those fed with addition of 6% fish oil up to the slauther revealed the greatest number of undesirable lessions.

Key words: pig, histopathological examination, alimentary tract, fish oil, rape oil

Introduction which, in consequence, may be related to diseases of blood circulation, nervous system, skin neoplasma, Because of the fatty acids profile, especially immune system disturbances or vision problems Essential Fatty Acids (EFA), including DHA and (Cichon 1998, Pike 1999, Kolanowski 2000). EPA, some plant and fish oils are popular in swine The significance and activity of eicosanoids in pro- feeding. A wide-scale study on fat in a human diet phylaxis and therapy of many diseases were the sub- showed that the consumption of fats rich in saturated jects of many studies but there is not much informa- fatty acids should be limited to 30 – 35% of daily tion about the influence of PUFA on alimentary tract energy intake (Grys 1998, Ziemlański 1998). condition. Profitable influence of diet rich in fish oil on human The aim of this study was to determine the effect and animal health was simultaneously observed. Fish of a 3 and 6% supplement of fish oil and a 3% supple- fat differs from animal fat and vegetable oils in the ment of rape oil with or without the carency on the composition of fatty acids and the concentration of histopathological pattern of alimentary tract in pig. EPA and DHA n-3 family (Kołakowska and Kołakowski 2001). Materials and Methods It has been repeatedly shown that a diet deficient in essential unsaturated acids or undesirable n-3 to The experiment was carried out with the use of fish n-6 acid ratio leads to disturbances in metabolism and rape oil. Fish oil originated from the drip formed

Correspondence to: T. Bakuła, e-mail: [email protected] 10 Bakuła T. et al. during fish heat treatment prior to tinning and from which makes it more biologically attractive in com- the fish meal production, specifically from fish waste parison with rape oil (Table 2). See fish oil are more and non-consumable fish. The fish oil was preserved biologically attractive since besides n-6 linoleic acid with liquid Rendox anti-oxidant (1 kg/t). Refined con- (LA) (C18:2) and n-3 α-linolenic acid (α-LNA) sumable rape oil under the brand name “Uniwer- (C18:3), it also contains other specific metabolites salny” was purchased in retail. The oil samples were such as n-3 eicosapentaenoic acid (EPA) (C20:5) and analysed for odour, consistency, acidity number, per- n-3 docosahexaenoic (DHA) (C22:6) which do not oc- oxide number, water content, petroleum benzine in- cur in rape oil (Cichon 1998, Ziemlański 1998, soluble compounds and protein content. Kołakowska and Kołakowski 2001). The qualitative and quantitative analyses of the The results of the histological analysis of parts of fatty acids were carried out using the Peisker method the alimentary tract are given in Table 3. Stomach (1964) in a gas chromatograph – PYE Unicam – series hyperaemia was found in one of the animals that were 104 with a flame-ionizing detector (FLD). fed with rape oil supplemented feed (group D1a) and The experiment was carried out on 24 crossbred in all the animals in group D1. Hyperaemia of the fattened pigs (F1: WBP and PBZ sows with Duroc and mucose membrane was observed in the duodenum in Pietrain boars) that weighted approximately 60 kg. groups K and D1. Jejunum hyperaemia occurred in The animals were kept in individual nonbedding grat- groups K, D1 and D1a (rape oil supplement) as well ing pens. They were divided into four groups: fatten- as in groups D3a and D3 (fed with the 6% fish oil ing pigs from control group (K) – were feeded without supplemented feed). Histiocytal infiltrations occurred oil supplements, pigs from the first experimental in the tunica mucosa of the duodenum and jejunum group (D1) – with 3% supplement of refined rape oil only in a few pigs of group K. Also in group K, (“Uniwersalny”), in the second experimental group eosinophil cell infiltration was found in mucose mem- (D2) – with 3% supplement of fish oil and in the third brane of the stomach, duodenum and jejunum. Simi- experimental group (D3) – with 6% supplement of lar infiltrations in jejunum were observed in all the fish oil. The animals were fed for 30 days with bal- animal groups, however, in groups D1a and D1 they anced feed produced in the industrial feed factory. were only present in some animals. Lymphocytal infil- Oils applied were mechanically mixed with feed direc- tration occurred in all the examined parts of the ali- tly before animal feeding in order to obtain mentary tract in animals representing the control homogenous consistency. Ten days prior to slaughter, group and groups D3a and D3. Enlargement of the the D1, D2 and D3 pig groups were divided into two lymphatic follicle was observed mainly in the wall of sub-groups. The pigs from groups D1a, D2a and D3a the stomach in group K. The excess of mucous and were fed oil-free feed, whereas the diet of the rest of exfoliation of epithelium were found in almost all the animals remained unchanged until the slaughter. groups, especially in the jejunum and stomach, except After the slaughter, the segments of the stomach, from the groups D2 end K and also in the duodenum duodenum, jejunum and colon were sampled for and colon, except from the group K. histopathological analyses. They were preserved in The histiocytal and lymphocytal infiltrations in the 10% neutralised formalin and embedded into the wall of the duodenum and jejunum correspond to the paraffin blocks. Microtome sections were stained with deformation and the atrophy of the villi, especially in haematoxylin and eosin (HE) and with the PAS the control group and in the animal group fed with method according to McManus. 6% fish oil supplemented feed. Necrosis of the peak part of the duodenum villi occurred in a few animals from group K and D1. This lesion was also observed Results and Discussion in the jejunum in one case from group D2. Micro- scopic examination also showed different organ The studied fish and rape oils, in spite of their lesions, which were found mainly in the control group different composition, fully met the qualitative and in the group fed 6% fish oil supplemented feed. requirements for the first choice feed fats (Table 1). The results obtained from animals fed with 3% fish or Fish oil contained a wider composition of fatty acids, rape oil supplement could suggest its beneficial effect

Table 1. Chemical analysis of the oils used in the experiment (according to the PN-R-64806:1997 norm).

Permissible value for feed Specification Fish oil Rape oil fats of Ist choice

Water content and volatile compounds % 0.55 0.04 up to 1.5 Naphtyl ether insoluble substances % 0.05 0.02 up to 5 Acid number as KOH/g 4.70 0.12 up to 50

Peroxide number meq O2/kg 4.14 1.03 up to 10 Protein content 0 0 up to 2 Energy MJ/kg 43.33 41.73 – Histopathological pattern ... 11

Table 2. The composition of the fatty acids of the oils used for the experiment.

Fatty acids SFA MUFA PUFA PUFA n-6 EFA n-3 EPA DHA Fish oil 23.59 44.35 32.06 8.72 16.78 6.60 12.34 Rape oil 34.16 32.98 32.00 29.77 3.02 – –

Explanation: SFA – saturated fatty acid, MUFA – monounsaturated fatty acid, PUFA – polyunsaturated fatty acid, EFA – essential fatty acid, EPA – eicosapentaenoic acid, DHA – decosahexaenoic acid.

Table 3. Microscopic lesions in the pigs alimentary tract.

Alimentary tract K D1 D1a D2 D2a D3 D3a Animal groups n=4 n=3 n=3 n=3 n=3 n=4 n=4

Average body mass of fattening pigs at a slaughter day 81 81.3 82 85 82.3 75 77.5 stomach –31–––– hyperaemia duodenum 1–1–––– jejunum 211––21 colon –––––––

stomach ––––––– histiocytal duodenum 1–––––– jejunum 1–––––– colon –––––––

stomach 2–1111– cell infiltrations eosinophil duodenum 3111232 jejunum 4233334 colon –1––––– stomach 1122124 lymphocytal duodenum 4322344 jejunum 2211342 colon 1111112

stomach 112–222 enlarged lymphatic follicle duodenum ––––1–– jejunum –21–1–– colon 1–1––––

stomach 1212–22 excessive mucous and epithelium exfoliation duodenum –21121– jejunum 1123232 colon –2––1–2

stomach ––––––– deformation and atrophy of villi duodenum 4221244 jejunum 2333344 colon –––––––

stomach ––––––– necrosis of villi peaks duodenum 1–1–––– jejunum ––1––1– colon –––––––

Explanation: a) K – control group, D1a, D2a, D3a– the experimental animal groups with 10 day waiting period, D1, D2, D3 – animal groups fed with oil supplemented feed until the day of slaughter, D1 and D1a – 3% rape oil supplement, D2 and D2a – 3% fish oil supplement, D3 and D3a – 6% fish oil supplement b) the number of animals with lesions is given in columns 12 Bakuła T. et al. on the condition of the alimentary tract of the animals Grys S (1998) Pathological events connected with disturbed examined. fatty acid metabolism of n-3 and n-6 series. Evening The most desirable therapeutic results (the lowest Primrose and Other Oils Containing N-6 or N-3 Fatty number of histopathological lesions in the examined Acids in Preventtion and Treatment. Proceedings of the Symposium May 15-16, Sulejów, Poland, pp. 89-107. parts of the alimentary tract) were obtained in the Kolanowski W (2000) Fish oil-unique nutritional value. animal group fed with 3% fish oil supplemented feed. Żywność Żywienie Prawo a Zdrowie 4: 430-434. Kołakowska A, Kołakowski E (2001) Particular nutritional values of fish. Przem Spoż 6: 10-13. Acknowledgments Peisker K (1964) Rapid semi-micro method for methyl es- ters from triglicerides using chloroform, methanol, sul- Study financed by the Scientific Research Commit- phuric acid. J Am Oil Chem Soc 41: 87-90. tee project no. 6 P06K 005 20. Pike IH (1999) Health benefits from feeding fish oil and fish meal. The role of long chain omega-3 polyunsaturated fatty acids in animal feeding. International Fishmeal and References Oil Manufacturers Association, Ifoma 28. Ziemlański Ś (1998) Physiological role of N-6 and N-3 fatty acids in the human body, with a particular regard to 1998 Cichon R ( ) N-3 and N-6 PUFA in Human Physiology prevention of the metabolic non-communicable dis- and Pathology. Evening Primrose and Other Oils Con- orders. Evening Primrose and Other Oils Containing taining N-6 or N-3 Fatty Acids in Prevention and Treat- N-6 or N-3 Fatty Acids in Preventtion and Treatment. ment. Proceedings of the Symposium May 15-16, Proceedings of the Symposium May 15-16, Sulejów, Po- Sulejów, Poland, pp. 116-124. land, pp. 11-30. Polish Journal of Veterinary Sciences Vol. 7, No. 3 (2004), Supplement, 13-15

Short original article Nitric oxide synthase (NOS) in dorsal root ganglia neurons associated with the porcine IMG: occurrence and reaction to mechanical injury

A. Bossowska, S. Gonkowski, J. Wojtkiewicz, C. Skobowiat, J. Kaleczyc1, J. Całka1, M. Majewski

Division of Clinical Physiology and 1 Animal Anatomy, Department of Functional Morphology, Faculty of Veterinary Medicine, University of Warmia and Mazury, Oczapowskiego St. 13, 10-717 Olsztyn, Poland

Abstract

The present study was aimed at disclosing the distribution, morphological features and nerve lesion-induced plasticity of NOS-immunoreactive sensory neurons associated with the porcine IMG by means of combined retrograde tract-tracing using Fast Blue (FB) and double-immunofluorescence. The vast majority (up to 90%) of FB-labelled, IMG-projecting afferent neurons were located

in L2 and L3 DRGs with the rest dispersed in Th14 to L5 DRGs. NOS-immunoreactivity was found in a small subset of DRG cells (approximately 2.5%), being predominantly confined to the medium- and small-sized perikarya. No changes in the number of FB-labelled neurons expressing NOS after nerve lesion were observed.

Key words: sensory neurons, inferior mesenteric ganglion, dorsal root ganglia, nitric oxide synthase (NOS), neuronal plasticity, pig

Introduction Luka´cova´ et al. 2003), in the present study, we decided to elucidate the distribution pattern of NOS-IR DRG It has been suggested that DRG neurons may in- neurons associated with IMG in the pig. Moreover, as fluence the activity of postganglionic sympathetic nerve lesion has in the recent past been shown to in- neurons including those found in IMG, in both direct duce a strong upregulation of nNOS in DRG neurons (through their dendritic arborizations within the gan- in rats (for references, see Bergman et al. 1999) and glia), as well as indirect way (i.e. through axonal col- monkey (Zhang et al. 1993), thought to represent ad- laterals forming synaptic contacts with either pregan- aptative responses serving to reduce the deleterious ef- glionic sympathetic neurons or interneurons within fects of the peripheral nerve damage and to promote the dorsal horn; for review, see Majewski 2000). Thus, survival and regeneration of the lesioned sensory path- as NO, a short-living, gaseous messenger molecule has ways,wehavealsoinvestigatedputativechangesinthe recently been shown to be widely used by mammalian expression pattern of NOS in DRG neurons supplying sensory and autonomic neurons (for references, see theporcineIMGafternerveinjury.

Correspondence to: A. Bossowska, tel.: +48 89 523-44-61, fax: +48 89 523-39-82, e-mail: [email protected] 14 Bossowska A. et al.

Materials and Methods fifth section (only neurons with clearly visible nucleus were included) and presented as mean ± SEM. More- Twelve female piglets (8 weeks old), divided into over, the size of the neurons studied was measured. two groups (n=6 each): control animals (CA) and ani- Pictures were captured by a digital camera connected mals undergoing right IMG ganglionectomy (GA to a PC, analyzed with AnalySIS software (version group) three weeks after FB application, were injec- 3.02, Soft Imaging System, FRG) and printed on ted with 5% aqueous solution of FB into the right a wax printer (Phaser 8200, Xerox, USA). IMG. Seven days after the ganglionectomy (performed in GA group), animals of both groups were sacrificed by an overdose of sodium pentobarbital Results and Discussion (Vetbutal, Biowet, PL). Ten µm thick cryostat serial + sections of Th6 –S2 DRGs were subjected to routine FB afferent neurons were found in Th15 to L5 single-labelling immunofluorescence using serum DRGs. As the vast majority of these perikarya (ap- raised in mouse and directed to NOS (1:1500, Sigma, proximately 90% of all retrogradely labeled cells) PL). Sections were then incubated with goat were located in L2 and L3 DRGs, the present data are anti-mouse IgG conjugated to biotin (DAKO, Dk) based on the results obtained from sections of L2 and + and afterwards, visualized by a streptavidin-CY3 com- L3 DRGs considering all counted FB neurons as plex (Jackson, USA). Retrogradely labeled/single-im- 100% population. NOS was present in 2.5 ± 1.2% of munostained perikarya were then evaluated under all FB-positive sensory neurons. The vast majority Olympus BX51 microscope equipped with epi-fluor- (63.5 ± 10.3%) of NOS-IR, IMG-projecting neurons escence and appropriate filter sets, counted in each belonged to the class of medium-sized cells. Neurons

Fig. 1. DRG L1, control pig. Retrogradely labeled NOS-IR neurons. x 200. + Fig. 2. DRG L1, axotomized pig. The number of FB /NOS-IR neurons appears unchanged. x 200. Nitric oxide synthase (NOS) ... 15 with a small diameter constituted 34.9 ± 9.3%, while References large NOS-IR, FB+ sensory neurons were found only occasionally (1.5 ± 1.5%). Changes in the Bergman E, Carlsson K, Liljeborg A, Manders E, Ho¨kfelt T, number of NOS-IR cells (4.0 ± 0.2%) induced by Ulfhake B (1999) Neuropeptides, nitric oxide synthase mechanical injury of the peripheral processes of and GAP-43 in B4-binding and RT97 immunoreactive FB-positive neurons were statistically not signifi- primary sensory neurons: normal distribution pattern and changes after peripheral nerve transaction and aging. cant when compared to CA group. Previously, the Brain Res 832: 63-83. presence of NOS has been reported in DRG neur- Cizkova´ D, Lukaćova´ N, Marsala M, Marsala J (2002) Neur- ons of humans (Terenghi et al. 1993) and many opathic pain is associated with alterations of nitric oxide others mammalian species (for references, see synthase immunoreactivity and catalytic activity in dorsal Lukaćova´ et al. 2003), including the pig (Majewski root ganglia and spinal dorsal horn. Brain Res Bull 58: et al. 1995). The present results are in agreement 161-171. with those mentioned above. It was shown that sci- Lukaćova´ N, Cizkova´ D, Krizanova´ O, Pavel J, Marsala M, atic nerve ligature induced an increase in the numb- Marsala J (2003) Peripheral axotomy affects nicotinam- ide adenine dinucleotide phosphate diaphorase and nitric er of NOS-IR DRG neurons in rats (Cizkovaétal. oxide synthase in the spinal cord of the rabbit. J Neurosci 2002) and rabbits (Lukaćova´ et al. 2003), but the Res 71: 300-313. same injury induced a distinct decrease in the Majewski M (2000) Synaptogenesis and structure of the au- number of NOS-IR cells in monkey DRGs (Zhang tonomic ganglia. Folia Morphol 58: 65-99. et al. 1993). Quite recently, Renganathan et al. Majewski M, Sienkiewicz W, Kaleczyc J, Mayer B, Czaja K, (2000) demonstrated that NO, implicated to be an Lakomy M (1995) The distribution and co-localization of autocrine regulator of neuronal survival, has been immunoreactivity to nitric oxide synthase, vasoactive in- up-regulated predominantly in small-sized c-type testinal polypeptide and substance P within nerve fibres supplying bovine and porcine female genital organs. Cell neurons. As the vast majority of NOS-IR afferent Tissue Res. 281: 445-464. neurons supplying porcine IMG were of medium Renganathan M, Cummins TR, Hormuzdiar WN, Black JA, size, the lack of injury-induced changes in the Waxman SG (2000) Nitric oxide is an autocrine regulator number of studied cells implicates that NO is not of Na+ currents in axotomized C-type DRG neurons. involved in the survival mechanisms of this subset of J Neurophysiol 83: 2431-2442. porcine afferent neurons. Terenghi G, Riveros Moreno V, Hudson LD, Ibrahim NB, Polak JM (1993) Immunohistochemistry of nitric oxide synthase demonstrates immunoreactive neurons in spinal cord and dorsal root ganglia of man and rat. J Neurol Sci Acknowledgements 118: 34-37. Zhang X, Verge V, Wiesenfeld-Hallin Z, Ju G, Bredt D, ¨ Supported by KBN, grant no. 0523-0802. The Synder SH, Hokfelt T (1993) Nitric oxide synthase-like immunoreactivity in lumbar dorsal root ganglia and spi- authors are indebted to Ms. A. Piękos and Mr. Z. nal cord of rat and monkey and effect of peripheral Liminowicz for their excellent technical help. axotomy. J Comp Neurol 335: 563-575. 16 Bossowska A. et al. Polish Journal of Veterinary Sciences Vol. 7, No. 3 (2004), Supplement, 17-19

Short original article Proliferative enteropathy (PE)-induced changes in the expression and co-incidence patterns of SP and/or CGRP in DRG neurons supplying the porcine descending colon

A. Bossowska, A. Józefowicz, S. Gonkowski, J. Wojtkiewicz, J. Kaleczyc1, Z. Pidsudko1, M. Majewski

Division of Clinical Physiology and 1Animal Anatomy, Department of Functional Morphology, Faculty of Veterinary Medicine, University of Warmia and Mazury in Olsztyn, Oczapowskiego St. 13, 10-717 Olsztyn, Poland

Abstract

This study aimed at disclosing the PE influence on the expression of SP and/or CGRP in porcine DRG neurons supplying descending colon. In healthy pigs, SP was found in 37.8 ± 0.9% and CGRP in 54.3 ± 2.4% of all FB+ neurons and co-localization of them was observed in 32.3 ± 2.3% of FB+ cells. PE induced an increase in the number of SP-IR perikarya (till 59.2 ± 1.3%), while the number of CGRP-IR ones decreased (till 36.8 ± 0.7%). 22.2 ± 4.3% of FB+ cells were SP/CGRP-IR. These results are contradictory to data concerning the axotomy- and chemical inflammation-evoked changes in the expression of SP and CGRP in porcine DRG neurons.

Key words: proliferative enteropathy, neuronal plasticity, substance P (SP), calcitonin gene-related peptide (CGRP), dorsal root ganglia neurons, pig

Introduction Bossowska et al. 2003) with profound changes in the synthesis rate and expression pattern of SP and In addition to intrinsic primary afferent neurons, CGRP. As some of these neurons may synthesize existence of which within the bowel wall has recently and utilize SP and/or CGRP as their messenger been unequivocally demonstrated (Furness et al. molecules released during “axon-reflexes” (Cooke 1998), there are also extrinsic (spinal) primary affer- 1998) and, furthermore, there is evidence that both ent neurons (Berthoud et al. 2004) involved in the substances may evoke slow EPSP in “second order” neural control of enteric functions, mediating both in- neurons involved in the intestinal secretion (Pan nocuous and noxious sensations. Recently, it has been and Gershon 2000, Cooke 1998), it may be of interest found that porcine spinal primary sensory neurons re- to investigate putative changes in the expression spond to transection of their peripheral processes patternofbothsubstancesinextrinsicprimary (Bossowska et al. 2004) or to chemical injury (chemi- afferent neurons challenged by Lawsonia intracel- cally-evoked inflammation of the bowel wall; lularis-infection.

Correspondence to: M. Majewski, 10-719 Olsztyn, Oczapowskiego 13, e-mail: [email protected] 18 Bossowska A. et al.

Materials and Methods labeled with biotinylated donkey anti-rabbit IgG (both from Jackson Immunochemicals, USA). The latter Six immature female pigs (approximately 8 weeks antibody was then visualized by a streptavidin-CY3 old), three control (C group) and three with clinically complex (Jackson). Retrogradely labeled/double-im- diagnosed Lawsonia intracellularis infection (PE munostained perikarya were then evaluated under group) were used. All animals were subjected to Olympus BX51 microscope equipped with epi-fluor- laparotomy and injected with 5% aqueous solution of escence and appropriate filter sets, counted in each Fast Blue (FB) into the wall of the descending colon. fifth section (only neurons with clearly visible nucleus After a three-week survival period, the animals were were included) and presented as mean ± SEM. Pic- sacrificed with an overdose of sodium pentobarbital tures were captured by a digital camera connected to (Vetbutal, Biowet, Poland) and perfused transcar- a PC, analyzed with AnalySIS software (version 3.02, dially with freshly prepared 4% paraformaldehyde in Soft Imaging System, FRG) and printed on a wax 0.1 M phosphate buffer (pH 7.4). Ten µm thick cryos- printer (Phaser 8200, Xerox, USA). tat sections of the L1 –L3 and S3 DRG were subjected to routine double-labelling immunofluorescence using combinations of antisera raised in different species Results and Discussion and directed towards SP (rat monoclonal) and CGRP (rabbit polyclonal). Anti-SP serum was visualized by Retrograde tract-tracing revealed that the vast ma- species-specific secondary antiserum conjugated to jority (more than 80%) of descending colon-projec- FITC, while the rabbit anti-CGRP antibody was ting extrinsic primary afferent neurons was present in

Fig. 1. Control DRG; FB+ neuron (a) exhibit co-localized SP (b) and CGRP-IR (c). x 200. Fig. 2. PE-affected DRG; SP (b) and CGRP (c) in a medium-sized, FB+ neuron (a). x 400. Fig. 3. PE-affected DRG; large, retrogradely labeled perikaryon (a) contained both SP (b) and CGRP (c). x 200. Proliferative enteropathy (PE)-induced ... 19

porcine L1 –L3 and S3 DRGs. The prevailing number and Timmermans 2004), to influence the electrolyte of the neurons belonged to the class of small- and secretion in PE-affected colon. medium-sized nerve cells (Fig. 1). By means of double-immunofluorescence it has been found that in healthy animals, SP was found in 37.8 ± 0.9% of all Acknowledgements FB+ neurons, while CGRP was encountered in 54.3 ± 2.4% of retrogradely labeled cells. Co-localiz- Supported by KBN, grant no. 6P06K 012 20. The ation of both substances was observed in 32.3 ± 2.3% authorsareindebtedtoMs.A.PiękosandMr.Z. of FB+ cells (Fig. 1). PE induced an increase in the Liminowicz for their excellent technical help. number of SP-IR perikarya (till 59.2 ± 1.3%), while the number of CGRP-IR ones simultaneously decreased (till 36.8 ± 0.7%). In contrast to control ani- References mals, only 22.2 ± 4.3% of FB+ cells still exhibited co-localized SP and CGRP; however, these cells be- Berthoud HR, Blackshaw LA, Brookes SJH, Grundy longed to the class of medium-sized (Fig. 2) and D(2004) Neuroanatomy of extrinsic afferents supplying large perikarya (Fig. 3). Results of the present study the gastrointestinal tract. Neurogastroenterol Motil 16: are thus contradictory to data concerning the mech- 28-33. anical injury – (a decrease in the number of SP-IR Bossowska A, Gonkowski S, Adriaensen D, Timmermans neurons and an increase in the number of CGRP-IR JP, Majewski M (2004) Changes in the chemical coding ones have been observed; Bossowska et al. 2004) and of spinal primary sensory neurons supplying the porcine acute chemical inflammation-evoked changes (both descending colon after transection of their peripheral substances were dramatically up-regulated; Boss- processes. Verh Anat Ges 99: 84. owska et al. 2003) in the expression pattern of SP Bossowska A, Gonkowski S, Majewski M (2003)Wpływ and CGRP in porcine descending colon-projecting procesu zapalnego na fenotyp neuronów czuciowych DRG neurons. Therefore, the results of the present zaopatrujących okrężnicę zstępującą u świni. Materiały XXXIX Sympozjum PTHiC, Wrocław, pp. 7. study suggest the activation of an adaptive mechan- Brown DR, Timmermans JP (2004) Lessons from the por- ism(s) of porcine sensory neurons affected by PE cine enteric nervous system. Neurogastroenterol Motil that were different from those induced by transec- 16: 50-54. tion of their peripheral processes or acute inflamma- Cooke HJ (1998) “Enteric tears”: chloride secretion and its tion of their target tissue. Furthermore, assuming neural regulation. News Physiol Sci 13: 269-274. that SP is able to evoke slow EPSP also in porcine Furness JB, Kunze WA, Bertrand PP, Clerc N, Bornstein submucous (both outer and/or inner) plexus neurons, JC (1998) Intrinsic primary afferent neurons of the in- this tachykinin, released from activated extrinsic pri- testine. Prog Neurobiol 54: 1-18. mary afferent c-fibres in an antidromic manner, may Pan H, Gershon MD (2000) Activation of intrinsic afferent be implicated, together with SP released from intrin- pathways in submucosal ganglia of the guinea pig small sic Dogiel type II primary afferent neurons (Brown intestine. J Neurosci 20: 3295-3309. 20 Bossowska A. et al. Polish Journal of Veterinary Sciences Vol. 7, No. 3 (2004), Supplement, 21-23

Short original article Influence of proliferative enteropathy on expression pattern of somatostatin (SOM) and galanin (GAL) in extrinsic primary afferent neurons supplying the porcine descending colon

A. Bossowska, A. Józefowicz, J. Wojtkiewicz, S. Gonkowski, J. Kaleczyc1, Z. Pidsudko1, M. Majewski

Division of Clinical Physiology and 1Animal Anatomy, Department of Functional Morphology, Faculty of Veterinary Medicine, University of Warmia and Mazury, Oczapowskiego St. 13, 10-717 Olsztyn, Poland in Olsztyn

Abstract

The present study was aimed, by immunofluorescence and retrograde tracing at disclosing changes in the number of GAL- and/or SOM-IR afferent neurons resulting from a chronic, proliferative inflammation of the porcine descending colon. In intact DRGs, GAL was found in 1.5 ± 0.3% of all traced perikarya, while SOM was expressed by approximately 0.6 ± 0.1% of these cells. Within the PE-affected DRGs, the number of GAL- or SOM-IR neurons slightly increased, reaching 7.3 ± 0.6% and 0.8 ± 0.1% of all traced afferent cells, respectively. These data suggest an involvement of GAL, but not SOM, in adaptive events in DRG neurons afflicted by chronic inflammatory process.

Key words: proliferative enteropathy, neuronal plasticity, somatostatin, galanin, dorsal root ganglia neurons, pig

Introduction most numerous in the L1 –L3 and S3 ganglia (Boss- owska et al. 2003a). Furthermore, as it has been In addition to IPANs (intrinsic primary afferent shown in the recent past, the transsection of the pe- neurons, located within the bowel wall; for a review, ripheral processes of afferent neurons supplying this see Clerc and Furness 2004), sensory modalities from segment of the porcine gut (Bossowska et al. 2004), or the intestine are also transmitted by quite numerous an acute inflammation of their target tissues (Boss- EPANs (extrinsic primary afferent neurons located owska et al. 2003b), lead to profound changes in the within the dorsal root ganglia or nodose/jugulare gan- expression pattern of GAL or SOM in retrogradely glion complex, a sensory component of the vagus; for labeled perikarya. However, there is no data dealing a most recent data, see Berthoud et al. 2004). In the with putative changes in the chemical coding of distal pig, EPANs connected with the descending colon bowel-projecting EPANs, induced by a chronic in- were found in Th13 –L4 and S3 –S4 DRGs, being the flammation. Therefore, the present study was aimed

Correspondence to: M. Majewski, tel.: +48 89 523-44-60, fax: +48 89 523-39-82, e-mail: [email protected] 22 Bossowska A. et al.

Figs. 1 and 2. Expression pattern of SOM (1b, 2b) and GAL (1c, 2c) in DRG neurons of control animals, with special regard to retrogradely labeled afferent neurons (1a, 2a). Note, that both substances are expressed by different neuronal populations. x 200. Figs. 3 and 4. Expression pattern of SOM (3b, 4b) and GAL (3c, 4c) in retrogradely labeled DRG neurons of PE animals (3a, 4a). Note, that also in these animals both substances are expressed by different neuronal populations. x 200 in Fig. 3, x 400 in Fig. 4.

at disclosing supposed changes in the number of Materials and Methods GAL- and/or SOM-immunoreactive (SOM-IR) afferent neurons, evoked by Lawsonia intracellularis infec- Six immature female pigs (approximately 8 weeks tion, resulting in a chronic, proliferative inflammation old), three control (C group) and three with clinically of the bowel segment of interest. diagnosed Lawsonia intracellularis infection (PE Influence of proliferative ... 23 group) were used. All animals were subjected to chemically induced acute inflammation, where GAL- laparotomy and injected with 5% aqueous solution of and SOM-IR neurons constituted 55.8 ± 2.7% and Fast Blue (FB) into the wall of the descending colon. 0.9 ± 0.3% of affected neurons, respectively (Boss- After a three-week survival period, the animals were owska et al. 2003b). Furthermore, data obtained in sacrificed with an overdose of sodium pentobarbital the present study are also contradictory to results of (Vetbutal, Biowet, Poland; 90 mg/kg b.w.) and per- experiment based on transsection of the nervi colici fused transcardially with freshly prepared 4% parafor- caudales comprising peripheral processes of EPANs: maldehyde in 0.1 M phosphate buffer (pH 7.4). Ten it has been shown that this kind of mechanical injury µm thick cryostat serial sections of the DRGs were elevated the number of GAL-IR cells up to subjected to routine double-labelling immunofluores- 53.8 ± 1.5% of all traced perikarya, while only cence using combinations of anisera raised in differ- 1.13 ± 0.4% of colon-projecting afferent neurons ex- ent species and directed towards GAL (rabbit poly- pressed SOM immunoreactivity (Bossowska et al. clonal, 1:16000, Bachem, CH), and SOM (rat mon- 2004). Thus, the present results suggest an involve- oclonal, 1:350, Affinity, UK). Primary antisera were ment of GAL in repair mechanism(s) of EPANs af- visualized by rat-specific secondary antisera con- flicted by bacterial inflammation; furthermore, the jugated to FITC or rabbit- specific antibodies con- relatively low increase in the number of GAL-IR cells jugated to biotin (all from Jackson Immunochemicals, observed in PE-affected DRG may be a sign of an USA). The latter antibodies were then visualized by exhaustion of compensative potential of these cells a streptavidin-CY3 complex (Jackson). Retrogradely during chronic challenge. labeled/double-immunostained perikarya were then evaluated under Olympus BX51 microscope equipped with epi-fluorescence and appropriate filter sets, Acknowledgements counted in each fifth section (only neurons with clearly visible nucleus were included) and presented as Supported by KBN, grant no. 6P06K 012 20. The mean ± SEM. Pictures were captured by a digital authors are indebted to Ms. A. Piękos and Mr. Z. camera connected to a PC, analyzed with AnalySIS Liminowicz for their excellent technical help. software (version 3.02, Soft Imaging System, FRG) and printed on a wax printer (Phaser 8200, Xerox, USA). References

Berthoud HR, Blackshaw LA, Brookes SJH, Grundy Results and Discussion D(2004) Neuroanatomy of extrinsic afferents supplying the gastrointestinal tract. Neurogastroenterol Motil 16: 28-33. In intact L1 –L3 and S3 DRG (the ganglia at these levels contained more than 80% of all retrogradely Bossowska A, Adriaensen D, Gonkowski S, Wojtkiewicz J, Timmermans JP, Majewski M (2003a) Distribution and labeled, descending colon-projecting afferent neur- ± neurochemical features of primary sensory neurons sup- ons), GAL was found in 1.5 0.3% of all traced plying the porcine colon descendens. Verh Anat Ges 98: perikarya (Fig. 1), while SOM was expressed by ap- 228. proximately 0.6 ± 0.1% of these cells (Fig. 2). Both Bossowska A, Gonkowski S, Adriaensen D, Timmermans JP, substances were present in totally different popula- Majewski M (2004) Changes in the chemical coding of tions of EPANs: while virtually all GAL-IR perikarya spinal primary sensory neurons supplying the porcine de- belonged to the class of small DRG neurons, im- scending colon after transection of their peripheral pro- munoreactivity of SOM was confined to cesses. Verh Anat Ges 99: 84. medium-sized perikarya. Within the DRGs collected Bossowska A, Gonkowski S, Majewski M (2003b) Wpływ from proliferative enteropathy-suffering animals, the procesu zapalnego na fenotyp neuronów czuciowych zaopatrujących okrężnicę zstępującą u świni. Materiały number of GAL- or SOM-IR neurons slightly in- ± ± XXXIX Sympozjum PTHiC, Wrocław, pp. 7. creased, reaching 7.3 0.6% and 0.8 0.1% of all Clerc N, Furness JB (2004) Intrinsic primary afferent neur- traced EPANs, respectively (Figs 3, 4). This run ones of the digestive tract. Neurogastroenterol Motil 16: I counter to the results obtained from animals with 24-27. 24 Bossowska A. et al. Polish Journal of Veterinary Sciences Vol. 7, No. 3 (2004), Supplement, 25-27

Short original article In vitro effects of leukocidin LukE/LukD on the rabbit immunocompetent cells

A. Bownik, A. K. Siwicki1, G. Prevost2

Department of Physiology and Toxicology, Catholic University of Lublin, Norwida St. 4, 20-061 Lublin, Poland 1 Department of Microbiology and Clinical Immunology, Faculty of Veterinary Medicine, University of Warmia and Mazury in Olsztyn, Oczapowskiego St. 13, Poland 2 Faculty of Medicine, University of Louis Pasteur in Strasbourg, Blaise Pascal St. 4, 67000, Strasbourg, France

Abstract

Staphylococcus aureus bacteria produces leukocidins – biomponent toxins with destructive influence on mammalian leukocytes. The purpose of the studies was to determine the in vitro effects of leukocidin LukE/LukD on the phagocyte activity and proliferative ability of lymphocytes isolated from rabbit blood. The results have shown the modulatory influence of the toxin on the immunocompetent cells activity. Cytolytic concentrations of leukocidin induced immunosuppression of metabolic and potential killing activity of phagocytes. Subcytolytic concentrations of leukocidin caused stimulation of phagocyte activities. No modulatory influence on the proliferative ability of T and B lymphocytes was observed.

Key words: staphylococcal leukocidin, rabbit, phagocyte activity, lymphocyte proliferation

Introduction the metabolic activity (RBA) and potential killing activity (PKA) of polymorphonuclear (PMN) and Leukocidins (Luk) are bicomponent pore forming mononuclear (MN) cells and proliferative ability of exotoxins produced by Staphylococcus aureus bacteria. T and B lymphocytes isolated from rabbit blood. The toxins are water soluble and consist of two protein subunits of S and F class. The up-to-date studies indicate that gamma-hemolysins exhibit leukotoxic Materials and Methods and hemolytic effects, leukocidins have destructive influence on polymorphonuclear leukocytes, monocytes Leukocidin LukE+LukD was added at concentra- and macrophages isolated from higher vertebrates tions 25000, 5000, 1000, 200, 40, 8, 1.6, 0.32 ng/ml (Pedelacq et al. 1999). A few studies on the effect of RPMI 1640 medium and incubated 30 min. with the leukocidins on leukocytes isolated from higher verte- prepared lymphocyte suspensions. The metabolic ac- brates were performed but there is little data on com- tivity (RBA) of blood PMN and MN cells described parative in vitro studies on the influence of leukocidin by Chung and Secombes (1988) and potential killing LukE/LukD on rabbit lymphocytes. The purpose of activity (PKA) of PMN and MN cells described by the in vitro study was to determine the influence of Rook et al. (1995) were measured. The blood leukocidin LukE/LukD at different concentrations on T (stimulated by concanavalin A) and B (stimulated

Correspondence to: A. Bownik, e-mail: [email protected] 26 Bownik A., Siwicki A.K., Prevost G. by bacterial lypopolysaccharide LPS) lymphocyte pro- Discussion liferation was determined by MTT assay (Mosmann 1983). The data was analysed statistically using pro- Little is known on the in vivo local effects and ge- gram Statistica 5.0. P<0.05 was assumed as significant. neral role of bicomponent toxins produced by Means and standard deviations are presented in the Staphylococcus aureus in the pathogenesis of fish and figures (n=10). mammalian diseases. Frequent isolation of different strains secreting leukocidins from mammalian necrotic lesions and abscesses suggests that these bicom- Results ponent toxins could be possibly important virulence factors in fish. Leukocidin LukE/LukD secreted by The studies have shown the modulatory influence the Newman strain of Staphylococcus aureus is a bi- of leukocidin LukE/LukD on the activity of phago- component cytotoxin of molecular weight 32.2 kDa cytes isolated from rabbit blood. The highest sup- for S and 34.3 for F class protein subunits (Gravet et pression of RBA and PKA of PMN and MN cells was al. 1998). Some studies with the use of leukocidin observed at concentrations 25000, 5000, 1000 and 200 were performed on mammalian phagocytic cells. ng/ml of the toxin (Fig. 1). Slight but statistically sig- Siwicki et al. (2004) observed the modulatory influence of leukocidin LukE/LukD on metabolic activity and killing activity of canine macrophages. The results

0.18 of our in vitro studies have shown modulatory influ- * * * 0.16 * ence of leukocidin LukE/LukD on phagocyte activity 0.14 isolated from rabbit blood. High cytolytic concentra- * 0.12 * tions of leukocidin LukE+LukD diminished RBA * * 0.1 * and PKA of PMN and MN cells. The inhibitory effect 0.08 * * was a direct result of lysis of phagocytes induced by 0.06 * 0.04 leukocidin what was documented by cell viability re- 0.02 sults (25 000 ng/ml – 3%). Leukocidin induced pores

Optical density (OD-620 nm) 0 cell membrane of PMN and MN cells consequently Control 25000 5000 1000 200 40 8 1.6 0.32 Leukocidin concentration (ng/ml RPMI) caused malfunction of ion exchange and cell integrity. RBA PKA Gravet et al. (1998) also noted the leukolytic effect of leukocidin LukE/LukD on PMN cells in rabbit and Fig. 1. In vitro influence of leukocidin LukE/LukD on meta- showed that in vitro the toxin is less cytotoxic in com- bolic activity (RBA) and potential killing activity (PKA) of parison to other staphylococcal leukotoxins in that PMN and MN cells isolated form rabbit blood (Oryctolagus cuniculus) (mean + SD, p<0.05, n=10). animal species. Our in vitro studies revealed that T and B cells were not susceptible to modulatory influence of leukocidin LukE/LukD. Suppression of nificant stimulation of phagocytic RBA and PKA was lymphocyte proliferation was not seen even at the seen at subcytolytic concentrations (1.6, 0.32 ng/ml) of highest concentration. On the other hand studies per- the toxin LukE/LukD. No modulatory influence of formed by Siwicki et al. (2003) have shown im- leukocidin LukE/LukD on proliferative ability of munosuppressive influence of leukocidin LukE/LukD T and B lymphocytes was observed at any concentra- on the proliferative ability of canine blood lym- tion used in the experiment (Fig. 2.). phocytes. Lack of sensitivity of rabbit lymphocytes to leukocidin could be a result of presence of specific receptors inactivating the toxin or no specific receptor to bind the S component of leukocidin. Pfannenberg et al. (1975) demonstrated that partially purified leukocidin secreted by bovine P83 strain of S. aureus induced lysis of 25% of bovine lymphocytes in vitro but no morphological changes were seen during contrast – phase observation. Loeffler et al. (1986) using 51Cr release assay noted a slight cytotoxic influence of partially purified leukocidin produced by P83 S. aureus strain on bovine lymphocytes. The toxic effect of leukocidin on mammalian lymphocytes observed by these two authors could be a result of contamination with other staphylococcal products like alpha- Fig. 2. In vitro influence of leukocidin LukE/LukD on prolif- -hemolysin that normally induces destruction of lym- erative ability of T and B lymphocytes isolated from rabbit phocytes. We need more in vitro and in vivo studies on blood (Oryctolagus cuniculus) (mean + SD, p<0.05, n=10). the effects of other leukocidins using different animal In vitro effects ... 27 species that have not been tested yet to determine mama JP, Mourey L (1999) The structure of possible sensitive species and possibly work out a Staphylococcus aureus leucocidin component methods for certain types of leukaemia treatment. (LukF-PV) reveals the fold of the water-soluble species of a family of transmembrane pore forming toxins. Struc- ture 7: 277-287. References Pfannenberg T, Blobel H, Schaeg W (1975) Cytotoxic effects of a leukocidin from Staphylococcus aureus. Zentral Bak- Chung S, Secombes SJ (1988) Analysis of events occurring teriol 233: 147-152. within teleost macrophages during the respiratory burst. Rook GAW, Steele J, Umar Dockrel HM (1995) A simple Comp Biochem Physiol 89B: 539-544. method for the solubilization of reduced NBT and its use Gravet A, Colin DA, Keller D, Giradot R, Monteil H, as a colorimetric assay for activation of human macro- Prevost G (1998) Characterization of a novel structural phages by gamma interferon. J Immunol Meth member, LukE-LukD of the bicomponent staphylococcal 82: 161-167. leucotoxins family. FEBS Lett 436: 202-208. Siwicki AK, Bownik A, Prevost G, Szmigielski S, Małac- Loeffler DA, Schat KA, Norcross NL (1986) Use of 51Cr zewska J, Mikulska-Skupień E, (2003) In vitro effect of release to measure the cytotoxic effects of staphylococcal staphylococcal leukocidins (LukE, LukD) on the prolif- leukocidin and toxin neutralization on bovine leukocytes. erative responses of blood lymphocytes in dog (Canis J Clin Microbiol 23: 416-420. familiaris). Bull Vet Inst Pulawy 47: 395-401. Mosmann T (1983) Rapid colorimetric assay for cellular Siwicki AK, Bownik A, Prevost G, Szmigielski S, Małac- growth and survival: Application to proliferation and zewska J, Mikulska-Skupień E (2004) In vitro influence cytotoxicity assays. J Immunol Meth 65: 55-63. of staphylococcal leukocidins (LukE, LukD) on the activ- Pedelacq JD, Maveyraud L, Prevost G, Baba-Moussa L, ity of blood phagocytes in dogs. Wien Tierarztl Mechr Gonzalez A, Courcelle E, Shepard W, Monteil H, Sa- 91: 1-5. 28 Bownik A., Siwicki A.K., Prevost G. Polish Journal of Veterinary Sciences Vol. 7, No. 3 (2004), Supplement, 29-31

Short original article Increased expression of NADPH-d/nitric oxide synthase after facial axotomy; ultrastructural and light microscopic analysis

J. Całka, G. Wolf 1, J. Kaleczyc, K. Wąsowicz

Department of Animal Anatomy, Veterinary Faculty, University of Warmia and Mazury in Olsztyn, Oczapowskiego St. 14, 10-719 Olsztyn, Poland 1 Institute of Medical Neurobiology, University of Magdeburg, Magdeburg, Germany

Abstract

Four weeks after transection of the facial nerve in rats, NADPH-diaphorase (NADPH-d) histochemistry was performed to analyze the induction of nitric oxide synthase (NOS) in injured facial motoneurons. Light microscopic examination of the affected facial motor nucleus revealed a statistically significant increase in the total number of stained motor neurons with a significant increase in staining intensity. Electron microscopic analysis disclosed the NADPH-d precipitation associated with bands of the endoplasmic reticulum, nuclear envelope, Golgi apparatus and mitochondrial membranes.

Key words: facial nerve, axotomy, NADPH-diaphorase, light and electron microscopy

Introduction histochemistry for electron microscopy (Wolf et al. 1992), we investigated the ultrastructural distribution Recently it became apparent that NOS, the en- of NOS expressed in the neuronal compartment of zyme synthesizing nitric oxide (NO), is dynamically the facial nucleus after facial nerve transection. expressed under pathological conditions (Schmidt et al. 1995) implying a role for NO in response to neuronal damage. Light microscopical studies revealed an Materials and Methods increased NOS expression in motor (Yu 1994), sensory (Hokfelt et al. 1994) and neurosecretory (Hokfelt Animals. The experimental material consisted of et al. 1994) neurons following traumatic injury. In our eleven adult male Wistar rats (200 – 250 g) of our own study we applied light and electron microscopy for breeding stock. All animals were treated in accord- a comparative assessment of the intensity of ance to the “Principles of Laboratory Animal Care” NADPH-d expression in control and axotomized rat (NIH publication No. 86-23, revised 1985). Following facial motor neurons. While the application of the facial axotomy or sham operation the rats were allow- NADPH-d histochemical reaction at light microscopi- ed to survive twenty eight days. Four of them were cal level enabled cellular localization of NOS, the processed for light microscopy and the remaining ultrastructural location of the enzyme in facial mo- three for electron microscopy. The light microscopy toneurons reflecting the subcellular source of NO re- control group included two sham-operated animals mains still unclear. Therefore, while using NADPH-d sacrificed twenty eight days after operation and two

Correspondence to: J. Całka, tel.: +48 89-523-49-22, fax: +48 89-523-49-86, e-mail: [email protected] 30 Całka J. et al. intact rats. Animals were anesthetized with chloral hy- evident increase in the neuronal NADPH-d express- drate (400 mg/kg i.p.) and subjected to unilateral tran- ion on the axotomized side was noticed (Fig. 1b). section of the left facial nerve (Yu 1994). In Among the motoneurons intensely, moderately and sham-operated rats the facial nerve was exposed but weakly stained profiles randomly distributed through not transected. All the animals were perfusion fixed the area of the nucleus were observed. and processed for light and electron microscopic Electron microscopy. Electron microscopic NADPH-d histochemistry (Calka et al. 1994). examination of subcellular distribution of the Quantitative study. Cell counting was carried out NADPH-diaphorase activity in intact controls, on four representative sections per animal with appli- sham-operated as well as axotomized animals re- cation of a three grade scale for neurons: intensely, vealed apparent histochemical staining of the facial moderately and weakly stained. The total number of motoneurons. The fine BSPT formazan precipitate stained neurons as well as numbers of intensely, mod- labeled numerous mitochondria, bands of endoplas- erately and weakly stained cells were compared be- mic reticulum, nuclear envelope and membranes of tween the affected versus unaffected side. Statistical the Golgi apparatus as well (Fig. 2a, b). differences were analyzed using Student’s t test and Cell counts. Following the nerve transection, considered significant if p<0.05. there was a significant increase in number of intensely and moderately stained neurons on the affected side versus contralateral unaffected side. These changes Results were accompanied by a distinct, but not statistically significant, decrease in population of weakly stained Light microscopy. Facial nuclei of all control, neurons (Fig. 3) and a significant increase in the total sham operated as well as experimental animals con- number of stained motoneurons (Fig. 4) on the tran- tained NADPH-d-positive motoneurons (Fig. 1a). An sected side comparing to the contralateral side.

1a 1b

2a 2b

Fig. 1. NADPH-d-positive staining (arrows) in control unaffected (a) and axotomized contralateral facial motoneurons (b). x 60. Fig. 2. Electron micrographs (a, b) illustrating NADPH-d activity in axotomized facial motoneurons. Note the NADPH-d-labeled membranes of the Golgi apparatus (bold arrows), endoplasmic reticulum (arrowheads), mitochondria (empty arrows), and nuclear envelope (bended arrows). x 7000. Increased expression ... 31

Discussion

The present study demonstrates an increased expression of NADPH-d/NOS in facial motoneurons following axotomy. Although the functional implications of this phenomenon are unknown, earlier studies have shown that lesion-induced neuronal NOS expression is related to the death of injured neurons (Kristensson et al. 1994). Accordingly, inhibition of NOS could effectively reduce motoneuronal death (Wu et al. 1993) and promotes nerve regeneration (Zochonde et al. 1997). Recent reports indicate that nNOS activity is regulated by PSD-95 and CAPON (Che et al. 2000), and plays a pivotal role in regulation of synaptic plasticity and synaptogeneses. Thus, it is likely that axotomy-induced NOS expression observed in the present study might play an important role in the recovery of synaptic function. Our electron microscopical findings showing formazan precipitation in the nuclear envelope, endoplasmic reticulum, mitochondria and Golgi apparatus fully confirm light Fig. 3. Effect of facial axotomy on the relative number of microscopical observations on high NOS activity in NADPH-d-positive (intensely, moderately or weakly facial motoneurons. stained) neurons per section on both the axotomized and the unoperated contralateral side of the facial nucleus. Each group represents mean ±S.E.M. from 4 rats, 4 sections per animal. *Denotes significant difference (p<0.05) against the References corresponding unoperated side by Student’s t-test. AX-axotomized, CON-control group. Calka J, Wolf G, Brosz M (1994) Ultrastructural demonstration of NADPH-diaphorase histochemical activity in the supraoptic nucleus of normal and dehydrated rats. Brain Res Bull 34: 301-308. Che YH, Tamatani M, Tohyama M (2000) Changes in mRNA for post-synaptic density-95 (PSD-95) and car- boxy-terminal PDZ ligand of neuronal nitric oxide synthase following facial nerve transection. Mol Brain Res 76: 325-335. Hokfelt T, Ceccatelli S, Gustafsson L, Hulting AL, Verge V, Villar M, Xu XJ, Xu ZO, Wiesenfeld-Hallin Z, Zhang X(1994) Plasticity of NO synthase expression in the nervous and endocrine system. Neuropharmacology 33: 1221-1227. Kristenson K, Aldskogius M, Peng ZC, Olson T, Aldskogius H, Bentivoglio M (1994) Co-induction of neuronal interferon-gamma and nitric oxide synthase in rat motor neurons after axotomy: A role in nerve repair or death? J Neurocytol 23: 453-459. Schmidt W, Wolf G, Calka J, Schmidt HHHW (1995) Evi- dence for bidirectional changes in nitric oxide synthase activity in the rat striatum after excitotoxically (quinolinic acid) induced degeneration. Neuroscience 67: 345-356. Wu W, Li L (1993) Inhibition of nitric oxide synthase reduces motoneuron death due to spinal root avulsion. Neur- osci Lett 153: 121-124. Wolf G, Wurdig S, Schunzel G (1992) Nitric oxide synthase in rat brain is predominantly located at neural endoplasmic reticulum: an electron microscopic demonstration of NADPH-diaphorase activity. Neurosci Lett 147: 63-66. Fig. 4. Effect of facial nerve transection on the number of Yu WH (1994) Nitric oxide synthase in motor neurons after NADPH-d-labeled facial motoneurons. Values shown repre- axotomy. J Histochem Cytochem 42: 451-457. sent means ±S.E.M. of the number of stained neurons per Zochonde DW, Mistra M, Cheng C, Sun H (1997) Inhibi- section. *Denotes significant difference (Student’s t-test) as tion of nitric oxide synthase enhances peripheral nerve compared to unoperated control, p<0.05. regeneration in mice. Neurosci Lett 228: 71-74. 32 Całka J. et al. Polish Journal of Veterinary Sciences Vol. 7, No. 3 (2004), Supplement, 33-35

Short original article The pathological investigation and analysis of the chicks cadmium chloride intoxication

X. Dang, S. Tian, D. Tian1, Z. Huang

Southwest University for Nationalities, Department of Life Science and Technology, Xiamianqiao St. 21, 610041 Chengdu, P.R. China 1 Chongqing Surgery Hospital, Chongqing P.R. China

Abstract

In the experiment 216 one day old chicks were divided into 6 contrasting groups at random and 5 groups were fed on provender with cadmium chloride of 2 × 10-7,1× 10-6,2× 10-6,1× 10-5,2× 10-5 respectively for 60 successive days. The observation records the changes of the 5 groups chicks in clinic presentation, pathological anatomy and pathological histology. The results indicated that the cadmium chloride intoxication led to growth restriction, serious anemia and caused the pathological changes of the chicks; livers, galls, stomachs and thyroid glands, especially the thyroid glands of the chicks were seriously harmed.

Key words: chick, cadmium chloride intoxication, growth control, anemia, pathomorphology

Introduction and histopathological changes in laboratory. The pathological investigation is the important basis in The compounds of cadmium, e.g. CdO, CdS, clinic diagnose and poisoning prevention. This article

Cd(NO3)2, Cd(CN)2 and CdSO4, etc. are of high toxic- gives a brief report on the pathological changes as ity. People can be poisoned by electroplate industry, a result of the chicks cadmium chloride intoxication. the coloring manufacture of cadmium alloy, the elec- trode manufacturing of accumulator, the insecticide Materials containing the element and the semiconductor and plastic manufacturing contacted with Cd compounds Animals: 216 chicks, male, the average weight was when taking cadmium compounds by accident or in- 38.75 g, from hennery of HuaYang ShuangLiu county. haling. No matter what form of the cadmium com- Poison used in experiment: cadmium chloride, pound has been taken into the body, it can lead to ShangHai Tingxin chemical plant (GB1285-94); Pass poisoning, and the compounds will be distributed to mark: 990801. every organ of the entire body. They are distributed Apparatus used: Rotary Cutting Microtome, mainly to the liver, kidney, lung and hypothyroid, and American AO Corporation drainaged by kidneys. The compounds enter the body by intestine, and 85% of them drainage by intestine too (Shulin 1989). Poison can be classified as acute and Methods chronic. Elicited by some intangible death of chickens andducksinsomeregionsweinvestigatedtoxicitiesof After one day observation of the newly break-shell a series of metals and recorded the poisoning reaction- chicks they were divided into 6 groups at random and

Correspondence to: X. Dang, tel.: 86028-85522150, fax: 86028-85522628, e-mail: [email protected] 34 Dang X. et al. were fed on provender with cadmium chloride of nomena of poor standing. All parameters were get- 2 × 10-7,1× 10-6,2× 10-6,1× 10-5,2× 10-5 respectively. ting worse with time. The feeding hours were as follows: 8 a.m., 2 p.m. and The Cadmium chloride intoxication affected the 8 p.m. without limit and with unlimited access to growth and the weight of chicks, the p<0.001. How- water every day. The weight of the chicks was es- ever, the fourth group showed the stimulating growth timated before adding feedstuffs at 8 a.m. Three phenomena.Theaverageweightofthefirstgroup chicks from each group were executed after picking was 282.29 g, the second one was 246.67 g, the third blood sample every five days. Then they were dissec- onewas208.33g,theforthonewas262.85g,thefifth ted and pathological changes were recorded. Samples one was 159.33 g on the thirtieth day; and on the of liver, spleen, intestine, proventriculus, gizzard, thy- sixtieth the average weight was 396.19 g (the first roid, lymph nodes were fixed by 10% formalin sol- group), 346.19 g (the second group), 292.39 g (the ution, cut by paraffin sectioning and stained by hae- third group), 368.39 g (the forth one), the fifth one matoxylin and eosin, then examined under the micro- was 222.22 g. The percent of dynamic restrain is scope. The hemoglobin (Hb) was determinated by Sa- showninFig.1. hli method, and the red blood cell packed volume was The effect of cadmium on the MCV (mean cor- measured by the Modified Wintrobe method (Minis- puscular volume) of RBC (red blood cell), on the try of Health P. R.China 1991). MCH (mean corpuscular hemoglobin) of RBC, on the MCHC (mean corpuscular hemoglobin concentration) and on the RBC packed volume is shown Results in Table1. The content of Hb in 100 ml blood is 12.6 g of the first group, 11.63 g of the second one, Clinical Presentation. After feeding on proven- 11.88 g of the third one, 10.93 g of the fourth one, der with addition of cadmium chloride a large number and 9.43 g of the fifth one on twenty day. After the of chicks showed low spirit, aversion to cold and poor sixtieth day, the content of Hb in 100 ml blood is 12.0 appetite after twelve hours. The observed effects were g of the first group, 10.56 g of the second one, 9.82 dependent on the quantity of provender. The results g of the third one, 8.63 g of the fourth one, 7.95 g of of accumulation were distinct in different experimental groups. In the sixth group accidental death had the fifth one, respectively. taken place at the fifth day and all chicks were dead Macroscopical lesions. The necropsy showed before the eight day. In the fifth group color lighten- that5%chicksdidnotabsorbtheyelkandhadpu- ing, blowzy feather, hoar and watery faeces were ob- trescence phenomena. The part of mucous mem- served. These phenomena were more distinct at the brane of gizzard broke off, necrosed and the twenty-fifth day, the beaks of the chicks poi- cholecyst got bigger. The color of thyroid in other soned were white and the faeces were hoar and wat- poisoned groups got lighter, and yellowish. Liver, ery. The fifth group of the chicks presented the phe- spleen and proventriculus were congested and the mucous membrane of the proventriculus fell off eas- 50 ily; the small intestine bloated, and the mucous mem- 45 brane disengaged; the liver and gall got bigger obvi- 40 35 ously and the gall took on dark green, the concentra- 30 tion of it was thicker than that of the control group. 25 Thebloodofthemostchickswasthick,whilethatof 20 15 few was thin. With the poisoning time, the corneal 10 layer of gizzard began to fall off on the fiftieth day 5 0 and gizzard began to have nodular focal necrosis -5 10 20 40 60 with calcification. After twenty days, all five groups -10 -15 showed weakness of legs. -20 Microscopical lesions. Blood smears from ter- -25 minally affected chicks showed that RBC was distor- -30 tional and leucocyte membrane was dissolved. In ad- group 1 group 3 dition we observed necrotic lesions in the thyroid and group 2 group 4 the proventricular mucosa etc. The experiment group 5 showed extensive necrosis in thyroid, superficial necrosis in mucous membrane of proventriculus; focal Explanation: the abscissa is time and the y-axis is the percent necrosis in gizzard. The jejunum, colon and rectum of restrain. The percent of restrain = (100-testing group/ congested in different degrees, and the spleen con- contrast group) × 100%. gested lightly, the liver congested, hepatocytes nec- Fig. 1. The influence of cadmiun chloride on the weight of rosed in portal zone of the hepatic lobules, the others chickens. atrophied, vacuolized and deteriorated. The pathological investigation ... 35

Table 1. The effect of cadmium on blood paramenters.

Group MCV(fl) MCH(pg) MCHC(g/L) RBC packed volume (%) 1 84.53 ± 3.93 30.15 ± 1.36 356.63 ± 18.96 34.49 ± 1.63 2 83.74 ± 3.12 28.46 ± 2.13 339.87 ± 19.26 32.66 ± 0.98 3 74.43 ± 3.31 25.59 ± 1.56 343.79 ± 20.96 31.56 ± 0.89 4 92.22 ± 3.03 26.15 ± 1.46 325.46 ± 30.16 30.05 ± 1.36 5 82.12 ± 3.26 20.69 ± 1.28 307.07 ± 23.63 28.30 ± 1.32

Explanation: MCV – mean corpuscular volume of RBC, MCH – mean corpuscular hemoglobin of RBC, MCHC – mean corpuscular hemoglobin concentration of RBC, RBC is red blood cell.

Discussion and rotted, produced a great deal of gas and lactic acid etc, leading to the phenomena of small The histopathological examination of cadmium intestine bloat. It harmed stomach and intestine, and chloride poisoned chicks revealed that the cadmium impairsed the absorption of iron. Second, stomach chloride intoxication will lead to growth restriction and intestine were harmed by cadmium chloride in (Shulin 1989). There are four main reasons. First, different degrees, resulting in hemorrhagic cadmium chloride harms thyroid gland of the chicks gastro-enteritis and petechiae in stomach and intes- and reduces the thyroxin coming from it, which re- tine of chick, which caused the reduction of iron im- stricts the growth of the chicks. Second, it can harm bibed. Third, cadmium chloride itself harmed blood the enteron of the chicks and impair the chicks imbi- corpuscle, eg. througn blood corpuscle and leucocyte bing alimentation. Third, the CdCl canrestraincal- 2 distortion, and the leucocyte membrane breaking cium imbibed by the chicks and accelerate venting of and dissolving. calciumfromthechick,whichalsoaffectsthegrowth Besides, the cadmium conpounds restricted the of chicks (Zhang 1994). Fourth, the compound can growth of animal and caused anemia probobaly be- harm liver and gall of the chicks. The cadmium causetheywererelatedtothesecretionofgrowth chloride can make excreting gall abnormal and cause hormone and thyroxin and they influenced the mar- bad digestion, and affects the growth of the chick. row cell. Cadmium compounds restrained the process The mechanism of the cadmium chloride stimulating of oxidatine phosphorylation in the mitochondria of the growth of the chick individually is under dis- hepatocytes. They influenced the activity of the en- cussion. zymes such as amino acid decarboxidase, histidine en- Cadmium chloride decreased the content of he- zyme, perocidare, dehydrogenase resulting in the moglobin in 100 ml blood of the chick remarkably < mis-function of tissue metabolism. This mechanism is (p 0.01) leading to anemia. As it did not affect the under the study. red blood count of chick obviously (p>0.05), the cause of anemia was lack of iron. MCV and MCH < decreased obviously (p 0.05), the PBC packed vol- References ume decreased and MCHC did not change remarkably. It was shown that the volume of the red blood Shulin L (1989) Toxicology and Analysis of Poison [M] Beij- cell was diminished and the content of Hb was de- ing: People’s Medical Publishing House, pp. 564. creased obviously, which was the signal of hypo- Ministry of Health P. R.China (1991) The Operation Regu- chromic microcytic anemia. The results of pathologi- lations of Clinic Inspection in China [M] Nanjing: South- cal analysis showed that the main reason of anemia east University Publishing House, pp. 26-29. had three points. First, the peptic glands of the Neimenggu Agriculture College, South China Agriculture College (1997) Cattle Pathology [M] Beijing: Agriculture chicks were harmed causing a decrease in secretion Publishing House, pp. 238. of both gastric juice and gastric acid, which resulted in Zhang Q (1994) The Whole Feed Additive [M] Beijing: the slowing of the motor activity of the stomach. This Beijing University Of Technology Publishing House, pp. caused the food stagnating in stomach fermented 126. 36 Dang X. et al. Polish Journal of Veterinary Sciences Vol. 7, No. 3 (2004), Supplement, 37-39

Short original article Changes in density of sympathetic nerve terminals and steroidogenic activity of porcine ovaries after dexamethasone-induced polycystic ovarian syndrome

A. Dzienis, M. Majewski1, J. Wojtkiewicz1, B. Jana

Institute of Animal Reproduction and Food Research of the Polish Academy of Sciences, Division of Reproductive Endocrinology and Pathophysiology, Olsztyn, Poland 1 Division of Clinicial Physiology, Department of Functional Morphology, Faculty of Veterinary Medicine, University of Warmia and Mazury, Oczapowskiego St. 13, 10-717 Olsztyn, Poland

Abstract

To establish the role of sympathetic nerves in the etiopathogenesis of ovarian cysts, we studied morphology and steroidogenic activity of the porcine ovaries after dexamethasone-induced polycystic status. In polycystic-changed ovaries, an increase in the density of TH/DβH- and/or NPY-IR nerve terminals were found; this was paralleled by significant changes in the content of progesterone,

androstedione and estradiol-17β, as well as in the expression rate of cytochromes P450scc and P450arom in the cysts and corpora lutea. These results suggest that sympathetic neurons supplying ovaries may participate in the etiopathogenesis of the ovarian cysts in gilts.

Key words: polycystic ovarian syndrome, ovary, neuropeptide Y, noradrenaline, steroidogenesis, dexamethasone, pig

Introduction associated with a derangement of the sympathetic control of the ovary. It is generally assumed that cystic ovarian disease (COD) is caused mainly by disturbances in the function hypothalamic-pituitary-ovarian axis, causing im- Materials and Methods pairment of the synthesis, release, and/or storage of various hormones of this functional unit. However, it The principles of animal care (NIH publication has also been suggested that an alternation in the ac- No. 86-23, revised 1985) as well as the specific nation- tivity of the sympathetic neurons innervating the al law on the protection of animals were followed. In ovary may contribute to the etiology and/or pro- this experiment 12 crossbred adult (7-8 months) gilts gression of cysts in women (Nakamura 1990) and rats were used. Animals were randomly assigned to one of (Parades et al. 1998). Therefore, we decided to study two groups: I, experimental (n=6) and II, control the possibility that cysts formation in gilts may be (n=6). In the group I, cysts were

Correspondence to: A. Dzienis, e-mail: [email protected] 38 Dzienis A. et al. induced by injections of dexamethasone (DXM; Dex- Results and discussion asone, Norbrook Lab., Newry, UK, 3.3 µg/kg b.m., in total volume of 6 ml, i.m.), every 12 h on days 7-21 of The ovaries of the control animals were heavier the first studied estrous cycle. Using the (p<0.05) than those after DXM administration same paradigm, the gilts of group II received 6 ml of (9.8 ± 0.9 vs. 4.3 ± 0.6 g; respectively). In both the con- saline. On day 11 of the next cycle, gilts were killed by trol and DXM-treated groups, the mean number of electrical shock and exsanguination. Afterwards, the follicles with diameter 1–3mmandCLwere not ovaries were dissected out, weighted, and the number significantly different (13.8 ± 2.5 vs. 7.75 ± 0.9 and of follicles, corpora lutea (CL) as well as cysts was 9.33 ± 1.0 vs. 5.5 ± 2.1; respectively). In the ovaries of counted. 10-µm-thick cryostat ovarian sections were the gilts treated with DXM, the follicles measuring stained by means of double-immunofluorescence to 3 – 6 mm in diameter were not found, while 2.2 ± 0.4 investigate the number and the distribution pattern of of follicular cysts, 1 – 3 cm in diameter, was encoun- tyrosine hydroxylase (TH), dopamine-β-hydroxylase tered per ovary. An increase in the number of (DβH) and/or neuropeptide Y-immunoreactive TH/DβH- and/or NPY-IR nerves was observed in the (NPY-IR) nerve fibers. The amount of progesterone vicinity of the follicles (Figs. 1b, 2b), CL, cysts (Fig. β (P4), androstendione (A4) and estradiol-17 (E2) was 2b) and blood vessels (Fig. 3b) in the experimental estimated by RIA in tissue probes taken out from CL, group, when compared to control animals (Figs. 1a, follicular and cystic wall, as well as in follicular and 2a, 3a). DXM injections lead to an increase (p<0.05) cystic fluids. Expression of cytochromes P450scc and in the mean content of P4 and a decrease of A4 and E2 P450arom in ovarian structures was determined by (p<0.05) in the cystic fluid, compared to 3 – 6 mm

Western blot. Immunoblots were quantitated by follicles in control group (P4: 347.2 ± 16.8 vs. scanning on Kodak 1D Image Analysis Software 258.1 ± 17.5 ng/ml; A4: 5.7 ± 1.3 vs. 24.3 ± 4.2 ng/ml; ± (USA). The mean ( SEM) weight of the ovaries and E2: 2.8 ± 1.7 vs. 10.5 ± 1.5 ng/ml; respectively). The the number of ovarian structures was estimated per concentrations of P4,A4 and E2 in the cystic wall were ovary, both in DXM-treated and control groups. Ob- higher (p<0.05 – 0.001) than those found in these fol- tained data were subjected to one-way analysis of vari- licles of control gilts (P4: 3398.7 ± 239.4 vs. ance (ANOVA). 1266.1 ± 234.2 ng/g of tissue; A4: 277.5 ± 1.5 vs.

4.2 ± 0.9 ng/g of tissue; E2: 7.8 ± 1.6 vs. 2.7 ± 1.4 ng/g

Fig. 1. Cortex ovarii,DβH-IR nerve terminals running in a close neighborhood of primary and primordial follicles in intact (a) and DXM-treated animal (b). x 200. Fig. 2. Cortex ovarii, noradrenergic nerve fibres running in the vicinity of follicles in control group (a) and a small cyst (b). F – follicle, C – cyst. x 200. Fig. 3. DβH-IR perivascular nerve fibres supplying intraovarian artery in control (a) and DXM-treated animal (b). A – artery. x 200. Changes in density ... 39 of tissue; respectively). In CL of the gilts receiving these structures. However, as there is a lack of data

DXM, an increase (p<0.001) in content of P4 and A4 dealing with the influence of NA on the content/ac- was observed, when compared to that detected in tivity of this enzyme, the relevance of this finding control group (P4: 70038 ± 1285 vs. 6521 ± 1664 ng/g remains obscure yet. Increased E2 concentration in of tissue; A4:45.1± 3.7 vs. 1.2 ± 0.2 ng/g of tissue; the polycystic ovaries can be explained by the stimu- respectively). Western blot analysis demonstrated lating effect NA on the production of P4 and A4 (pre- that the expression of cytochromes P450scc and cursors for estrogens), which was also elevated. In-

P450arom in CL of DXM-treated animals was higher jections of DXM resulted in a decrease in the A4 and

(130% and 104.7% of control values, respectively). E2 concentrations in the cystic fluid. This observation

In the cystic wall, the staining intensity of the P450scc was in agreement with earlier study in which a reduc- band was lowered (by 8.2%), whereas intensity of tion of androgens and estrogens amounts in the cys- cytochrome P450arom staining was enhanced by 7.9%, tic fluid in gilts was also observed (Babalola et al. as compared to the 3 – 6 mm follicles of control gilts. 1990). The present study revealed that in the poly- Our study revealed that the formation of ovarian cystic-changed ovaries in gilts an increase in the cysts caused a decrease in the ovarian weight, what number of the sympathetic nerve terminals was asso- correspond well with earlier observations in rats with ciated with changes of the steroidogenic activity, COD (Lara et al. 1993). In ovaries with DXM-in- what indicates the important role of the sympathetic duced polycystic status, a dramatic increase in the neurons in the etiology and/or progression of ovarian number of TH/DβH- and/or NPY-IR nerves was ob- cysts. served in close vicinity of follicles, CL, cysts and blood vessels. Increased density of sympathetic nerves, paralleled by elevated content of noradrenaline (NA) was References found in the polycystic-changed ovaries of women (Nakamura 1990) and rats (Lara et al. 1993). Thus, it Babalola GO, Shapiro BH (1990) Sex steroid changes in porcine cystic ovarian disease. Steroids 55: 319-324. appears possible that the increase in the P4,A4 and E2 contents in the cystic wall and CL, observed in the Kotwica J, Bogacki M, Rękawiecki R (2002) Neural regulation of the bovine corpus luteum. Domest Anim Endoc- present experiment, may be a consequence of the in- rinol 23: 299-308. creased density of sympathetic intraovarian nerves. It Lara HE, Ferruz JL, Luza S, Bustamante DA, Borges Y, was reported that NA stimulated the P4 synthesis in Ojeda SR (1993) Activation of ovarian sympathetic the porcine granulosa (Wiesak et al. 1990) and bovine nerves in polycystic ovary syndrome. Endocrinology luteal cells (Kotwica et al. 2002). Moreover, express- 133: 2690-2695. ion of cytochrome P450scc in the CL was considerably Nakamura Y (1990) Treatment of polycystic ovary syn- increased in ovaries investigated in the present study. drome: an overview. Horm Res 33 (Suppl. 2): 31. It has been shown that NA activated cytochrome Parades A, Galvez A, Leyton V, Aravena G, Fiedler JL, Bustamante D, Lara HE (1998) Stress promotes devel- P450 in the bovine luteal cells (Kotwica et al. 2002). scc opment of ovarian cysts in rats: the possible role of sym- In the present study, in the gilts treated with DXM, an pathetic nerve activation. Endocrine 8: 309-315. increase in the E2 accumulation in the cystic wall and Wiesak T, Przała J, Muszynska A, Hunter MG (1990) Effect CL was associated with a higher content of P450arom in of catecholamines and FSH on progesterone secretion by pig granulosa cells. Endocrinol Exp 24: 449-456. 40 Dzienis A. et al. Polish Journal of Veterinary Sciences Vol. 7, No. 3 (2004), Supplement, 41-43

Short original article The study on the NADPHd-positive innervation of the porcine mammary gland

A. Franke-Radowiecka, J. Całka, K. Wąsowicz, P. Podlasz, J. Kaleczyc

Department of Functional Morphology, Division of Animal Anatomy, Faculty of Veterinary Medicine, University of Warmia and Mazury in Olsztyn, Oczapowskiego St. 13, 10-719 Olsztyn, Poland

Abstract

The origin and distribution of NADPH-diaphorase-(NADPHd)-positive nerve fibres supplying the porcine mammary gland have been investigated. Retrograde tracing studies have revealed that NADPHd-positive innervation of the porcine last abdominal mamma originates from L1-L4 DRG. The NADPHd-positive nerves were found in the dermis of the nipple, with single nerve terminals penetrating into the epidermis. Smooth muscle cells (SMC) located in the nipple were supplied with few NADPHd-positive fibres. Small arteries of this region received abundant NADPHd innervation, while large arteries, veins and lactiferous ducts (LD) possessed relatively weaker supply. Similarly, the parenchyma was provided with moderate number of NADPHd-positive nerve fibres which surrounded SMC, blood vessels and LD.

Key words: NADPH-diaphorase, neurons, retrograde tracing, mammary gland, pig

Introduction Materials and Methods

Accumulating evidence suggests that NO may par- Three sexually immature female pigs (3 months, 50 ticipate in the regulation of morphological and func- – 55 kg body weight) of the Large White Polish breed tional features of the mammary gland (Onoda and were anaesthetized with sodium pentobarbital (Vet- Inano 1998). Although, application of the NADPHd butal, Biovet, Poland; 20 mg/kg, i.v.). The retrograde histochemistry revealed NOS-positive, non-neuronal tracer FB was injected into the nipple (10 µl) and structures in the mammary gland of human (Thomsen parenchyma (20 µl) of the right last abdominal 1995), goat (Lacasse et al. 1996), cow (Lacasse et al. mamma. After three weeks, the animals were reanaes- 1996) and rat (Onoda and Inano 1998) there is a com- thetized and transcardially perfused with 4% buffered plete lack of information on the NO-ergic innervation paraformaldehyde solution (pH 7.4). Subsequently, of the gland. Therefore, while applying Fast Blue the right and left last abdominal mamma, and DRG (FB) retrograde tracing method and NADPHd his- including bilateral thoracic, lumbar and sacral ganglia tochemistry, the origin and distribution of NOS nerve were dissected out. The tissues were postfixed and supply in the porcine mammary gland were the objec- transferred into 18% buffered sucrose solution tive of this study. (pH 7.4). The samples were cut into 10 µm thick

Correspondence to: A. Franke-Radowiecka, tel.: +48 89 523-37-33, fax: +48 89 523-49-86, e-mail: [email protected] 42 Franke-Radowiecka A. et al. cryostat sections and processed for NADPHd his- The parenchyma was supplied with moderate tochemical staining procedure (Scherer-Singler et al. number of NADPHd-positive nerve fibres. SMC 1983). located in the parenchyma were poorly innervated by these nerves (Fig. 2). Arteries were supplied with moderate number of NADPHd-positive nerve fibres Results that were mostly longitudinally arranged and some of them formed bundles. Veins were supplied with only Generally, the porcine mammary gland was moder- single NADPHd-positive nerves. Some ately supplied with NADPHd-positive nerve fibres. NADPHd-positive processes were observed around The most abundant distribution of NADPHd-positive the LD and single fibres penetrated into the epi- nerve fibres was observed in the dermis of the nipple thelium of the ducts. (Fig. 1). These fibres occurred singly or in fascicles. Retrograde tracing studies have revealed that Single nerve terminals penetrated into the epidermis some DRG neurons projecting to the last right ab- as well. SMC located in the nipple were supplied with dominal mamma were NADPHd-positive (Fig 3a, b). few NADPHd-positive nerve fibres running between These neurons were located in right L1-L4 DRG. and in parallel to myocytes. Small arteries of the nipple were abundantly innervated by the nerves; however, less numerous NADPHd-positive fibres Discussion were associated with larger arteries. Veins were also supplied with few NADPHd-positive nerves which This is the first study reporting on the were arranged mostly along the vessels. Additionally, NADPHd-positive innervation of the porcine mam- some NADPHd-positive nerves were encountered mary gland. Although, Thomsen et al. (1995) ob- around the LD. served NOS-I activity in vascular endothelial and my-

1 2

3a 3b

Fig. 1. Single NADPHd-positive nerve fibre (arrow) located in the dermis of the nipple of the porcine mammary gland. x 400. Fig. 2. NADPHd-positive nerve fibres (arrows) associated with smooth muscle cells in the parenchyma of the porcine mammary gland. x 200. Fig. 3. FB-positive DRG neuron (arrow) (a) projecting to the porcine mammary gland. The same neuron in a consecutive section display NADPHd activity (arrow) (b). x 200. The study on the NADPHd-positive ... 43 oepithelial cells of human breast cancer and normal References tissue, there is no data on expression of NOS in the neural supply of the gland. Additionally, NADPHd Bredt DS, Hwang PM, Snyder SH (1990) Localization of activity was revealed in the secretory epithelium of nitric oxide synthase indicating a neural role for nitric cow and goat mammary gland, but no specific staining oxide. Nature 347: 768-70. was obtained using antibodies to NOS-I (Lacasse et Franke-Radowiecka A (2003) Distribution and chemical al. 1996). On the other hand, Onoda and Inano (1998) coding of dorsal root ganglia (DRG) neurons supplying not only revealed the NOS-I in the epithelial cells of the porcine last abdominal mamma. Annals of Anatomy the rat mammary parenchyma, but also proved ident- 185: 242-243. ity of the NOS-immunocytochemical staining with Hope BT, Michael GS, Kinigge KM, Vincent SR (1991) NADPHd histochemistry. Neuronal NADPH diaphorase is a nitric oxide synthase. Application of the retrograde tracing method re- Proc Natl Acad Sci USA 88: 2811-4. vealed that neural perikarya supplying the mammary Lacasse P, Farr VC, Davis SR, Prosser CG (1996) Local secretion of nitric oxide and the control of mammary gland originate from the right L1-L4 DRG and some blood flow. J Dairy Sci 79: 1369-74. of them are NADPHd-positive. These results fully Onoda M, Inano H (1998) Localization of nitric oxide syn- correspond to the former findings dealing with the thase and nitric oxide production in the rat mammary distribution and chemical coding of DRG neurons gland. J Histochem Cytochem 46: 1269-78. supplying the porcine mammary gland (Franke-Ra- Scherer-Singler U, Vincent SR, Kimura H, McGreer EG dowiecka 2003). The acquired data concerning the (1983) Demonstration of a unique population of neurons origin of NADPHd innervation of the gland indicate with NADPH-diaphorase histochemistry. J Neurosci that it may be sensory in nature; however, the dis- Methods 9: 229-234. tribution of the fibres may suggest its role in motor Thomsen LL, Miles DW, Happerfield L, Bobrow LG, function and control of the local blood flow in the Knwles RG, Moncada S (1995) Nitric oxide synthase ac- mammary gland, as well. tivity in human breast cancer. Br J Cancer 72: 41-44. 44 Franke-Radowiecka A. et al. Polish Journal of Veterinary Sciences Vol. 7, No. 3 (2004), Supplement, 45-47

Short original article Proliferative enteropathy-induced de novo synthesis of NPY in intramural ganglia neurons of the porcine descending colon

S. Gonkowski, J. Wojtkiewicz, A. Bossowska, J. Kaleczyc1, J. Całka1, M. Majewski

Division of Clinical Physiology and 1Animal Anatomy, Department of Functional Morphology, Faculty of Veterinary Medicine, University of Warmia and Mazury, Oczapowskiego St. 13, in 10-717 Olsztyn, Poland

Abstract

This study investigated the influence of proliferative enteropathy (PE) on the distribution pattern of NPY-immunoreactive neurons in the descending colon. NPY was present in 0.3 ± 0.3%, 0.5 ± 0.3% and 0.7 ± 0.1% of neurons in the myenteric (MP), outer submucous (OSP) and inner submucous (ISP) plexus in control animals, respectively. In PE-influenced colon, NPY was found in 2.9 ± 0.7%, 1.1 ± 0.1% and 18.4 ± 2.7% of MP, OSP and ISP neurons, respectively. Furthermore, NPY-IR neuroendocrine-like cells were observed in the colonic epithelium. The increase in the number of NPY-IR ISP neurons/intraepithelial neuroendocrine-like cells may suggest a neuro- and/or epithelioprotective role of NPY during chronic colitis.

Key words: proliferative enteropathy, neuronal plasticity, neuropeptide Y, enteric neurons, pig.

Introduction bowel were found to contain this peptide (Brown and Timmermans 2004). Thus, it is widely accepted that in Neuropeptide Y, a 36 amino-acid residues-long the pig, the vast majority of NPY-IR nerve fibres brain-gut neuropeptide (Tatemoto et al. 1982) is within the wall of the small and large intestine are of thought to be one of the most important peptidergic extrinsic origin (for review, see Brown and Timmer- transmitters in both the sympathetic (Lindh et al. mans 2004). However, in a preliminary experiment 1989, Klimaschewski et al. 1996) and parasympathetic concerning inflammation-induced plasticity of enteric nervous system (O’Donohue et al. 1985). On the neurons, we have unexpectedly encountered quite nu- other hand, under physiological conditions, it is vir- merous population of NPY-IR cells within the inner tually absent from sensory (Weihe 1990) and intrinsic submucous plexus. It should be noted that NPY has enteric neurons. It should be stressed, however, that been proposed as a potent neuroprotective/neurop- while NPY was observed in guinea-pig inhibitory cir- roliferative agent (Hansel et al. 2001). Therefore, we cular muscle motor neurons with short projections decided to study the dynamic of its presence in neur- and in descending interneurons subserving local re- ons/nerve fibres in the porcine proliferative en- flexes (Furness 2000), no neurons in the porcine teropathy (PE)-affected descending colon.

Correspondence to: M. Majewski, tel.: +48 89 523-44-60, fax: +48 89 523-39-82, e-mail: [email protected] 46 Gonkowski S. et al.

Materials and Methods antibody conjugated to FITC (Jackson Im- munochemicals, USA). Single-immunostained Six immature female pigs (approximately 8 weeks perikarya were evaluated under Olympus BX51 old), three control (C group) and three with clinically microscope equipped with epi-fluorescence and ap- diagnosed Lawsonia intracellularis infection (PE propriate filter sets, counted in each ganglionated group) were used. The animals were housed and plexus ([i.e., the myenteric – (MP), outer submucous treated in accordance with the rules approved by the – (OSP), and inner submucous – (ISP) plexus]), found local Ethical Commission. All the animals were sac- in the section studied (6 sections per animal; only rificed with an overdose of sodium pentobarbital neurons with clearly visible nucleus were included) (Vetbutal, Biowet, Poland; 90 mg/kg b.w.) and per- and presented as mean ± SEM. Pictures were cap- fused transcardially with freshly prepared 4% parafor- tured by a digital camera connected to a PC, analyzed maldehyde in 0.1 M phosphate buffer (pH 7.4). In with AnalySIS software (version 3.02, Soft Imaging order to exclude any putative cross-reactivity with System, FRG) and printed on a wax printer (Phaser other antibodies, 10 µm thick cryostat sections of the 8200, Xerox, USA). descending colon wall were subjected to routine single-labelling immunofluorescence, using well-char- Results and Discussion acterized polyclonal antiserum raised in rat and directed towards NPY (Affinity, UK,; 1:240). Primary NPY was present in 0.3 ± 0.3%, 0.5 ± 0.3% and antiserum was visualized by species-specific secondary 0.7 ± 0.1% of neurons in the MP, OSP and ISP plexus

Fig. 1. Expression of NPY in control and PE-influenced ISP (arrows). Note the de novo synthesis of NPY in a number of ISP neurons in pigs suffering from adenomatosis. x 200. Fig. 2. De novo synthesis of NPY in neuroendocrine cells of porcine PE-influenced colon (arrows). x 200. Proliferative enteropathy-induced ... 47 in the control animals, respectively, what corresponds References well with previous studies (Timmermans et al. 1992). On the contrary, in PE-influenced colon, NPY was Brown DR, Timmermans JP (2004) Lessons from the por- found in 2.9 ± 0.7%, 1.1 ± 0.1% and 18.4 ± 2.7% of cine enteric nervous system. Neurogastroenterol Motil MP, OSP and ISP neurons, respectively (Fig. 1). Thus, 16: 50-54. the results obtained clearly suggest that this chronic, Furness JB (2000) Types of neurons in the enteric nervous proliferative, bacteria-driven inflammation is not only system. J Auton Nerv Syst 81: 87-96. Hansel DE, Eipper BA, Ronnett GV (2001) Neuropeptide able to induce the de novo synthesis of neuropeptide Y functions as a neuroproliferative factor. Nature 410: Y by a subset of enteric neurons, but also accom- 940-944. plishes this in a plexus-depending way. Interestingly, Klimaschewski L, Kummer W, Heym C (1996) Localization, this process was neither accompanied by a boost in regulation and functions of neurotransmitters and neur- the density of intraganglionic or intramuscular omodulators in cervical sympathetic ganglia. Microsc Res NPY-IR nerve terminals nor by an increase in the Tech 35: 44-68. number of such coded nerve fibres in the submucosal Lindh B, Lundberg JM, Ho¨kfelt T (1989) NPY-, galanin-, and mucosal layers. Hence, this may further, albeit VIP/PHI-, CGRP- and substance P-immunoreactive neuronal subpopulations in cat autonomic and sensory indirectly, substantiate the hypothesis of neuroprotec- ganglia and their projections. Cell Tissue Res 256: tive function of NPY in challenged ISP neurons. 259-273. Moreover, NPY-IR neuroendocrine-like cells were O’Donohue TL, Chronwall BM, Pruss RM, Mezey E, Kiss also observed in the colonic epithelium (Fig. 2), an JZ, Eiden LE, Massari VJ, Tessel RE, Pickel VM, event not reported in normal counterparts studied. DiMaggio DA, Hotchkiss AJ, Crowley WR, Zukowska Thus, an elevation in the number of NPY-IR ISP Grojec Z (1985) Neuropeptide Y and peptide YY neur- neurons/intraepithelial neuroendocrine-like cells may onal and endocrine systems. Peptides 6: 755-768. be suggestive for an additional epithelioprotective Tatemoto K, Carlquist M, Mutt V (1982) Neuropeptide Y – novel brain peptide with structural similarities to (epithelioproliferative?) role of NPY during chronic peptide YY and pancreatic polypeptide. Nature 296: colitis. 659-660. Timmermans JP, Scheuermann DW, Stach W, Adriaensen D, De Groodt-Lasseel MHA (1992) Functional morphol- Acknowledgements ogy of the enteric nervous system with special reference to large mammals. Eur J Morphol 30: 113-122. Supported by KBN, grant no. 6P06K 012 20. The Weihe E (1990) Neuropeptides in primary afferent neurons. authors are indebted to Ms. A. Piękos and Mr. Z. In: Zenker W, Neuhuber WL (eds) The primary afferent neuron. Plenum Publishing Corp., New York, pp. Liminowicz for their excellent technical help. 127-159. 48 Gonkowski S. et al. Polish Journal of Veterinary Sciences Vol. 7, No. 3 (2004), Supplement, 49-51

Short original article Co-incidence pattern of SP and CGRP in neural structures of the porcine descending colon affected by proliferative enteropathy

S. Gonkowski, J. Wojtkiewicz, A. Bossowska, J. Kaleczyc1, W. Sienkiewicz1, M. Majewski

Division of Clinical Physiology and 1Animal Anatomy, Department of Functional Morphology, Faculty of Veterinary Medicine, University of Warmia and Mazury, Oczapowskiego St. 13, 10-717 Olsztyn, Poland

Abstract

In control animals, SP-IR neurons constituted 3.7 ± 0.5%, 3.4 ± 0.4% and 6.6 ± 1.1%, while CGRP-IR perikarya amounted to 1.6 ± 0.2%, 1.1 ± 0.4% and 1.2 ± 0.5% of all neurons in myenteric (MP), outer (OSP) and inner submucous plexus (ISP), respectively. Co-localization was found in 26.1 ± 3%, 39.3 ± 10.7% and 23.3 ± 0.2% of all MP, OSP and ISP perikarya, respectively. In animals suffering from PE, SP-IR neurons contributed to 7.1 ± 0.8%, 9.1 ± 0.6% and 22.2 ± 1.6%, while CGRP-IR formed 4.0 ± 0.0%, 4.0 ± 0.8% and 11.7 ± 0.5% of all neurons found in MP, OSP and ISP, respectively. Co-localization was observed in 7.7 ± 0.6%, 23.8.3 ± 9.5% and 14.1 ± 4.1% of MP, OSP and ISP perikarya, respectively. These results may suggest a neuroprotective role of SP and CGRP. Key words: proliferative enteropathy, neuronal plasticity, substance P (SP), calcitonin gene-related peptide (CGRP), enteric neurons, pig

Introduction pathological processes including inflammation (Sharkey and Kroese 2001), it appears to be of inter- Sensory information from the intestinal lumen is est to study the expression pattern of SP and/or conducted by two subsets of sensory neurons. First CGRP in neural structures of porcine descending co- division of them is composed of intrinsic primary af- lon affected by proliferative enteropathy (adenomato- ferent neurons of the Dogiel II type, located in OSP sis suis), a chronic proliferative inflammatory process and ISP (Clerc and Furness 2004), while the second evoked by Lawsonia intracellularis. subpopulation consists of extrinsic (spinal) primary afferent neurons located in DRGs (Berthoud et al. 2004). Both subsets of these neurons have been shown Materials and Methods to contain SP and/or CGRP (Bossowska et al. 2003, Brown and Timmermans 2004). However, SP and/or Six immature female pigs (approximately 8 weeks CGRP have also been demonstrated in the se- old), three control (C group) and three with clinically cretomotor and excitatory motor neurons, both popu- diagnosed Lawsonia intracellularis infection (PE lations crucial for maintaining bowel homeostasis. As group) were used. The animals were housed and enteric neurons are now widely accepted to be par- treated in accordance with the rules approved by the ticularly highly adaptive in their response to various local Ethical Commission. All the pigs were sacrificed

Correspondence to: M. Majewski, tel.: +48 89 523-44-60, fax: +48 89 523-39-82, e-mail: [email protected] 50 Gontkowski S. et al.

Fig. 1. Distribution and co-incidence patterns of SP (a, c, e) and CGRP (b, d, f) in MP (a, b), OSP (c, d) and ISP (e, f) of porcine descending colon in control animals. Figs. a, b x 250, c-f x 200. Fig. 2. Changes induced by proliferative enteropathy in the distribution and co-incidence patterns of SP (a, c, e) and CGRP (b, d, f) in MP (a, b), OSP (c, d) and ISP (e, f) of porcine descending colon. Figs. a-f x 200.

with an overdose of sodium pentobarbital (Vetbutal, ted to routine double-labelling immunofluorescence, Biowet, Poland; 90 mg/kg b.w.) and perfused transcar- using a mixture of antisera raised in different species dially with freshly prepared 4% paraformaldehyde in and directed to substance P (rat monoclonal, Biogen- 0.1 M phosphate buffer (pH 7.4). Ten µm thick cryos- esis, UK,; 1:500) and CGRP (rabbit polyclonal, Bi- tat sections of the descending colon wall were subjec- ogenesis, UK,; 1:8000). Primary antisera were visualiz- Co-incidence pattern ... 51 ed by species-specific secondary antisera conjugated spectively. Co-localization of the substances studied to FITC or biotin (all from Jackson Immunochemi- was found in 7.7 ± 0.6%, 23.8.3 ± 9.5% and cals, USA). The latter antibodies were then visualized 14.1 ± 4.1% of MP, OSP and ISP somata, respectively by a streptavidin-CY3 complex (Jackson). Double-im- (cf. Figs. 2a-f). The number of nerve fibres exhibiting munostained perikarya were then evaluated under immunoreactivity to the peptides studied was slightly Olympus BX51 microscope equipped with epi-fluor- lower in PE colon, when compared to the control in- escence and appropriate filter sets, counted in each testine, however, a virtually total co-localization of ganglionated plexus found in section studied (6 sec- both peptides has been observed. The present results tions per animal; only neurons with clearly visible nu- may be indicative for a neuro- and/or epithelioprotec- cleus were included) and presented as mean ± SEM. tive role of SP and CGRP (by intensification of mu- Semi-quantitative evaluation of the density of nerve cous secretion?) during Lawsonia intracellularis infec- terminals within the muscular or mucosal layers has tion, as may be judged from dramatic up-regulation in been based on counting of all terminals immunoreac- the expression rate of these substances within the in- tive to given antigen per observation field. Nerve pro- flamed bowel. However, the exact physiological rel- files were counted in 6 sections per animal, (in 6 fields evance of adaptive changes observed remains to be per section) and obtained data were pooled and pres- elucidated in detail. ented as mean. Pictures were captured by a digital camera connected to a PC, analyzed with AnalySIS software (version 3.02, Soft Imaging System, FRG) and printed on a wax printer (Phaser 8200, Xerox, Acknowledgements USA). Supported by KBN, grant no. 6P06K 012 20. The authors are indebted to Ms. A. Piękos and Mr. Z. Results and Discussion Liminowicz for their excellent technical help.

In the control animals, SP-IR neurons have been shown to constitute 3.7 ± 0.5%, 3.4 ± 0.4% and References 6.6 ± 1.1% of all neurons in MP, OSP and ISP, while CGRP-IR perikarya amounted to 1.6 ± 0.2%, Berthoud HR, Blackshaw LA, Brookes SJH, Grundy 1.1 ± 0.4% and 1.2 ± 0.5% of all MP, OSP and ISP D(2004) Neuroanatomy of extrinsic afferents supplying neurons, respectively. Co-localization of both substan- the gastrointestinal tract. Neurogastroenterol Motil 16: ces was found in 26.1 ± 3%, 39.3 ± 10.7% and 28-33. Bossowska A, Adriaensen D, Gonkowski S, Wojtkiewicz J, 23.3 ± 0.2% of all MP, OSP and ISP perikarya, re- Timmermans JP, Majewski M (2003) Distribution and spectively. Furthermore, in the muscular layer of the neurochemical features of primary sensory neurons sup- normal colon, much more SP- (approximately 28.4 per plying the porcine colon descendens. Verh Anat Ges 98: field of observation) than CGRP-IR nerve profiles 228. (2.3 per field) were observed (Figs. 1a-f). It has been Brown DR, Timmermans JP (2004) Lessons from the por- shown that Lawsonia intracellularis infection evoked cine enteric nervous system. Neurogastroenterol Motil profound changes in the number of SP- and/or 16: 50-54. CGRP-IR neurons in particular plexuses: within MP, Clerc N, Furness JB (2004) Intrinsic primary afferent neur- 7.1 ± 0.8% and 4.0 ± 0.0% of all neurons were SP- or ones of the digestive tract. Neurogastroenterol Motil 16: CGRP-positive, respectively. In the OSP, SP was 24-27. Sharkey KA, Kroese AB (2001) Consequences of intestinal found in 9.1 ± 0.6% and CGRP – in 4.0 ± 0.8% of all inflammation on the enteric nervous system: neuronal ac- ganglionic cells, while these peptides were present in tivation induced by inflammatory mediators. Anat Rec ± ± 22.2 1.6% and 11.7 0.5% of all ISP perikarya, re- 262: 79-90. 52 Gontkowski S. et al. Polish Journal of Veterinary Sciences Vol. 7, No. 3 (2004), Supplement, 53-55

Short original article Proliferative enteropathy (PE)-induced changes in the number of vasoactive intestinal polypeptide-immunoreactive (VIP-IR) neural elements in the porcine descending colon

S. Gonkowski, J. Wojtkiewicz, A. Bossowska, J. Kaleczyc1, W. Sienkiewicz1, M. Majewski

Division of Clinical Physiology and 1Animal Anatomy, Department of Functional Morphology, Faculty of Veterinary Medicine, University of Warmia and Mazury, Oczapowskiego St. 13, 10-717 Olsztyn, Poland

Abstract

In normal descending colon, VIP-IR varicose nerve terminals were moderately dense, while in PE-gut they were almost twice as numerous. VIP-IR neurons in control animals constituted approximately 0.9 ± 0.2%, 0.91 ± 0.4 and 7.7 ± 1.0% of all perikarya in the myenteric (MP), outer (OSP) and inner submucous plexus (ISP), respectively. In contrast, these neurons amounted to approximately 17.7 ± 1.6%, 11.8 ± 1.3% and 35 ± 3.3% of all ganglionic perikarya within MP, OSP and ISP of PE colon, respectively. As VIP is well known to be a potent neuroprotective agent, the up-regulation in its synthesis may reflect a repair mechanism developed by neurons affected by PE.

Key words: proliferative enteropathy, neuronal plasticity, vasoactive intestinal polypeptide (VIP), enteric neurons, pig

Introduction (Klimaschewski 1997) as well as enteric neurons (Sandgren et al. 2003). It has been also proved to act Vasoactive intestinal polypeptide (VIP) is abun- as an important endogenous anti-inflammatory sub- dantly present within the porcine enteric nervous sys- stance, which, on the one hand, down-regulates the tem, being confined to inhibitory motor, secretomotor synthesis rate of pro-inflammatory cytokines and, on and vasomotor neurons, located within all three gan- the other hand, promotes the synthesis of anti-inflam- glionated plexuses (for a review, see Brown and Tim- matory interleukins, like IL-10, from, e.g. the myen- mermans 2004). Recently, the up-regulation of VIP teric ganglia (for review, see Ekblad and Bauer 2004, within intrinsic enteric neurons have been shown to be Mawe et al. 2004). Therefore, we decided to investi- a key-feature of adaptive changes in the inflamed gate the expression pattern of VIP in porcine de- bowel (Ekblad and Bauer 2004). Thus, in the recent scending colon affected by proliferative enteropathy, past, VIP has been suggested to be one of the most as it may be of interest to better understand the mech- potent neuroprotective agents for sympathetic anisms of enteric neurons plasticity.

Correspondence to: M. Majewski, tel.: +48 89 523-44-60, fax: +48 89 523-39-82, e-mail: [email protected] 54 Gonkowski S. et al.

Fig. 1. Distribution and co-localization pattern of the pan-neuronal marker PGP 9.5 (a, c, e) and VIP (b, d, f) in nerve fibres within the circular muscle layer (CML; a, b), MP (c, d) and ISP (e, f) of porcine control descending colon. x 200. Fig. 2. Changes in distribution and co-localization pattern of the pan-neuronal marker PGP 9.5 (a, c, e) and VIP (b, d, f) in nerve fibres within the circular muscle layer (CML; a, b), MP (c, d) and ISP (e, f) of porcine descending colon from animals suffering from proliferative enteropathy. x 200.

Materials and Methods treated in accordance with the rules approved by the local Ethical Commission. All the animals were sac- Six immature female pigs (approximately 8 weeks rificed with an overdose of sodium pentobarbital  old), three control (C group) and three with clinically (Vetbutal , Biowet, Poland; 90 mg/kg b.w.) and per- diagnosed Lawsonia intracellularis infection (PE fused transcardially with freshly prepared 4% parafor- group) were used. The animals were housed and maldehyde in 0.1 M phosphate buffer (pH 7.4). Proliferative enteropathy ... 55

Ten µm thick cryostat sections of the descending co- synthesis in a dramatic manner, similar to that ob- lon wall were subjected to routine double-labelling served after axotomy or colchicine application (Ek- immunofluorescence, using combinations of anisera blad et al. 1996), surgery (Schwarz et al. 1999) or in- raised in different species and directed towards pro- testinal hypertrophy (Ekblad et al. 1998). Thus, be- tein gene-product 9.5 (PGP9.5; mouse monoclonal, cause VIP is well recognized as a potent neuroprotec- Sigma, PL, 1:2000) and VIP (rabbit polyclonal, Bi- tive agent (for review, see Ekblad and Bauer 2004), it ogenesis, UK, 1:8000). Primary antisera were visualiz- appears possible that the up-regulation in its synthesis ed by species-specific secondary antisera conjugated may reflect a repair mechanism developed by neurons to FITC or biotin (all from Jackson Immunochemi- affected by pathological processes accompanying cals, USA). The latter antibodies were then visualized Lawsonia intracellularis infection. by a streptavidin-CY3 complex (Jackson). Double-immunostained perikarya were evaluated under Olym- pus BX51 microscope equipped with epi-fluorescence Acknowledgements and appropriate filter sets, counted in each ganglionated plexus found in the section studied (6 sec- Supported by KBN, grant no. 6P06K 012 20. tions per animal; only neurons with clearly visible nu- The authors are indebted to Ms. A. Piękos and cleus were included) and presented as mean ± SEM. Mr. Z. Liminowicz for their excellent technical help. Pictures were captured by a digital camera connected to a PC, analyzed with AnalySIS software (version 3.02, Soft Imaging System, FRG) and printed on References a wax printer (Phaser 8200, Xerox, USA). Brown DR, Timmermans JP (2004) Lessons from the porcine enteric nervous system. Neurogastroenterol Motil Results and Discussion 16: 50-54. Ekblad E, Bauer AJ (2004) Role of vasoactive intestinal peptide and inflammatory mediators in enteric neuronal Within the muscular layer of the control gut seg- plasticity. Neurogastroenterol Motil 16: 123-125. ment, VIP-IR varicose nerve terminals were moder- Ekblad E, Mulder H, Sundler F (1996) Vasoactive intestinal ately dense (approximately 13 terminals per area of peptide expression in enteric neurons is upregulated by microscopic investigation), while in the PE-gut, they both colchicine and axotomy. Regul Pept 63: 113-121. were almost twice as numerous (approx. 24 per area). Ekblad E, Sjuve R, Arner A, Sundler F (1998) Enteric neur- In contrast, the number of VIP-IR nerve profiles onal plasticity and a reduced number of interstitial cells within the colonic crypts slightly decreased, from ap- of Cajal in hypertrophic rat ileum. Gut 42: 836-844. prox. 42 per area in control specimens, to approx. 35 Klimaschewski L (1997) VIP – a “very important peptide” in the sympathetic nervous system? Anat Embryol per area in PE-influenced colon. VIP-IR neurons in ± 196: 269-277. the control animals constituted approx. 0.9 0.2%, Mawe GM, Collins SM, Shea-Donohue T (2004) Changes in ± ± 0.91 0.4 and 7.7 1.0% of all neurons in the myen- enteric neural circuitry and smooth muscle in the in- teric (MP), outer (OSP) and inner submucous plexus flamed and infected gut. Neurogastroenterol Motil (ISP), respectively. In contrast, a distinct increase in 16: 133-136. the number of VIP-IR perikarya was observed in Sandgren K, Lin Z, Svenningsen AF, Ekblad E (2003) chronically inflamed colon, where these neurons Vasoactive intestinal peptide and nitric oxide promote made up approx. 17.7 ± 1.6%, 11.8 ± 1.3% and survival of adult rat myenteric neurons in culture. J Neur- 35 ± 3.3% of all ganglionic perikarya within the MP, osci Res 72: 595-602. OSP and ISP, respectively. The present results clearly Schwarz NT, Tweardy DJ, Billiar TR, Bauer AJ (1999) In- terleukin-6 mediates an increase in VIP mRNA within demonstrate that proliferative enteropathy, induced the jejunal muscularis: a neural mechanism for pos- by Lawsonia intracellularis, is able to up-regulate VIP toperative ileus. Neurogastroenterol Motil 11: 288. 56 Gonkowski S. et al. Polish Journal of Veterinary Sciences Vol. 7, No. 3 (2004), Supplement, 57-59

Short original article Clinical evaluation of fillings made with glass-ionomer and composite materials in sandwich technique in canine molar teeth

M. Janeczek1, W. Janeczek2, A. Sender-Janeczek3, A. Chrószcz1, A. Czerski4, R. Henklewski5

1 Department of Anatomy and Histology, Agricultural University of Wrocław, Kożuchowska St. 1/3, 51-163 Wrocław, Poland 2 Department of Animal Hygiene, Agricultural University of Wrocław, Chełmońskiego St. 31c, 51-630 Wrocław, Poland 3Academical Stomatological Polyclinic, Krakowska St. 25, 50-425 Wrocław, Poland 4 Department of Animal Physiology, Agricultural University of Wrocław, C.K. Norwida St. 31, 50-375 Wrocław, Poland 5 Department and Clinic of Veterinary Surgery, Agricultural University of Wrocław, Plac Grunwaldzki St. 51, 50-366 Wrocław, Poland

Abstract

The study concern 15 fillings of the carietic defects in molar teeth in 15 dogs. The carious defects were located in grooves of the masticatory surface of molar teeth. The fillings were made in sandwich technique, with glass-ionomer material used as the base for the composite material. After 12 months from the manipulation the restorations have been evaluated according to modifited Ryge’s scale. In this evaluation above mentioned materials obtained the results: marginal adaptation 95% – A, anatomical shape 95%, surface smoothness A – 100%, marginal discolouration A – 100%, colour assortment A – 60%.

Key words: caries, tooth, composit material, glass-ionomer, sandwich technique, dog

Introduction cement Kavitan and composite XRV Herculite,as well the point-evaluation of executed filling after 12 The dental caries (caries dentis) occures rarely in month interval. dogs. The medical treatment in case of the caries consists in mechanical ablation and following filling of the defect with siutable material (Bieniek and Bieniek Materials and Methods 1993, Hale 1997). The sandwich technique, known also as second binding technique or double-layer, is The treatment was made on 12 dogs in various age wide used in the human and veterinary stomatology and breeds. The symetrical caries defects of two molar (Gończowski and Krupiński 2003, Janeczek et al. teeth from the the left and right mandible were detec- 2004). The present work presents the caries defects ted in 3 dogs. All from the defects were located in treatment and the filling way using glass-ionomer grooves of the molar teeth masticatory surface. All de-

Correspondence to: W. Janeczek, tel: +48 71 328-48-34, fax: +48 71 320-57-48, e-mail: [email protected] 58 Janeczek M. et al. fects were classified in the 1st degree (according to Disscusion Black). After the dental calculus ablation the caries defects were performed next and they were filled. The The denatal caries are not popular in dogs. Hale operation was executed in the deep general anesthesia (1998) reported caries in 5.3% from 435 investigated using xylasine (1 mg/kg) and atropine (0.02 mg/kg) dogs. Bieniek and Bieniek (1993) observed caries in i.m. for premedication. The introduction of general 6% of dogs. anesthesia was secured with ketamine (5 mg/kg) and The stomatological treatment of caries consist in diazepam (0.25 mg/kg) i.v. afterwards maintained with the mechanical removal of pathological tissue until ketamine and diazepam i.v. accordance with an effect the caries defect is complitely removed and the appro- production. The restoration treatment of caries defect priate conditions for filling achieved (Hale 1998). was applied with the contra-angle hand piece NSK Next the base material is applied into the defect. The activated by Carlo de Giorgi microengine. The ma- constant ion-change process between hardned nipulations were executed in the following sequence: glass-ionomer, tooth tissue and saliva begin after the the preparation of caries defect, dentine and enamel end of the hardninig process. The chemical compound were subjected to the dentine-enamel conditioner (10 of fluorine releasing from the cement is important for sec.), the irrigation and desiccation of defect, the car- the secondary caries prevention (Mount and Ngo ies defect filling with glass-ionomer chemohardening 2003). The ion-change ability is the most important  material (Kavitan ) – 6 minutes hardening time, 10 peculiarity, because the materials take part not only in minutes cessation time, the dental surface was subjec- the restoration of anatomical tooth structure but also ted to etcher (10 min.), 10 minutes cessation time, in the healing of tissues (Ruchała-Tyszler et al. 2003).  Opti Bond Solo plus binding system application, The next of very important glass-ionomer materials polimerization, the filling covering with photoharden- propriety is very strong adhesion to the dentine, the  ing composite material XRV Herculite in one or two low thermal expansibility index, relative neutrality for layers, polimerization and filling preparation. the dental pulp and high volume reduction of compo- The glass-ionomer cement was prepared according sitive (Gaintantzopoulou at al. 1994). The composi- to producer’s prescription. The polimerization process tive materials are characterized by the very high crush was made with halogen lampligth Hansadent Hilux resistance and famous elastic proprieties. The prep- 250. The filling has been reexaminated after one year aration system used in our work and the following period. The marginal adaptation, marginal discolour- caries defect fillings are known as the conventional ation, anatomical shape, surface smoothness and col- sandwich technique (Gończowski and Krupiński our assortment have been also reevaluated according 2003). The method used in above-mentioned work to modifited Ryge’s scale (Ruchała-Tyszler 2003). allows for the optimal utilisation of the materials. Our work describes the reinvestigation 12 months after the treatment. The selected parameters which were classi- Results fied according to modifited Ryge’s scale are generally use in the evaluation of fillings in human stomatology The above mentioned glass-ionomer cement was of and allows for exact qualification of used materials ductile consistency enabling exact application into the proprieties in clinical treatment (Ruchała-Tyszler cavity. The results of clinical examination after 12 2003). months were fully satisfying (Tab. 1). Especially high mark was given to low appearance of marginal losses and marginal discolouration which characterize Conclusion binding of the filling with tooth tissue and assure low level of secondary caries occurance. The results of The work proved that the sandwich technique us- anatomical shape and surface smoothness were also ing glass-ionomer and compositive materials is fully positive. The esthetic of filling was a little worse but adequate for the canine molar teeth fillings. The we have to notice that the filling was made in the rare modifited Ryge’s scale used in our clinical investiga- exposed dog’ molar teeth. None of fillings was missed tions gives strict information, which allows for the ob- after 12th months, the secondary caries was not jective evaluation of filling. It is noticeable, the evalu- investigated. ation of selected parameters is possible in ambu-

Table 1. The results of clinical investigation after 12 months.

Anatomical Marginal Marginal Smoothness Colour Criterion shape adaptation discolouration of surface selection ABCABCABCABCABC % 95 5 – 95 5 – 100 – – 100 – – 60 30 10 Number 14 1 14 1 15 15 9 4 2 Clinical evaluation of fillings ... 59 latory conditions and it needs nothing except Gończowski K, Krupiński J (2003) Marginal adaptation of elementar stomatological equipment. The results the restorations made with the conventional sandwich of our investigation allow for statement that the technique and simplified sandwich technique witch glass- materials used in our work may be also use success- ionomer bonding system Fuji Bond LC (GC) – laboratory investigation. Stom Wsp 2 (suppl): 13-19. fully in animal stomatology. Hale F (1997) Dental caries in the dog. J Vet Dent 15: 79-83. Janeczek M, Janeczek W, Sender-Janeczek A, Henklewski References R(2004) Postępowanie w przypadku złamania kła z długotrwałą ekspozycją miazgi zęba u psa. Acta Sc Pol, Bieniek HJ, Bieniek KW (1993) Zahnheilkunde fu¨r die Med Vet 3: 37-43. Kleintierpraxis. Ferdinand Enke Verlag, Stuttgart. Mount GJ, Ngo H (2003) Biologic activity of the glass- Gaintantzopoulou MD, Willis GP, Kafrawy AH (1994) Pulp ionomer. Stom Wsp 2 (suppl): 20-24. reactions to light-cured glass ionomer cements. Am Ruchała-Tyszler A, Tyszler Ł, Weyna E (2003) Esthetics of J Dent 7: 39-42. cervical fillings. Stom Wsp 4: 40-43. 60 Janeczek M. et al. Polish Journal of Veterinary Sciences Vol. 7, No. 3 (2004), Supplement, 61-63

Short original article The level of selected antioxidative indices and testosterone in the blood of rats injected with vitamin E and selenium under an increased ozone concentration in the air

M. Jedlińska-Krakowska, K. Jakubowski, A. Kowalski

Division of Pathophysiology, Department of Pathology and Pharmacology, Faculty of Veterinary Medicine, University of Warmia and Mazury in Olsztyn, Oczapowskiego St. 13, 10-719 Olsztyn, Poland

Abstract

In the blood of the ozone-exposed rats testosterone concentration was observed to decrease, whereas both the contents of glucose and malondialdehyde and the activity of glutathione peroxidase were found to increase. In animals exposed to ozone and injected with vitamin E and selenium the above-mentioned changes were significantly less intensive which suggests that those compounds reduce, at least to some extent, the effects of the harmful activity of oxidative stress.

Key words: rat, ozone, malondialdehyde, glutathione peroxidase, testosterone

Introduction effect of the oxidative stress, simultaneously reduces an organism’s demand for selenium. As a major component of photochemical smog Although the available literature lacks the detailed ozone evokes oxidative stress in living organisms data on the impact of ozone on the functioning of which is linked with the production of free oxygen gonads in males, still it is known that free oxygen rad- radicals (Pryor et al. 1995, Salgo et al. 1995). Living icals may disturb their reproductive processes. Taking organisms have been demonstrated to develop into account the metabolic relationships between vit- a number of defence mechanisms capable of preven- amin E and selenium as well as the fact that ozone is ting free-radical reactions, thus providing a balance often applied in medicine for therapeutic purposes, between oxidative and antioxidative processes. One of this study was undertaken to determine whether, and the agents of these mechanisms is glutathione per- to what extent, selenium and vitamin E supplementa- oxidase (GPx) that protects cellular membranes by cation prevents the negative effects of the ozone activity talysing the reaction of H2O2 reduction. A key-compo- on the testes. nent of that enzyme is selenium which is a part of the active centre of GPx (Arteel and Sies 2001). Selenium metabolism is closely linked to the metabolism of an- Materials and Methods other recognised antioxidant, vitamin E. Its antioxidative properties are based on the reaction with The experiment was performed on 30 adult male free radicals which, by protecting a cell against the Wistar Hannower rats with the average body weight

Correspondence to: M. Jedlińska-Krakowska, tel.: +48 89 523-32-96, e-mail: [email protected] 62 Jedlińska-Krakowska M., Jakubowski K., Kowalski A. of 405 ± 10 g and age of 12 months. All animals were Results randomly divided into 3 groups (10 rats each) denoted by the following abbreviations: In blood of animals exposed to ozone (III KO) I (K) – control animals; II (OEv) – animals ex- a statistically significant decrease in testosterone con- posed to ozone and injected intramuscularly with vit- centration, an increase in the contents of glucose and amin E (22.5 mg/rat) and selenium (0.33 mg/rat) prep- malondialdehyde (MDA) as well as an increased ac- aration Evetsel (Pliva, Kraków); injections were made tivity of glutathione peroxidase were observed for 25 days in 5-day intervals. Evetsel was adminis- (Table1). In the animals exposed to ozone and injec- tered to the animals 2 weeks prior to the experiment ted with vitamin E and selenium (II OEv), the level of to increase and stabilise the levels of vitamin and sele- MDA was also statistically significantly higher, how- nium in their bodies; III (KO) – rats exposed to ozone ever, it was apparently lower compared to rats ex- without vitamin protection. All rats from groups II posed to ozone only. In that group, an increasing ten- and III were exposed to 0.5 ± 0.2 ppm of ozone dency for glucose level and GPx activity with refer- 5 h a day for the period of 25 days. ence to the control group (I K) was observed. Ozone was generated from compressed air in an IMPOZ-4 ozoniser (Institute of Precise Mechanics, Warsaw) and supplied to a chemically-sealed (with Discussion neutral polyethylene foil) room to be mixed sponta- neously with air. The concentration of ozone in the In all ozonated animals, including those receiving exposure chamber was controlled with the iodometric vitamin E and selenium, the blood level of glucose method (Saltzman et al. 1959). During ozonation the was found to increase, which is a typical body re- animals had free access to water but feed was response to stress. Under stressing conditions, the symphatetic-adrenomedullary system is activated followed moved due to an oxidative effect of ozone. Apart from by the pituitary-corticomedullary system, which is the 5 hours of ozone-exposure the animals were kept connected through an increased demand of the body under identical conditions in terms of air composition, for directly available energetic substrates. An in- temperature, and feeding regime. After termination creased glucose concentration in blood is accom- of the experiment the maximal amount of blood was panied by an elevated activity of glutathione per- collected from all animals in halothane anaesthesia oxidase and an increased level of malondialdehyde (Narcotan, Leciva, Czech Republic) by a heart punc- may indicate oxidative stress. At the same time, a high ture. Immediately after sampling the activity of GPx activity proves the intensification of free-radical glutathione peroxidase was determined in whole processes and antioxidative defence reactions of the blood (Paglia and Valentine 1967), whereas concen- organism (Arteel and Sies 2001). On the other hand, trations of testosterone (Kotwica and Wiliams 1982), an elevated level of highly toxic malondialdehyde is malondialdehyde (Ward et al. 1985), and glucose were linked with lipid peroxidation generated by free oxy- assayed in blood plasma. gen radicals with MDA being one of its main A statistical analysis of results involved calculations end-products (Pryor et al. 1995, Kim et al. 2000). of arithmetic means, standard error of the mean and Oxidative stress produces changes in the spermato- significance of differences compared to the control genic epithelium and disturbs spermatogenesis group. (Sharma and Agarwal 1996), however, a decreased The experimental animals were handled according testosterone concentration is often observed upon dif- to the provisions of the Act on Animal Protection as ferent stressing conditions. It may also result from se- well as to recommendations of the Local Committee cretory disorders of the pituitary gland and hy- for Animal Experiments at the University of Warmia pothalamus and from changes in the gonads. An in- and Mazury in Olsztyn, Poland. creased ozone concentration in the air (the state of

Table 1. Testosterone, glucose and element concentrations in the blood of rats ( ± SEM).

Groups/indices I K (n=10) II OEv (n=10) III O (n=10) testosterone [ng/ml] 1.01 ± 0.09 0.90 ± 0.31 0.73 ** ± 0.09 glucose [mg/dl] 77.02 ± 3.22 82.4 ± 5.09 92.42 * ± 6.47 malondialdehyde [µm/l] 9.18 ± 0.38 10.64 ** ± 0.24 12.26 ** ± 0.22 glutathione peroxidase [U/l] 2073 ± 165 2274 ± 87 2708* ± 168

Explanation: * p≤0.05 – significance in relation to the control group (I K), ** p≤0.01 The level of selected antioxidative ... 63 oxidative stress) has been reported to diminish the Kotwica J, Wiliams GL (1982)Relationshipplasmatesto- activity of 17 beta-hydroxysteroid dehydrogenase and sterone concentrations to pituitary-ovarian hormone se- alkaline phosphatase in the gonad tissue in rats cretions during the ovine estrous cycle and and the ef- (Jedlińska-Krakowska 1998). fect of testosterone propionate administred during In the rats receiving injections of vitamin E and luteal regression. Biol Reprod 27: 790-801. Paglia DE, Valentine WN (1967) Studies on quantitative selenium the changes referring to ozone-exposure and qualitative characterization of erythrocyte were apparently less intensive. An exception was glutathione peroxidase. J Lab Clin Med 70: 158-169. a high, statistically significant level of malondial- Pryor WA, Squadrito GL, Friedman M (1995)Thecascade dehyde. The results suggest that Evetsel reduces, at mechanism to explain ozone toxicity: the role of lipid least to some extent, the results of the detrimental ozonation products. Free Radic Biol Med 19: 935-941. activity of oxidative stress in male rats. Salgo MG, Cueto R, Pryor WA (1995) Effect of lipid ozonationproductsonliposomalmembrabesdetected by Laurdan fluorescence. Free Radic Biol Med 19: References 609-616. Saltzman BE, Gilbert N (1959) Iodometric microdeter- Arteel GE, Sies H (2001) The biochemistry of selenium and mination of organic oxidants and ozone. Anal Chem 31: the glutathione system. Environ Toxicol Pharmacology 1914-1920. 10: 153-158. Sharma RK, Agarwal A (1996)Roleofreactiveoxygen Jedlińska-Krakowska M (1998) Blood plasma testosterone species in male infertility. Urology 48:835-850. level, alkaline phosphatase and 17-β-hydroxysteroid de- Ward A.P, Till OG, Hatherill JR, Annersley TM, Kunkel hydrogenase activity in rats exposed to ozone and injec- RG (1985) Systematic complement activation, lung in- ted vitamin E. Bromat Chem Toksykol 31: 281-285. jury and products of lipid peroxidation. J Clin Invest 76: Kim J, Chehade J, Pinnas JL, Mooradian AD (2000) Effect 517-527. of selected antioxidants on malondialdehyde modifica- tions of proteins. Nutrition 16: 1079-1081. 64 Jedlińska-Krakowska M., Jakubowski K., Kowalski A. Polish Journal of Veterinary Sciences Vol. 7, No. 3 (2004), Supplement, 65-68

Short original article Lafora’s bodies in borzoi dog. Case report

M. Katkiewicz, B. Osińska

Department of Clinical Sciences, Faculty of Veterinary Medicine, Warsaw Agricultural University, Nowoursynowska St. 159c, 02-776 Warsaw

Abstract

Lafora bodies were diagnosed in female borzoi dog, 14 years old. There were no CNS clinical symptoms, except of peripheral neurogenic skeletal muscle atrophy and the pain. Post mortem examination revealed blood circulatory disturbances in the liver, lungs and spleen. The heart bicuspid and tricuspid valves had shown endocardiosis changes. The microscopic examination of the brain hemi- spheres and cerebellum revealed the presence of disseminated Lafora bodies. They were dark-blue in haematoxylin and eosin staining and PAS positive as well. The liver had slight features of the jaun- dice, with advanced hyperaemia and necrosis of hepatocytes. Lafora’s bodies were also found in degenerated/necrotic hepatocytes.

Key words: Lafora’s body, borzoi dog, histopathology, brain, liver

Introduction throughout the brain, seen in neuronal perikaryon and neuropil. These inclusions are rounded, in hae- The disease syndrome manifested by the presence matoxylin and eosin (HE) staining strongly basophilic of Lafora’s bodies in the brain is extremely rare in the and positive in periodic acid Schiff reaction (PAS). dog (Davis at al. 1990, Kaiser et al. 1991). For years The presence of the inclusion bodies in the skin sweat the etiopathology of this disease, which was observed glands argued for using the skin biopsy in the early in humans and animals, was the mystery. In the 80’s, procedure and in the differential diagnosis of Lafora’s Japanese authors presented the opinion that Lafora’s disease in patients with neurological signs. bodies appeared in the brains of aging animals In this paper the results of post mortem examin- (Kamiya and Suzuki 1989). Now it is documented ation of borzoi dog with Lafora’s disease are de- that the syndrome has the inherited genetic back- scribed. ground due to Lafora gene mutation and is manifested by extralysosomal storage of polyglucosan substance. In human medicine it was proved that Materials and Methods Lafora’s disease is the result of autosomal recessive defect in carbohydrate metabolism (Serratosa at al. Female dog, borzoi breed (Russian chart), 14 years 1995). The stored material is present in the brain, old was presented for post mortem examination. For liver, striated skeletal muscles and myocardium, and histopathological examination the following organ the skin tubular epithelium of apocrine sweat gland and tissue specimens were fixed in 10% buffered for- (Busard et al. 1994, Tamura et al. 1995, Thom and malin: heart, liver, lung, spleen, kidney, skeletal Revesz 1996). Lafora’s bodies are disseminated muscle, brain and cerebellum, thyroid with para-

Correspondence to: M. Katkiewicz, e-mail: [email protected] 66 Katkiewicz M., Osińska B. thyroid gland and adrenal glands. The paraffin em- found. Occasionally in the myocardial fibres bedded tissue sections were routinely stained with basophilic degeneration was observed. The micro- haematoxylin and eosin. Additionally cerebellum scopic changes in the liver were typical to chronic specimens were stained with PAS reagent. passive hyperaemia, followed by centrolobular trabecular atrophy with the bile retention in hepatocytes. The liver cells of the lobule periphery showed Results and Discussion lipid infiltration. There were numerous areas of hepatocyte necrosis, in which the numerous round haema- The dog’s owner, veterinary surgeon had informed, toxylin stained corpuscles were found (Fig. 2). In the that the dog showed the symptoms of heart insufficiency since she had been born. The dog died with signs of leg muscle atrophy, mild icterus and lethargy. In post mortem examination the following macroscopic changes were found: the mucous membranes were yellowish stained, lungs were swollen and congested, both heart valves were thickened, the liver showed features of post hyperaemic lipid degeneration, kidney parenchyma was yellowish with disseminated cortical small scares, the brain and menin- ges were hyperaemic and leg skeletal muscles appeared atrophic. In the microscopic examination of internal organs the following changes were noted. In HE stained cerebellum specimens the numerous, dark-blue, round corpuscles, disseminated in the white substance were found (Fig. 1). Their diameter varied Fig. 2. The liver of the dog with numerous Lafora’s bodies seen in degenerated/necrotic hepatocytes, and the high degree of trabecular atrophy. Haematoxylin and eosin staining, x 20.

Fig. 1. Cerebellum of the dog with disseminated inclusion bodies typical for Lafora’s disease. Haematoxylin and eosin staining, x 20.

from point-like to the size of Purkinje cells nucleus. Fig. 3. Skeletal muscles of the dog with Lafora’s disease with high degree of atrophic degenerative changes Haematoxylin The corpuscles were PAS positive. The inclusions and eosin staining, x 40. were present in the cytoplasm of Purkinje cells and in the cerebellum neuropil. The Purkinje cells showed signs of degeneration and necrobiosis. The brain had lungs the high degree hyperaemia, proliferation of the features of oedema (status cribrosus) and the degen- interstitial tissue, focal calcium deposits, numerous erative changes in the neurons. In the brain cortex syderocytes, dysplastic changes in cells of the bron- some focal calcium deposits were observed. The inclu- chial epithelium and the focal oedema were observed. sion bodies were not so numerous, as was observed in In the spleen the circulatory disturbances were mani- the cerebellum. In the heart muscle focal purulent fested by the white pulp atrophy. In the kidneys inflammation with necrotic focus in muscle cells were interstitial and membranous-proliferative glomerular Lafora’s bodies ... 67 inflammation, with necrosis of tubular epithelium and eccrine glands located in this skin region. But and focal calcification were noticed. In the thyroid the liver biopsy is considered to be the most reliable gland the presence of adenoma, adipose tissue prolif- site for the early diagnosis of Lafora’s disease, es- eration and focal interstitial calcium deposits were pecially in juvenile human patients. There are no noted. The disturbances of calcium metabolism ob- data concerning the application of the liver and skin served in above described organs were related to biopsy in Lafora’s disease of any animal species. parathyroid glandular hyperplasia. In the adrenal The microscopic changes found in other internal glands the adenoma located in the zona fasciculata organs of the affected dog were not typical for the was found. In skeletal muscles of the etremities the described disease syndrome, excluding the myocar- advanced atrophy of muscle fibers and degenerative dium and skeletal muscles. The human Lafora’s dis- changes were found (Fig. 3). This was accompanied ease is characterized by the presence of typical by adipose tissue hyperplasia. Discrete, small cytoplasmic inclusions in the myocardial fibres, ac- Lafora’s bodies were also found and skeletal and companying by their basophilic degeneration. In the heart muscle fibers, but comparing to the cerebellum dog myocardium the inclusions were very scarce and and liver they were not so well visible. In the myocar- small but the degenerative changes were observed. dium dystrophic changes accompanied by circulatory The advanced histopathological changes, manifested blood disturbances were observed. by membranous-proliferative glomerular inflamma- The inclusion bodies characteristic for Lafora’s tion, followed by tubular degenerative and necrotic changes were found in the kidneys. These changes disease were found in the central nervous system and seemed to be the result of the long lasting generaliz- liver and their presence was the basis of the diag- ed metabolic disturbances and liver damage which nosis. Unfortunately, the genetic trait of the exam- additionally were intensified by the dogs advanced ineddogwasnotpossibletofollowup,sothede- age. scribed case was unique. However, the central nerv- The above mentioned biopsy procedures are used ous system lesions were typical for Lafora’s body syn- in the routine diagnosis in the early stages of drome – the dog clinical ailments, as was stated by Lafora’s disease in human medicine. Nowadays the the owner, were mainly reflected by circulatory and biopsy techniques are well developed in veterinary metabolic liver and striated muscle disturbances. The medicine, so, may be used in Lafora’s disease diag- only neurological symptoms were lethargy and the nosis when signs of myoclonus epilepsy are observed. skeletal muscle atrophy but the metabolic disturban- The familiar character of Lafora’s disease is the in- ces proved by the presence of Lafora’s bodies were dication for having special attention in the animal responsible also for the injury of parenchymatous or- breeding program. Very small number of publica- gans and muscle cells. As compared to human clini- tions on Lafora’s disease cases in dogs diagnosed in cal manifestation of Lafora’s disease, in dogs case no various breeds suggest no breed dependent predis- epilepsy symptoms were observed. In the majority of position of this genetic anomaly inheritance. human cases Lafora’s disease is manifested by seiz- ures typical for progressive myoclonous epilepsy and this symptom is considered as the significant for this References disease syndrome. However, the similar brain lesions were described in the beagle dogs showing the signs Busard HL, Span JP, Renkawek K, Renier WO, Gabreels of epilepsy (Montgomery, Lee 1983), but the cere- FJ, Slooff JL, Van’tHof MA (1994)Polyglucosanbodies bellar typical lesions were found in some sick dogs in the brain tissue: a systematic study. Clin Neuropathol only. Among 68 beagle dogs, which had died with 13: 60-63. symptoms of myoclonous epilepsy, the cerebellar in- Busard HL, Gabreels-Festen AA, Renier Wo, Gabreels FJ, clusions bodies localized in neurons, typical for hu- Stadhouders AM (1987) Axilla skin biopsy: a reliable man Lafora’s body diseases were detected in six dogs test for the diagnosis of Lafora disease. Ann Neurol only. 21: 599-601. The presence of numerous basophilic inclusions Davis KE, Finnie JW, Hooper PT (1990)Lafora’sdisease in the cytoplasm of the liver cells or being free-laying in a dog. Aust Vet J 67: 192-193. in necrotic areas was found in the examined dog. Dias-Tosta E, Vieira LF, Alves A, Miziara HL (1995) These lesions were similar to changes described in Lafora’s disease: a possible diagnosis of juvenile demen- tia Arq Neuropsiquiatr 53(3-A): 455-463. human Lafora’s disease. The description of Lafora’s Kaiser E, Krauser K, Schwartz-Porsche D (1991) Lafora’s bodies in the liver (Yokota et al. 1987) as well as in disease (progressive myoclonic epilepsy) in the Basset tubular cells of skin glands (Busard et al. 1987, White hound – possibility of early diagnosis using muscle bi- and Gomez 1988) had made the liver and skin biopsy opsy? Tieraztl Prax 19: 290-295. very useful in the clinical and laboratory differential Kamyia S, Suzuki Y (1989) Polyglucosan bodies in the diagnosis procedures. For the skin biopsy the speci- brain of the cat. J Comp Pathol 101: 263-267. mens are taken from axillary region, because the Montgomery DL, Lee AC (1983)Braindamageintheepi- Lafora’s bodies are present in cells of the apocrine leptic beagle dog. Vet Pathol 20: 160-169. 68 Katkiewicz M., Osińska B.

Serratosa JM, Delgado-Escueta AV, Posada I, Shih S, cal, immunohistochemical and immuno-electronmicros- DruryI,BercianoJ,ZabalaJA,AntunezMC,Sparkes copoc studies. Histopathology 26: 501-508. RS (1995) The gene for progressive myoclonusepilepsy Thom M, Rewesz T (1996) Typical polyglucosan bodies are of the Lafora type maps to chromosome 6q. Hum Mol present in the sweat gland lumina in Lafora’s diseases. Genet 4: 1657-1663. Acta Neuropathol (Berlin) 92: 1102-1103. Tamura S, Takahashi M, Kawamura S, Ishibara T (1995) White JW, Gomez MR (1988) Diagnosis of Lafora’s dis- Basophilic degeneration of the myocardium: histologi- ease by skin biopsy J Cutan Pathol 15: 171. Polish Journal of Veterinary Sciences Vol. 7, No. 3 (2004), Supplement, 69-72

Short original article Pathomorphological characteristic of supraspinal muscles in rabbits after short-term electrostimulation

I.M. Kowalski, J. Szarek1,I.Babińska1,J.Lipińska1,J.Fabczak1

Voivodeship Children Hospital and Rehabilitation Center, 11-015 Ameryka, Poland 1 Department of Forensic and Administration of Veterinary Medicine, University of Warmia and Mazury in Olsztyn, Oczapowskiego St. 13, 10-717 Olsztyn, Poland

Abstract

This experiment was performed on developing rabbits (aged 3.5 months), divided into 2 groups (n=5): 1 – animals treated with electrostimulation for 2 h/day, 2 – control rabbits without electrostimulation of the supraspinal musculature. Stimulation was carried out using an electric stimulator SCOL-2, according to the method modified by Kowalski. The animals were sacrificed after 3 months of electrostimulation. The microscopic examination of supraspinal muscles of rabbits revealed the hypertrophy of muscle fibres, an increase in their congestion and a significant rise in the level of glycosaminoglycanes. The obtained results indicate that the short-term electrostimulation has strengthened the structure of the muscles stabilizing the spine had strengthened.

Key words: scoliosis, lateral electrical surface stimulation (LESS), muscle, rabbit

Introduction Machidaetal.,1994),havebeendescribedforthe last ten years. However, the long-term LESS method The idea of applying Lateral Electrical Surface which has been applied so far, based on nine hours of Stimulation (LESS) in the treatment of idiopathic therapy per day, has not proven effective enough and scoliosis(IS)isthestimulationofthesupraspinal has produced undesired morphological lesions muscles area, which causes the impulsation of the (Szarek et al. 2003). These observations served as the preserved structures of spinal cord and mobilization basis for establishing the author’s own Lateral Elec- of the supplementary system of control over the trical Surface Stimulation programme, shortened to function at this level (Machida et al. 1994). two hours a day (Kowalski 1999). The effects of treating scoliosis in people with the application of the LESS method, in Poland also known by the name of the SCOL apparatus and in Materials and Methods western countries by the name of the ScoliTron apparatus or by the name of Electro Spinal Orthosis Ten male, pure-bred (New Zealand White), – E.S.O., or the abbreviation of Surface Electrical age-matched (born within 5 days) and clinically Stimulation – SES, and also as Transcutaneous Ele- healthy rabbits were used in this study. The animals ctrical Stimulation – TCES (Wright et al. 1992, were kept indoors in the room with controlled tem-

Correspondence to: I. Kowalski, tel.: +48 89-519-48-14, e-mail: [email protected] 70 Kowalski I.M. et al. perature (180C) and humidity (70%). Each animal Results was placed in a metal cage (50 x 50 x 50 cm) and received dry feed and water ad libitum.A30-daype- In all rabbits from group 1 (animals subjected to riod was allowed for adaptation to new environment. LESS), wave-like muscle fibers were observed (Fig. At the beginning of the treatment, the rabbits were 1). It affected small sections of fibers and was slightly aged approximately 3.5 months and weighed be- more evident in the scraps derived from the muscle tween 2000 – 2200 g. located nearer the electrodes. The experiment was conducted according to the Atrophy of fragments of muscle fibers in MLT on previously described methodology, with the use of an the right side (stimulated) was observed occasionally electrical stimulator (SCOL-2, Elmech, Warsaw, Po- within two scraps (Fig. 2a). Atrophy of cross striation land) of the technical specifications previously pre- took place in two rabbits. Granular degeneration was sented (Kowalski 1999, Szarek et al. 2003). The rab- present only in short sections of muscle fibers, in bits were randomly assigned to one of the two groups three scraps of MLT (Fig. 1a) and in one case of (each n=5): group 1, where the rabbits were treated MIT. Occasionally the examined material revealed for 2 hours/day and group 2 (control) – where ani- single vacuoles. Several tiny foci of necrosis were ob- mals were not electrostimulated. served in one scrap of MLT on the right side. Sections of right and left musculus longissimus Almost half of the examined muscles revealed dorsi (MLD): musculus longissimus thoracis (MLT) a slight congestion. Additionally, regeneration of and m. iliocostalis thoracis (MIT) – muscles stabili- muscle fibres was seen in the analyzed material zing rabbit spinal column were microscopically exa- (Fig. 1). Proliferation of nuclei in the muscle fibres mined. The material was taken from areas targeted has been revealed in almost a half of the examined with electrostimulation on the right side and analo- scraps, where occurred sporadically, and affected gous areas on the left side of the spine. The material only small fragments of fibres. Infiltration of mono- was fixed in 10% neutral formalin and paraffin slices nuclear cells were observed relatively rarely. were routinely stained with hematoxylin and eosin In the examined material of the left side of the (HE). To show the glycosaminoglycane level, micro- rabbits from group 1 (not stimulated) pathomor- scopic muscle preparations were stained by the phological lesions were observed similar to those ob- PAS-method according to McManus. Digestion with served on the right side, but no foci of atrophy and diastase was used as a control sample. The analysis necrosiswerefound.Thelesionsvisibleherewereof was carried out in compliance with the criteria given a vestigial character and they affected very small by Szarek et al. (1985). fragments of fibres.

Fig. 1. A – Musculus longissimus thoracis dexter of the electrostimulated rabbit – wave-like muscle fibres (arrows), granular degeneration (asterisk), regenerating muscle fibers with chain arrangement of nuclei (arrow heads); B – Musculus longissimus thoracis sinister of the electrostimulated rabbit – wave-like of muscle fibres (arrows) and regenerating muscle fibres with chain arrangement of nuclei (arrow head). HE stain., x 290. Pathomorphological characteristic ... 71

Fig. 2. Musculus longissimus thoracis dexter A – electrostimulated rabbit – PAS+ reaction indicates concentration of glicosaminoglycanes close to sarcolemma and in undergoing atrophy muscle fibres (arrows), as well as the hypertrophy of muscle fibres (H); B – control rabbit – uniform PAS+ reaction. PAS according to McManus staining, x 290.

The analyzed muscle fibres on the right side in muscles – conditions of dystonia – is widely accepted rabbits from group 1 most frequently were characte- by the authors as the crucial factor in IS rized by a large and very large content of etiopathogenesis (Wright et al. 1992). Therefore, the glicosaminoglycanes. Positive PAS reaction was par- role of LESS in the IS correction is the stimulation of ticularly clearly seen at the periphery of muscle the weakened neuromuscular system. As a result of fibres, while the atrophying fibres revealed excessive this stimulation on the side of the developed scolio- accumulation of glycogen (Fig. 2a). The level of sis, the strengthening of the muscles stabilizing the glicosaminoglycanes in muscle fibres on the left side spine occurs, and subsequently the correction in rabbits from group 1 was characterized as large. (Kowalski 1999). The polysaccharides content in these cases varied Our own research proved that short-term LESS from average to very large. occasionally caused unfavorable morphological di- In rabbits from group 2 (the control group), the sorders of small intensification within the area of microscopic structure of muscles appeared normal. musculus longissimus dorsi in the form of regressive Granular degeneration, proliferation of nuclei fibres lesions. However, there were frequently observed and infiltration of mononuclear cells were observed positive effects in supraspinal muscles: muscle fibre only occasionally, in a small number of muscle fibres. hypertrophy, the rise in their congestion and the rise Muscle fibres of animals from group 2 were predomi- in the polysaccharide level. These lesions indicated nantly characterized by a small amount of that the muscle tissue structure of the muscles glicosaminoglycanes; this substance was evenly distri- stabilizing the spine had strengthened. buted (Fig. 2b). In the present experiment the new program of the LESS therapy, shortened to 2 hours a day, has proven to be devoid of unfavorable muscle disorders. Discussion

The tension of supraspinal muscles stabilizing the Acknowledgements spinal column depends on the spinal regulation (deep and superficial reflexes) and on complex Supported by grant 4 T11E 009 25. The authors mechanisms controlled by supraspinal nervous sys- are indebted to Ms. K. Dublan, Msc and Mr. A. tem centers (Wright et al. 1992, Machida et al. 1994). Penkowski, Msc for their excellent technical assi- However, the reduction of tension of supraspinal stance. 72 Kowalski I.M. et al.

References spinal muscles of rabbits after long-term experimental electrostimulation. Pathol Res Pract 199: 613-618. Kowalski IM (1999) A new shortened method of electros- Szarek J, Piekut A, Kalinowska ZE (1985) Experimental lar- timulation in animal experiment. Post Rehab 13: 113-119. val ascaridiosis (Ascaris suum Goeze, 1782) in white mice. Machida M, Dubousset J, Immamura Y, Iwaya T, Yamada I. Histochemical studies on the liver. Acta Vet Hung 33: T, Kimura J, Torriyama S (1994) Pathogenesis of 25-32. idiopathic scoliosis: SEPs in chicken with experimentally Wright J, Herbert MA, Velasquez R, Bobechko WP (1992) induced scoliosis and in patients with idiopathic scoliosis. Morphologic and histochemical characteristics of skeletal J Pediatr Orthop 14: 329-335. muscle after long-term intramuscular electrical stimula- Szarek J, Kowalski IM, Van Dam F, Zarzycki D, Pawlicki R, tion. Spine 17: 767-770. Fabczak J (2003) Pathomorphological pattern of supra- Polish Journal of Veterinary Sciences Vol. 7, No. 3 (2004), Supplement, 73-75

Short original article NOS-, VIP- or GAL-immunoreactivity disappear from proliferative enteropathy-affected inferior mesenteric ganglion (IMG) neurons supplying porcine descending colon

M. Majewski, J. Wojtkiewicz, S. Gonkowski, A. Bossowska, Z. Pidsudko1, J. Kaleczyc1

Division of Clinical Physiology and 1Animal Anatomy, Department of Functional Morphology, Faculty of Veterinary Medicine, University of Warmia and Mazury, Oczapowskiego St. 13, 10-717 in Olsztyn, Poland

Abstract

This study investigated the influence of proliferative enteropathy (PE) on porcine descending colon-projecting IMG perikarya immunoreactive to NOS, VIP and/or GAL. Fast Blue (FB) tract-tracing combined with immunofluorescence revealed that in control animals, a low number of FB+ IMG neurons were NOS- (2.6 ± 0.8%), VIP- (0.9 ± 0.2%) or GAL- immunoreactive (IR) (2.3 ± 0.6%). In contrast, in animals suffering from PE none of retrogradely traced cells were IR to studied substances. These results suggest the existence of a Lawsonia intracellularis inflammation-specific response of affected prevertebral neurons.

Key words: proliferative enteropathy, neuronal plasticity, nitric oxide synthase (NOS), vasoactive intestinal polypeptide (VIP), galanin (GAL), inferior mesenteric ganglion, pig

Introduction in the pig to mechanical injury (axotomy) or to acute inflammation of the bowel wall have previously been This study investigated the influence of prolif- presented (Majewski et al. 2002), virtually nothing is erative enteropathy (PE), a chronic proliferative in- known about the influences of a chronic bacteria-inflammatory process evoked by Lawsonia intracel- duced colitis on the expression pattern of putative lularis, on neurochemical features of the porcine in- neuroprotective substances in affected sympathetic ferior mesenteric ganglia neurons, especially those ex- neurons. hibiting immunoreactivity to NOS, VIP and/or GAL, the substances previously suggested to be able to exert a neuroprotective action on injured sympathetic neur- Materials and Methods ons (Klimaschewski et al. 1996; Majewski et al. 1996). However, while the preliminary data concerning the Six immature female pigs (approximately 8 weeks responses of IMG neurons supplying the distal colon old), three control (C group) and three with clinically

Correspondence to: M. Majewski, tel: +48 89 523-44-60, fax: +48 89 523-39-82, e-mail: [email protected] 74 Majewski M. et al.

Fig. 1 and 2. In control animals retrogradely labeled neurons (1a, 2a) exhibited different combinations of NOS (1b, 2b) and VIP (1c) or GAL (2c) co-incidence. Note, that virtually all NOS-IR cells were simultaneously VIP-IR, smaller FB+ neurons were usually devoid of NOS, VIP and/or GAL. x 200. Fig. 3 and 4. Retrogradely labeled neurons in IMG from animals suffering from PE were devoid of NOS- (3b, 4b) and VIP- (3c) or GAL-immunoreactivity (4c). x 200.

 diagnosed Lawsonia intracellularis infection (PE (Vetbutal , Biowet, Poland; 90 mg/kg body weight) group) were used. All animals were subjected to and transcardially perfused with freshly prepared 4% laparotomy and injected with 5% aqueous solution of paraformaldehyde in 0.1 M phosphate buffer (pH Fast Blue (FB) into the wall of the descending colon. 7.4). Ten µm thick cryostat sections of the IMG were After a three-week survival period the animals were subjected to routine double-labelling immunofluores- sacrificed with an overdose of sodium pentobarbital cence, using combinations of anisera raised in differ- NOS-, VIP- or GAL-immunoreactivity ... 75 ent species and directed towards NOS (mouse mon- labeled GAL-IR neurons was observed in the IMG oclonal, rabbit polyclonal), VIP (mouse monoclonal, affected by axotomy, numbers of NOS- and VIP-IR rabbit polyclonal), GAL (rabbit polyclonal). Primary perikarya remained unchanged. On the other hand, antisera were visualized by species-specific secondary an acute chemically-induced inflammation of the de- antisera conjugated to FITC or biotin (all from Jack- scending colon wall was not able to either up- or son Immunochemicals, USA). The latter antibodies down-regulate the synthesis ratio of substances were then visualized by a streptavidin-CY3 complex studied by ganglionic neurons. However, it resulted in (Jackson). Retrogradely labeled/double-immunos- a distinct increase in the density of intraganglionic tained perikarya were then evaluated under Olympus terminals of viscerofugal enteric neurons (Majewski et BX51 microscope equipped with epi-fluorescence and al. 2002). Based on the data obtained, an existence of appropriate filter sets, counted in each fifth section a Lawsonia intracellularis inflammation-specific re- (only neurons with clearly visible nucleus were in- sponse of affected prevertebral neurons may be sug- cluded) and presented as mean ± SEM. Pictures were gested. Furthermore, it appears that NO and VIP captured by a digital camera connected to a PC, ana- does not fulfill the criterion of being neuroprotective lyzed with AnalySIS software (version 3.02, Soft Imag- agents in the porcine nervous system, at least in its ing System, FRG) and printed on a wax printer sympathetic subdivision controlling the bowel-func- (Phaser 8200, Xerox, USA). tion. Further studies are needed to elucidate both the exact physiological relevance, as well as the exact mechanism(s) of these observations. Results and Discussion

Retrograde tract-tracing combined with Acknowledgements double-immunofluorescence staining revealed a low number of retrogradely labelled perikarya containing Supported by KBN, grant no. 6P06K 012 20. The immunorectivity to NOS (2.6 ± 0.8%; Figs. 1, 2), VIP authors are indebted to Ms. A. Piękos and Mr. Z. (0.9 ± 0.2%; Figs. 1, 2) or GAL (2.3 ± 0.6%) in the Liminowicz for their excellent technical help. IMGs of control animals. Furthermore, dense, moderately dense or scarce nerve fibres exhibiting immunoreactivity to NOS, VIP or GAL, respectively, References were observed in the control IMG (Figs. 1, 2). These fibres were patchily distributed throughout the gan- Klimaschewski L, Obermu¨ller N, Majewski M, Bachmann S, glion, being more numerous around large, TH/DfH- Heym C (1996) Increased expression of nitric oxide syn- and/or SOM-IR perikarya than around smaller, thase in a subpopulation of rat sympathetic neurons after NPY-containing somata. When ganglia from PE-suf- axotomy – correlation with vasoactive intestinal peptide. fering animals were studied, it has been found that Cell Tissue Res 285: 419-425. retrogradely labeled, PE-influenced IMG neurons Majewski M, Bossowska A, Gonkowski S, Wojtkiewicz J, were devoid of detectable amounts of any of the three Brouns I, Scheuermann DW, Adriaensen D, Timmer- substances studied (Fig. 3, 4). In contrast, the distribu- mans JP (2002) Neither axotomy nor target-tissue in- tion pattern and the density of intraganglionic NOS-, flammation changes NOS- or VIP-synthesis rate in the porcine distal bowel-projecting inferior mesenteric gan- VIP- or GAL-IR nerve fibres remained virtually un- glion (IMG) neurons. Folia Histochem Cytobiol 40: changed. Hence, these results run counter to data 53-54. dealing with axotomy- or acute chemical inflamma- Majewski M, Kaleczyc J, Klimaschewski L, Heym C, Lakomy tion-evoked changes in the expression of above men- M(1996) Axotomy-induced changes in chemical pheno- tioned substances (Majewski et al. 2002). Thus, while types of porcine IMG “ovarian” neurons. Verh Anat Ges a distinct up-regulation in the number of retrogradely 91: 62-63. 76 Majewski M. et al. Polish Journal of Veterinary Sciences Vol. 7, No. 3 (2004), Supplement, 77-79

Short original article Proliferative enteropathy (PE)-induced changes in the expression pattern of DβH, NPY and/or SOM in porcine IMG neurons supplying the descending colon

M. Majewski, J. Wojtkiewicz, S. Gonkowski, A. Bossowska, W. Sienkiewicz1, J. Kaleczyc1

Division of Clinical Physiology and 1Animal Anatomy, Department of Functional Morphology, Faculty of Veterinary Medicine, University of Warmia and Mazury, Oczapowskiego St. 13, 10-717 in Olsztyn, Poland

Abstract

The present study aimed at disclosing PE-driven changes in the number of retrogradely labelled descending colon-projecting, noradrenergic (i.e., DβH-IR), NPY- and/or SOM-IR IMG neurons in the pig. In control animals, 89.6 ± 1.8% of all retrogradely labeled neurons exhibited DβH-IR; 57.7 ± 2.3% of them co-expressed SOM-IR, while 35.7 ± 3.7% of DβH-IR perikarya were NPY-positive. In PE-animals, 77.9 ± 1.3% of all retrogradely traced perikarya were DβH-IR, while 22.7 ± 3.4% of them were NPY-IR. Only 2.6 ± 0.3% of DβH-containing retrogradely labeled neurons were SOM-IR. Thus, it appears that the chronic form of the PE resulted in a down-regulation of synthesis rate of “main transmitters” in affected IMG neurons.

Key words: proliferative enteropathy, neuronal plasticity, noradrenaline, neuropeptide Y (NPY), somatostatin (SOM), inferior mesenteric ganglion, pig

Introduction cell are able to profoundly change biochemical pathways of affected sympathetic neurons (Klimaschewski Inferior mesenteric ganglion (IMG) is the virtually et al. 1996, Zigmond 2000). However, there is lack of sole source of sympathetic prevertebral neuronal in- data dealing with bowel chronic proliferative inflam- put to the neural circuits controlling the porcine de- mation as a factor influencing the homeostasis of sym- scending colon. As revealed previously (Pidsudko pathetic neurons involved in the control of the gut et al. 2001), the vast majority of normal colon-projec- function(s). Therefore, the present study was aimed at ting neurons use different combination of noradrena- disclosing putative Lawsonia intracellularis infec- line (visualized by the presence of its synthesis- tion-driven (i.e., proliferative enteropathy (PE)-in- -rate limiting enzymes, tyrosine hydroxylase (TH) duced) changes in the relative frequency of retro- and dopamine-β-hydroxylase (DβH)), NPY and/or gradely labelled, descending colon-projecting, norad- SOM as their “main messenger” molecule(s). It is renergic (i.e., DβH-IR), NPY- and/or SOM-IR IMG well-known that pathological processes including neurons in the pig. inflammation of the target tissue of affected

Correspondence to: M. Majewski, tel.: +48 89 523-44-60, fax: +48 89 523-39-82, e-mail: [email protected] 78 Majewski M. et al. Proliferative enteropathy ... 79

Materials and Methods spared by SOM-IR terminals. NPY-IR varicose nerve terminals were sporadically found in the IMG neur- Six immature female pigs (approximately 8 weeks opil. In animals suffering from Lawsonia intracel- old), three control (C group) and three with clinically lularis infection, TH/DβH-IR neurons comprised diagnosed Lawsonia intracellularis infection (PE 77.9 ± 1.3% of all FB-containing cell bodies; while the group) were used. The animals were housed and relative frequency of neurons containing simulta- treated in accordance with the rules approved by the neously NPY reached approximately 23% local Ethical Commission. All pigs were subjected to (22.7 ± 3.4%; Fig. 3), there was a dramatic decrease in laparotomy and injected with 5% aqueous solution of the number of noradrenergic SOM-IR perikarya Fast Blue (FB) into the wall of the descending colon. – only 2.6 ± 0.3% of FB/DβH-containing neurons After a three-week survival period, the animals were were simultaneously SOM-IR in affected IMG (Fig. sacrificed with an overdose of sodium pentobarbital 4). It has been found that in PE-suffering animals, the (Vetbutal, Biowet, Poland; 90 mg/kg b.w.) and trans- number of SOM-IR nerve terminals was slightly in- cardially perfused with freshly prepared 4% parafor- creased, whilst the delicate NPY-IR intraganglionic maldehyde in 0.1 M phosphate buffer (pH 7.4). Ten nerve fibres appeared to be stronger fluorescing, what µm thick cryostat sections of the IMG were subjected may suggest an arrest in the orthodromic axoplas- to routine double-labelling immunofluorescence, us- matic transport/release of these neuropeptides by the ing different combinations of anisera raised in differ- affected neurons. Thus, as may be judged from ent species and directed towards TH (mouse mon- the present results, the chronic form of the PE result- oclonal, rabbit polyclonal), DβH (mouse monoclonal, ed in a drastic down-regulation of “main transmitter” rabbit polyclonal), NPY (rat polyclonal, rabbit poly- synthesis rate in affected neurons, especially with re- clonal) or SOM (rat monoclonal, rabbit polyclonal). gard to SOM. It may be thus indicative of Primary antisera were visualized by species-specific a PE-evoked “exhaustion” of compensatory mechan- secondary antisera conjugated to FITC or biotin (all ism(s) in colon-projecting sympathetic neurons, what, from Jackson Immunochemicals, USA). The latter in turn, may lead to an exacerbation in the deregula- antibodies were then visualized by a streptavidin-CY3 tory processes within the bowel. However, the exact complex (Jackson). Retrogradely labeled/double-im- mechanism of the process(es) described, as well as munostained perikarya were then evaluated under its/their patophysiological relevance, remains to be Olympus BX51 microscope equipped with epi-fluor- elucidated. escence and appropriate filter sets, counted in each fifth section (only neurons with clearly visible nucleus were included) and presented as mean ± SEM. Pic- Acknowledgements tures were captured by a digital camera connected to a PC, analyzed with AnalySIS software (version 3.02, Supported by KBN, grant no. 6P06K 012 20. The Soft Imaging System, FRG) and printed on a wax authors are indebted to Ms. A. Piękos and Mr. Z. printer (Phaser 8200, Xerox, USA). Liminowicz for their excellent technical help.

Results and Discussion References

In control animals, up to 90% of all retrogradely Klimaschewski L, Kummer W, Heym C (1996) Localization, labeled neurons exhibited TH- and DβH-IR regulation and functions of neurotransmitters and neur- (89.6 ± 1.8%); 57.7 ± 2.3% of them co-expressed omodulators in cervical sympathetic ganglia. Microsc Res SOM-IR (Fig. 1), while 35.7 ± 3.7% of TH/DβH-IR Tech 35: 44-68. Pidsudko Z, Kaleczyc J, Majewski M, Lakomy M, Scheuer- perikarya were simultaneously NPY-positive (Fig. 2). mann DW, Timmermans JP (2001) Differences in the In accordance with previous report (Pidsudko et al. distribution and chemical coding between neurons in the 2001), virtually all the SOM-IR cells belonged to the inferior mesenteric ganglion supplying the colon and rec- class of large neurons, being approached by dense to tum in the pig. Cell Tissue Res 303: 147-158. moderately dense, patchily distributed SOM-IR nerve Zigmond RE (2000) Neuropeptide action in sympathetic terminals; in contrast, smaller NPY-IR perikarya, ganglia. Evidence for distinct functions in intact and located mainly at the ganglionic periphery, were axotomized ganglia. Ann N Y Acad Sci 921: 103-108. ← Figs. 1 and 2. In the IMG of control animals a large population of retrogradely labeled (1a, 2a), DβH-positive neurons (1b, 2b) were simultaneously SOM- (1c) or NPY-IR (2c). Only a few of FB+ cells were DβH-immunonegative (double-headed arrow in Fig. 2). Appropriate arrows/arrowheads point out to differently chemically coded IMG neurons. x 200. Figs. 3 and 4. In PE-suffering animals the number of retrogradely labeled (3a) DβH/NPY-IR neurons was only slightly reduced (3b, c), while nearly all FB+ cells were devoid of SOM-immunoreactivity (4a-c). Appropriate arrows/arrowheads point out to differently chemically coded IMG neurons. x 200. 80 Majewski M. et al. Polish Journal of Veterinary Sciences Vol. 7, No. 3 (2004), Supplement, 81-84

Short original article Effect of Methisoprinol, KLP-602 and NDV infection on the selected biochemical indices of the allantoic fluid of chicken embryos

J. Małaczewska, Z. Rotkiewicz, A.K. Siwicki

Department of Infectious and Invasive Diseases, Chair of Clinical Microbiology and Immunology, Faculty of Veterinary Medicine, University of Warmia and Mazury in Olsztyn, Oczapowskiego St. 13, 10-719 Olsztyn, Poland

Abstract

The effect of Methisoprinol, KLP-602 and NDV infection on the reactivity of lymphocytes and selected biochemical parameters of allantoic fluid of chicken embryos was tested. Only KLP-602 positively affected the reactivity of lymphocytes. The administration of KLP-602 to infected with NDV embryos made lymphocytic reactivity increase to the level noted in the control group. The drug increased also the activity of ceruloplasmin in allantoic fluid of chicken embryos.

Key words: chicken embryos, NDV, Methisoprinol, KLP-602

Introduction Materials and Methods

The process of introducing new drugs into the mar- The experiment was performed on chicken ket includes preclinical tests performed on cell cul- embryosaged10daysandolder.Tendaysold tures and chicken embryos. Chicken embryos consti- embryos were inoculated by the allantoic cavity tute good experimental material, because they contain route. The drugs examined were introduced by the immune system elements. Despite the fact that this same route. Allantoic fluids were collected for bio- system is morphologically immature and its functions chemical analyses 6, 12, 24, 48 and 72 hours after are not fully developed yet, it allows to obtain positive inoculation. results of in ovo inoculations (Sharma and Burmester Two antiviral agents KLP-602 (active substance 1982, Ahmad and Sharma 1993, Sharma 1997, Stone – lysozyme dimer), manufactured by Nika Health et al. 1997, Mast and Goddeeris 1999). The results of Products, Ltd, USA at the following doses: 20, 10, 5, such studies suggest that drug administration to em- 2 and 1 mg/embryo, and Methisoprinol (active sub- bryos may lead to changes in the biochemical par- stance – isoprinozine), manufactured by Polfa Grod- ameters of their body fluids, and that simultaneous zisk, at the following does: 50, 25, 12.5, 6 and application of drugs and microbes can stimulate anti- 3 mg/embryo were used in the studies. A physiological viral immunity. buffer solution (PBS) was applied as a solvent.

Correspondence to: A.K. Siwicki, tel.: +48 89 523-32-17, e-mail: [email protected] 82 Małaczewska J., Rotkiewicz Z., Siwicki A.K.

Two strains of Newcastle disease virus (NDV) level of this reactivity was increasing according to were used: mesogenic Roakin strain the age. On the 3th day of incubation, IS was 3.2 -9.5 (EID50=10 /ml) and lentogenic inoculation LaSota (Table 1). -9.5 strain (Sotasec vaccine, EID50=10 /ml). The activity of ceruloplasmin in the allantoic In order to determine lymphocytic reactivity 10 fluid increased as soon as after 6 hours from the days old chicken embryos were given the maximum administration of KLP-602 or inoculation with the tolerable doses of both drugs in the form on injec- LaSota strain or the Roakin strain of Newcastle dis- tions into the allantoic cavity. The other groups were ease virus, as well as after the application of the 4 inoculated by the same route with 10 EID50 of the LaSota strain and Methisoprinol, and the Roakin LaSota strain of Newcastle disease virus, combined strain and Methisoprinol or KLP-602. Methisop- with the maximum tolerable dose of KLP-602 or Me- rinol applied alone had no effect on the activity of thisoprinol or without the drugs. The control group this acute phase protein (Table 2). comprised chicken embryos treated with PBS. The inoculated embryos were incubated for five consecutive days. Then their lymphocytic reactivity was de- Table 1. Index of lymphocyte stimulation in chicken em- termined with a blastic transformation test on the bryos under the influence of PHA. day of inoculation (time 0), and after 72 and 120 Time of the test hours (according to the method described by Mała- Groups czewska et al. 2003). 0h 72h 120h To determine biochemical parameters 10 days Control 1.5 2.1 4.5 old chicken embryos were allocated to 9 groups, 25 KLP–602 – 3.2 5.5 embryos each. Then they were inoculated with: Me- Methisoprinol – 2.5 3.9 thisoprinol (6 mg/embryo), KLP-602 (5 mg/em- LaSota + Methisopr. – 0.8 1.8 2 LaSota + KLP – 1.5 2.7 bryo), LaSota strain of NDV (10 EID50)orthe 2 LaSota – 0.7 1.5 Roakin strain of NDV (10 EID50), injected into the allantoic cavity (Table 1). The next two groups were inoculated with the LaSota strain and treated with Methisoprinol or KLP-602, and another two were The highest increase in lysozyme activity in the allantoic fluid was noted after 48 and 72 hours from inoculated with the Roakin strain and treated with inoculation with the Roakin strain or the LaSota both drugs. The control group was given PBS. strain of NDV, as well as after inoculation with the Allantoic fluids were collected 6, 12, 24, 48 and LaSota strain and simultaneous administration of 72 hours after inoculation to determine ceruloplas- Methisoprinol. When applied alone Methisoprinol min activity by the spectrophotometric method de- cause a slight increase in lysozyme activity only in scribed by Rice et al (1986), and lysozyme activity the 6th hour after injection. In successive hours the by the turbidimetric method described by Parry et activity of this enzyme in this group of embryos was al. (1965), modified by Siwicki and Anderson lowerthaninthecontrolgroup(Table3). (1993). The results obtained were subjected to a two-factor analysis of variance and Tukey test, determining means, standard deviations and significance of dif- Discussion ferences at p≤0.05 and p≤0.01. The objective of the present study was to determine the effects of Methisoprinol and KLP-602 on the reactivity of embryo lymphocytes, and the activ- Results ity of ceruloplasmin and lysozyme in the allantoic fluid. Themaximumtolerabledoseswere:6mg/em- The highest reactivity of lymphocytes was ob- bryo of Methisoprinol and 5 mg/embryo of served in the group receiving KLP-602 (IS=5.5), KLP-602, and such doses were applied in the experi- andthelowest–inthegroupofembryosinoculated ment. Higher doses of both drugs were toxic to the with the LaSota strain of Newcastle disease virus embryos; their administration resulted in growth in- and treated with Methisoprinol. The administration hibition or even embryo death. Furthermore, high of KLP-602 to infected with NDV embryos made doses of Methisoprinol caused urate salt deposition lymphocytic reactivity increase to the level noted in on the embryos’ bodies, chorioallantoic membrane the control group. and in the kidneys. Enhanced ceruloplasmin activity in the allantoic The lymphocytes of 10 days old embryos of the fluidoftheembryosinoculatedwithbothstrainsof control group incubated with phytohemagglutinin NDV, as well as of those treated with the drugs responded by slight simulation (IS was 1.5) and the examined, was observed starting from the 6th hour Effect of Methisoprinol, KLP-602 ... 83

Table 2. Ceruloplasmin activity in the allantoic fluid of chicken embryos (mg%).

Sampling. Group (administered Time after inoculation (hrs) Significance of differences drug, virus) I II III IV V VI between samplins 0 6 12 24 48 72 Control M 6.19 6.49a 5.59 6.61 7.11 6.43 No statistically significant SD 2.55 1.01 1.94 1.94 1.77 1.05 differences Methisoprinol M 5.94 6.11c 6.29 6.34 6.12 5.97 SD 0.95 2.22 1.62 1.67 1.45 1.17 d b KLP-602 M 6.24 9.02 7.41 6.92 8.10 8.11 I

Explanation: *abcd – statistically significant differences at p≤0.05 **ABCD – statistically significant differences at p≤0.01

Table 3. Lysozyme activity in the allantoic fluid of chicken embryos (mg%).

Sampling. Group (administered Time after inoculation (hrs) Significance of differences drug, virus) I II III IV V VI between samplins 0 6 12 24 48 72 Control M 0.58 0.78AC 0.62Aa 1.02a 1.58A 2.37A < SD 0.34 0.33 0.21 0.50 0.57 0.92 I,II,III,IV VI** Methisoprinol M 0.78 1.68A 1.47 0.65Ac 1.15A 1.44A < < < SD 0.47 0.12 0.50 0.19 0.41 0.50 I II*, IV III,VI* IV II** Roakin M 0.62 3.66B 2.81B 2.00d 7.11BD 7.60B < < SD 0.36 0.67 0.90 0.75 2.09 1.23 I II,V,VI** II,III,IV V,VI** Roakin+Methisoprinol M 0.51 1.58Aa 2.20b 2.17Bb 3.54Ca 2.86A < < < SD 0.23 0.38 1.07 0.86 0.76 0.70 I III,IV* I V,VI**, II V** LaSota M 1.02 2.81Db 1.90 1.77d 6.35Bb 6.42B < SD 0.13 1.23 0.75 0.46 1.80 1.38 I,II,III,IV V,VI** LaSota+Methisoprinol M 0.74 1.69A 2.47b 1.12 7.28BD 7.64B < < SD 0.28 0,30 0.92 0.37 0.93 1.53 I III* I,II,III,IV V,VI**

Explanation: *abcd – statistically significant differences at p≤0.05 **ABCD – statistically significant differences at p≤0.01 after inoculation. Higher activity of this acute phase group was increasing in successive hours of observa- protein was observed until the end of the experi- tions. ment, but a statistically significant increase in this Lysozyme activity in the allantoic fluid of the con- parameter, compared with the control group, was trol embryos was increasing with their age. After in- recorded only in the 6th hour after inoculation and oculation with both strains of NDV it reached a sig- only in the group inoculated with the Roakin strain nificantly higher level than in the control group. Its of Newcastle disease virus and treated with highest activity was observed after 48 and 72 hours in KLP-602. The activity of this enzyme in the control the embryos inoculated with both strains of NDV and 84 Małaczewska J., Rotkiewicz Z., Siwicki A.K. in those inoculated with the LaSota strain and References treated with Methisoprinol. In the groups receiving KLP-602 lysozyme activity was very high due to its Ahmad J, Sharma JM (1993) Protection against hemor- external application, so, the data for these groups rhagic enteritis and Newcastle disease in turkeys by em- were ignored. bryo vaccination with monovalent and bivalent vaccines. The available literature on the topic provides no Avian Dis 37: 485-491. information on the level of the above parameters in Małaczewska J, Rotkiewicz Z, Siwicki AK (2003) Effect of chicken embryos. Mazur-Gonkowska (2004), who Methisoprinol and KLP-602 on the development of immunocompetent organs and selected biochemical indices analyzed acute phase proteins and non-specific im- of the allantoic fluid of chicken embryos. Polish J Vet Sci munity in turkeys inoculated with bacteria and vi- 3 (suppl): 21-24. ruses, reported similar values of the above pa- Mast J, Goddeeris BM (1999) Development of immunocom- rameters. However, a comparative analysis seems im- petence of broiler chickens. Vet Immunol Immunopathol possible in this case due to considerable differences 70: 245-256. in the type of experimental material (allantoic fluids Mazur-Gonkowska A, Koncicki A, Krasnodębska-Depta of chicken embryos vs. blood serum of adult turkeys). A(2004) Assessment of acute phase response in turkeys This author observed a slight increase in the serum experimentally infected with Escherichia coli or haemor- concentration of ceruloplasmin in turkeys inoculated rhagic enteritis virus. Bull Vet Inst Pulawy 48: 19-23. withHEvirusaslateas24hoursafterinoculation. Parry RM, Chandau RC, Shahani RM (1965) A rapid and sensitive assay of muramidase. Proc Soc Exp Biol Med The serum activity of lysozyme in turkeys inoculated 119: 384-386. with this virus also increased 24 hours after inocula- Rice EW, Wagman E, Takenaka Y (1986) Ceruloplazmin tion reaching the highest level after 72 hours. En- assay in serum: standarization of ceruloplazmin activity in hanced activity of this enzyme was also observed in terms of international enzyme units. Diag Lab 12: 39-53. the control group after 48 and 72 hours (data not Sharma JM (1997) The structure and function of the avian shown). immune system. Acta Vet Hung 45: 229-238. It may be concluded that Methisoprinol Sharma JM, Burmester BR (1982) Resistance to Marek’s and KLP-602 had some positive effects on chicken disease at hatching in chickens vaccinated as embryos embryos in the aspects described above. This con- with turkey herpesvirus. Avian Dis 25: 134-149. cerns first of all KLP-602, which had positively af- Siwicki AK, Anderson DP (1993) Immunostimulation in fish: Measuring the effects of stimulants by serological fected lymphocytic reactivity in chicken embryos and immunological methods. US Fish and Wildl Service under the influence of PHA, and on the develop- -IFI, Olsztyn, 1, pp. 17. ment of immunocompetent organs (Małaczewska et Stone H, Mitchell B, Brugh M (1997) In ovo vaccination of al. 2003). chicken embryos with experimental Newcastle disease and avian influenza oil-emulsion vaccines. Avian Dis 41: 856-863. Polish Journal of Veterinary Sciences Vol. 7, No. 3 (2004), Supplement, 85-87

Short original article Levels of C-reactive protein and interferon gamma in pigs vaccinated with deleted vaccine against Aujeszky’s disease after experimental infection with virulent Herpesvirus suis type 1

E. Mikulska-Skupień, W. Szweda, Z. Procajło, M. Bigoszewski1

Department of Infectious and Invasive Diseases and 1Department of Clinical Sciences, Faculty of Veterinary Medicine, University of Warmia and Mazury in Olsztyn, Oczapowskiego St. 13, 10-719 Olsztyn, Poland

Abstract

The purpose of the present study was to evaluate the dynamics of the C-reactive protein (CRP) and interferon gamma (IFN-γ) level formation in sera of pigs vaccinated intramuscularly versus intradermally with a deleted vaccine against Aujeszky’s disease after experimental infection with virulent Herpesvirus suis typ 1 (SHV-1). Three groups, seven piglets each, were formed. At the age of 12 weeks two groups were vaccinated twice, 4 weeks apart, with deleted Porcilis Begonia vaccine 6.0 5.0 (Intervet) at a dose of 2.0 ml (10 TCID50) intramuscularly or 0.2 ml (10 TCID50) intradermally. Pigs of control group were injected with 2.0 ml of PBS intramuscularly. Seventy days after the first vaccination all pigs were intranasally infected with Northern Ireland Aujeszky-3 strain of SHV-1 at 5.5 a dose of 10 TCID50. The increase of CRP level in pig sera from both vaccinated groups was statistically significantly lower (p<0.01) than in control group in the most terms of blood sampling, showing good protective action of the vaccine through quick stimulation of mechanisms of cell-mediated and humoral immunity. Intradermal vaccination, ensuring the high activity of lymphocytes T, induces higher level of IFN-γ after exposition to virulent NIA-3 strain of SHV-1, when compared to intramuscular vaccination.

Key words: Aujeszky’s disease, deleted vaccine, experimental infection, CRP, IFN-γ

Introduction most important signal inducing trancriptive factors of APP genes are proinflammatory cytokines – IL-1, IL-6 One of the components of the non-specific body and TNFα, produced by various types of immune as response causing a restriction of inflammation, elim- well as epithelial cells and fibroblasts, as a result of ination of damaging or infectious factors and restor- activation by viruses, bacteria, fungi, substances re- ing the homeostasis is so called “acute phase releasing from destroyed tissues and several other fac- sponse”. Changes in the concentration of several tors (Kostro et al. 2003). In the pig, C-reactive protein serum proteins, i.e. acute phase proteins (APP) took (CRP), haptoglobin (Hp) and pig Major Acute Phase place during this process (Kostro et al. 1996). The Protein (MAPP) are the main APP (Kostro et al.

Correspondence to: E. Mikulska-Skupień, tel./fax: +48 89 523-35-75, e-mail: [email protected] 86 Mikulska-Skupień E. et al.

2002). Non-specific humoral immunity, as an answer Interferon gamma (IFN-γ). The levels of IFN-γ to viral antigen, is however mainly represented by syn- in pig serum were determined by immunoenzyme test thesis of interferons – INF (Gołąb et al. 2002). The using ready to use diagnostic Kit – Swine Interferon-γ mammalian cells are able to produce three main (swIFN-γ) (BioSource International, Inc., USA). forms of interferons: α, β and γ (La Bonnardiere et al. Blood samples were taken immediately before infec- 1994). Antiviral action of IFN depends also on their tion and on 1, 2, 3, 5, 7 and 14 dpi. influence on immunological system through stimula- Statistical evaluation. The results were statisti- tion of specific and non-specific mechanisms partici- cally evaluated using one-way analysis of variance pating in the response to viral antigens. Production of (ANOVA) to compare several means (NIR test) at IFN plays also an extremely important role in protec- p<0.05 and p<0.01 and presented as mean and SD. tion against virulent Herpesvirus suis type 1 (SHV-1) (Chinsakchai and Molitor 1994). The most actively influencing the immune system is IFN-γ, produced by Results and Disscusion activated lymphocyte T belonging to all subpopulations and NK cells (Chinsakchai and Molitor 1994, The fluctuations in the level of CRP in porcine Gołąb et al. 2002). sera after experimental infection is presented in Table The purpose of our study was to evaluate the dy- 1. On 1 dpi highly statistically significant increase in namics in the formation of the levels of CRP and the CRP level was observed in control group, as com- IFN-γ in pigs vaccinated intramuscularly versus in- pared to both immunized groups. Statistically signifi- tradermally with a deleted vaccine against Aujeszky’s cant differences between immunized and control disease after experimental infection with virulent groups were observed until 5 dpi. The formation of SHV-1. CRP levels in pig sera of both vaccinated groups was very similar, only on 3 dpi statistically significantly higher level of CRP was noticed in group II, when Materials and Methods compared to group I. The formation of IFN-γ levels in pig sera after ex- Animals. An experiment was carried out using 21 perimental infection is shown in Table 2. Analyzing piglets, 8 week old, divided into three groups, 7 ani- the degree of non-specific immune response to vac- mals each, kept in the isolation units. Piglets were free cinal antigen, evaluated by the IFN-γ level in sera from anti-gE antibodies checked by Pseudorabies Vi- after experimental infection of piglets with virulent rus gp I Antibody Test Kit (Herd Chek Anti-PRV gp SHV-1, one can see that there were statistically sig- I, IDEXX Lab Inc, USA). nificant increase in IFN-γ level on 3 and 5 dpi in pigs Vaccine and vaccination. Deleted, gE-and vaccinated I.D., when compared to pigs vaccinated TK-negative, live attenuated Porcilis Begonia vaccine I.M. (Intervet, The Netherlands) with adjuvantive diluent In this study the levels of CRP in pig sera after Diluvac Forte was used. At the age of 12 weeks pigs experimental infection with NIA-3 strain of SHV-1 were vaccinated twice, 4 weeks apart, at a dose of 2.0 were determined. CRP belongs to that kind of APP, 6.0 ml (10 TCID50) intramuscularly – group I or 0.2 ml whose level increases just 6-8 hours after affecting the 5.0 (10 TCID50) intradermally (I.D.) using needleless organism by damaging stimulus and reaches its high- apparatus SERENA model SD 1-2, (Emaplast, Italy) est value during 24-48 hours (Kostro et al. 2003). In – group II. Pigs of group III (control) were injected our study the increase of CRP level in pig sera from with 2.0 ml of PBS. both vaccinated groups was statistically significantly Experimental infection. Seventy days after the lower than in control group in the most terms of blood first vaccination all pigs were experimentally infected sampling, showing good protective action of the vac- with virulent Northern Ireland Aujeszky-3 (NIA-3) cine through quick stimulation of mechanisms of 5.5 strain of SHV-1 at a dose of 10 TCID50 by intranasal cell-mediated and humoral immunity. The highest instilling of 0.5 ml of virus suspension into each nos- CRP level was observed in control group on 3 dpi, tril. while in groups vaccinated I.M. and I.D. on 4 and C-reactive protein (CRP). The levels of CRP in 6 dpi, respectively. The obtained results were corre- pig serum were evaluated by nephelometric method lated with clinical signs observed at that time. using Beckman Array 360 System apparatus (USA) It is accepted, that acute phase response is parallel- and anti-human CRP antibodies (CRP kit p/n 449760) ed by behavioral changes, like a decrease in the appe- and human standards, according to the manufacturer tite and life activity, depression, elongation of the recommendation and Heegaard et al. (1998) consider- sleep time, slowering of digestion processes, as well as ations. Blood samples were taken immediately before occurrence of fever as a result of disorders in the ther- infection and every day in the period 1 to 7 days post moregulation centre, induced by the action of IL-1, infection (dpi). IL-6 and/or TNFα (Bigoszewski et al. 2001, Kostro Levels of C-reactive protein ... 87

Table 1. The CRP level (mg/l) in pig sera after experimental infection.

Days after infection Groups 01234567 I (I.M.) X 4.9 4.9A 10.3A 7.8Aa 20.0a 17.5A 18.5 6.3A SD 0.7 0.8 1.6 0.5 1.6 1.0 3.3 2.4 II (I.D.) X 3.3 8.9A 14.0a 19.1Ab 19.0a 17.9A 19.9 11.8 SD 0.8 3.1 6.7 0.41 3.3 1.9 2.2 3.7 III (Control) X 5.1 19.5B 25.7Bb 34.9B 30.2b 31.8B 21.7 17.4B SD 1.2 3.6 3.5 7.9 6.3 1.5 1.3 2.9

Explanations: differences between groups A, B at p < 0.01; a, b at p < 0.05

Table 2. The IFN-γ (pg/ml) in pig sera after experimental infection.

Days after infection Groups 01235717 I (I.M.) X 12.23Aa 70.56A 74.61A 61.93A 63.38A 64.58A 61.14A SD 0.77 2.82 2.52 1.36 4.56 3.14 2.60 II (I.D.) X 11.10Ab 87.50B 85.08B 109.33B 100,29B 73.72B 64.15A SD 0.71 1.90 0.82 3.07 0.66 1.35 0.84 III (Control) X 15.29B 31.05C 40.34C 41.57C 52.70C 34.92C 33.84B SD 0.46 0.96 0.64 2.47 5.27 1.81 1.82

Explanations: differences between groups A, B, C at p < 0.01; a, b at p < 0.05

et al. 2003). In the control group, where intensifica- References tion of clinical signs as well as increase of body temperature were observed from 3rd dpi up, simulta- Bigoszewski M, Rychlik A, Depta A (2001) Białka ostrej neously the highest CRP levels were noticed, while in fazy u zwierząt. Medycyna Wet 57: 151-155. both vaccinated groups, where clinical signs were Chinsakchai S, Molitor TW (1994) Immunobiology of weakly expressed, the levels of CRP in sera were also pseudorabies virus infection in swine. Vet Immunol Im- munop 43: 107-116. statistically significantly lower. Interferon plays an Gołąb J, Jakóbisiak M, Zagożdżon R, Obłąkowski P (2002) important role in e.g. increasing cytotoxicity of lym- Cytokiny. W: Gołąb J, Jakóbisiak M, Lasek W (eds): Im- phocytes Tc and NK cells, MHC expression, macro- munologia. Wydawnictwo Naukowe PWN, pp. 198-248. phages activation and phagocytosis enhancement, as Heegaard PMH, Klausen J, Nielsen JP, Gonzales-Ramon N, well as in the induction of expression of other Pin˜eiro M, Lampreave F, Alava MA (1998) The Porcine cytokines, like IL-1, IL-6 or TNFα (Chinsakchai and Acute Phase Response to infection with Actinobacillus Molitor 1994, Gołąb et al. 2002). In pigs infected pneumoniae. Haptoglobin, C-Reactive Protein, Major with SHV-1 interferon appears in sera and urine 24 Acute Phase Protein and Serum Amyloid A Protein Are hours after infection, reaching its maximum level be- Sensitive Indicators of Infection. Comp Biochem Physiol tween 3 and 4 dpi. Then, its level gradually decreases 119B(2): 365-373. Kostro K, Gliński Z, Piliszczyński M (2002) Monitoring (Wittmann et al. 1980, La Bonnardiere et al. 1994). γ chorób zakaźnych trzody chlewnej. Magazyn Wet 72: In our study considerably higher level of IFN- was 31-34. observed in group II than in groups I and control. Kostro K, Luft-Deptuła D, Gliński Z, Miazga A (2003) Rola The increase of IFN-γ level was observed just on białek ostrej fazy w patologii zwierząt. Życie Wet 78: 1 dpi, but the highest value was noticed on 3 dpi in 19-25. group of I.D. vaccinated pigs. Obtained results sug- Kostro K, Sobieska M, Wiktorowicz K, Wołoszyn S (1996) gest, that I.D. vaccination, by ensuring the high activ- Białka ostrej fazy u zwierząt – występowanie i charak- ity of lymphocytes T, induces higher level of IFN-γ terystyka. Medycyna Wet 52: 152-155. after exposition to virulent NIA-3 strain of SHV-1, La Bonnardiere C, Lefevre F, Charley B (1994) Interferon when compared to I.M. vaccination. Thus, I.D. route response in pigs: Molecular and biological aspects. Vet Immunol Immunop 43: 29-36. of administration of deleted AD vaccine can be Wittmann G, Jakubik J, Ahl R (1980) Multiplication and agoodalternativeandcanbeusedinplaceofor distribution of Aujeszky’s disease (pseudorabies) virus in parallelly to traditional I.M. route in AD “vaccina- vaccinated and non-vaccinated pigs after intranasal infection-eradication programme” in Poland. tion. Arch Virol 66: 227-240. 88 Mikulska-Skupień E. et al. Polish Journal of Veterinary Sciences Vol. 7, No. 3 (2004), Supplement, 89-92

Short original article Vimentin – the marker in dog kidney injury

B. Osińska, M. Katkiewicz

Department of Clinical Sciences, Faculty of Veterinary Medicine, Warsaw Agricultural University, Nowoursynowska St. 159c, 02-787 Warsaw

Abstract

The aim of this work was to perform the histopathological evaluation of 50 dog kidneys with special attention to vimentin immunocytochemical expression. In examined kidneys glomerulonephritis (50 cases), degeneration and/or necrosis of tubular epithelial cells (49 cases), acute or chronic interstitial nephritis with kidney fibrosis (39 cases) were diagnosed. Glomerular tuft atrophy and/or glomerular sclerosis was found in almost all kidneys. In 34 cases thickening of the blood vessel wall was also observed. In 47 cases vimentin expression was noted in low degree in podocytes of glomerular and basal membranes. In 49 cases the focal vimentin expression was found in tubular epithelial cells of kidney cortex and/or medulla. The weak expression of vimentin was also observed in the interstitium. In 46 dogs various degree of vimentin expressions were seen in blood vessel walls. Decrease in vimentin expression was correlated with glomerular damage and their appearance in tubular cells, as a symptom of the alteration of their cytoskeleton. The thickening of blood vessel walls seen in routine stained sections corresponded to changes in vimentin expressions. The vimentin expression is a very good marker of the early injury of cells in glomerules, tubules and blood vessels.

Key words: dog, kidney disease, vimentin expression

Introduction very important role in filtration process, cell adhesion, cell migration and differentiation, so their defects may There are many factors which play the important be engaged in pathogenesis of glomerular and tubu- role in the physiological kidney function. Some lo-interstitial diseases (Miner 1999). authors considered that for normal kidney functions Podocytes participate also in kidney filtration pro- the most important among other factors are the nor- cesses (Yaoita et al. 1999), so their alteration in dis- mal blood supply and circulation. eased kidney is followed by hypertrophy of still The nephron is the kidney functional unit, so the healthy podocytes. These hypertrophic podocytes are primary glomerular diseases are responsible for sec- more susceptible for pathological stimulation, so in ondary tubular damage. The primary glomerular dis- consequence it leads to chronic kidney insufficiency eases are caused by deposition of immunological com- (Yaoita et al. 2002). In Mammals, including Canidae plexes, thrombosis, emboli, and due to bacterial and glomelural podocytes express very strong vimentin im- viral infections. The long lasting noxious disease is munocytochemical reaction. The intensity of this ex- followed by atrophy, or glomerular sclerosis, and sec- pression is also considered as the marker of their ondary appearing regressive changes in tubular epi- health status and functional ability (Yaoita et al. thelium. The glomerular changes reciprocally may re- 1999). flect in various tubular pathological changes with de- The pathogenesis of extraglomerular kidney creased blood supply. Kidney basal membranes play lesions are complicated and only partially recognized.

Correspondence to: B. Osińska, e-mail: [email protected] 90 Osińska B., Katkiewicz M.

The noxious agents are causing tubular cell degener- pression was noted in some glomerular capsules, in ation, necrosis and/or atrophy. The compensatory tu- podocytes of all glomerular and basal membranes. In bular mechanism is hypertrophy and regeneration of 5 cases the focal vimentin expression was found in epithelium, mediated by various hormones and tubular epithelial cells of kidney cortex and medulla. growth factors (Klahr and Morrissey 2000). Vimen- The weak vimentin expression was also observed in tin expression may indicate the proliferative the interstitium. In 4 dogs various degree expressions podocyte activity, described in human and rat kid- were seen in blood vessel walls. neys (Gro¨ne et al. 1987). In all dogs older then 1 year membrano-prolif- The chronic kidney diseases with tubular atrophy erative or proliferative glomerulonephritis was pres- are accompanied by proliferation of the interstitial ent. Glomerular tuft atrophy and/or glomerular tissue. According to recent data, fibroblasts of the sclerosis was found in almost all kidneys. In all the interstitial tissue may originate from transformed tu- examined kidneys reversible injury and necrosis were bular epithelial cells in diseased kidney (Klahr and seen in tubular epithelium. In almost all kidneys Morrissey 2000, Okada et al. 2000, Stahl and Felsen glomerular tuft atrophy and glomerular sclerosis 2001). were found. In some cases acute or chronic inter- Vimentin has very strong expression in glomerular stitial nephritis with kidney fibrosis was observed. structures and endothelium and smooth muscles of Thickening of the blood vessel walls were found. thebloodvesselwallinthehealthykidney.There- Vimentin expression in Bowman’s capsule was seen sults of research performed on homozygotic mice focally. In all the examined kidneys vimentin express- with “knock-out” vimentin gene indicated its crucial ion was present in podocytes and in glomerular base- role in vascular adaptation capacity. The loss of this ment membranes, but in various degree of intensity. capacity was leading to the developing of so called Vimentin expression in tubular epithelial cells was end-stage kidney in those mice (Terzi et al. 1997). observed focally in the kidney cortex and in kidney The aim of this work was to perform the his- medulla. In the interstitial tissue focal vimentin ex- topathological evaluation of dog kidneys, with pression was noted. Vimentin expression in blood special concern on vimentin immunocytochemical vessel walls was present and showed different inten- expression. sity. In some cases vimentin expression was only in endothelial cells or completely absent in kidneys (Fig. 1, 2). Materials and Methods The histopathological lesions of kidneys observed indogs15-22yearsofagewereofthesametypebut Kidneyswereobtainedfrom50dogsofboth more advanced, as in younger dogs. It was also mani- sexes, different breeds, from 4 months to 22 years fested in changes of vimentin expression. Vimentin old, sent for routine necropsy examination. The car- expression was found only in single glomerules, as in casses were selected according to anamnesis and post some blood vessel walls. In the majority of mortem lesions indicated for kidney diseases as the glomerules and blood vessel walls there was no main cause of death. The time of collection of the vimentin expression. These vimentin expression al- material did not exceed 6 hours from the death of terations were accentuated by the appearing in a dog. Both kidneys were fixed in 10% buffered for- multiple tubule cells of kidney cortex and medulla. malin. From each kidney 2 – 4 specimens were col- Histopathological lesions and vimentin expression lected, embedded in paraffin and stained with hae- in kidneys of all examinated cases are summarized in matoxylin and eosin. Vimentin cytochemical express- table 1. ion was visualised on sections mounted on Silane- coated slides by using monoclonal anybodies clone V 9 (Novocastra). Table 1. Histopathological lesions and vimentin expression The examined dogs were divided into 5 groups, in examined kidneys. according to the age of animals, as follows: I (0 Number of cases > > Vimentin –1year),II(1–5years),III( 5–10years),IV( 10 with Part of nephron expression –15years)andV(>15 years). histopathological (IPOX) lesions (HE) Glomerules 50 47↓ Results Tubular epithelium cells 49 49↑ Blood vessel walls 34 46↓ In dogs up to 1 year old focal glomerulonephritis wasfound.Theinflammatorychangeswereaccom- Explanation: panied by focal hydropic and parenchymatous de- ↓ – reduced vimentin expression generation seen in tubular epithelium. Vimentin ex- ↑ – increase in vimentin expression Vimentin – the marker ... 91

Fig. 1. Vimentin expression found in tubular cells (→) and focally in blood vessel endothelium (→) in kidney of the IIIrd group. IPOX, x 100.

Fig. 2. High degree of vimentin expression visible in tubular cells, but the low degree of vimentin expression present in glomerules (→). There is no vimentine expression in blood vessel wall (→). IPOX, x 100. 92 Osińska B., Katkiewicz M.

Discussion expression was seen in the blood vessel cells earlier then the development of the histopathological Vimentin expression in dog healthy nephron is changes were noted. This observation suggests, that limited to glomerules and it appears in tubular epi- molecular disturbances present in the cell metabolism thelium in chronic kidney diseases (Vilafranca et al. were followed by structural changes. 1994). The tubular damage may be manifested by the The results of the presented work may be applied increase in mitoses with transient expression of in veterinary practice, as well as in comparative pa- vimentin (Okoń 2003). Klahr and Morrisey (2000) thology research. observed that tubular cells undergoing early pathological changes transformed them into fibroblasts, which possesed expression of vimentin. The similar References lesion was observed in tubular cells of rats exposed to ¨ ¨ carcinogens (Ward et al. 1994). Grone HJ, Weber K, Grone E, Helmchen U, Osborn In all the examined kidneys of dogs up to 1 year M(1987) Coexpression of keratin and vimentin in damaged and regenerating tubular epithelia of the membranous-proliferative glomerulonephritis was kidney. Am J Pathol 129: 1-8. found. The tubular nephropathy was accompanied by Klahr S, Morrissey JJ (2000) The role of vasoactive the glomerular changes. Vimentin expression ap- compounds, growth factors and cytokines in the pears in degenerated tubules, in spite to glomerules, progression of renal disease. Kidney International in which its expression decreased proportionally to 57: 7-14. the degree of glomerular damage. Vimentin express- Miner JH (1999) Renal basement membrane compo- ioninbloodvesselwallswasnormal,indicatingtheir nents. Kidney International 56: 2016-2024. good functional status (Terzi et al. 1997). nd Okada H, Inoue T, Suzuki H, Strutz F, Nelson EG In the II group the histopathological changes (2000) Epithelial-mesenchymal transformation of were observed in all parts of the nephron, as well as renal tubular epithelial cells in vitro and in vivo. inbloodvesselwalls.Thedegreeofthesechanges Nephrol Dial Transplant 15 (suppl 6): 44-46. correlated with vimentine expression. These findings Okoń K (2003) Tubulo-interstitial lesions in were in accordance with previously presented data glomerulopathy. I. Pathogenesis. Pol J Pathol (Vilafranca et al. 1994, Terzi et al. 1997, Klahr and 54: 87-94. Morrisey 2000). Stahl PJ, Felsen D (2001) Transforming growth fac- rd th In the III and IV groups the further enhance- tor-β, basement membrane, and epithelial-mesen- ment of glomelural pathological changes was mani- chymal transdifferentiation. Implications for fested by appearing of so called semilunar crescents, fibrosis in kidney disease. Am J Pathol which stimulated glomerular sclerosis or caused 159: 1187-1192. glomerular atrophy. These lesions were also respon- Terzi F, Henrion D, Colucci-Guyon E, Federici P, sible for the high degree of blood circulatory disturb- Babinet Ch, Levy BI, Briand P, Friedlander G, ances which were followed by the dystrophy of tubu- (1997) Reduction of renal mass is lethal in lacking lar epithelial cells. This was also accompanied by vimentin. Role of endothelin-nitric oxide imbal- changesinvimentinexpression.Therewasade- ance. J Clin Invest 100: 1520-1528. crease in vimentin expression correlating with Vilafranca M, Domingo M, Ferrel L (1994) Tubular glomerular damage and their appearance in tubular vimentin metaplasia in canine nephropathies. Res cells, as a symptom of the alteration of their cytos- Vet Sci 57: 248-250. keleton. The thickening of blood vessel walls seen in Ward JM, Stevens JL, Konishi N, Kurata Y, Uno H, routine stained sections corresponded to changes in Diwan BA, Ohmori T (1992) Vimentin metaplasia vimentin expressions. The alteration of vascular walls in renal cortical tubules of preneoplastic, neoplas- and interstitial fibrosis were the dominant type of tic, aging, and regenerative lesions of rats and hu- lesions in the Vth group. The changes in vimentin mans. Am J Pathol 141: 955-964. expression were the most remarkable in those dogs Yaoita E, Franke WW, Yamamoto T, Kawasaki K, kidneys, and were observed in nephrons and inter- Kihara I (1999) Identification of renal podocytes stitial tissue and blood vessel walls. in multiple species: higher vertebrates are vimentin In conclusion the results of the study have shown positive/lower vertebrates are desmin positive. His- that kidney pathological changes increase with the tochem Cell Biol 111: 107-115. age of a dog, but disease course is usually character- Yaoita E, Yao J, Yoshida Y, Morioka T, Nameta M, ized by a silent, subclinical progression. The vimentin Takata T, Kamiie J-I, Fujinaka H, Oite T, expression is a very good marker of the early cell al- Yamamoto T (2002) Up-regulation of connexin43 terations in glomerules, tubules and blood vessels. It is in glomerular podocytes in response to injury. Am important to notice that the decrease in vimentin J Pathol 161: 1597-1606. Polish Journal of Veterinary Sciences Vol. 7, No. 3 (2004), Supplement, 93-95

Short original article T-zone lymphoma in a dog. Preliminary morpho-immunohistochemical study

E. Pavlidou

Department of Pathobiology, College of Veterinary Medicine, University of Illinois at Urbana-Champaign, Urbana, Illinois, USA

Abstract

The author presents a case of canine T-zone lymphoma, correlating histopathological and immunohistochemical features. This lymphoid neoplasm, known in human medicine, is not contem- plated in the actual World Health Organization (WHO) Classification of 2002, regarding the Haema- topoietic Tumours of Domestic Animals.

Key words: dog, T-zone lymphoma, histopathology, immunohistochemistry

Introduction Results

As Pinkus and Said (2001) and Ralfkiaer et al. Histologically, the architecture of the lymph node (2001) have shown, T-zone lymphomas are character- was effaced by a mass with a follicular (i.e. not dif- ized by an infiltrate that involves lymph node’s fuse) histological pattern consistent with follicles of paracortex. In human medicine they are included small, dark, central areas surrounded by large, ex- in the category of “Peripheral T-cell lymphoma, panding, pale areas (Fig. 1-3). Cytologically, two unspecified” and considered as low grade. types of cell population were identified. The pale areas were composed of small to intermediate, generally monomorphic, neoplastic lymphoid cells, with Materials and Methods increased nuclear details, round to irregularly int- ended nuclei, irregularly thickened nuclear mem- Haematoxylin and eosin stained sections of for- branes, distinct chromocenters, irregular para- malin fixed, paraffin embedded lymph node tissue of chromatin clearing, and pale cytoplasm. Intracellular a four year old, mix breed, dog, with persistent sub- boundaries were generally distinct. Mitoses were mandibular lymphadenomegaly and no other signs, rare. The dark areas were composed of small, in- were examined to investigate the histopathological dented, dark, relatively mature, non-neoplastic lym- features of the lesion. Immunohistology, using the phocytes (Fig. 4). Immunohistochemically, the neo- streptavidin-biotin-peroxidase method, was per- plastic cells of the pale areas were CD3+ and formed. As primary antibodies, mouse anti-human CD79α-, demonstrating T-cell lineage (Fig. 5), CD3 (T-cell marker, diluted 1:100, DAKO) and whereas the non-neoplastic cells of the dark areas CD79α (B-cell and plasma cell marker, diluted 1:150, were CD79α+ and CD3-, demonstrating B-cell lin- BIOGENEX) were used. eage (Fig. 6).

Correspondence to: E. Pavlidou, Tuderte St. 47, 06126 Perugia, Italy, e-mail: [email protected] 94 Pavlidou E.

Fig. 1. The paracortex of the lymph node is effaced by a mass with a follicular (i.e. not diffuse) histological pattern consistent with follicles (spherical to oval structures) of small, dark, central areas surrounded by large, expanding, pale areas. Haematoxylin and eosin staining, x 1.25. Fig. 2. The benign mantle cells in the centre of the follicles are CD79α+ (brown); i.e. they are positive for a marker (CD79α) specific for the cells of the B lineage. Immunohistochemistry CD79α, x 1.25. Fig. 3. The neoplastic cells in the expanded paracortex (or T-zone area) are CD3+ (brown); i.e. they are positive for a marker (CD3) specific for the cells of the T lineage. Immunohistochemistry CD3, x 1.25. Fig. 4. Cytological detail of the same canine lymph node. In the follicular structures, two different cell populations are present. The malignant T cells of the pale areas (upper slide) are small to intermediate, generally monomorphic, with increased nuclear details, round to irregularly indented nuclei, irregularly thickened nuclear membranes, distinct chromocenters, irregular parachromatin clearing, and pale cytoplasm. Intracellular boundaries are generally distinct. The benign mantle cells of the dark areas are small, indented, dark, relatively mature, benign lymphocytes (low slide). Haematoxylin and eosin staining, x 40. Fig. 5. Detail of the follicular architecture of the mass. The malignant T cells of the expanded paracortex (or T-zone area) are CD3+. Immunohistochemistry CD3, x 40. Fig. 6. Detail of the follicular architecture of the mass. The non-neoplastic mantle cells (B cells) of the dark areas of the nodular (follicular) structures are CD79α+. Immunohistochemistry CD79α, x 40. T zone lymphoma in a dog ... 95

Discussion cal structure. In the centre there are the benign, lighter staining, follicular centre cells. These are sur- In the first stages, follicular centre cell lymphomas rounded by a rim of dark staining, benign, mantle (FCCL), mantle cell lymphomas (MCL), marginal cells. Around and outside of the mantle cells there is zone lymphomas (MZL), and T-zone lymphomas as- a continuous cuff of light staining, marginal zone cells. sume a follicular histological pattern, characterized by The sections examined in this case appear with the the presence of spherical (nodular) structures. follicular histological pattern of the T-zone lymphoma Nevertheless, these spherical structures are differ- described in human medicine. Cytopathology and im- ently defined in each particular neoplasm. In FCCL, munohistochemistry reinforce and confirm the diag- MCL and T-zone lymphomas, the follicular histologi- nosis based on the histological pattern consistent with cal pattern generally is consistent with a two layer T-zone lymphoma in this dog, and this is the first time spherical structure. In FCCL there is an expansion of that this tumour is described in the veterinary medi- the inner compartment of the follicle, because of pro- cine. More specific immunohistochemical studies, and liferation of lighter staining, malignant, follicular molecular investigations need to be performed to better define the characteristic clinicopathological fea- centre cells (centrocytes and centroblasts). These are tures of canine T-zone lymphoma and further updat- surrounded by a thinned perimeter of dark staining, ing of the WHO system 2002 is required. benign mantle cells. In MCL there is an expansion of the outer compartment of the follicle, because of proliferation of dark staining, malignant mantle cells. References These surround lighter staining, shrunken, almost de- pleted germinal centres. In T-zone lymphoma, there is Pinkus GS, Said JW (2001) Peripheral T-Cell Lymphoma. an expansion of the compartment of the paracortex In: Knowls DM (ed) Neoplastic Hematopathology, 2nd area, because of proliferation of lighter staining, ma- ed, Lippincott Wiolliams and Wilkins, Philadelphia, pp. lignant T-cells. These surround the dark staining, be- 1091-1125. nign cells of the mantle zone, forcing them to usually Ralfkiaer E, Mueller-Hermelink HK, Jaffe ES (2001) Pe- aggregate as a single dense cluster at the centre of the ripheral T-cell lymphoma, uspecified. In: Jaffe ES, Lee follicle with consequent depletion of the follicular Harris N, Stein H, Vardiman JW (eds) Pathology and centre cells. Because the dark mantle cells appear at Genetics. Tumours of Hematopoietic and Lymphoid Tis- sues, IARC Press, Lyon, pp. 227-229. the centre rather than at the periphery of the lesion, Valli VE, Jacobs RM, Parodi AL, Verneau W, Moore PF as in follicular lymphomas, the appearance of these (2002) Histological Classification of Haematopoietic Tu- structures in T-zone lymphoma is referred to as mours of Domestic Animals, Second Series, Vol VIII, “inverted follicles”. Finally, MZL has a characteristic AFIP-American Registry of Pathology-WHO, Washin- follicular pattern consistent with a three layer spheri- gton, DC. 96 Pavlidou E. Polish Journal of Veterinary Sciences Vol. 7, No. 3 (2004), Supplement, 97-99

Short original article The distribution and chemical coding of porcine urinary bladder trigone-projecting neurons located in prevertebral ganglia other than IMG

Z. Pidsudko, M. Majewski1

Division of Animal Anatomy and 1Clinical Physiology, Department of Functional Morphology, Faculty of Veterinary Medicine, University of Warmia and Mazury in Olsztyn, Oczapowskiego St. 13, 10-719 Olsztyn, Poland

Abstract

Combined retrograde tracing and double-labelling immunofluorescence were used to investigate the distribution and chemical coding of neurons in prevertebral ganglia supplying the urinary bladder trigone (UBT) in juwenile pigs (n=5). The urinary bladder trigone-projecting neurons (UBT-PN) were mainly located in the celiac-superior mesenteric ganglion complex (C-SMG), as well as in the ovarian (OG), aortico-renal (ARG) and adrenal (ADG) ganglia. Immunohistochemistry disclosed that the vast majority of UBT-PN were noradrenergic (i.e. TH-positive). Many noradrenergic neurons contained NPY or, less frequently, SOM and/or GAL. This study has revealed a relatively large population of differently coded UBT-PN located in porcine PVG other than the IMG. Thus, as may be judged from their somatotopic organization and neurochemical coding, sympathetic pathway to the UBT is more complex than suggested hitherto.

Key words: prevertebral ganglia, urinary bladder trigone, immunohistochemistry, retrograde tracing, neuropeptides, pig

Introduction lower urinary tract function is to provide an excitatory input to the bladder to maintain closure of the outlet. The function of the urinary bladder is to store In addition, there is evidence that the sympathetic urine and, at appropriate intervals, to evacuate it. supply to the bladder serves to inhibit parasym- What appears to be a relatively simple sequence of pathetic activity during the accommodation phase of events is on deeper analysis a sophisticated process urine storage (De Groat and Steers 1988). The inner- requiring close coordination between the different vation of these organs has been found to originate components of the lower urinary tract. It is well recog- from three sets of peripheral nerves: sacral parasym- nized yet that an intact sympathetic supply to the phatetic (pelvic nerves), thoracolumbar symphatetic bladder is not essential for micturition to take place. (hypogastric nerves and prevertebral ganglia – PVG), The primary role of the sympathetic nervous system in and sacral somatic (primarily the pudendal nerves)

Correspondence to: Z. Pidsudko, tel.: +48 89 523-39-53, fax: +48 89 523-49-86, e-mail: [email protected] 98 Pidsudko Z., Majewski M.

(for references see Pidsudko 2004). Since our knowl- Specificity of both the tracing and labelling techniques edge on the distribution and chemical coding of the was checked as described previously (Pidsudko et al. urinary bladder trigone-projecting neurons in the pig 2001). is, at the moment, limited only to the IMG (Pidsudko 2000), we have combined retrograde tracing and Results and Discussion double-immunolabelling to elucidate: 1) the involvement of PVG other than IMG in this neural pathway The porcine PVG were found to contain many and 2) neurochemical features of their UBT-PN. FB-positive (FB+) neurons projecting to the urinary bladder trigone which were distributed within both the left and right ganglia. The PVG complexes con- Materials and Methods tained 1181 ± 120 (mean ± S.E.) of FB+ neurons. The majority of them (about 90% of all FB+ neurons) was The study was performed on 5 juvenile pigs of the localized in the OG. 921 ± 98 of FB+ neurons were Large White Polish breed. The animals were housed found in OG, 216 ± 19 in ARG and 44 ± 3 in ADG, and treated in accordance with the rules approved by considering both the left and right PVG. Immunohis- the local Ethical Commission. In the experimental tochemistry revealed that the vast majority of the FB+ animals, the fluorescent retrograde neuronal tracer UBT-PN were TH/DβH-IR (approx. 88%). A promi- Fast Blue (FB) was injected into both the left and nent proportion of these neurons contained also im- right side of the urinary bladder trigone during munoreactivity to NPY (18%; Fig. 1.) and a smaller laparatomy performed under pentobarbital anes- number was SOM- (5%; Fig. 2.) or GAL-IR (0.6%). thesia. After a survival period of three weeks the pigs The present study has shown that the efferent inner- were reanaesthetised and transcardially perfused with vation of the porcine urinary bladder trigone originate 4% buffered paraformaldehyde. The collected prever- not only from the IMG, but also from other PVGs. tebral ganglia (i.e., C-SMG, ADG, ARG and OG) This corresponds well with findings obtained in lab- were postfixed by immersion in the same fixative for oratory and domestic animals, in which it has been several hours and finally stored in 18% sucrose until shown that not only sympathetic chain ganglia, but sectioning. The left and right PVG were cut into 10 also PVG, as sources of the efferent nerve supply, are µm thick cryostat serial sections. FB-labelled cell crucial for the maintenance of the lower urinary tract counts were done prior to the immunohistochemistry. functions (Downie et al. 1984, De Groat and Steers To determine the relative number of the UBT-PN, 1988). Immunohistochemistry has revealed that many the neurons were counted in every fourth section of UBT-PN is noradrenergic in nature; however it is from both the left and right ganglia in all the animals. apparent that a lot of them contain neuropeptides as Only neurons with a clearly visible nucleus were con- well. It should be stressed that some of them contain sidered. All the sections containing retrogradely label- NPY- and, additionally, in a smaller proportion SOM- led neurons were processed for double-labelling im- or GAL- IR (Ja¨nig and McLachlan 1987). The precise munofluorescence with antibodies listed in Table 1. role of these different sub-populations of neurons has

Table 1. Antisera used in the study.

Antigen Host Code Dilution Supplier Primary Antisera DβH rabbit DZ 1020 1:500 Affiniti, UK CGRP rabbit RPN 1842 1:1600 Amersham, UK TH mouse 1017381 1:40 Boehringer Mannheim, GER GAL rabbit 4600-5004 1:1600 Biogenesis, UK SOM rat 8330-0009 1:30 Biogenesis, UK SP rat 8450-0505 1:250 Biogenesis, UK VIP rabbit 20077 1:200 Incstar, MO, USA NPY rabbit NA 1233 1:400 Affiniti, UK NPY rat NZ 1115 1:200 Affiniti, UK Secondary Reagencies FITC-conjug. goat anti-rabbit IgG 1:400 Jackson Immunores. Lab., USA FITC-flourolink. goat anti-mouse IgG 1:400 Jackson Immunores. Lab., USA FITC-flourolink. goat anti-rat IgG 1:400 Jackson Immunores. Lab., USA Biotinylated goat anti-rabbit IgG 1:400 Dako, DK Biotinylated goat anti-rat IgG 1:400 Amersham, UK Biotinylated goat anti-mouse IgG 1:400 Amersham, UK Cy3-conjugated streptavidin 1:4000 Dianova, Hamburg, GER The distribution and chemical ... 99

Fig. 1a-c. Urinary bladder trigone-projecting neurones in the porcine OG. Some FB-positive neurons (a) co-express TH- (b) and NPY-immunoreactivity (c; arrow point out NPY-positive neuron). Scale bar 25 µm. Fig. 2a-c. A loose cluster of FB-positive neurons (a) in the porcine OG, one of them co-localizing DβH (b; arrows) and SOM (c). Scale bar 25 µm. yet to be defined. The adrenergic activation of the References smooth muscle of the urinary bladder was shown to be mediated via α-adrenoreceptors, and release both De Groat WC, Steers WD (1988) Neural control of the noradrenaline (NA; acting here as a relaxant agent) urinary bladder and sexual organs: experimental and a neurotransmitter that contracts these muscles. studies in animals. In: Autonomic Failure: A Textbook SOM-IR was seen in sparse to dense meshwork of of Clinical Disorders of the Autonomic Nervous System, nerve fibres throughout the pig urinary tract, but little ed. E. Bannister, Oxford: Oxford University Press, pp. is known of this peptide role. It is possible that neur- 196-222. ons containing SOM have a local modulatory or in- Downie JW, Champion JA, Nance DM (1984) A quantitat- hibitory effect, as SOM is capable to powerfully block- ive analysis of the afferent and extrinsic efferent inner- ing the release and interfere with the action of other vation of specific regions of the bladder and urethra in transmitters (Ja¨nig and McLachlan 1987, Pidsudko the cat. Brain Res Bull 12: 735-740. 2004). In summary, the porcine PVG have been found Ja¨nig W, McLachlan EM (1987)Organizationoflumbar to contain many neurons projecting to the urinary spinal outflow to distal colon and pelvic organs. Physiol bladder trigone. This study has also revealed a rela- Rev 67: 1332-1404. Pidsudko Z (2000) Distribution and chemical coding of tively large population of differently coded PVG neurons in the porcine inferior mesenteric ganglion pro- UBT-PN, that are probably involved in the neural jecting to the urinary bladder trigone. Verh Anat Ges control of the urinary bladder. 95: 52-53. Pidsudko Z, Kaleczyc J, Majewski M, Łakomy M, Scheuer- Acknowledgements mann DW, Timmermans J-P (2001) Differences in the distribution and chemical coding between neurons in Supported by KBN grant 5 P06K 047 17. The the inferior mesenteric ganglion supplying the colon and authors wish to thank Prof. J. Kaleczyc for critical rectum in the pig. Cell Tissue Res 303: 147-158. reading of the manuscript, as well as M. Marczak, Pidsudko Z (2004) Distribution and chemical coding of G. Greniuk and A. Penkowski for their excellent tech- neurons in intramural ganglia of the porcine urinary nical assistance. bladder trigone. Folia Histochem Cytobiol 42: 3-11. 100 Pidsudko Z., Majewski M. Polish Journal of Veterinary Sciences Vol. 7, No. 3 (2004), Supplement, 101-103

Short original article Caffeic acid feeding of pregnant mice influences the prenatal development of their offspring

E. Rogala, E. Skopińska-Różewska1, E. Wojtasik2, J. Chorostowska-Wynimko1, E. Sommer1, M. Filewska1, A.K. Siwicki3

III Department of Lung Diseases and 1Department of Laboratory of Diagnostic Immunology, National Institute of Tuberculosis and Lung Diseases, Warsaw, Poland 2 Department of National Medicinal Products, Office Med. Prod., Med. Devices and Bioacides, Warsaw, Poland 3 Department of Microbiology and Clinical Immunology, University of Warmia and Mazury in Olsztyn, Oczapowskiego St. 13, 10-719 Olsztyn, Poland, e-mail: [email protected]

Abstract

The aim of the present study was to evaluate the effect of one of polyphenols, caffeic acid (CA), administered to the pregnant Balb/c mice on the embryos growth, angiogenic activity of embryos tissue, VEGF and bFGF production. The study was performed on 2-3 months old Balb/c mice fed during pregnancy caffeic acid (1 or 6 mg/day). The pregnancy was terminated at the 18th day, embryos were extracted, weighted and homogenized. Content of CA in embryos homogenates was estimated by HPLC. Concentration of angiogenic factors was tested by ELISA. Homogenates angiogenic activity was assessed in mice cutaneous angiogenesis assay. Feeding pregnant mice caffeic acid (6 mg/day) decreased the embryos weight and bFGF concentration (close to significance). CA did not decrease the angiogenic activity of embryos tissue, in higher dose this activity was elevated. The influence on VEGF concentration was not observed.

Key words: caffeic acid, pregnant mouse, angiogenesis, VEGF, bFGF, HPLC

Introduction Skopińska-Różewska et al. 2003). Moreover, we reported that pregnant mice chocolate feeding resulted Common articles of consumption like tea, coffee, in reduction of relative length of limbs and thigh cocoa, or chocolate, are rich in methyloxantines and bones in their progeny. VEGF content in offspring phenolic acids (i.e. caffeic acid, chlorogenic acid). bones was also lowered (Skopiński et al. 2003). Hu- Our previous studies have shown that these com- man daily diet contains about 1 g of phenolic acids. pounds exert antiangiogenic effect suppressing an- We put forward a hypothesis that consumption of giogenic factors production (VEGF, bFGF) and tu- phenolic acids during pregnancy may influence em- mor-induced angiogenesis (Skopińska-Różewska et bryogenesis. Therefore, the aim of the present study al. 1998, Barcz et al. 1998, Bałan et al. 1999, is to estimate the effect of one of polyphenols, caffeic

Correspondence to: E. Rogala, e-mail: [email protected] 102 Rogala E. et al. acid, administered to the pregnant Balb/c mice on the tion was estimated by the ELISA method in the em- embryos growth, angiogenic activity of embryos tissue bryos homogenates, according to the manufacturer’s and proangiogenic factors (VEGF, bFGF) produc- instructions. The cutaneous angiogenesis assay was tion. performed according to method modified by Sidky and Auerbach. Briefly, 0.05 ml embryos homogenate samples were injected intradermally into anaesthe- Materials and Methods tized Balb/c mice. After 72 hours mice were sacrificed and new blood vessels were identified and counted in The study was performed on 2-3 months old inbred the dissection microscope (6 x magnification) in the Balb/c mice fed during pregnancy (from 1st day to 18th central 1/3 of the microscopic field. The caffeic acid day) 1 or 6 mg/day of caffeic acid (Sigma Aldrich, content in embryos homogenates was estimated by the Poznań, Poland) served on wheat flakes. Control mice high performance liquid chromatography (HPLC) sys- were fed only wheat flakes. Doses of caffeic acid used tem. Briefly, samples were diluted with 0.1 M NaOH, in this study were taken basing on our previous experi- shaken on ultrasonic bath, passed through 0.45 µm ments (Bałan et al. 1999). The pregnancy was termin- filter and injected into HPLC system consisting of ated on the 18th day using Morbital, embryos were Luna C18 analytical column (Phenomenex, USA) extracted, weighted, suspended in phosphate buffer with the mobile phase consisted of saline – 1 g/ml (PBS, Polfa, Kutno), homogenized us- methanol-water-acetic acid delivered at the flow-rate ing ultrasound sonicator (VirSonic, Virtis, USA) and of 1.0 ml/min, UV-Vis detector SPD-6 AV operated frozen in -75oC for further evaluation. Experiments at 327 nm (Shimadzu, Germany) and a Rheodyne were approved by the Local Ethical Committee. Model 7125 injection valve (Berkeley, USA) equipped Proangiogenic cytokines VEGF and bFGF concentra- with 20 µl loop. Quantitative results were obtained by

Table 1. The effect of caffeic acid administered to pregnant mice on the 18 old day embryos growth.

Caffeic acid Number of Mean number of ± mg/day litters embryos/litter ± SE Mean embryo weight/litter SE

0 (control) 6 8.7 ± 0.38 0.64 ± 0.06 1 5 11.2 ± 0.95 0.34 ± 0.03 p<0.05 p<0.01 6 6 6.7 ± 1.56 0.42 ± 0.05 n.s. p<0.02

Table 2. The effect of caffeic acid administered to pregnant mice on the angiogenic activity (LIA test) and proangiogenic cytokines concentration estimated in the 18 old day embryos homogenates (n=number of LIA test).

Caffeic acid Number of Mean number of blood VEGF concentration bFGF concentration mg/day litters vessels ± SE (pg/ml) ± SE (pg/ml) ± SE 0 (control) 6 15.7 ± 0.88 393 ± 45 897 ± 105 n=89 1 5 16.5 ± 0.92 326 ± 8.5 978 ± 107 n=72 n.s. n.s. 6 6 18.2 ± 0.51 352 ± 65 603 ± 105 n=60 n.s. 0.05

Table 3. Caffeic acid concentration in 18 day old embryos homogenates evaluated by HPLC.

Caffeic acid Number of Caffeic acid concentration Statistical significance of difference mg/day litters (µg/g) ± SE from control 0 (control) 5 0.16 ± 0.04 – 1 4 0.45 ± 0.10 p<0.05 6 4 1.01 ± 0.16 p<0.01 Caffeic acid feeding ... 103 comparing samples and standard (caffeic acid, Acknowledgements Sigma-Aldrich, Poznań, Poland) peak areas. This work was supported by KBN grant 3PO5EO3722. Results and Discussion

Caffeic acid feeding exerted significant inhibitory References effect on the embryos weight in both examinated Azuma K, Ippoushi K (2000) Absorption of chlorogenic acid doses (1 and 6 mg/day). The mean number of embryos and caffeic acid in rats after oral administration. J Agric in litter was significantly elevated in dose of 1 mg/day Food Chem 48: 5496-5500. < of caffeic acid (p 0.05) (Table 1). Caffeic acid feeding Bałan B, Skopińska-Różewska E, Barcz E, Sokolnicka I, in dose 6 mg/day, stimulated the angiogenic activity of Gawrychowski K, Strzelecka H (1998) Wpływ wybranych embryos homogenates (p<0.05) (Table 2). The same kwasów fenolowych na aktywność angiogenną komórek dose decreased bFGF concentration, the difference raka jajnika-doniesienie wstępne. Onkol Pol 2: 203-208. was close to significance (0.05

0.1). Caffeic acid in Barcz E, Sommer E, Janik P, Marianowski L, Skopińska- dose of 1 mg/day did not affect these parameters. The -Różewska E (2000) Adenosine receptor antagonism influence of both doses of caffeic acid on VEGF con- causes inhibition of angiogenic activity of human ovarian centration was not observed (Table 2). The positive cancer cells. Oncol Rep 7: 1285-1291. Demeule M, Michaud-Levesgue J, Annabi B (2002) Green correlation between angiogenic activity of tea catechins as a novel antitumor and antiangiogenic homogenates and caffeic acid concentration (HPLC compounds. Curr Med. Chem Ant-Canc Agents analysis) was observed (r=0.5819). Caffeic acid con- 2: 441-463. centration in embryos homogenates was proportional Glinkowska, Bałan B, Sommer E, Demkow U, Sokolnicka I, to applied doses (Table 3). Strzelecka H, Skopińska-Różewska E (1997) The effect In the available literature we did not find any data of phenolic compounds of poplar extract on cutaneous about the influence of phenolic acids on embryonic angiogenesis reaction induced in mice by human mono- angiogenesis. In earlier study on the effect of choc- nuclear leukocytes. Acta Pol Pharm 54: 151-154. olate feeding of pregnant mice, significant inhibition Olthof MR, Hollman P (2001) Chlorogenic acid and caffeic of embryos tissue angiogenic activity was observed acid are absorbed in humans. J Nutr 131: 66-71. Skopiński P, Skopińska-Różewska E, Sommer E, Choros- (Skopińska-Różewska et al. 2003). Chocolate contains towska-Wynimko J, Rogala E, Cendrowska I, Chrys- several active substances, among them methyloxan- towska D, Filewska M, Białas-Chromiec B, Bany J (2003) tines and polyphenols, so described by these authors Chocolate feeding of pregnant mice influences length of inhibitory effect may depend on combined action of limbs of their progeny. Pol J Vet Sci 3: 57-59. these compounds. In our study caffeic acid did not Skopińska-Różewska E, Chorostowska-Wynimko J, Sommer decrease the angiogenic activity of embryos tissue, in E, Rogala E, Demkow U, Filewska M, Radomska- higher dose this activity was elevated. Moreover, caf- -Leśniewska D, Skurzak H, Bany J, Siwicki AK (2003) feic acid did not influence VEGF concentration and Wpływ diety wzbogaconej w czekoladę na angiogenezę bFGF concentration was not statistically significantly embrionalną i nowotworową u myszy. Rola immuno- reduced. Some authors reported that phenolic acids, modulatorów pochodzenia naturalnego w zapobieganiu i leczeniu chorób. Praca zbiorowa pod red. among them caffeic acid, stimulate angiogenic activity Skopińska-Różewska E, Siwicki AK, wyd. Medyk, of human mononuclear leukocytes (MNL) (Glin- Warszawa. kowska et al. 1997). The present study showes that Skopińska-Różewska E, Janik P, Przybyszewska M, Sommer caffeic acid influences prenatal development. But it is E, Białas B (1998) Inhibitory effect of theobromine in necessary to evaluate further the mechanism of caffeic induction of angiogenesis and VEGF mRNA expression acid action on embryogenesis and its influence on in v-raf transfectants of human urothelial cells HCV-29. postnatal development. Int J Mol Med. 2: 649-652. 104 Rogala E. et al. Polish Journal of Veterinary Sciences Vol. 7, No. 3 (2004), Supplement, 105-107

Short original article In vitro influence of synthetic pyrethroid – cypermethrin on phagocytes of heterothermic and homeothermic animals

A. Rymuszka, A.K. Siwicki1

Department of Physiology and Toxicology, Catholic University of Lublin, Al. Racławickie St. 14, 20-950 Lublin, Poland 1Department of Microbiology and Clinical Immunology, Faculty of Veterinary Medicine, University of Warmia and Mazury in Olsztyn, Oczapowskiego St. 13, 10-719 Olsztyn, Poland

Abstract

The in vitro effect of cypermethrin on fish and rabbit polymorphonuclear and mononuclear phagocytic cells was determined. Experimental studies were carried out on cells isolated from pronephros and spleen of carp and from the rabbit blood. Cypermethrin was added at concentrations of 0.0001, 0.0005, 0.001, 0.005 µg/ml medium to the fish phagocyte cell suspension, and to the rabbit phagocyte cell suspension was added at concentrations of 1, 2.5, 5, 10 µg/ml medium. The results indicate that cypermethrin in in vitro studies has a greater suppressive effect on carp than rabbit phagocytes.

Key words: cypermethrin, metabolic activity of phagocytes, carp, rabbit

Introduction vity of carp (Cyprinus carpio L.) and rabbit (Oryc- tolagus cuniculus). Pyrethroid compounds are commonly used in households to eradicate pests and insects. Cypermeth- rin – alpha cyano pyrethroid, is an example of a syn- Materials and Methods thetic pyrethroid, that has short stability in the environment. Half-live period in soil is 2-4 weeks and Pure cypermethrin was used in the study (Sigma, 2 weeks in natural estuaries. Cypermethrin exhibits Aldrich). Material for the in vitro study was taken a very toxic influence on fish. The insecticide easily from five healthy carps and rabbits. Carps were killed comes to tissues from water and its bioaccumulation by an anaesthetising overdose of 0.2% Propiscin prep- index is about 1000. That group of chemical com- aration (IRS, Żabieniec, Poland). Pronephros and pounds is quite safe in use, quickly metabolised and spleen were dissected mechanically and pressed excreted from the organism of higher vertebrates. Cy- through a nylon mesh. Cells were suspended in cul- permethrin affects the nervous system mainly leading ture medium RPMI 1640 (Sigma, Aldrich). In order to disturbances in potassium, calcium and ion regula- to isolate the phagocytes the suspension (mainly mac- tion in neurons. rophage and neutrophils) was put on a gradient The purpose of the studies was to determine in Gradisol G (Aqua- Medica, Łódź, Poland) and centri- vitro the influence of cypermethrin on metabolic acti- fuged at 400 g for 30 min. at a temperature of 4oC.

Correspondence to: A. Rymuszka, e-mail: [email protected] 106 Rymuszka A., Siwicki A.K.

The phagocytes were collected and suspended in cant increase of metabolic activity of phagocytes iso- culture medium RPMI 1640. The blood was taken lated form rabbit blood was found. from rabbit ear veins. Isolation of blood phagocytes (mainly neutrophils) was performed using gradient 0.45 Gradisol G solution. RBA Cypermethrin was added at concentrations of 0.4 * 0.0001, 0.0005, 0.001, 0.005 µg/ml medium to the cell 0.35 suspension (3-5 x 106 cells ml-1) isolated from 0.3 pronephros and spleen. The preparation was added to 0.25 * rabbit phagocytes isolated from the blood at concen- 0.2 µ trations of 1, 2.5, 5, 10 g/ml medium. 0.15 * In the purpose to determine the metabolic activity 0.1

of phagocytes the RBA – Respiratory Burst Activity Optical Density (630 nm) 0.05 method described by Secombes (1990) was used. Each 0 experimental and control determination was per- control 1 2.5 5 10 formed in six fold repetition. Concentration of Cypermethrin (µ g/ml medium) The results were analysed statistically by ANOVA at a difference of means p≤0.05. The post-hoc Duncan Fig. 2. In vitro effects of cypermethrin on metabolic activity ¯ ± test was performed to assess the differences of means. of neutrophiles isolated from rabbit blood (x SD, n=10, *differences statistically significant).

Results Discussion

The results of the in vitro studies show the sup- Several studies conducted in aquatic environmen- pressive effect of cypermethrin on phagocytes isolated tal have demonstrated that immune functions in fish form carp and rabbit. After addition of cypermethrin collected from contaminated environments were se- at concentrations of 0.005, 0.001, 0.0005 µg/ml to the verely impaired. The presented results showed that carp cell suspension, a statistically significant decrease cypermethrin affects more suppressively on the meta- of RBA test values was found (Fig. 1). bolic activity of the cells isolated from pronephros and spleen of carp than on the metabolic activity of the cells isolated from rabbit blood. It is probably a conse- 0.6 quence of higher sensitivity of fish to pyrethroids. Fur- Pronephros Spleen thermore the results indicate that the modulatory in- 0.5 fluence of cypermethrin on phagocytic cells depends on the applied concentration (suppression at higher 0.4 * and stimulation at the lowest concentrations used). * The results of the earlier studies also show that cyper- 0.3 * methrin possess a strong immunotoxic effect on fish immunocompetent cells, manifested by a decreased 0.2 * Opticall density (630 nm) * number of antibody secreting cells – ASC (Rymuszka * et al. 2002). These results are consistent with those 0.1 obtained by other authors. Sopińska and Guz (1998) investigated the effect of permethrin (synthetic 0 control 0.0001 0.0005 0.001 0.005 pyrethroid) on immune cells of carp. Permethrin de- Concetration of Cypermethrin (µ g/ml medium) creases the phagocytic ability of neutrophils and macrophages residing in blood and pronephros. O’Hal- Fig. 1. In vitro effects of cypermethrin on metabolic activity loran et al. (1996) showed the immunotoxic influence of phagocyte cells isolated from carp pronephros and spleen of esfenvalerate – a synthetic pyrethroid on rainbow (RBA) (x¯ ±SD, n=10, *differences statistically significant). trout lymphocyte T and B mitogenesis. The pyrethroid insecticides are not free from ad- Various, a dose – dependent activity of phagocytes verse effects on immune system of higher vertebrates. isolated from rabbit was found. Cypermethrin at The specific immunological effects include changes in a concentration of 5, 10 µg/ml medium causes sup- the humoral response rate, thymus weight, and overall pression of metabolic and phagocytic activity on rab- system resistance to infections (Tulinska et al. 1995, bit phagocytes (Fig. 2). After application of cyper- Madsen et al. 1996, Santoni et al. 1997). Cypermeth- methrin at a concentration of 2.5 µg/ml the pa- rin and permethrin also decrease the division rates of rameters were not changed significantly in comparison T – and B – lymphocytes in laboratory cultures, sug- to the control group. After administration of prepara- gesting a functional impairment of these cells (Stelzer tion in a concentration of 1 µg/ml statistically signifi- and Gordon 1984, Blaylock et al. 1995). In vitro influence of synthetic ... 107

In conclusion, the results of this study show that Rymuszka A, Siwicki AK, Studnicka M, Sierosławska A, cypermethrin possess immunotoxic effect both on fish Bownik A, Kazuń K (2002) In vitro and in vivo studies on and rabbit phagocytes in vitro. the effect of selected pesticides on the number of antibody secreting cells (ASC) and specific immune response in carp (Cyprinus carpio L.). Acta Pol Toxicol 10: 181-189. References Santoni G, Cantalamessa F, Mazzucca L, Romagnoli S, Poc- coli M (1997) Prenatal exposure to cypermethrin modu- Blaylock BL, Abdel-Nasser M, McCarty SM, Knesel JA, Tol- lates rat NK cell cytotoxic functions. Toxicology 120: son KM, Ferguson PW, Mehendale HM (1995) Sup- 231-242. pression of cellular responses in BALB/c mice following Secombes Ch J (1990) Isolation of salmonid macrophages oral exposure to permethrin. Bull Environ Contam Toxi- and analysis of their killing activity. In: Stolen JS, col 54: 768-774. Fletcher TC, Anderson D P, Roberson BS, van Muiswin- Madsen C, Claesson MH, Ropke C (1996) Immunotoxicity kel WB (eds) Techniques in fish immunology. SOS Publi- of the pyrethroid insecticides deltamethrin and alpha-cy- cations, Vol. 1, Fair Haven, USA, pp. 137-154. permethrin. Toxicology 18: 219-227. Sopińska A, Guz L (1998) Influence of permethrin on phag- O’Halloran K, Ahokas JT, Wright PFA (1996) In vitro re- rocytic activity in carps. Medycyna Wet 54: 126-128. sponses of fish immune cells to three classes of pesticides. Stelzer KJ, Gordon MA (1984) Effects of pyrethroids on In: Stolen JS, Fletcher TC, Bayne CJ, Secombes CJ, lymphocyte mitogenic responsiveness. Res Commun Zelikoff JT, Twerdok LE, Anderson DP (eds) Modula- Chem Pathol Pharmacol 46: 137-150. tors of Immune Responses. SOS Publications, Vol. 2, Tulinska J, Kubova J, Janota S, Nyulassy S (1995) Investiga- Fair Haven, NJ, USA, pp. 535-538. tion of immunotoxicity of supercypermethrin forte in the Wistar rat. Hum Exp Toxicol 14: 399-403. 108 Rymuszka A., Siwicki A.K. Polish Journal of Veterinary Sciences Vol. 7, No. 3 (2004), Supplement, 109-113

Short original article The influence of dexamethason on the ovarian and uterine structure and ER, PR and alfa-inhibin expression in pseudopregnant rabbits

D. Saurek, M. Katkiewicz

Department of Clinical Science, Faculty of Veterinary Medicine, Warsaw Agricultural University, Nowoursynowska St. 159c, 02-776 Warsaw, Poland

Abstract

Dexamethason given in therapeutic doses to pseudopregnant rabbits caused marked increase in the ovarian interstitial tissue transformation into luteinized cells. These changes were proved by the immunocytochemical expression of alfa-inhibin. The degree of interstitial luteinisation was proportional to the dose of dexamethason. Dexamethason effect was seen also in ovarian epithelium by the increase in their nuclear expression of ER. Simultaneously, this hormone caused changes in the uterine horn structures, with dose-dependent effect. It was manifested by epithelial dystrophy, loss of secretion ability and loss of nuclear ER/PR receptors expression in endometrial cells. In summary, dexamethason given in therapeutic doses to pseudopregnant rabbits caused significant changes in the ovarian and uterine microscopic structure, which were expressed by molecular cellular changes too.

Key words: pseudopregnant rabbit, dexamethasone, uterus, ovary, pathomorphology, im- munocytochemistry

Introduction and Aron 1981). It is also proved, that cortisol in- hibits LH secretion. Dexamethason is the popular corticosteroid hor- The aim of the work was to investigate the structure mone used in the therapy of human and animal dis- of reproductive organs of pseudopregnant rabbits ex- eases. According to the recently published data there posed to the therapeutic doses of dexamethason. may be the close interaction between adrenal and gonadal steroids (Doufourny and Skinner 2002). As well the research provided on the stress influence Materials and Methods on the animals reproduction indicated the interaction between adrenal function and hy- The experiment was performed on 9 female pothalamus-pituitary secretion which play the basic healthy conventional white Polish female rabbits, role in regulating gonadal function (Plas-Roser divided into 3 groups: A – 3 rabbits of the control

Correspondence to: M. Katkiewicz, e-mail: [email protected] 110 Saurek D., Katkiewicz M. group; B – 3 rabbits, which received 0.5 mg/kg b.w. of observed in theca cells. The changes were manifested dexamethason (Dexafort, Intervet, Holland) i.m.; and by the appearing of very numerous polygonal cells C – 3 rabbits, which received 1.5 mg/kg b.w. of dex- among normal theca cells. The theca externa/inter- amethason i.m. After 16 days all the rabbits were stitium cells also looked like luteinised cells, however, mated twice in 3 day intervals by vasectomised male corpora lutea were also present. In uterine horns atro- rabbit. All the rabbits were euthanized on the 22th day phic changes were observed with the simultaneous de- of the experiment (euthanasia was performed by i.v. crease in GAG secretion (Fig. 2). injection of barbiturates). The ovaries and uterine 1 C – experimental rabbits, which received 1.5 horn specimens were fixed in 10% buffered formalin. mg/kg b.w. i.m. of dexamethason. In HE stained Paraffin sections were stained haematoxylin and eosin ovaries the apoptotic changes were present almost in (HE) and AB/PAS histochemical reaction. The paraf- all follicles. The follicular microcysts formation were fin sections put on Silan covered glasses were used for observed too. The luteinisation of theca and inter- immunocytochemical reaction for ER receptors stitial cells described in B group of experimental rab- (murine monoclonal antibodies IgG1 class, clone CC bits were of high degree in this group. Almost all of 4-5, Novocastra NCL-ER-LH 2), for PR receptors the ovarian interstitium cells were transformed into (murine antibodies IgG1, clone 1 A6, Novocastra large polygonal cells with similar microscopic appear- NCL-PGR), and for alfa-inhibin expression (mon- ance to the luteal cells (Fig. 6). The microscopic struc- oclonal antibodies inhibin alfa subunits R1 MCA 951 ture of uterine horns were similar to the rabbits of S, Serotec). The specimens of ovary and uterus were B experimental group, however, hydropic degener- routinely used for positive controls. The receptors ex- ation of epithelial cells were additionally found. The pression was evaluated by the scale: 0 – negative, faint affected cells lost almost completely their secretory – very low, + – low, ++ – weak, +++ – strong, ability, as was seen in AB/PAS reaction (Fig. 3). ++++ – very strong. 2. The immunocytochemical expression of alfa-inhibin, ER and PR receptors. 2 A – rabbits of the control group. In normal Results ovary the follicular cells posses expression of alfa-inhibin in the cytoplasm. In the ovary of control 1. Microscopic structure of the ovary and uter- pseudopregnant rabbit it was seen also in cytoplasm of ine horns in HE and AB/PAS staining methods. follicular cells, in various intensity of expression. The 1 A – rabbits of the control group. In HE stained faint alfa-inhibin expression was also observed in ovaries there were numerous corpus luteum antral single theca cells, as well as in small groups of theca primary follicles, also with numerous apoptotic fol- externa/interstitium cells. Some marginally localized licles (Fig. 4). The main changes were localized in corpus luteum cells also showed faint expression of ovarian interstitial tissue manifested by hyperplasia alfa-inhibin (Fig. 7). The expression of ER nuclear and hypertrophy of theca interna cells and appearance receptors was found in ovarian epithelial cells. The of the nests of luteinised interstitial cells. In uterine faint ER expression was found in some follicular cells horns endometrial proliferation, hyperaemia and of secondary follicles and cumulus oophorus cells. oedema with transformation of epithelial cells into se- Also very few cells of corpus luteum possessed faint cretory phase were found. Glycosaminoglicans ER expression. In the uterus the faint ER expression (GAG) secretion was seen in the uterus surface and were seen in basal glandular cells, interstitial and my- by glandular epithelium (Fig.1). ometrium cells. The expression of PR nuclear recep- 1 B – experimental rabbits, which received 0.5 tors was observed in follicular cells of secondary and mg/kg b.w. i.m. of dexamethasone. In ovaries of tertiary follicles. In the uterus very weak expression of these rabbits (Fig. 5) numerous antral follicles were PR nuclear receptors was observed in glandular epi- present but their oocytes were undergoing necrobiotic thelium. changes. The same changes were observed in second- 2 B – experimental rabbits, which received 0.5 ary follicles. However, on the first place changes were mg/kg b.w. i.m. of dexamethason. In comparison to

Table 1. Alfa-inhibin expression in ovaries of the control and the dexamethason-exposed pseudopregnant rabbits.

Follicular cells Corpus luteum Rabbit Theca Theca extrena/ group Primary Secondary Tertiary Peripheral Luteal interna interstitium follicles follicles follicles cells cells A + ++ +++ (cumulus faint 0 faint faint oophorus cells) B faint ++ +++ + faint ++ ++ C faint ++ +++ faint 0 + +++ Explanation: 0 – negative, faint – very low expression, + – low expression, ++ – weak expression, +++ – strong expression, ++++ – very strong expression The influence of dexamethason ... 111

Fig. 1. The uterine horn of the control pseudopregnant rabbit with well marked secretion of AB positive GAG in endometrial epithelium cells. AB/PAS reaction, x 10. Fig. 2. The uterine horn of the pseudopregnant rabbit, which obtained 0.5 mg/kb b.w. i.m. of dexamethason. The endometrial proliferation and the decrease in AB positive GAG secretion in uterine epithelium are visible. AB/PAS reaction, x 10. Fig. 3. The uterine horn of the pseudopregnant rabbit, which obtained 1.5 mg/kg b.w. i.m. of dexamethason. There are endometrial proliferation with the loss of secretory acivity and advanced hydropic degeneration of uterine epithelium cells. AB/PAS reaction, x 10. Fig. 4. The ovary of the control psudopregnant rabbit. Haematoxylin and eosin staining, x 10. Fig. 5. The ovary of the pseudopregnant rabbit exposed to 0.5 mg/kg b.w. i.m. of dexamethason. The remarkable ovarian interstitial cells transformation and the follicular apoptosis are visible. Haematoxylin and eosin staining, x 10. Fig. 6. The ovary of the pseudopregnant rabbit exposed to 1.5 mg/kg b.w. i.m. of dexamethasone. The transformed into luteal-like cells are almost all interstitial cells. Haematoxylin and eosin staining, x 10. Fig. 7. The ovary of the pseudopregnant rabbit with follicular cell alfa -inihibin expression. IPOX, x 10. Fig. 8. The ovary of the pseudopregnant rabbit exposed to 0.5 mg/kg b.w. i.m. of dexamethasone. The expression of alfa-inhibin is visible in follicular and in theca cells. IPOX, x 20. Fig. 9. The ovary of the pseudopregnant rabbit exposed to 1.5 mg/kg b.w. i.m of dexamethason. The very advanced changes in alfa-inhibin expression are present in the stromal cells. The expression of various intensity is observed in almost whole ovarian interstitium cells. IPOX, x 20. 112 Saurek D., Katkiewicz M. control rabbits, the expression of alfa-inhibin was in- cantly intensified with the dose-dependent effect in creased in follicular cells of secondary and tertiary rabbits which received dexamethasone. In high dex- follicles. The increase in alfa-inhibin expression was amethasone therapeutic dose the process of inter- also observed in theca interna and theca externa/interstitial cells luteinisation was found in almost all ovar- stitium cells (Fig. 8). In corpus luteum cells the in- ian interstitium cells, however, of the various inten- crease in the expression of this cytoplasmic hormone sity.AstheprocessofcellluteinisationisLH-de- was faint. Ovarian ER expression was of high intensity pendent, it may be concluded that massive stroma with concomitant hyperplasia of the ovarian epi- cell luteinisation in pseudopregnant rabbits were un- thelium. In the uterus there was not found ER ex- der influence of this pituitary hormone. It is proved pression. In the ovary, the faint expression of nuclear that alfa-inhibin may influence LH-dependent an- PR receptors was noted in cells which had features of drogen synthesis in theca interna cells. So the in- luteinisation, and in nuclei of follicular cells of sec- crease in alfa-inhibin expression in theca/interstitial ondary follicles. There was no expression of PR nu- cells observed in pseudopregnant rabbits may reflect clear receptors in the uterine horns of this experimen- its stimulatory effect on androgen synthesis. tal rabbit group. There is no answer, how long the observed 2 C – experimental rabbits, which received dex- changes in ovarian cells of pseudopregnant rabbit ex- amethason in doses of 1.5 mg/kg b.w. i.m. In this posed to dexamethasone persisted, and if this pro- group of rabbits alfa-inhibin expression was signifi- cess is totally reversible. The observed ovarian and cantly increasing in theca externa/interstitium cells, ap- uterine changes are responsible for the important pearing in almost all ovarian interstitial cells but it was side-effect of dexamethasone. This must be taken in various degree of expression (Fig. 9). The alfa-in- into account in veterinary clinical practice, as cor- hibin expression in the follicular and corpus luteum ticosteroids are the very popular hormones used in cells behaved similarly as in B experimental rabbit the therapy of different animal diseases. The further group. The ER expression in the ovarian epithelium research is needed to elucidate the corticosteroids was of very high intensity. In some follicular corona participation of eventual etiopathology of reproduc- radiata cells low intensity of ER expression was ob- tion organ diseases. served. Similarly to B group of rabbits ER expression The expression of ER and PR receptors in ovaries was not found in the uterus. The expression of nuclear and uterus of both experimental pseudopregnant PR receptor examined in ovaries and uterine horns rabbitgroupprovedthemodulatory,dose-dependent were similar to the B experimental group of rabbits. dexamethasone effect. The increase in ER nuclear expression in ovarian epithelium cells after dexamethasone exposure may be interpreted as the oes- Discussion trogenic effect of dexamethasone. In spite the effect of dexamethasone stimulation on the uterus was The results of the present experiment indicated manifested by blockade of ER receptors, as com- that both therapeutic dexamethason doses caused pared to control rabbits. the changes in structure and function of the ovaries The high intensity of nuclear PR expression was and uterus of pseudpregnant rabbits. The expression observed in ovarian interstitial luteinised cells of of the examined immunocytochemical markers that pseudopregnant rabbits which were exposed to both is: alfa-inhibin, ER and PR receptors, allows to con- therapeutic dexamethasone doses. clude, that dexamethasone was responsible for sig- The side-effect of dexamethason visible in epi- nificant functional changes in both examined repro- thelial and muscle cells is well known. The observed ductive organs. The most remarkable were the ovar- changesinrabbituterusprovedthistypeofdex- ian changes seen in alfa-inhibin expression. The amethason side-effect. physiological role of alfa-inhibin in the ovary is to Insummary,theresultsofthepresentedresearch regulate folliculogenesis by inihibition of FSH secre- proved the alteration of ovaries and uterus of tion (Erickson and Hsueh 1978). Alfa-inhibin pres- pseudopregnant rabbits after exposure to dex- ent in follicular cells regulates also their oestrogen amethasonegivenintherapeuticdoses. synthesis by autocrine mechanism. This cytoplasmic hormone is mainly secreted by follicular cells but may be present in small amount in theca and luteal cells. The effect of dexamethason on the ovarian References functional morphology in pseudopregnant rabbit were manifested in considerable changes in alfa-in- D’Agostino J, Valadka RJ, Schwartz NB (1990)Differen- hibin expression which were increasing with the in- tial effects on in vitro glucocorticoids on luteinizing hor- crease in hormone doses. The process of luteinisa- mone and follicle-stimulating hormone secretion: de- tion in interstitial cells was observed also in control pendence on sex pituitary donor. Endocrinology pseudopregnant rabbits but this process was signifi- 127: 891-899. The influence of dexamethason ... 113

Doufurny L, Skinner DC (2002) Progesterone receptor, Plas-Roser S, Aron C (1981) Stress related effects in the estrogen receptor alpha and the type II glucocorticoid controlofsexualreceptivityandthesecretionofproges- receptor are coexpressed in the same neurons of the terone by the adrenals in cyclic female rats. Physiol ovine preoptic area and arcuate nucleus: a triple immu- Behav 27: 261-264. nolabelling study. Biol Reprod 67: 1605-1612. Suter DE, Schwartz NB, Ringstorm SJ (1988)Dual Erickson GF, Hsueh AJW (1978) Secretion of inhibin role of glucocorticoides in regulation of pituitary con- by rat granulose cell in vitro. Endocrinology tent and secretion of gonadotropins. Am J Physiol 103: 1960-1962. 254: 595-600. 114 Saurek D., Katkiewicz M. Polish Journal of Veterinary Sciences Vol. 7, No. 3 (2004), Supplement, 115-118

Short original article Somatostatin-immunoreactive nerve structures in the ileum and large intestine of pigs undergoing dysentery

W. Sienkiewicz, J. Kaleczyc, M. Łakomy

Division of Animal Anatomy, Department of Functional Morphology, Faculty of Veterinary Medicine, University of Warmia and Mazury, Olsztyn, Poland

Abstract

The present study investigated the chemical coding of nerve structures of the ileum and large intestine in pigs suffering from dysentery. Cryostat sections of intestines were processed for double-labelling immunohistochemistry using antisera against PGP 9.5 and SOM. Increased number of SOM-IR perikarya was encountered in dysenteric animals as compared to controls in both myenteric plexus (MP) of all intestine segments studied and in outer submucous plexus (OSP) of cecum and centripetal turns. In OSP of ileum and centrifugal turns the percentage of SOM-IR neurons didn’t change, whereas in OSP of descending colon number of SOM-IR perykarya decreased. Inner submucous plexus (ISP) of all intestines studied showed decreasing number of SOM-IR nerve cell bodies except ileum, where percentage of SOM-IR neurones was not changed. In all layers of intestines under investigation, namely: muscular coat, all plexuses (MP, OSP, ISP) and mucous membrane the density of observed SOM-IR nerve fibres decreased in dysenteric animals.

Key words: pig, dysentery, intestine innervation, somatostatin

Introduction known, but information on neuronal elements containing this peptide in enteric nervous system in ani- Among mammalian viscera, the gastrointestinal mals undergoing inflammation of the digestive tract, tract is unique with respect to the arrangement of its especially in pigs, is very limited. Therefore, the aim innervation. In contrast to many other organs, the in- of the present study was to investigate immunohis- nervation of the gut is accomplished by numerous in- tochemical properties of SOM-IR nerve structures in trinsic neurons forming intestinal plexuses. The mor- the ileum and large intestine in pigs undergoing dys- phology of this system, its neurochemistry and func- entery. tion is relatively well known, but most of the data were obtained in healthy animals. Data regarding properties of the somatostatin-IR intestinalstructures Materials and Methods were obtained in humans (Keast et al. 1984), laboratory animals (Vinik et al. 1981) and pigs (Timmer- The study was performed on 9 five-months-old pigs mans et al. 1990). The localisation and functions divided into two groups. Control group (n=3) consis- (Lamers 1987) of somatostatin are relatively well ted of clinically healthy animals. Experimental ani-

Correspondence to: W. Sienkiewicz, tel.: +48 89 523-39-53, fax: +48 89 523-49-86, e-mail: [email protected] 116 Sienkiewicz W., Kaleczyc J., Łakomy M. Somatostatin-immunoreactive nerve ... 117 mals (n=6) were infected per os with Brachyspira hy- ileum and centrifugal turns, the percentage of odysenteriae bacterium. Then the animals showing dis- SOM-IR neurons not changed, whereas in OSP of the tinct symptoms of disease were deeply anaesthetised descending colon number of SOM-IR perykarya was and transcardially perfused with 4% paraformal- decreased (Fig. 2a, 2b). The inner submucous plexus dehyde. Then, the tissues (ileum, cecum, centripetal (ISP) of all intestines studied contained decreased and centrifugal turns of the ascending and descending number of SOM-IR nerve cell bodies (Fig. 3a, 3b) colon) were collected. The tissues were sectioned with except ileum, where percentage of SOM-IR neurons a cryostat and processed for double-labelling im- was not changed. In all layers of intestines under munohistochemistry using antisera against PGP 9.5 study, namely: muscular coat, all plexuses (MP, OSP, and SOM (for details see Kaleczyc et al. 2003). The ISP) (Fig. 1, 2, 3a, 1, 2, 3b) and mucous membrane, sections were analysed using confocal microscope the density of SOM-IR nerve fibres was decreased in Bio-Rad MRA-2. Then, the percentage of SOM-IR dysenteric animals. Exact data regarding percentage perykarya was calculated as a fraction of total number of SOM-IR neurones and density of nerve fibres are of neurons labelled with PGP. All the animals were shown in Table 1. treated in accordance with the rules approved by the Local Ethical Commission (conforming to Principles of Laboratory Animals Care, NIH publication no. Discussion 86-23, revised 1985). This study has revealed changes in the number of SOM-IR structures (nerve fibres and perikarya) in Results porcine intestines of animals undergoing dysentery. Many studies dealing with innervation of the intes- In the myenteric plexus (MP) of all intestines tines disclosed the presence of SOM-IR structures in studied and in the outer submucous plexus (OSP) of these organs. They were found in the digestive tract of the cecum and centripetal turns of dysenteric animals, humans (Keast et al. 1984), laboratory animals (Vinik an increased number of SOM-IR perykarya was found et al. 1981) and pigs (Timmermans et al. 1990). as compared to controls (Fig. 1a, 1b). In OSP of the Somatostatin plays an important, mainly inhibitory

Table 1. Relative density of nerve fibres (NF) and percentage of the SOM –IR neurons (NCB) in intestines studied.

Outer Inner Muscular Myenteric submucous submucous Mucous coat plexus plexus plexus layer Control NF ++ +++++ +++ ++++ ++ ± ± ± Ileum pigs NCB – 5.07 0.98 7.32 0.03 10.98 0.78 – Dysenteric NF + – +++ +++ ++ ++ pigs NCB – 11.76 ± 1.18 7.51 ± 0.72 10.72 ± 1.28 – Control NF ++ ++++ +++ ++ + pigs NCB – 3.71 ± 0.27 1.78 ± 0.17 1.17 ± 0.08 – Cecum Dysenteric NF + – ++ + + – + – pigs NCB – 6.38 ± 1.76 3.54 ± 0.08 0.54 ± 0.04 – Control NF ++ +++ +++ ++ + – ± ± ± Centripetal turns pigs NCB – 2.30 0.21 0.50 0.19 1.15 0.09 – Dysenteric NF + + ++ + – – pigs NCB – 7.61 ± 0.96 5.61 ± 0.40 0.75 ± 0.09 – Control NF ++ ++++ +++ ++ + Centrifugal turns pigs NCB – 2.77 ± 0.19 3.55 ± 0.24 2.83 ± 0.36 – Dysenteric NF +– + + +– – pigs NCB – 5.58 ± 0.31 3.73 ± 0.23 0.47 ± 0.06 – Control NF ++ ++++ +++ +++ + Descending colon pigs NCB – 1.44 ± 0.27 1.36 ± 0.21 4.02 ± 0.31 – Dysenteric NF +– ++ +– +– +– pigs NCB – 6.53 ± 1.74 0.85 ± 0.22 0.81 ± 0.06 –

← Fig 1. Myenteric plexus in the cecum of the control (a) and dysenteric pig (b). Fig 2. Outer submucous plexus in centripetal turns of the control (a) and dysenteric pig (b). Fig 3. Inner submucous plexus in the descending colon of the control (a) and dysenteric pig (b). 118 Sienkiewicz W., Kaleczyc J., Łakomy M. role in the regulation of many different intestinal glion in the male pig. Folia Histochem Cytobiol 41: functions including motility, secretion, blood flow, ab- 201-211. sorption and growth (Lamers 1987). SOM-IR struc- Keast JR, Furness JB, Costa M (1984) Somatostatin in hu- tures were described also in inflamated intestines man enteric nerves. Distribution and characterization. Cell Tissue Res 237: 299-308. (Koch et al. 1988). Our findings are in accordance Koch TR, Carney JA, Morris VA, Go VL (1988) Somatos- with results obtained by Koch et al. (1988) dealing tatin in the idiopathic inflammatory bowel diseases. Dis with innervation of the intestine in idiopathic inflam- Colon Rectum 31: 198-203. matory bowel disease. Decreased number of SOM-IR Lamers CB (1987) Clinical and pathophysiological aspects nerve fibres in dysenteric porcine intestine and in- of somatostatin and the gastrointestinal tract. Acta En- creased number of SOM-positive neurons can indi- docrinol Suppl (Copenh) 286: 19-25. cate an inhibition of SOM release from intestinal Ruskone A, Rene E, Chayvialle JA, Bonin N, Pignal F, nerve endings which may escalate symptoms of diar- Kremer M, Bonfils S, Rambaud JC (1982) Effect of rhea (lack of SOM neutralizes inhibitory effects of somatostatin on diarrhea and on small intestinal water this peptide on enteric functions, especially on motil- and electrolyte transport in a patient with pancreatic cholera. Dig Dis Sci 27: 459-466. ity and absorption). This conclusion is strongly con- Timmermans JP, Scheuermann DW, Stach W, Adriaensen firmed by application of SOM or its analogues in D, De Groodt L (1990) Distinct distribution of CGRP-, treatment of diarrhea with very positive results (Rus- enkephalin-, galanin-, neuromedin U-, neuropeptide Y-, kone et al. 1982). somatostatin-, substance P-, VIP- and serotonin-containing neurons in the two submucosal ganglionic neural net- works of the porcine small intestine. Cell Tissue Res 260: References 367-379. Vinik AI, Gaginella TS, Dorisio TM, Shapiro B, Wagner Kaleczyc J, Sienkiewicz W, Klimczuk M, Czaja K, Lakomy L(1981) The distribution and characterization of M(2003) Differences in the chemical coding of nerve somatostatin-like immunoreactivity in epithelial cells, fibres supplying major populations of neurons between submucosa, and muscle of the rat stomach and intestine. the caudal mesenteric ganglion and anterior pelvic gan- Endocrinology 109: 1921-1926. Polish Journal of Veterinary Sciences Vol. 7, No. 3 (2004), Supplement, 119-121

Short original article In vitro immunosuppressive effects of oxytetracycline on phagocytic cells and lymphocytes isolated from rainbow trout (Oncorhynchus mykiss) and carp (Cyprinus carpio L.) head kidney

A. Sierosławska, A.K. Siwicki1

Department of Physiology and Environmental Toxicology, Faculty of Mathematics and Natural Sciences, Catholic University of Lublin, Norwida St. 4, 20-061 Lublin, Poland 1 Department of Microbiology and Clinical Immunology, Faculty of Veterinary Medicine, University of Warmia and Mazury in Olsztyn, Oczapowskiego St. 13, 10-719 Olsztyn, Poland

Abstract

The in vitro influence of oxytetracycline (OTC) on the metabolic activity and potential killing activity of phagocytes and the proliferative response on the mitogens of lymphocytes isolated from rainbow trout and carp head kidney were assessed. It was found that the cell ability to produce reactive oxygen species and potential killing activity were suppressed by OTC in the highest used concentrations (40-60 µg/ml), both in rainbow trout and carp. The suppressive effects of OTC were also seen on fish lymphocyte reactivity at drug concentrations from 20-30 µg/ml to 60 µg/ml.

Key words: oxytetracycline, fish, lymphocyte, phagocytic cells, immunosuppression

Introduction Moreover some immunosuppressive effects of OTC were suggested in treated animals. The aim of the Oxytetracycline (OTC) is an antibiotic with work was to assess the in vitro effects of oxytetracyc- a broad spectrum of activity, often used in human and line on the selected mechanisms of rainbow trout and veterinary medicine. It belongs to the group of anti- carp leucocytes. biotic drugs the most commonly used in farmed fish therapy, where was introduced in 1951. Since that time some negative aspects of its usage have been Material and methods reported. It has been found to persist for several months in the fish farm sediments, where could be In the studies the principles of laboratory animal detected in an active form. That results in an induc- care and the national laws on the protection of ani- tion of drug resistance in the sediment bacteria. mals were followed. The studies were conducted on

Correspondence to: A. Sierosławska, e-mail: [email protected] 120 Sierosławska A., Siwicki A.K. ten healthy rainbow trouts (Oncorhynchus mykiss) 140 and ten carps (Cyprinus carpio L.) weighing 300 ± 50 120 g. The rainbow trouts were maintained in 500 l tanks with aerated, flowing water at a mean temperature of 100 14oC and fed with diet Safir containing 45% of pro- 80 tein (Aller Aqua, Nożynko) for over two months be- * * * * fore sampling. The carps were maintained in 20 60

% of control * l tanks with aerated water at a mean temperature of trout RBA * * * 20oC and fed with diet AT Starter containing 35% of 40 carp RBA protein (Cargill, Siedlce) for four weeks before samp- 20 trout PKA ling. The fish were treated with a lethal dose of carp PKA Propiscin (IRS, Żabieniec) and then organs, head kid- 0 neys, were taken. Isolation of leucocytes from fish control 1 5 10 20 30 40 60 µ head kidneys was performed by centrifugation on the OTC concentrations ( g/ml) density gradients according to Rowley (1986). Briefly, Fig. 1. In vitro effects of OTC on metabolic activity and po- separating mixture was prepared by building up 2.5 ml tential killing activity of fish phagocytes (x¯ ± SD, n=6, of Gradisol L (1.077 g/ml, Aqua-Medica, Poland) on *statistically significant, p<0.05). 2.5 ml of Gradisol G (1.115 g/ml, Aqua-Medica, Po- land). After centrifugation (1000 x g, 40 min., 10oC) two bands were formed. The first band was rich with phological changes in cells, as smaller flexibility of cell lymphocytes (80-85% of isolated cell population of membrane, a loss of pseudopodia and, in the result, the band), and the second one was rich with neutrophiles (85 – 95%). Phagocytic ability of phagocytes worse ability to adhere to the base. OTC may affect was measured by the respiratory burst activity (RBA) the assembly of F-actin by interfering with calcium-dependent mechanisms involved in the forma- with oxygen burst activator PMA (phorbol 12-myris- tion of actin dimers and trimers (Hoeben et al. 1997). tate acetate, Sigma) and potential killing activity Neutrophiles are of special susceptibility on low cal- (PKA) with Staphylococcus aureus 209P after 24 h ex- cium level, which stops phagosome maturing and their posure of the cells on oxytetracycline HCl (Pacific fusion with lisosomes (Kwiatkowska et al. 1999). Pharmachem, Ltd.) at the concentrations of 1, 5, 10, When incubating fish lymphocytes with OTC for 20, 30, 40, 60 µg/ml. To determine the influence of the 72 hours, lower proliferation was seen from 20 µg/ml drug on lymphocyte proliferative activity, the MTT in case of rainbow trout T cells (activated by ConA) test was used. The cells were exposed to the drug at and B cells (activated by LPS). Similarly, suppressed the same concentrations as mentioned above for 72 proliferation of carp T cells was observed from 20 h and stimulated with the mitogens – ConA (con- µg/ml and 30 µg/ml of B cells (Fig. 2). canavalin A, Sigma) as a T cell activator, and LPS (lipopolysaccharide, Sigma) as a B cell activator. Sta- tistical analysis of the results was performed by ANOVA. Differences between means were deter- 120 mined by Duncan test and considered statistically significant at p<0.05. 100

80 * * * * Results and discussion * 60 * * * The effects of OTC on metabolic activity of phago- * * % of control trout B cell cytes, mainly neutrophiles, were examined by measur- 40 * carp B cell * ing NBT reduction. It was reported, that OTC in- * hibited respiratory burst activity of fish phagocytic 20 trout T cell * * cells at a concentration of 40 and 60 µg/ml. Similar carp T cell results were seen when potential killing activity of the 0 cells was tested (Fig. 1). control 1 5 10 20 30 40 60 µ Because of the high activity of OTC in chelating Ca OTC concentrations ( g/ml) ions, both extra- and intracellular levels of calcium Fig. 2. In vitro effects of OTC on fish lymphocyte prolife- become lower in the presence of the drug. It may be ration (x¯ ± SD, n=6, *statistically significant, p<0.05). the reason of lower activity of phagocytes, since Ca ions are of great importance for respiratory burst. It Suppressive effects of OTC on proliferative activity was also shown (Paape et al. 1991, Hoeben et al. 1997) of lymphocytes were also reported by others. Lunden that higher concentrations of OTC may cause mor- et al. (2000) in in vivo study, after OTC treatment of In vitro immunosuppressive effects ... 121 rainbow trout at a dose of 75 mg/kg b.w. per os for 10 However, it is not well documented if modulated IL-2 days, have shown decrease of mitogen – stimulated secretion is the effect or the cause of weaker cell pro- proliferation by 40% and 63%, respectively, for B and liferation (Myers et al. 1995b). T cells isolated from fish head kidney. They also observed strong immunosuppressive activity of OTC in References in vitro studies. In that study drug concentrations from 0.1 µg/ml to 100 µg/ml inhibited T cell reaction on Hoeben D, Dosogne H, Heyneman R, Burvenich Ch (1997) PHA. In case of B cells similar suppression was seen Effect of antibiotics on the phagocytic and respiratory from a concentration of 1 µg/ml. Stronger than ob- burst activity of bovine granulocytes. Europ J Pharmacol served in our own study susceptibility of rainbow trout 332: 289-297. Kroon AM, Van den Bogert C (1983) Antibacterial drugs cells on OTC may have been a result of different used and their interference with the biogenesis of mitochon- protocols, but probably age of used fish was the most dria in animal and human cells. Pharm Weekbl 5: 81-85. important factor. In the mentioned study rainbow Kwiatkowska K, Sobota A (1999) Przekazywanie sygnału trout fry was used (mean b.w. 42 g) and the isolated fagocytarnego: od agregacji receptorów do przebudowy cells were pooled, while our fish were older (mean cytoszkieletu. Post Biol Kom 26: 59-81. b.w. 200 g) and all determinations were done individ- Lunden T, Bylund G (2000) The influence of in vitro an in ually for each fish. vivo exposure to antibiotics on mitogen induced prolifer- Probably OTC can influence the cell proliferation ation of lymphoid cells in rainbow trout (Oncorhynchys mykiss). Fish Shellfish Immunol 10: 395-404. in two ways. At first, as a calcium ion chelator, it Myers MJ, Farrell DE, Henderson M (1995a) Oxytetracyc- reduces their concentration in cell surrounding, line-mediated alteration of murine immunocompetence. which makes impossible the calcium influx into the Pathobiol 65: 270-271. cells after mitogen stimulation and thus interrupts Myers MJ, Farrell DE, Henderson M (1995b) Oxytetracyc- withDNAandRNAsynthesis(Myersetal.1995a). line-mediated alteration of murine immunocompetence. The second interaction can proceed at relatively low Pathobiol 65: 270-271. OTC concentrations. It is associated with sup- Paape MJ, Miller RH, Ziv G (1991) Pharmacological enhan- pression of mitochondrial protein synthesis which cement or suppression of phagocytosis by bovine neutrophils. Am J Vet Res 52: 363-366. disturbs mitochondrial biogenesis (Kroon et al. Rowley AF (1990) Collection, separation and identification 1983). of fish leucocytes. In: Stolen JS, Fletcher TC, Anderson Changes in lymphocyte proliferative ability and DP, Roberson BS, Van Muiswinkel WB (ed) Techniques their lower immunoglobulins production can also be in Fish Immunology. SOS Publications, USA, pp. an effect of modulation by OTC of IL-2 production. 113-136. 122 Sierosławska A., Siwicki A.K. Polish Journal of Veterinary Sciences Vol. 7, No. 3 (2004), Supplement, 123-125

Short original article Immunopathogenesis of herpesviruses: influence of channel catfish herpesvirus (CCHV) on macrophage and lymphocyte activity – in vitro comparative study

A.K. Siwicki1,3, M. Morand2, B. Kazuń3, S. Trapkowska1

1 Department of Microbiology and Clinical Immunology, Faculty of Veterinary Medicine, University of Warmia and Mazury in Olsztyn, Oczapowskiego St. 13, 10-719 Olsztyn, Poland 2 Laboratoire Departemental d’Analyses, LDA 39, Conseil General du Jura, France 3 Department of Fish Pathology and Immunology, Inland Fisheries Institute in Olsztyn, Poland

Abstract

Several DNA viruses infecting fish have been studied for analysis the pathogenesis and for developing the effective methods of prevention and therapy. Also some of fish viruses have been used as models for studying disease mechanisms. In this paper, we present the in vitro influence of channel catfish herpesvirus (CCHV) on the phagocytic ability and potential killing activity of splenic phagocytes and proliferative response of pronephric lymphocytes stimulated by mitogens ConA isolated from european catfish (Silurus glanis) and carp (Cyprinus carpio). The results showed that channel catfish herpesvirus (CCHV) induced suppressive effect on the phagocytic ability and potential killing activity of splenic phagocytes and decreased the lymphocyte activity isolated from pronephros in european catfish, compared to the carp where the immunostimulating effect was observed. The analyses of this study suggested that CCHV inhibited of cell-mediated immunity in european catfish.

Key words: fish, CCHV, phagocyte activity, lymphocyte proliferation

Introduction Herpesviridae are the most extensively studied fish viruses. First of all because they are ubiquitous in aqua- Interest in lower vertebrates viruses has recently tic organisms, since they have been isolated all over increased for a variety of reasons despite the fact that the world from both marine and freshwater fishes of none of them infect warm-blooded animals and man. different species, and is responsible for severe losses Some of lower vertebrates viruses have been used as in aquaculture. Several viruses isolated from different models for studying disease mechanisms. The family species of fish have been classified as herpesviruses Herpesviridae contains the three subfamilies: Alpha- (Eaton et al. 1989, Jung 1995, Hedrick 2000, Essbauer herpesvirinae, Betaherpesvirinae, Gammaherpesvirinae and Ahne 2001). The icosahedral capsid is made of and the unassigned genus “Ictalurid herpes-like vi- 162 capsomeres that are embedded in a protein ruses” (Ward 1993, Essbauer and Ahne 2001). The matrix and are surrounded by an envelope, which

Correspondence to: A.K. Siwicki, tel.: +48 89 523-32-17, e-mail: [email protected] 124 Siwicki A.K. et al. includes virus-encoded glycoproteins. The capsid con- herpesviruses have recently been recognized in Euro- tains a single linear double-stranded DNA molecule, pe, Asia, USA and Australia. A unifying theme which is replicated in the nuclei of the infected cells. among the herpesviruses is the intimate interrelation- In fish 25 types of herpes-like viruses have been detec- ship of virus infection with host cellular immunocom- ted but only few are well characterised. Channel cat- petence. Actually, we have a few informations about fish herpesvirus (CCHV) is a one of the most effect of herpesvirus on the cell-mediated immunity in pathogenic virus for catfish (Davison 1992). Experi- fish. In present study the influence of the channel cat- mental infection of catfish fingerlings with CCHV in- fish herpes virus (CCHV) on spleen macrophage me- duces haemorrhagic, oedematous and anaemic dis- tabolism was assessed by examining their respiratory ease and strong mortality (Davison 1992, Essbauer burst activity. The effects of CCHV on the respiratory and Ahne 2001). burst activity in european catfish and carp are pres- The aim of the present study was to determine the ented in Fig. 1. The results showed that channel cat- in vitro influence of channel catfish herpesvirus fish herpes virus significantly (p<0.05) decreased the (CCHV) on the phagocytic ability of splenic phago- macrophage activity level in european catfish, com- cytes and proliferative response of pronephric lym- pared to the control and carp. In carp the stimulating phocytes stimulated by mitogen in european catfish influence of CCHV on the macrophage respiratory (Silurus glanis) and carp (Cyprinus carpio). burst activity was observed.

0.8 Materials and Methods Carp European Catfish The spleen and pronephros were isolated from 10 healthy european catfish and carp with mean weight 0.6 of 100 g and the macrophages or lymphocytes were separated and cell culture prepared. Single cell suspension was obtained by placing the spleen and pronephros in medium RPMI-1640 and teasing it 0.4 through a steel meash. The macrophages were isolated by density-gradient centrifugation using Gradisol (Polfa) and lymphocytes were isolated by RBA (OD 620 nm) centrifugation using Histopaque-1077 (Sigma) gradi- 0.2 ent. A modification of the Secombes (1990) method was used to study the respiratory burst activity of spleenic macrophages stimulated by phorbol myristate 0 Control CCHV acetate (PMA, Sigma). Cells suspension (1x106/ml of medium) were incubated with CCH virus (1x105 Fig. 1. In vitro influence of CCHV on the splenic macro- p.f.u./ml of medium) for 2 h at 22oC. The plates were phages respiratory burst activity (RBA) in european catfish read on a micro-reader at OD 620 nm. and carp (mean ± SD, n=10). The proliferative response of the pronephric lym- 0.6 phocytes was determined by the MTT colorimetric as- ConA say method according to Carmichael et al. (1987) as ConA+CCHV 0.5 CCHV modified for fish by Siwicki et al. (1999). Con- Control canavalin A (ConA, Sigma) at a concentration of 64 fg/ml was used for stimulation of lymphocyte prolifer- 0.4 ation. The lymphocytes suspension (5 x 106/ml of medium) were incubated 3 days with CCH virus (1 x 105 0.3 p.f.u./ml of medium), with mitogen + CCH virus and

only with mitogen. The plates were read on a micro- LP (OD 6200.2 nm) reader (OD 620 nm). Statistical analyses were performed using the Stu- 0.1 dent t-test. Differences in mean were considered stat- < istically significant at p 0.05. 0 European Catfish Carp Fig. 2. In vitro influence of CCHV on the proliferative re- Results and Discussion sponse (LP) of pronephric lymphocytes stimulated by ConA (ConA), ConA and CCHV (ConA+CCHV), only by CCHV Herpesviruses are very pathogenic for lower verte- (CCHV) and in non-stimulated cells (Control) in european brates. Systemic infections of fish farming caused by catfish and carp (mean ± SD, n=10). Immunopthogenesis of herpesviruses ... 125

The proliferative ability of pronephric lymphocytes References showed a similar pattern. The influence of channel catfish herpes virus on the proliferative response of Carmichael J, DeGraff WG, Gazdar AF, Minna JD, Mitchell pronephric lymphocytes are presented in Fig. 2. In JB (1987) Evaluation of a tetrazolium-based european catfish the CCHV decreased the prolif- semiautomated colorimetric assay. Assessment of erative response of lymphocytes, compared to the chemosensitivity testing. Cancer Res 47: 936-942. carp and control. In carp the stimulating influence of Davison AJ (1992) Channel catfish virus: a new type of her- CCHV on lymphocyte proliferation induced by ConA pesvirus. Virology 186: 9-14. was observed. Essbauer S, Ahne W (2001) Viruses of lower vertebrates. A channel catfish herpes virus was tested in vitro J Vet Med 48: 403-475. Eaton WD, Wingfield WH, Hedrick RP (1989) Prevalence on cell-mediated immunity in two species of fish: carp and experimental transmission of the steelhead herpes- and european catfish. In our study, a strong sup- virus in salmonid fishes. Dis Aquat Org 7: 23-39. pressive influence of the CCHV on macrophage re- Hedrick RP, Gilard O, Yun S, Spangenberg JV, Nordhausen spiratory burst activity and lymphocyte proliferation RW, Kebus MJ, Bercover H, Eldar A (2000) A herpes- was observed in european catfish, compared to the virus associated with mass mortality of juvenile and adult carp where CCHV increased the macrophage and koi, a strain of common carp. J Aquat Anim Health lymphocyte activities. The effect of DNA viruses on 12: 44-57. the european catfish and carp leucocytes has been Jung SJ, Miyazaki T (1995) Herpesviral hematopoietic nec- previously reported by Siwicki et al. (1999, 2000). The rosis of goldfish, Carassius auratus (L.). J Fish Dis 18: authors clearly demonstrated a suppressive effect of 211-220. iridovirus on the phagocyte and lymphocyte activity Secombes CJ (1990) Isolation of salmonid macrophages and and hypothesised that this suppression led to a reduc- analysis of their killing activity. Techn Fish Immunol tion in the number of cells or to an impairment of 1: 137-154. phagocytic function. This preliminary in vitro study Siwicki AK, Pozet F, Morand M, Volatier C, Terech-Majewska E (1999) Effects of iridovirus-like also demonstrated a strong inhibitory influence of the agent on the cell-mediated immunity in sheatfish (Silurus CCH virus, but on selected species. In carp the im- glanis) – an in vitro study. Virus Res 63: 115-119. munostimulatory influence was observed, compared Siwicki AK, Morand M, Pozet F, Bernard D, Budrewicz to the european catfish where the strong suppression B(2000) Influence of iridovirus on lymphocyte activity: was observed. These results suggested that the CCHV in vitro comparative study in fish. Pol J Vet Scien suppressed the intracellular metabolism of the euro- 3: 93-96. pean catfish macrophages and lymphocytes and has Ward CW (1993) Progress towards a higher taxonomy of immunomodulatory effect on the cell-mediated im- viruses. Res Virol 144: 419-453. munity dependent to the species of fish. 126 Siwicki A.K. et al. Polish Journal of Veterinary Sciences Vol. 7, No. 3 (2004), Supplement, 127-129

Short original article Pathogenesis of Birnaviridae – influence of infectious pancreatic necrosis virus (IPNV) on cell-mediated immunity, total Ig level and lysozyme activity in Salmonid

A.K. Siwicki1,4, E. Terech-Majewska2, J. Szarek3, S. Trapkowska1, B. Kazuń4

1 Department of Microbiology and Clinical Immunology and 2 Department of Epizootiology and 3 Department of Forensic and Administration of Veterinary Medicine, University of Warmia and Mazury in Olsztyn, Oczapowskiego St. 13, 10-719 Olsztyn, Poland 4 Department of Fish Pathology and Immunology, Inland Fisheries Institute in Olsztyn, Poland

Abstract

The Birnaviridae are the most extensively studied viruses in lower and higher vertebrates. Infec- tious pancreatic necrosis virus (IPNV) is the prototype of the Birnaviridae. In our study we examined the influence of IPNV on the splenic macrophage and lymphocyte activity, lysozyme activity and Ig ]level in serum of rainbow trout. Healthy fish a mean weight of 10 g were inoculated intraperitoneally 7 with 50 µl of a viral suspension of the IPNV with a titre of 10 TCID50/ml, and 3 and 5 days after infection fish were scarified and dissected. Samples of pronephros and spleen were taken for histological and immunological assays. The results showed that IPNV strongly decreased pronephric macrophage and lymphocyte activity in infected fish, compared to the control. Also the lysozyme activity and total Ig levels were significantly decreased. The results showed strong suppressive effect of IPNV on immunocompetent cells and humoral defence mechanisms.

Key words: fish, IPNV, macrophage and lymphocyte activity, lysozyme and Ig levels

Introduction of birnaviruses contains two segments (A and B) of duble-stranded RNA which are circularized by VP1, The Birnaviridae are ubiquitous in aquatic organ- a viral protein (genome-linked protein, VPg). Infec- isms and they have been isolated all over the world tious pancreatic necrosis virus (IPNV) is the proto- from both marine and freshwater fish of different spe- type of the Birnaviridae (Brown 1986). This group also cies. They are responsible for severe losses in aquacul- includes the oyster virus (OV), the Drosophila X virus ture. The Binnaviridae dsRNA genome is easily purifi- (DXV), the infectious bursal disease virus (IBDV) ed and is resistant to common RNases. The bi- from poultry and a rotifer virus (RBV). These viruses rnaviruses have developed unusual coding strategies. differ from the bisegmented dsRNA viruses isolated The single-shelled, naked, icosahedral, viral particle from mammals which are tentatively classified as

Correspondence to: A.K. Siwicki, tel.: +48 89 523-32-17, e-mail: [email protected] 128 Siwicki A.K. et al.

Picobirnaviruses, because of the size of their particle cytopatic effect (CPE) was extensive, the cells were (55-65 nm) and their genome. The segments of IPNV harvested and centrifuged to eliminate cell debris. and IBDV have been sequenced. The organization of The virus stock was titered and stored at -70oC until these genomes is similar. Terminal repeats -AAGAG- use. The fish from experimental group were in- are present at the 5’ and 3’ ends of the A segment and oculated intraperitoneally with 50 µl of a viral suspen- 7 these are inverted on the B segment. These terminal sion of the IPNV with a titre of 10 TCID50/ml. The sequences are identical for IPNV and IBDV and may control group was inoculated with cell culture me- play a role in replication or packaging. The motif dium (RPMI-1640, Sigma). Fish were maintained in GLPYIGKT (IPNV) and GLPYVGRT (IBDV), 500 l tanks with recilculation system of water at tem- reminiscent of the GXXXXGKS/T ras-type GTP perature 15oC. For the immunological and histologi- binding proteins found on VP1, may relate to guanylyl cal assays, 3 and 5 days after infection, 10 fish from transverase activity demonstrated from both IPNV each group were bled by syringe from the caudal vein and IBDV (Dobos 1993). Hybrid arrested translation and dissected. Simples of liver and spleen were taken (HAT) shows that the protein order is for histological assays and pronephros for immu- 5’-VP2-NS-VP3-3’. The autocatalytic proteolytic en- nological study. The pronephros was removed and zyme is identified as NS and the active site maps to its single cell suspension obtained by teasing the tissue in carboxyl-terminus between amino acids 693 and 720 medium through a nylon mesh. The cells were then (Manning 1990) which results in the formation of washed in heparinized Hank’s balanced salt solution VP1-pG and VP1-pGpG. (HBSS) and isolated by gradient-density centrifu- Very little information exists about the replication gation on a Percoll (Pharmacia, Upsala, Sweden). strategy of birnaviruses and their pathogenesis. VP1 is A modification of the technique of Secombes found in the viral particle both as a free form and (1990) was used to study the potential killing activity bound to the RNA (Vpg) and is thought to be the (PKA) and respiratory burst activity (RBA) of viral RNA dependent RNA polymerase. However, at- pronephric macrophages stimulated by phorbol myris- tempts to search for consensus sequences in the VP1 tate acetate (PMA, Sigma). protein fail to find any known motifs such as the GDD The lymphocyte proliferation (LP) was determined motif, characteristic for the RNA polymerases. Com- using the MTT colorimetric assay method of Car- parison with the mammalian Picobirnaviridae would michael et al. (1987). The cells were stimulated by be useful, but no sequences have been published yet. concanavaline A (ConA, Sigma). Also very few information exists about the inµluence The lysozyme activity in serum was measured using of IPNV on the defence mechanisms in fish. In this a turbidimetric assay (Studnicka and Siwicki 1986) study, we examine the effects of IPNV on the im- and total immunoglobulin (Ig) levels in serum were munocompetent cells activity and nonspecific humor- determined using colorimetric assay (Anderson and al defence mechanisms in rainbow trout (Oncorhyn- Siwicki 1994). chus mykiss).

Results and Discussion Materials and Methods In the present experimental study, we continued Two groups (50 fish per group) of healthy rainbow the in vivo experiments to examine the effects of trout, with a mean body weight of 10g, were used in IPNV on macrophage and lymphocyte activity in rain- our study. The IPNV isolated from rainbow trout was bow trout. The respiratory burst activity (RBA) and used and virus stock was prepared in the chinook potential killing activity (PKA) of pronephric macro- salmon embryo cell line (CHSE-214). When the phages isolated 3 and 5 days from fish infected and

Table 1. Effects of the infectious pancreatic necrosis virus (IPNV) on the macrophage respiratory burst activity (RBA) and potential killing activity (PKA), proliferative response of lymphocytes (LP), lysozyme activity and total Ig levels in serum 3 and 5 days after experimental infection of rainbow trout and in control non-infected fish (mean ± SD, n=10).

Time after experimental infection with IPNV Parameters 3 days 5 days Control IPNV Control IPNV RBA (OD 620 nm) 0.42 ± 0.05 0.24 ± 0.04* 0.45 ± 0.05 0.18 ± 0.02* PKA (OD 620 nm) 0.38 ± 0.06 0.20 ± 0.03* 0.39 ± 0.05 0.12 ± 0.03* LP (OD 620 nm) 0.58 ± 0.05 0.31 ± 0.05* 0.56 ± 0.05 0.24 ± 0.04* lysozyme activity (mg/l) 32.5 ± 1.5 17.5 ± 1.4* 34.5 ± 2.0 15.8 ± 1.6* total Ig level (g/l) 12.4 ± 2.2 8.5 ± 1.5* 12.8 ± 1.8 6.5 ± 1.2* Explanation: * – statistically significant to the control p<0.05 Pathogenesis of Birnaviridae ... 129 control are presented in Table 1. The results of this References experimental study showed the strong suppression effects of IPNV on the phagocytic ability and in- Brown F (1986) The classification and nomenclature of vi- tracellular killing activity of pronephric macro- ruses. Intervirology 25: 140-143. phages. Also the 3 and 5 days after infection the Dobos P (1993) In vitro guanylylation of infectious pancre- proliferativeresponseofpronephriclymphocytes atic necrosis virus polypeptide VP1. Virology 193: 403-413. stimulated by ConA was statistically lower (p<0.05), Manning DS, Mason CL, Leong LC (1990) Cell-free transla- compared to the control IPNV-free fish (Table 1). tional analysis of the processing of infectious pancreatic The results presented that IPN virus has also de- necrosis virus polyprotein. Virology 179: 9-15. creasinginfluenceontheproliferativeresponseof Carmichael J, DeGraff WG, Gazdar AF, Minna JD, Mitchell pronephric lymphocytes. JB (1987) Evaluation of a tetrazolium-based The 3 and 5 days after infection, the lysozyme semiautomated colorimetric assay. Assessment of activity and total Ig levels in serum were significantly chemosensitivity testing. Cancer Res 47: 936-942. decreased (p<0.05), compared to the control fish Secombes CJ (1990) Isolation of salmonid macrophages and analysis of their killing activity. Techn Fish Immunol (Table 1). The results showed that IPNV has influ- 1: 137-154. ence on the lysozyme and Ig production by im- Siwicki AK, Anderson DP, Rumsey GL (1994) Dietary in- munocompetence cells. The similar results were ob- take of immunostimulants by rainbow trout affects served in rainbow trout naturally infected with IPNV non-specific immunity and protection against furunculo- (Siwicki et al. 1998). The present experimental in sis. Vet Immunol Immunopathol 41: 125-139. vivo study strongly suggested that infectious pancre- Siwicki AK, Morand M, Klein P, Kiczka W (1998) Treat- atic necrosis virus has direct influence on the ment of infectious pancreatic necrosis virus (IPNV) cell-mediated immunity and nonspecific humoral de- disease using dimerized lysozyme (KLP-602). J Appl Ichthyol 14: 229-232. fence mechanisms in fish. Studnicka M, Siwicki AK (1986) Lysozyme level in carp (Cy- prinus carpio). Bamidgeh 2: 22-26. 130 Siwicki A.K. et al. Polish Journal of Veterinary Sciences Vol. 7, No. 3 (2004), Supplement, 131-133

Short original article Chocolate feeding of pregnant mice resulted in epigallocatechin-related embryonic angiogenesis suppression and bone mineralization disorder

P. Skopiński1,2, E. Skopińska-Różewska3, A. Kamiński4, A. Dziedzic-Gocławska4, E. Sommer3, J. Chorostowska-Wynimko3, I. Cendrowska5, D. Chrystowska5, J. Sadło6, A.K. Siwicki7

1 Department of Histology and Embryology, Biostructure Center, Medical University, 02-004 Warsaw, Poland 2 Department of Ophtalmology, Medical University, Sierakowskiego St. 13, 03-709 Warsaw, Poland 3 Department of Laboratory of Diagnostic Immunology, National Institute of Tuberculosis and Lung Diseases, 01-138 Warsaw, Poland 4 Department of Transplantology, Medical University, Chałubińskiego St. 5, 02-004 Warsaw, Poland 5 Pharm. Manufact. Enterprise GEMI, 05-480 Mickiewicza St. 36, Karczew, Poland 6 Department of Radiation Chemistry and Technology, Institute of Nuclear Chemistry and Technology, 03-195 Warsaw, Poland 7 Department of Microbiology and Clinical Immunology, University of Warmia and Mazury in Olsztyn, Oczapowskiego St. 13, 10-719 Olsztyn, Poland

Abstract

Our previous studies have shown inhibitory action of some phenolic compounds and theobromine on tumour and embryonic angiogenesis. We also observed significant inhibition of embryos growth and limbs and femoral bones shortening in 4 weeks old offspring of female mice fed chocolate during pregnancy and lactation. The aim of the present study was to evaluate: 1) the effect of chocolate given to mice during the pregnancy and lactation period on the angiogenic activity of 18 days embryos tissues (cutaneous angiogenesis test) and their epigallocatechin (EGC) content (HPLC method); 2) bones mineralization in 30 days old offspring by electron spin resonance spectroscopy (ESR). Results: 1) angiogenic activity of embryonic tissues correlated negatively with their epigallocatechin content; 2) crystallinity of femoral bones was higher in offspring of chocolate fed mothers than in control ones.

Key words: mice, pregnancy, chocolate, angiogenesis, EGC, bones mineralization

Introduction mice, inhibition of VEGF led to inhibition of angiogenesis and to a decrease in the levels of chondroc- Vascular endothelial growth factor (VEGF) is the lasts and osteoblasts at the growth plates (Gerber and key regulator of capillary invasion and cartilage re- Ferrara 2000). We previously reported that feeding modeling during pre- and post-natal development. In pregnant mice with chocolate resulted in a decrease in

Correspondence to: P. Skopiński, e-mail: [email protected] 132 Skopiński P. et al. the relative length of limbs and femoral bones in spectrometry, ESR (Ostrowski et al. 1980, Dzie- 4 weeks old progeny as well as decrease in their dzic-Gocławska et al. 1984), 6 weeks after irradiation VEGF content (Skopiński et al. 2003). The aim of the with a dose of 100 kGy using 60Co source. present study was to evaluate the mineralization process of these bones measuring crystallinity by electron spin resonance spectrometry (ESR). We also reported Results and Discussion previously that embryonic tissues obtained from chocolate-fed mothers induced lower neovascular reaction The results of HPLC analysis are presented on the in cutaneous angiogenesis test than embryonic tissues Table 1. Epigallocatechin content of embryos ob- obtained from control group of pregnant mice tained from chocolate-fed mice was significantly high- (Skopińska-Różewska et al. 2003). The aim of the er than in corresponding controls, and significant present study was to evaluate content of catechins in negative correlation was found between epigal- 18 days embryos and to establish its possible relation locatechin content of homogenates and their an- to angiogenic activity of their tissues. giogenic activity (Fig. 1). Tissue concentrations of other catechins did not correlate negatively or positively with angiogenic activity. Evaluation of bone Materials and Methods mineralization revealed that the value of crystallinity of compact bone of diaphyses was about 17% higher The study was performed on 2 months old female in chocolate group than in the controls, of cancellous Balb/cxC3H F1 and female Balb/c mice fed during bone of epiphyses was about 30% higher in chocolate pregnancy and lactation, in addition to normal labora- group than in offspring of control mothers. tory chow, 400 mg/day bitter chocolate (Wedel, Po- land). Some mice were sacrificed by lethal dose of 0.25 chloral hydrate on 18 day of pregnancy, embryos were r=-(0.6226) extracted, suspended in PBS (1 g/1 ml), homogenized 0.2 p<0.05 (VirSonic ultrasound sonicator) and frozen at -80oC. 0.15 A high performance liquid chromatographic method for the simultaneous determination of four catechines 0.1 (catechin, epigallocatechin, epigallocatechin gallate, 0.05 epicatechin) was developed. The method was used to determine the levels of these catechins in the biologi- Epigallocatechin (mg/g) 0 21 cal material and food sample. Analysis was carried out 91113151719 mean number of newly-formed blood vessels using a Phenomenex Luna C18 column with an iso- cratic solvent of methanol-water-acetic acid and UV Fig. 1. Negative correlation between angiogenic activity of detection (204 nm). Quantification was carried out by embryos tissue homogenates and their EGC concentration. the external standard method. The cutaneous angiogenesis assay was performed to assess homogenates angiogenic activity (Rogala et al. 2001). Shortly, 0.05 ml homogenate samples (4-6) were injec- Our results of bone crystallinity measurements sug- ted intradermally to anaesthetized Balb/c mice. After gest that chocolate feeding of pregnant mice may dis- 72 hours mice were sacrificed. Newly-formed blood turb the processes of bone mineralization in offspring. vessels were identified and counted on the inner skin It is in accordance to our previous findings, obtained side, in dissection microscope, at the 6x magnification, from experiments performed on progeny of choc- in the central 1/3 of the microscopic field. The rest of olate-fed mice, where we observed shortening of limbs progeny was sacrificed at the 4th week after birth. and bones and lowering of their VEGF content Femoral bones were separated into dia- and epiph- (Skopiński et al. 2003). yses. Evaluation of the amount of crystallinity of bone The chocolate at dose 400 mg daily per mice dur- mineral was performed by electron spin resonance ing pregnancy significantly suppressed angiogenic

Table 1. Angiogenic activity and epigallocatechin concentration of 18 day embryos tissue homogenates.

Mean number of bloodEpigallocatechin Statistical significance Mothersvessels +/-se (A) concentration (mg/g) +/-se of difference (B) Controls (n=8) 18.1 +/- 0.6 0.06 +/- 0.02 – Chocolate fed 11.4 +/- 0.5 0.16 +/- 0.02 A – p<0.01 (n=6) B–p<0.05 Chocolate feeding of pregnant ... 133 activity of 18 day embryos tissues (Skopińska- betics and in patients with normal carbohydrate metab- -Różewska et al. 2003) and, as revealed by our present olism. Basic Appl Histochem 28: 21-26. study, this effect correlated with epigallocatechin con- Gerber HP, Ferrara N (2000) Angiogenesis and bone tent. This daily dose corresponds to 200 g of bitter growth. TCM 10: 223-228. Kondo T, Ohta T, Igura K, Hara Y, Kaji K (2002) Tea chocolate consumed by person of 70 kg body mass. catechins inhibit angiogenesis in vitro, measured by hu- We suppose, that malformations of limbs, reported in man endothelial cell growth, migration and tube forma- our previous work (Skopiński et al. 2003) and bone tion, through inhibition of VEGF receptor binding. Can- mineralization disorder observed in the present study cer Lett 180: 139-144. might be connected with anti-angiogenic action of Ostrowski K, Dziedzic-Gocławska A, Stachowicz W (1980) chocolate catechins, reported as anti-angiogenic Radiation induced paramagnetic entities in tissue mi- agents in in vitro and in vivo models (Cao et al. 2002, neral and their use in calcified tissue research. In: Free Kondo et al. 2002). Attention should be made to poss- Radicals in Biology, W Pryor ed, Acad Press, New York, IV: 321-344. ible harmful effects of catechines-rich food and bever- Rogala E, Skopińska-Różewska E, Sommer E, Pastewka K, ages during pregnancy and lactation. Chorostowska-Wynimko J, Sokolnicka I, Kazoń M(2001) Assessment of the VEGF, bFGF, aFGF and IL-8 angiogenic activity in urinary bladder carcinoma, us- Acknowledgements ing the mice cutaneous angiogenesis test. Anticancer Res 21: 4259-4264. This work was supported by grant from Polish Skopiński P, Skopińska-Różewska E, Sommer E, Choros- Scientific Committee, 3PO5EO3722. towska-Wynimko J, Rogala E, Cendrowska I, Chrys- towska D, Filewska M, Białas-Chromiec B, Bany J (2003) Chocolate feeding of pregnant mice influences length of limbs of their progeny. Pol J Vet Sci 6 (suppl): 57-59. References Skopińska-Różewska E, Chorostowska-Wynimko J, Sommer E, Rogala E, Demkow U, Filewska M, Radomska- Cao Y, Cao R, Brakenhielm E (2002) Anti-angiogenic -Leśniewska D, Skurzak H, Bany J, Siwicki AK (2003) mechanism of diet-derived polyphenols. J Nutr Biochem Wpływ diety wzbogaconej w czekoladę na angiogenezę 13: 380-390. embrionalną i nowotworową u myszy. w: Rola Im- Dziedzic-Gocławska A, Fuchs U, Ostrowski K, Stachowicz munomodulatorów Pochodzenia Naturalnego W, Michalik J (1984) Crystallinity of mineral deposited w Zapobieganiu i Leczeniu Chorób, ed. Medyk, War- in arterial walls in the course of arteriosclerosis in dia- szawa, 245-250. 134 Skopiński P. et al. Polish Journal of Veterinary Sciences Vol. 7, No. 3 (2004), Supplement, 135-138

Short original article Ultrastructural pattern of the muscle fibres of turkeys fed a diet containing oxidized fat

and infected with the O78K80H9 pathogenic serotype of Escherichia coli

J. Szarek1, A. Koncicki2, Z. Zduńczyk3, J. Jankowski4, A. Andrzejewska5, I. Babińska1, J. Lipińska1

1 Department of Forensic and Administration of Veterinary Medicine, 2 Department of Avian Diseases, University of Warmia and Mazury in Olsztyn, Oczapowskiego St. 13, 10-719 Olsztyn, Poland 3 Institute of Animal Reproduction and Food Research, Polish Academy of Sciences, Tuwima St. 10, 10-747 Olsztyn, Poland 4 Department of Poultry Science, University of Warmia and Mazury in Olsztyn, Oczapowskiego St. 5, 10-719 Olsztyn, Poland 5 Department of Clinical Pathomorphology, Medical Academy, Waszyngtona St. 13, 15-269 Białystok, Poland

Abstract

This experiment involved 40 one-day-old BUT-9 turkeys. For the first 8 weeks, the birds were fed

a mixture supplemented with a fat of peroxide value (PV) of 5 – 150 mEq O2/kg and 10 turkeys from

each PV level were taken and infected through the air sacs with a 1 ml dose of O78K80H9 pathogenic serotype of Escherichia coli suspension representing the 8 x 106 bacteria count based on McFarland’s scale. After a subsequent 4 days, all the birds were sacrificed and analysed macroscopically. Medial gluteal muscles from 5 turkey hens from each group were examined ultrastructurally.

It was found that the feeding turkey hens diets supplemented with fat of PV 150 mEq O2/kg for 8 weeks resulted in the development of ultrastructural lesions most often in mitochondria, rarely in smooth and rough endoplasmic reticulum and sporadically in other organelles of the muscles. Such feeding also caused a tendency to steatosis. The birds fed diets supplemented with oxidized fat and then infected with E. coli exhibited a more explicit tendency to ultrastructural lesions in the above-mentioned organelles than the healthy birds.

Key words: turkey, oxidized fat, Escherichia coli, muscle

Introduction Both the genetic basis and the intensive rearing conditions pose a threat to birds’ health (Jankowski et al. Rapidly growing turkeys, genetically selected for 2000, Koncicki et al. 2000). Peroxidized fat has a simi- high meat yield, were obtained at the cost of decrea- lar effect through its peroxide lipids which inhibit sing their natural immunity (Koncicki et al. 2001). body metabolism. Studies in this respect were carried

Correspondence to: J. Szarek, tel./fax: +48 89 523-32-52, e-mail: [email protected] 136 Szarek J. et al. out mainly on rats and, to a lower extent, on pigs, Results chickens or turkeys (Kanazawa et al. 1985, Dibner et al. 1996, Renerre et al. 1999, Szarek et al. 1999, Kon- Based on the macroscopic examinations, catarrhal cicki et al. 2000, 2001). Therefore, the aim of the inflammation of the intestine, parenchymatous dege- study was to examine the muscle ultrastructural neration of the liver and, in few cases, also kidneys, lesions in turkeys fed diets supplemented with diffe- were found in the majority of turkey hens from groups rent levels of peroxide fat and infected with Es- II and IV. These lesions were more explicit in the cherichia coli. birds from group IV than in those from group II. Hy- peraemia of the liver and kidneys and steatosis of the liver were sporadically observed in the turkey hens Materials and Methods from groups III and IV. No macroscopic lesions were found in birds representing group I. This study involved 40 one-day-old BUT-9 turkey Ultrastructural examination of the medial gluteal chicks. The birds were divided into 4 groups (n=10) muscle showed the regular structure of the muscle and fed ad libitum. The diet fed was described by fibres in all the turkey hens from group I (Fig. 1) while Koncickiego et al. (2001). The feed was divided into in some birds from group II proliferation of week portions and supplemented with a mixture of mitochondria and sporadically mitochondrial swelling rapeseed oil and poultry fat at the proportion of 66 were observed. : 34% and with a different peroxide value (PV): in The examined muscles of the experimental birds groups I and II – below 5 mEq O2/kg and in groups III fed diets supplemented with fat of the PV of 150 mEq and IV – 150 mEq O2/kg. Until week 4 the diet was O2/kg (groups III and IV) contained varied sizes of supplemented with 2% and then with 3% of oxidized lipid droplets situated both in interstitial tissue and in fat (Jankowski et al. 2000). intermyofibril fibre spaces (Fig. 2, 3). The prolife- The 8 weeks old turkeys in groups II and IV were ration of rough and smooth endoplasmic reticulum infected through the air sacs with a 1 ml dose of the was quite frequently observed in sarcoplasm. The tur- O78K80H9 pathogenic serotype of Escherichia coli re- key hens from group IV with such a condition very presenting the 8 x 106 bacteria count based on McFar- frequently exhibited vesicle transformation (Fig. 3). land’s scale. The bacteria for infection were obtained Small areas of myofibrils with perturbations in the in the lyophilized form from the National Veterinary course of the Z line were found in the majority of the Research Institute in Puławy. The bacteria were birds from groups III and IV (Fig. 4). multiplied in a broth medium BHI (brain heart infu- Lesions in mitochondria occurred in the muscles of sion) prepared according to the producer’s instruc- the birds from groups III and IV. They were: prolife- tions (Dico Laboratories, Detroit, Michigan, USA) ration, rarefaction of matrix, swelling and degradation and then transferred on the McConkey medium. The of crista, including particularly clearly visible in the birds were slaughtered 72 h after the infection. From turkey hens from group IV (Fig. 5). In the birds from each group, 5 turkey hens were selected for mor- the above groups, small fragments of the muscle fibres phological studies. For ultrastructural examinations, with degradation of the myofibrils and disorder of the segment of medial gluteal muscles were taken. Sec- myofibrils arrangement were sporadically observed. tions were fixed in a mixture of 1% paraformaldehyde Swelling of the endothelium cells in the blood vessel and 2% glutaraldehyde in a 0.2 M phosphate buffer walls in the majority of the birds from group IV were (pH 7.4) at 4oC for 2 h and post fixed in 2% osmium found (Fig. 6). tetroxide for 1 h. Specimens were embedded in Epon 812. The ultrathin sections were cut on a LKB ultra- Discussion microtome and contrasted with uranyl acetate and lead citrate and examined in an Opton 900 PC TEM Koncicki et al. (2001) showed the effect of oxidized (FRG). fat on susceptibility of turkeys to infection with hae-

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Fig. 1. Medial gluteal muscle of a turkey hen from group I – regular ultrastructure of the myofibrils. TEM, magn. x 12 000. Fig. 2. Medial gluteal muscle of turkey hen from group III – varied sizes of lipid droplets in the interstitial tissue (asterisks). TEM, magn. x 8 000. Fig. 3. Medial gluteal muscle of turkey hen from the group IV – fat droplet in the intermyofibril space (asterisk), beside well developed mitochondria, in sarcoplasm – vesicle transformation of the rough endoplasmic reticulum. TEM, magn. x 6 000. Fig. 4. Medial gluteal muscle of turkey hen from the group III – perturbations in the course of the Z line. TEM, magn. x 12 000. Fig. 5. Medial gluteal muscle of turkey hen from the group IV – lesions in mitochondria: proliferation (P), swelling, rarefaction of matrix and degradation of crista (asterisks). TEM, magn. x 6 000. Fig. 6. Medial gluteal muscle of turkey hen from the group IV – swelling of the vessel endothelium (S), to the right – a fragment of nucleus of the erythrocyte. TEM, magn. x 12 000. Ultrastructural pattern of the muscle ... 137

morrhagic enteritis virus (HEV). In this experiment, tibility to HEV infection. Additionally, it is known the greater spleen index in the birds fed diets supple- that the first effect of peroxide decomposition is the mented with oxidized fat (1.77 – 2.61) in comparison to damage of intestinal mucosa which accelerates the ab- the control birds (1.07 and 1.55), showed that the pre- sorption (Kazanawa et al. 1985). sence of peroxide lipids in feed increases birds suscep- In the previous experiments, the authors of this 138 Szarek J. et al. paper observed a correlation between the body re- effect on enterocyte turnover, hepatocyte proliferation sponse of turkeys exposed to prolonged feeding diets and the gut associated lymphoid tissue. Anim Feed Sci supplemented with peroxide fat of the Lea number of Tech 62: 1-13. Jankowski J, Zduńczyk Z, Juśkiewicz J, Koncicki A, Fal- 150 mEq O2/kg and the infection with O78K80H9 pathogenic serotype of Escherichia coli.Thiswasre- kowska A, Faruga A (2000) The response of turkeys to flected in the morphological lesions in mitochondria diets containing fat differing in degree oxidation. J Anim and smooth and rough endoplasmic reticulum as well Feed Sci 9: 363-370. as in vessel endothelium. The birds infected with this Kanazawa K, Kanazawa E, Natake M (1985) Uptake of secondary auto-oxidation products of linoleic acid by the pathogen and fed diets supplemented with oxidized fat rat. Lipids 20: 412-419. for 8 weeks showed a clearer tendency to ultrastru- Koncicki A, Krasnodębska-Depta A, Zduńczyk Z, Jankowski ctural lesions in these organelles than the healthy birds. J, Szarek J (2000) Biological response of turkeys fed on Previous papers indicated that diets supplemented mixtures with differentiated fat oxidation levels. Zesz with small amounts of oxidized fat and containing Nauk Akad Rol Wrocław 376: 63-76. adequate levels of antioxidants (vitamin E and sele- Koncicki A, Krasnodępska-Depta A, Zduńczyk Z, Jankowski nium) do not pose threat to birds’ health or evoke only J, Szarek J, Mazur-Gonkowska B, Guiro S (2001) Influ- subclinical lesions in their bodies (Lea et al. 1966, ence of feed mixtures with differentiated degree of fat Renerre et al. 1999, Jankowski et al. 2000, Koncicki oxidation degree on Hemorrhagic Enteritis Virus (HEV) et al. 2000). However, larger doses have a negative ef- in turkeys. Medycyna Wet 57: 441-443. fect on animals (Dibner et al. 1996, Szarek et al. 1999). Lea CH, Parr J, L’Estrange JL, Carpenter KJ (1966) Nutri- Based on the present experiment, feeding turkey tional effect of auto-oxidized fats in animal diets. 3. The hens diets supplemented with oxidized fat with a PV of growth of turkeys on diets containing oxidized fish oil.

150 mEq O2/kg for 8 weeks results in the development Brit J Nutr 20: 123-133. of ultrastructural lesions most often in mitochondria, Renerre M, Pincet K, Mercier Y, Gatellier P, Metro rarely in smooth and rough endoplasmic reticulum and B(1999) Influence of dietary fat and vitamin E on anti- sporadically in other organelles in the medial gluteal oxidant status of muscles of turkey. Influence of dietary muscle. It also causes a tendency to develop steatosis. fat and vitamin E on antioxidant status of muscles of turkey. J Agric Food Chem 47: 237-244. Szarek J, Zduńczyk Z, Andrzejewska A, Juśkiewicz J (1999) References The effect of prolonged feeding of rats with a diet containing oxidized fat on liver ultrastructural pattern. Pro- Dibner LJ, Atwell CA, Kitchell ML, Shermer WD, Ivey FJ ceedings of the International Conference, Kaunas: (1996) Feeding of oxidized fats to broilers and swine: 165-169. Polish Journal of Veterinary Sciences Vol. 7, No. 3 (2004), Supplement, 139-142

Short original article Ultrastructural characteristics of supraspinal muscles in rabbits after short-term electrostimulation

J. Szarek, I.M. Kowalski1, A. Andrzejewska2, J. Lipińska, I. Babińska, M.Z. Felsmann

Department of Forensic and Administration of Veterinary Medicine, University of Warmia and Mazury in Olsztyn, Oczapowskiego St. 13, 10-717 Olsztyn, Poland 1 Provincial Childrens’ Hospital and Rehabilitation Centre, 11-015 Ameryka, Poland 2 Department of Clinical Pathomorphology, Medical Academy, Waszyngtona St. 13, 15-269 Białystok, Poland

Abstract

This study was conducted on ten rabbits, aged 3.5 months. The animals were divided into 2 groups (n=5 each). In group 1, the supraspinal muscles were electrostimulated for 2 h/day for three months (by means of SCOLL-2 apparatus) according to the Lateral Electrical Surface Stimulation (LESS) method modified by Kowalski. In group 2 (control rabbits) no electrostimulation was conducted. An ultrastructural examination of the supraspinal muscles that were influenced by LESS showed generally a considerable increase in the number of mitochondria and their enlargement, and large clusters of the glycogen. These features indicated an intensified metabolism in muscle fibres caused by short-term electrostimulation. The disturbances in the course of the Z line were much less frequently observed. Other sub-cellular lesions in the stimulated muscles were observed relatively rarely.

Key words: scoliosis, lateral electrical surface stimulation (LESS), ultrastructural characteristics of muscle, rabbit

Introduction negative side-effects of the long-term application of LESS on humans and animals are known (Kowalski Paediatric rehabilitation studies on the shortening 1997, Kowalski et al. 2002, Szarek et al. 2003) there of the period of use of the Lateral Electrical Surface is a need to examine the ultrastructure of the muscles Stimulation (LESS) method (from 8-10 h/day to that were subjected to electrostimulation, including 2 h/day) in the treatment of idiopathic scoliosis (IS) in a morphological examination at the sub-cellular children and adolescents gave positive preliminary re- level, in order to applying the modification of the sults (Xiong et al. 1994). It proved the effectiveness of method. the method, particularly at an initial angle of the spi- Thus in the light of the introduced facts, a study nal scoliosis of less than 20o, according to Cobb’s identifying the ultrastructural features of supraspinal method (Kowalski 1997). Nevertheless, since the muscles after short-term LESS is fully justified.

Correspondence to: J. Szarek, tel./fax: +48 89 523-32-52, e-mail: [email protected] 140 Szarek J. et al. Ultrastructural charecteristics of supraspinal ... 141

Materials and Methods (Fig. 2). In all animals, the muscle fibres with a large number of mitochondria in sarcoplasma were found Ten male, pure-bred (New Zealand White), (Fig. 1, 3). Giant mitochondria were relatively rare, age-matched (born within 5 days) and clinically with transformation vesicle of the crests (Fig. 2). In healthy rabbits, obtained from a farm in Gdańsk (G-1 the majority of the animals, large clusters of glycogen on the list of Reproductive and Breeding Rabbit within the sarcoplasm were found (Fig. 2). In three Farms of the Ministry of Agriculture and Food Econ- rabbits, an irregular and sometimes zig-zagging course omy, Poland) were used in the experiment. The ani- of the Z line was observed (Fig. 4, 5). In some of these mals were adapted for 30 days to the experiment con- cases, the Z line made thin processes running along ditions. Rabbits were kept indoors in a room with con- the sarcomere (Fig. 4). Its atrophy was also observed, trolled temperature (18oC) and humidity (70%). Each with accompanying enlargement of endoplasmic re- animal was placed in a metal cage (50 x 50 x 50 cm) ticulum channels (Fig. 5, 6) and a loss of myosin and and received dry feed and water ad libitum. At the actin filament (Fig. 6). Occasionally, the thickening of beginning of the LESS treatment, the rabbits were the Z line took place (Fig. 1). aged approximately 3.5 months and their average MLT on the left side in the rabbits subjected to body mass was 2 100 g. LESS of the spine was normal as observed in an elec- The study was conducted in two groups (n=5), tron microscope. It is remarkable that the fibre struc- where the rabbits in group 1 were subjected to LESS ture of actin and myosin filaments was particularly for 2 h/day, and those in group 2 (control) were not distinct, with relatively frequent thickening of the subjected to electrostimulation. Z line (Fig. 3, 7). The muscle fibres were usually rich The ultrastructural examination was done on seg- in mitochondria (Fig. 3). ments of musculus longissimus dorsi (MLD): m. lon- The ultrastructural examination of MLT on the gissimus thoracis (MLT) and m. iliocostalis thoracis right and left side in the control rabbits revealed the (MIT) – the muscles which stabilise the rabbit’s spine. morphological features within the norm (Fig. 8). The samples were taken both from places which were The ultrastructural lesions observed in the supra- subjected to LESS (on the right side) and from spinal muscles in rabbits, caused by short-term LESS non-stimulated ones (on the left of the spine). The (proliferation of mitochondria, their enlargement, muscles were fixed in glutaraldehyde in phosphatic large clusters of glycogen in sarcoplasm, thickening of buffer of pH 7.2 and sealed in Epon 812. Semi-thin the limiting membrane) were positive. They are proof sections were stained according to the method of of intensified correct metabolism in the muscle fibres Levis and Knight’s (1977); the appropriate place for subjected to short-term LESS (Hadley-Miler et al. preparing ultrathin sections was established under 1994). The character of the changed cell organelles a light microscope. Structural analysis was conducted indicates a correct muscle reaction to the training, using an Opton 900 PC electron microscope (FRG). caused by repeated electric stimuli with the frequency of 30-40 Hz that produces tetanic contraction (Wright et al. 1992). Similar results were observed by Bigard et Results and Discussion al. (1993) in their experiments with monkeys. It should be noted that short-term LESS causes The ultrastructure of MLT in rabbits on the right sporadic low intensity disturbances; these included side of the spine, which was subjected to LESS, was a loss of single filaments of myosin and actin, and an normal. irregular course of the Z line. The intensity of those The most frequently observed mitochondria were lesions did not indicate their negative effect on the spherical (Fig. 1, 2), and relatively often enlarged physiology of muscles. Thus, the author’s own re- ← Fig. 1. Musculus longissimus thoracis dexter (MLTD) of a rabbit subjected to lateral electrical surface stimulation (LESS). Proliferation of mitochondria, thickening of the Z line and its irregular course. TEM, magn. x 27 600. Fig. 2. MLTD of a rabbit subjected to LESS. Enlarged mitochondria with vesicular widening of the crests (asterisks), the presence of glycogen granules in sarcoplasm. TEM, magn. x 15 800. Fig. 3. Musculus longissimus thoracis sinister (MLTS) of a rabbit subjected to LESS. Thickening of the Z line, numerous rod-shaped and spherical mitochondria. TEM, magn. x 15 800. Fig. 4. MLTD of a rabbit subjected to LESS. Irregular course of the Z line in places (asterisks) and its processes (arrows). TEM, magn. x 15 800. Fig. 5. MLTD of a rabbit subjected to LESS. A zig-zagging course of the Z line, enlarged endoplasmic reticulum channels (RS). TEM, magn. x 15 800. Fig. 6. MLTD of a rabbit subjected to LESS. Irregular course of the Z line, atrophy of myofilaments (encircled), enlarged endoplasmic reticulum channels (RS). TEM, magn. x 19 800. Fig. 7. MLTS of a rabbit subjected to LESS. Thickened Z line. Visible clusters of glycogen granules and crosswise T channels (arrows). TEM, magn. x 15 800. Fig. 8. MLTD of a control rabbit. Normal structure. TEM, magn. x 15 800. 142 Szarek J. et al. search emphasises the positive effect of short-term Kowalski IM, Szarek J, Zarzycki D, Talar J (2002) Disorders LESS on the muscle fibres of the supraspinal muscles, in course of experimental long-timed electrostimulation. which stabilise and correct the spine. Chinese J Clin Rehab 6: 1526-1527. Kowalski IM (1997) Clinical and experimental evaluation of an effect of the electrical surface muscle stimulation on spinal equilibrium. Ph.D. thesis, Collegium Medicum Acknowledgements Universitas Jagellonica Cracoviensis. Levis R, Knight DP (1997) Staining methods for sectioned Supported by grant 4 T11E 009 25. material. North Holland Publishing Company. Amster- dam, New York, Oxford. Szarek J, Kowalski IM, Van Dam F, Zarzycki Zarzycki, Paw- References licki R, Fabczak J (2003) Pathomorphological pattern of paravertebral muscles of rabbits after long-term experimental electrostimulation. Pathol Res Pract 199: 613-618. Bigard AX, Lienhard F, Merino D, Serrurier B, Guezennec Xiong B, Sevastik J, Hedlund R, Sevastik B (1994) Sagittal CY (1993) Effects of surface electrostimulation on the configuration on the spine and growth of the posterior structure and metabolic properties in monkey skeletal elements in early scoliosis. J Orthop Res 12: 113-118. muscle. Med Sci Sports Exerc 25: 355-362. Wright J, Herbert MA, Velasquez R, Bobechko WP (1992) 1994 Hadley-Miller N, Mims B, Milewicz D ( ) The potential Morphologic and histochemical characteristics of skeletal role of the elastic fiber system in adolescent idiopathic muscle after long-term intramuscular electrical stimula- scoliosis. J Bone Joint Surg Am 76: 1193-1206. tion. Spine 17: 767-770. Polish Journal of Veterinary Sciences Vol. 7, No. 3 (2004), Supplement, 143-146

Short original article Pathomorphological pattern of the liver and kidney in polar fox fed a diet supplemented with a new generation of feed

J. Szarek, M.O. Lorek1, J. Lipińska, A. Gugołek1, I. Babińska, M. Truszczyńska, M.Z. Felsmann

Department of Forensic and Administration of Veterinary Medicine, Faculty of Veterinary Medicine, University of Warmia and Mazury in Olsztyn, Oczapowskiego St. 13, 10-719 Olsztyn, Poland Chair of Fur-bearing Animal Breeding, Faculty of Animal Bioengineering, University of Warmia and Mazury in Olsztyn, Oczapowskiego St. 5, 10-719 Olsztyn, Poland

Abstract

In order to improve fox productivity, a new generation feed supplement containing strains of yeast (facilitating digestion), plant extract from Yucca schidigera (binding ammonia in the manure), lactic acid and zinc bioplex known under the commercial name as the DigDeo-Korektor (DDK) was added to their diet for a period of 3.5 months. The experiment was conducted on 60 young polar foxes in the period from weaning to pelting. The animals were divided into two groups (n=30) containing equal numbers of males and females. Group 1 was fed standard diet and group 2 received an addition of 5 g of DDK per day per animal. Based on the morphological examinations, it can be concluded that in foxes consuming their diets supplemented with the DDK, an increased blood supply to the organs, lower storage of lipid in the liver and an increased activity of the macrophage-histiocyte system were observed.

Key words: fox, DigDeo-Korektor, liver, kidney, pathomorphology

Introduction improvement of the sanitary state of foxes and increasing their performance. The current fox rearing techniques aim at the maximum animal growth and thus producing the largest possible sizes of hide and best quality of fur. In Materials and Methods achieving such results, feeding plays an important role, especially having appropriate protein, fat and en- The examination was conducted on 60 young polar ergy levels in their diets (Skrede and Ahlstrom 1992, foxes (30 females and 30 males), born at about the Ahlstrom 1995). Additionally fox breeders try to im- same time, in the period from weaning to pelting on prove animal productivity by controlling the digestion a production farm. The experimental foxes were kept processes. Such activities improve nutrient utilisation in cages designed for rearing young animals installed and eliminate pathogens. These are the methods for in one room. The animals were divided into two

Correspondence to: J. Szarek, tel./fax: +48 89 541-10-20, e-mail: [email protected] 144 Szarek J. et al. groups (n=30) containing equal numbers of males Parenchymatous degeneration of the renal tubule and females. The foxes of group 1 were fed a standard epithelium was found in three cases of the paraffin diet and the animals of group 2 additionally received liver sections of the foxes from group 1 (Fig. 2). In a new generation feed supplement containing strains one animal, a few cells of the renal tubule epithelium of yeast (facilitating digestion), plant extract from had features of necrosis. Glomerulonephritis was ob- Yucca schidigera (binding ammonia in the manure), served in 2 foxes and congestion was observed in one lactic acid and zinc bioplex known under the commer- animal (Fig. 2). cial name as the DigDeo-Korektor (DDK) in the dose Fat droplets in hepatocyte cytoplasm (usually 5 g per day per animal. The feed was prepared from smaller than a cell nucleus) were found in the micro- typical feed components by balancing the diets ac- scopic preparations of livers of the all foxes fed with cording to the requirements of the growing animals the DDK feed supplement (Fig. 3). It was lipid infil- (Lorek et al. 2000, Szarek et al. 2003). tration which covered from about 30% to 60% hepa- Animal body mass gain was analysed individually tocytes (Fig. 3). It was accompanied by paren- based on the measurement of the parameters taken chymatous degeneration most often in limited areas every two weeks. The results were statistically ana- to a half of the analysed animals. Individual hepa- lysed using a single-factor variance analysis in the or- tocytes with the features of necrosis were seldom ob- thogonal system (Ruszczyc 1981). served. In six foxes, congestion of the liver was ob- After 3.5 months of polar fox feeding in the served, which most frequently was exhibited by above-described manner, the animals were sacrificed. dilatation of large blood vessels, filling them with Five males and 5 females were randomly selected blood cells and, occasionally, by excessive storage of from each of the two groups for morphological ana- blood morphotic elements in the Disse’s spaces. In lyses (liver and kidney macro- and microscopic ana- one case, the described lesion was accompanied by lyses). The experimental material was fixed in 10% a trail extravasation. The swelling of the walls of neutralised formalin and the liver and kidney paraffin some blood vessels was observed in the livers of two sections were stained with haematoxylin and eosin foxes and hyperplasia of the connective tissue within (HE) and the livers were additionally stained by the liver was found in two other foxes. Dispersed Sudan III according to Lillie Ashburn. infiltration of mononuclear cells occurred with varied intensity in 7 animals. Infiltration of stellate cells occurred relatively frequently. In some cases, hyper- Results and Discussion trophy of these cells was found (Fig. 3). In the majority of animals, individual hepatocytes with mitotic di- The average fox body mass at the beginning of the visions were observed. experiment was 2.02 kg in group 1 and 1.92 kg in In the kidneys of three foxes from group 2, foci of group 2. A similar difference was maintained until parenchymatous degeneration of the renal tubule week 8 and after 10 weeks of the experiment the aver- epithelium were found and, in one case, hyaline de- age fox body mass equalised and was 7.82 kg. By the generation was found. Only few tubules’ epithelium end of the rearing period, the foxes from group cells became necrotic. In 5 animals, kidney conges- 2 weighed slightly more. At the end of the experiment tion was reported and, in one, extravasation was ob- (after 14 weeks) the average body mass was 13.21 kg served. In the kidney sections of 4 foxes, and 14.84, respectively. However, the differences in glomerulonephritis was observed. On occasion, it was the average body masses were statistically insignifi- accompanied by infiltration of macrophage-his- cant. tiocyticals. Based on the macroscopic analysis, the foxes from The experimental results indicate that the foxes group 1 were diagnosed for congestion of kidneys (in fed diets supplemented with DDK had a better blood one case) and the animals from group 2 for conges- supply to the liver and kidney in comparison to the tion of liver (in two cases) and congestion of kidney animals fed unsupplemented diets. Additionally, the (in one case). effectoftheanalysedfactorsonthefoxmetabolism Lipid infiltration was found in all the livers of was visible. It resulted in a decrease of lipid infiltra- group 1 foxes (Fig. 1). Relatively frequently fat drop- tion by half. The size of the lipid droplets in hepa- lets were larger than hepatocytes nuclei and they were tocyte cytoplasm was decreased and the number of found in 50%-70% of parenchymatous liver cells (Fig. lipid non-storing cells increased. 1). Parenchymatous degeneration of hepatocytes was The DDK also stimulated the macrophage-his- found in 4 animals covering each time a small area tiocytical system. This was exhibited by cell infiltra- (Fig. 1). Congestion of the liver with varied intensity tionsinliversandinkidneysaswellasbyhyper- was found in 50% of the analysed foxes (Fig. 1). Infil- trophy of the stellate cells in liver. tration of mononuclear cells was found in the livers of Szarek et al. (2003) showed that the DDK admin- three animals and proliferation of the stellate cells istered to foxes in the same manner as in the de- was observed in two animals. scribed experiment induces a better blood supply of Pathomorphological patterns ... 145

Fig. 1. Liver of a polar fox from group 1 – lipid infiltration in the form of numerous and large lipid droplets in hepatocytes, parenchymatous degeneration and congestion. Haematoxylin and eosin staining, x 460.

Fig. 2. Kidney of a polar fox from group 1 – parenchymatous degeneration, glomerulonephritis. Haematoxylin and eosin staining, x 460. 146 Szarek J. et al.

Fig. 3. Liver of a polar fox from group 2 – lipid infiltration in the form of small and less numerous lipid droplets in hepatocytes, hypertrophy of stellate cells. Haematoxylin and eosin staining, x 460.

tunica mucosa of the stomach and small intestine References and limits excessive accumulation of epithelium on the surface of the mucous membrane of the alimen- Ahlstrom O (1995) Fordoyelighet av for med ulike fettniva tary tract. Additionally, it was found that the de- hos blarev og mink. Norsk Pelsdyrblad 3: 12-13. scribed supplement had a positive effect on the rear- Gugołek A, Lorek MO, Lipiński K (1999)Attempttouse ing and production performance of foxes (Gugołek unconventional supplements in growing polar fox nutri- et al. 1999). tion. Scientifur 3: 187-195. Lorek MO, Gugołek A, Szarek J, Przeździecka D (2000) Effects of a new generation of feed supplement on some performance indices and health states in mink. Scien- Conclusions tifur 24: 86-88. Ruszczyc Z (1981) Metodyka doświadczeń zootechnic- Based on the morphological examinations, it can znych. PWRiL, Warszawa. be concluded that diets supplemented with the Dig- Skrede A, Ahlstrom O (1992) Fett og karbohydrater i for Deo-Korektor increase blood supply to the organs, til rev og mink. Norsk Pelsdyrblad 6: 11-12. decrease storage of lipid in the liver and increase the Szarek J, Lorek MO, Przeździecka D, Gugołek A, Lipińska activity of the macrophage-histiocytical system in po- J, Kostiuk S (2003) Pathomorphological pattern of the lar foxes. alimentary tract in fox fed with a diet supplemented with a new generation feed. Scient Messenger Lviv Nat Acad Vet Med 5: 185-188. Polish Journal of Veterinary Sciences Vol. 7, No. 3 (2004), Supplement, 147-150

Short original article Pathomorphological pattern of the kidney in European sheatfish (Silurus glanis)after iridovirus infection and lysozyme dimer application in a bath

J.Szarek,A.K.Siwicki1,A.Andrzejewska2,I.Babińska,J.Lipińska, M. Truszczyńska, E. Terech-Majewska1

Department of Forensic and Administration of Veterinary Medicine, 1 Department of Ichtiopathology, University of Warmia and Mazury, Oczapowskiego 13, 10-717 Olsztyn, Poland 2 Department of Clinical Pathomorphology, Medical Academy, Białystok, Poland

Abstract

An experiment was conducted with european sheatfish (Silurus glanis)infectedwithiridovirus using fish which had been earlier immersed in lysozyme dimer (KLP-602) and then infected with iridovirus. The head kidney was evaluated microscopically and ultrastructurally. The results were compared to the observations of control fish. The lysozyme dimer was found to cause a rise in the activity of polymorphonuclear and mononuclear cells and inhibited the multiplication of iridoviruses.

Key words: sheatfish, kidney, iridovirus, lysozyme dimer

Introduction Materials and Methods

The lysozyme dimer (KLP-602, Nika Health Prod- An experiment was conducted with fry of european ucts, USA) is an active and efficient product in the sheatfish (Silurus glanis) with body weight of 80-100 g, treatment of many diseases of various animal species obtained at the Experimental Farm IRŚ in Zatorze, (Klein et al. 1997, Kołodziejczyk et al. 2002). Poland. The fish were adapted for 14 days to the ex- Considering its immunostimulative and im- perimental conditions. The examinations were carried munocorrective action (Siwicki et al. 1996, Kolman out in plastic pools of 500-1000 l capacity with the et al. 2002), the effect of dymerized lysozyme closed water circulations and a temperature of (KLP-602) on the morphology of european sheat- 20-22oC. At the time of experiments fish were fed with fish kidney after infection with iridovirus was exam- commercial feed containing 35% of protein. The ined. sheatfish were divided into three groups (n=12). The

Correspondence to: J. Szarek, tel./fax: +48 89 523-32-52, e-mail: [email protected] 148 Szarek J. et al. first group contained the control fish, not subjected to The microscopic structure of the kidney of all the immersion in lysozyme dimer (KLP-602) and not in- control fish in histological preparations was normal. fected with iridovirus. The sheatfish in the second In the kidney of the control sheatfish, an ultra- group were not immersed in the lysozyme dimer but structural pattern characteristic of the species under were infected with iridovirus (a virus isolated from study was observed. sheatfish – 62.90 – was used), 0.1 ml of which was In the kidney of the sheatfish in group two, numer- introduced to the organism intraperitoneally. The fish ous epithelial cells with necrotic features were found from the third group were immersed in a 0.2% sol- (Fig. 1). Some epithelium cells contained an increased ution of lysozyme dimer (KLP-602) for 0.5 h, and number of mitochondria and proliferation of rough after 72 hours they were infected with iridovirus, like endoplasmatic reticulum was observed in them. Myel- the fish in the previous group. The fish were subjected in-like structures of various sizes were found with to clinical examination and anaestheizated by Propis- relatively high frequency in the cytoplasm of these cin (IRŚ Żabieniec) 96 hours later. During a micro- cells. The presence of virions was observed in a con- scopic evaluation, sections of head kidney were taken siderable number of epithelial cells. They were usually for microscopic and ultrastructural examination. The situated in the nuclei cells, whose structure in such material for microscopic examination was embedded cases was damaged (Fig. 2). Only sporadically the in paraffin and that for ultrastructural examination virions were situated in the cytoplasm. They were was embedded in Epon 812. Sections of paraffin were gathered in large clusters, were usually mature stained with hematoxylin and eosin, and the ultrathin and well-formed. Macrophages were found more ones were contrasted with uranyl acetate and lead ci- frequently and plasmatic cells slightly more frequently trate. The kidney was observed in an Opton 900 PC than in the control fish. electron microscope, made in Germany. Epithelial cells in the kidney of the third group became necrotic only sporadically. In many of them there was a complex rough endoplasmatic reticulum Results and in some of them, the smooth endoplasmatic reticulum became transformed into vesicles. Virions During the clinical observation, it was found that were present mainly in cytoplasm, where they formed all the sheatfish in all groups behaved correctly small clusters (Fig. 3). They were found in the cells thoughout the time of the experiment. Their post whose number was half as large as in the case of the mortem macroscopic pattern also did not deviate previous group. Plasmatic cells and macrophage infil- from standard. trations, and sometimes granulocytes with mitotic

Fig. 1. Sheatfish 24 hours after an infection with iridovirus – necrosis of renal tubules epithelium. Haematoxylin and eosin staining, x 500. Pathomorphological pattern ... 149

Fig. 2. Sheatfish 24 hours after an infection with iridovirus – renal tubule epithelium cell with large clusters of virions present in the cell nucleus (arrows) and single virions present in cytoplasm (arrowheads). TEM, x 14 500.

Fig. 3. Sheatfish 24 hours after an infection with iridovirus, the fish was previously (72 hours earlier) immersed for 0.5 hour in a 0.2% aqueous solution of lysozyme dimer – renal tubule epithelium cells with a small number of virions present in cytoplasm (arrows) and single virions situated outside of the cell (arrowheads). TEM, x 14 500. 150 Szarek J. et al. division, were found much more often than in the References sheatfish of the second group. Klein P, Siwicki AK, Morand M (1997) Immunomodilating Discussion activity of dimerized lysozyme (KLP-602) after suppression induced by infectious pancreatic necrosis virus It was found that lysozyme dimer significantly in- (IPNV). In: Siwicki AK (ed) Biological monitoring of creased the activity of polymorphonuclear and mono- environmental contamination. Effects of xenobiotics on nuclear cells (Siwicki et al. 1996). It accelerated the the immune system. Wydawnictwo IRŚ, Olsztyn, pp. phagocytic activity of neutrophils and macrophages, 195-205. lymphocyte proliferation, lysozyme and immunog- Kolman H, Kolman R, Siwicki AK, Szczepkowska B (2002) lobulin levels in plasma. These processes were reflected Some humoral effects in Siberian sturgeon (Acpenser in the experiment conducted for this study. The activity baeri Brandt) after lysozyme dimer application in a bath. of these cells was more distinct in the sheatfish, which Arch Ryb Pol 8: 171-180. were subjected to the lysozyme dimer and iridoviruses Kołodziejczyk P, Stankiewicz I, Porowski M, Pejsak Z(2002)EffectivenessofLydiumKLPintreatingcoli- than in the fish, which were not immersed in water with form mastitis in sows and mixed respiratory infections in the addition of lysozyme dimer (KLP-602). In addition, swine. Bull Vet Inst Pul 58: 270-274. the microscopic and ultrastructural image of the sheat- SiwickiAK,KleinP,KiczkaW,MorandM,Studnicka fish kidney showed the inhibiting action of the ly- M(1996) In vitro effects of monomer and dimerized sozyme dimer (KLP-602) on the multiplication of lysozyme on the polymorphonuclear (PMN) and mono- iridoviruses and on their activity and the stimulation of nuclear (MN) cells activity. In: Stolen JS, Fletcher TC, the activity of plasmatic and phagocytic cells. Bayne CJ, Secombes CJ, Zelikoff JT, Twerdok LE, An- derson DP (ed) Modulators of immune responses – the Acknowledgements evolutionary trail. SOS Publications, Fair Haven, NJ, pp. 221-232. The authors are indebted to Ms. K. Dublan, MSc and Mr. A. Penkowski, MSc for their excellent technical assistance. Polish Journal of Veterinary Sciences Vol. 7, No. 3 (2004), Supplement, 151-154

Short original article Rhodococcus equi infection in foals – clinical and pathomorphological changes

L. Witkowski, M. Sobczak-Filipiak, J. Kaba, M. Rzewuska1,J.Kita

Department of Clinical Sciences and 1Department of Preclinical Sciences, Faculty of Veterinary Medicine, Warsaw Agricultural University, Nowoursynowska 159, 02-776 Warsaw, Poland

Abstract

Rhodococcus equi is one of the most important cause of disease in foals. Seldom it is observed in other animal species and in humans. Rhodococcosis is an endemic disease causing serious economi- cal loses in a certain number of study farms in Poland. A number of inquiries devoted to this disease have been performed in Poland yet its occurrence is still not adequately explored. The aim of this study is to recognize the present situation of R. equi infectioninselectedstudyfarmsinPolandwith a particular emphasiss on the clinical course of the disease.

Key words: Rhodococcus equi, horses

Introduction mune response, readily develop disease, if exposed aerogenously to sufficient numbers of Rhodococcus Rhodococcus equi is one of the most important equi (Yager 1987). Therefore, management and envi- cause of disease in foals seldom observed in other ronmental circumstances play a major role in deter- species of animals and in humans. The disease has mining the magnitude of this challenge and in the worldwide distribution. Although all horse farms are prevalence of the disease. likely to be infected with R. equi the clinical disease Although Rhodococcus equi infection can produce is enzootic and devastating on some farms, sporadic life-threatening pyogranulomatous pneumonia, most on others, and unrecognized on most (Takai et al. horses develop a protective immune response, which 1991). lasts throughout life (Kohler et al. 2003). Most commonly it is isolated from humans with In 1-6 months old foals Rhodococcus equi is an acquired immunodeficiency syndrom (AIDS), but important cause of pneumonia. About half pneu- there were also cases of infection in renal transplant recipients, adult humans with hemolymphatic tu- monic foals have also ulcerative colitis (necrotizing mours (i.e. acute leukemia) or after corticosteroids enterocolitis) or the other intestinal lesions (Yager therapy and in an immunocompetent persons as 1987, Prescott 1991, Giguere and Prescott 1997, Hon- a posttraumatic infection (Prescott 1991). dalus 1997). Rhodococcus equi is ubiquitous in the soil. The As it is known Rhodococcus equi infection could main routes of infection are respiratory and alimen- have some different clinical and pathomorphological tary tracts. The particular susceptibility of the foals is forms. The aim of this study was to determine forms not clearly understood. Foals, whose maternal im- of infection, which are present in four farms with en- munity wanes before generation of their own im- demic rhodococcosis in Poland.

Correspondence to: L. Witkowski, tel.: +48 22 847-58-18, e-mail: [email protected] 152 Witkowski L. et al.

Materials and Methods ranged usually from 0.1 to 3.0 cm, but the largest diameter measured 10 x 25 cm. Histopathologically, the The study was performed in four farms in Poland lesions in lungs were predominantly pyog- on seventeen foals died with clinical signs of rhodo- ranulomatous. In one horse the giant cells were ob- coccosis. The foals were both sexes, 2-4 months old served. Neutrophils were numerous; lymphocytes and and of different breeds (xo – 3, xx – 8, oo – 1, cross- plasma cells were present in moderate numbers. In six breed – 5). All animals were treated with different cases there were also inflamatory cell infiltrates in in- antibiotics (also erythromycin and rimfampin). Clini- terstitial tissue of lungs. Other anathomopathological cal signs and dates were collected by interview with changes were also present, as follows: one case of local veterinarians. Post mortem examination, his- pleuritis, one of pericarditis, twelve of hydropericar- topathological and bacteriological diagnosis were per- dium. In one case (with clinically observed diarrhoea) formed in all cases. Histopathological examination there was colitis connected with other intestinal (lungs and other pathological changed organs; paraf- lesions (Fig. 2.). The pyogranulomatous changes have fin slides stained haematoxylin and eosin) and bacteri- been observed in a colon (and R. equi was isolated). ological examinations (from suppurative changes; se- There was also clinically diagnosed cornear opacity lective medium CAZ-NB agar) were conducted using (two cases) and edema of joints (two cases). Rhodo- routine methods. coccus equi was isolated from nine cases (app. 53%).

Results Discussion

All seventeen foals suffered from pneumonia. They The Rhodococcus equi infection has been con- had dyspnoea and fever (39.5-41oC), rarely nasal dis- firmed by bacteriological examination only from nine charge and cough. Four of them died after very short cases, but the clinical and anathomopathological period (1-3 days) of clinical signs of disease. In fifteen symptoms were evident and similar in all foals. Prob- cases there were solitary or numerous abscesses in lems with isolation of R. equi could be caused by long lungs parenchyma (Fig. 1). The diameters of abscesses antibiotic treatment and difficult bacteriological

Fig. 1. Lungs of foal with abscesses caused by R. equi. Rhodococcus equi infection ... 153

Fig. 2. Colon with abscesses caused by R. equi.

examination of suppurative changes. In some samples foals could have also uveitis or panophtalmitis, and in there was intensive growth of proteolytic bacterias two horses there were clinical changes in cornea. (Proteus spp.). The predominant clinical symptom of In conclusion, Rhodococcus equi infection in Polish the disease was pneumonia and it correlated very well horses features different morphological and clinical with the results of post mortem examination. This is in forms, just like the cases studied elsewhere. This pre- accordance with previous publications (Prescott 1991, liminary study shows however that, contrary to other Giguere and Prescott 1997, Hondalus 1997). In our publications (Yager 1987, Prescott 1991, Giguere and study the pathological changes in a digestives tract Prescott 1997, Hondalus 1997), in the course of rho- was diagnosed only in one case (app. 6%); however, dococcosis the pathological changes in digestive tract the data from other publications differ and estimate are observed seldom. its prevalence for 30-50% (Yager 1987, Prescott 1991, Giguere and Prescott 1997, Hondalus 1997). We did not observe other symptoms reported in the literature. Chaffin et al. (1995) described the cauda equina References syndrome with urinary incontinence, discospondylitis and paravertebral abscess in a case of Rhodococcus Chaffin MK, Honnas CM, Crabil MR, Schneiter HL, Brum- equi infection in 4 months old male of Quarter Horse. baugh GW, Brinner RP (1995) Cauda equina syndrome, In this case Rhodococcus equi was isolated from speci- discospondylitis, and a paravertebral abscess caused by mens of bone and fluid samples from abscess. Firth et Rhodococcus equi in a foal. J Am Vet Med Assoc 206: al. (1993) reported osteomyelitis, connected with this 215-220. infection in two foals. Giguere and Prescott (1997) Firth EC, Alley MR, Hodge H (1993) Rhodococcus equi described non septic polisynovitis, particularly of the – associated osteomyelitis in foals. Aust Vet J 70: tibiotarsal and stifle joints and septic arthritis and/or 304-307. Giguere S, Prescott JF (1997) Clinical manifestations, diag- osteomyelitis. In our material there were two clinical nosis, treatment, and prevention of Rhodococcus equi in- cases of joint edema. fections in foals. 56: 313-334. Pedrizet and Scott (1987) described subcutaneous Hondalus MK (1997) Pathogenesis and virulence of Rhodo- abscess formation and cellulitis in a three months old coccus equi. Vet Microbiol 56: 257-268. thoroughbred filly, but we have not proved such cases Kohler AK, Stone DM, Hines MT, Byrne BA, Alperin DC, of infection. Norton LK, Hines SA (2003) Rhodococcus equi secreted Giguere and Prescott (1997) reported that some antigens are immunogenic and stimulate a type 1 recall 154 Witkowski L. et al.

responses in the lungs of horses immune to Rhodococcus Takai S, Ohbushi S, Koike K, Tsubaki S, Oishi H, Kamada equi infection. Infect Immun 71: 6329-6337. M(1991) Prevalence of virulent R. equi in isolates from Pedrizet JA, Scott DW (1987) Cellulitis and subcutaneous soil and feces of horse from horse-breeding farms with abscesses caused by Rhodococcus equi infection in a foal. and without endemic infection. J Clin Microbiol J Am Vet Med Assoc 190: 1559-1561. 29: 2887-2889. Prescott JF (1991) Rhodococcus equi: an animal and human Yager JA (1987) The pathogenesis of Rhodococcus equi pathogen. Clin Microbiol Rev 4: 20-34. pneumonia in foals. Vet Microbiol 14: 225-232. Polish Journal of Veterinary Sciences Vol. 7, No. 3 (2004), Supplement, 155-157

Short original article CGRP-immunoreactive structures in the porcine superior cervical ganglion (SCG) – decentralization/deafferentation- -induced changes

J. Wojtkiewicz, S. Gonkowski, A. Bossowska, J. Kaleczyc1, J. Całka1, M. Majewski

Division of Clinical Physiology and 1Animal Anatomy, Department of Functional Morphology, Faculty of Veterinary Medicine, University of Warmia and Mazury, Oczapowskiego St. 13, 10-717 Olsztyn, Poland

Abstract

The occurrence of CGRP-containing nerve structures was studied in intact and decentralized/deafferented porcine SCG. A low number of CGRP-IR perikarya, supplied by patchily distributed moderate to dense meshwork of varicose LENK-IR fibres, was found in the ganglion; number of these cells remained constant after decentralization/deafferentation. Three subpopulations of CGRP- and/or SP-IR nerve fibres have been found to exist in porcine SCG: SP/CGRP-IR, solely SP- and solely CGRP-IR (48%, 45% and 7% of all studied fibres, respectively). Virtually all SP- and SP/CGRP-IR nerves disappeared after the decentralization/deafferentation. However, solely CGRP-IR fibres were still present in SCG, what suggests their intraganglionic origin.

Key words: superior cervical ganglion (SCG), calcitonin gene-related peptide (CGRP), substance P (SP), decentralization, deafferentation, pig

Introduction a most recent references, see Hisa et al. 1997). CGRP-IR sympathetic neurons have been found also Calcitonin gene-related peptide (CGRP) was in the porcine stellatum (Happola et al. 1993) and found to be widely distributed throughout both the inferior mesentetric (Majewski and Heym 1992) central and peripheral nerve system. In the periphery, ganglia. As there is no data concerning the CGRP and substance P (SP) are thought to be the distribution of CGRP-IR neurons/nerve fibres most important peptides involved in the transmission in the porcine SCG, the present study was aimed by of sensory modalities. However, apart from the affer- means of double and triple-labelling immunofluores- ent collaterals present in the ganglionic neuropil cence at unravelling the distribution and neur- (Heym et al. 1993), CGRP-IR postganglionic neurons ochemical coding of CGRP-IR structures in this gan- have also been described in sympathetic ganglia of glion of the pig. man (i.e. Tajti et al. 1999) and various mammals (for

Correspondence to: M. Majewski, tel.: +48 89 523-44-60, fax: +48 89 523-39-82, e-mail: [email protected] 156 Wojtkiewicz J. et al. CGRP-immunoreactive structures ... 157

Materials and Methods areinlinewithdataconcerningthehuman,felineand canine ganglion, but are contradictory to the findings Six immature female pigs, divided into two groups: regarding rodents SCG (for a most recent references, intact animals (control group; C) and animals with see Hisa et al. 1997). As revealed by double- and cervical sympathetic trunk transected 1.5 cm below triple-immunofluorescence labelling, sympathetic the SCG (decentralized/deafferented group, D), were CGRP-IR neurons of the porcine SCG belonged to the used for the present study. Ten µm thick cryostat sec- subset of non-adrenergic neurons targeted by opioider- tions were incubated with two or three primary anti- gic nerve terminals in a very specific manner. This bodies raised in different species, including rabbit agreed well with neurochemical characteristics of the anti-CGRP (1:4000, Affiniti, UK, Bachem, CH), rat CGRP-IR nerve cells found in other species (i.e. cat, anti-SP (1:350, Biogenesis, UK), rat anti-NPY (1:150, dog, for a most recent references, see Hisa et al. 1997), Affiniti, UK) and mouse anti-TH (1:60, Chemicon, but not in human SCG, where a relatively large numb- USA) and mouse anti-LENK (1:600, Biogenesis, UK) er of noradrenergic neurons contained immunoreactiv- antiserum. Double- or triple-labelled sections were in- itytoCGRP0(Baffietal.1992).Furthermore,asre- cubated with goat anti-rabbit biotinylated IgG vealed by decentralization/deafferentation, virtually all (DAKO, Dk) and antigen-antibody complexes visual- of the solely CGRP-IR nerve terminals were of in- ized then by secondary antisera/reagencies coupled to traganglionic origin, while the SP/CGRP- and solely FITC, CY-3 or AMCA (all from Jackson, USA). Pic- SP-IR nerve fibres were processes of spinal primary tures were captured by a digital camera connected to afferent neurons. a PC, analyzed with AnalySIS software (version 3.02, Soft Imaging System, FRG) and printed on a wax Acknowledgments printer (Phaser 8200, Xerox, USA). This study was supported by the UWM grant no. Results and Discussion 05090207. References CGRP-IR but TH-, NPY- and LENK-immunonegative neurons were sporadically observed in Baffi J, Gorcs T, Slowik F, Horvath M, Lekka N, Pasztor E, the porcine SCG (Figs 1, 4, 5). These cells were asso- Palkovits M (1992) Neuropeptides in the human superior ciated with moderate to dense meshwork of cervical ganglion. Brain Res 570: 272-278. Happola O, Lakomy M, Majewski M, Wasowicz K, Yanaihara LENK-IR, varicose nerve terminals (Fig. 4) which dis- N(1993) Distribution of neuropeptides in the porcine appeared after cervical sympathetic trunk (CST) stellate ganglion. Cell Tissue Res 274: 181-187. transsection. SP-IR principal ganglionic neurons were Heym C, Common B, Klimaschewski L, Preissler U, Kummer found in porcine SCG neither in intact, nor in decen- W(1993) Immunohistochemical evidence from co-localiz- tralized/deafferented ganglia (Fig. 3, 5). Similarly, no ation and denervation studies for four types of substance changes in the number and immunofluorescence in- P-containing nervous structures in the rat superior cervical tensity of CGRP-IR neurons were observed after CST ganglion. Anat Embryol 187: 485-492. transsection. Double-immunofluorescence revealed HisaY,KoikeS,UnoT,TadakiN,BambaH,OkamuraH, three populations of CGRP- and/or SP-IR nerve Tanaka M, Ibata Y (1997) Coexistence of calcitonin fibres within the ganglion: SP/CGRP-IR, solely SP- gene-related peptide and NADPH-diaphorase in canine superior cervical ganglion. Neuroscience 228: 135-138. and solely CGRP-IR (Fig. 3, constituting up to 48%, Majewski M, Heym C (1992)Immunohistochemicallocaliz- 45% and 7% of all fibres studied, respectively). While ation of calcitonin gene-related peptide and cotransmitters virtually all SP- and SP/CGRP-IR nerve fibres disap- in a subpopulation of post-ganglionic neurons in the por- peared from the porcine SCG after the decentraliz- cine inferior mesenteric ganglion. Acta Histochem 92: ation/deafferentation, a small population of solely 138-146. CGRP-IR nerve terminals was still present in the gan- Tajti J, Moller S, Uddman R, Bodi I, Edvinsson L (1999)The glionic neuropil. Thus, the results dealing with the oc- human superior cervical ganglion: neuropeptides and currence of CGRP-IR neurons in the porcine SCG peptide receptors. Neurosci Lett 263: 121-124.

← Fig. 1. CGRP-IR (a; arrow) but NPY- (b) and TH-immunonegative (c) neuronal cell body in the control SCG. Triple-immunofluorescence labeling, x 200. Fig. 2. The control SCG. CGRP-IR nerve fibres (a) supplied a subset of NPY- (b) and TH-IR (c) neurons of a medium and small diameter. Triple-immunofluorescence staining, x 200. Fig. 3. Three populations of CGRP- (a) and/or SP-IR (b) nerve fibres were found between TH-IR (c) neurons in the porcine SCG. Triple-immunofluorescence staining, x 400. Fig. 4. Interrelationship between preganglionic LENK-IR nerve fibres (a) and CGRP-IR neuron (b) in the intact porcine SCG. Double-immunofluorescence staining, x 200. Fig. 5. CGRP-IR (a), but SP-immunonegative perikaryon (b) in the decentralized/deafferented porcine SCG. Note a complete lack of SP-IR structures. Double-immunofluorescence staining, x 150. 158 Wojtkiewicz J. et al. Polish Journal of Veterinary Sciences Vol. 7, No. 3 (2004), Supplement, 159-161

Short original article Proliferative enteropathy (PE)-induced changes in the distribution pattern of vesicular acetylcholine transporter-immunoreactive (VAChT-IR) nerve terminals in the porcine inferior mesenteric ganglion (IMG)

J. Wojtkiewicz, S. Gonkowski, A. Bossowska, J. Kaleczyc1,M.Majewski

Abstract

This study was aimed at disclosing density of (both viscerofugal and preganglionic sympathetic) VAChT-IR nerve terminals in the IMG of pigs suffering from PE. In the control, healthy pigs virtually all IMG neurons were supplied by moderate to dense meshwork of VAChT-IR nerves. In PE-affected pigs, both the number and fluorescence intensity of these nerve terminals were distinctly increased. In conclusion, an increase in the number of VAChT-IR nerve fibres observed within the porcine IMG may reflect sprouting of viscerofugal cholinergic nerve terminals or may result from the inhibition of ACh release during PE.

Key words: proliferative enteropathy, noradrenaline, vesicular acetylcholine transporter (VAChT), inferior mesenteric ganglion (IMG), pig

Introduction matory process of the bowel wall it may be of interest to investigate cholinergic nerve terminals in the porcine The present study focused on the cholinergic nerve IMG to verify the assumption that the organization of terminals supplying descending colon-projecting, both the intestino-intestinal reflexes can be deregulated by DβH-IR and DβH-immunonegative neurons in the this pathological process. In the present study, we de- IMG of pigs undergoing poliferative enteropathy (PE, cided to use the antibody against VAChT for the detec- induced by Lawsonia intracellularis infection). Till now, tion of the cholinergic structures within the porcine the search of pre- and/or postganglionic cholinergic IMG, as it is more reliable marker than ChAT for structures in the peripheral nerve system was based on axons/their arborizations originating from the spinal the use of markers of acetylcholine (ACh) synthesis cord (intermediolateral nucleus, the source of sympath- (choline acetyletransferase, ChAT, or vesicular acetyl- etic preganglionic fibres) or from the viscerofugal neur- choline transporter, VAChT). Since PE is known to be ons located in the myenteric and submucosus plexuses associated with a severe, chronic, proliferative inflam- (Brown and Timmermans 2004) of the porcine bowel.

Correspondence to: M. Majewski, tel.: +48 89 523-44-60, fax: +48 89 523-39-82, e-mail: [email protected] 160 Wojtkiewicz J. et al.

Fig. 1-4. IMG in the control (Figs. 1, 3) and PE-suffering pigs (Figs. 2, 4).The images demonstrate the distribution pattern of VAChT-IR (Figs 1-4b) varicose nerve terminals (largely of viscerofugal origin as revealed by axotomy – (not shown)), around FB+,DβH-IR, neurons. Note a lower intensity of DβH immunofluorescence and, simultaneously, a higher intensity of VAChT-immunofluorescence. x 200 (Figs. 1, 2) and x 400 (Figs. 3, 4).

Materials and Methods IMG (A group) were used. All pigs were subjected to median laparotomy and injected with 5% aqueous sol- Nine immature female pigs (approximately 8 weeks ution of Fast Blue (FB) into the wall of the descend- old), three control (C group), three with clinically di- ing colon. After a there-week survival period, the ani- agnosed Lawsonia intracellularis infection (PE) and mals of C and PE groups were sacrificed with an over- three subjected to bilateral transsection of the caudal dose of sodium pentobarbital (Vetbual, Biowet, colonic nerves, the sole pathway for all viscerofugal Polad: 90 mg/kg b.w.) and transcardially perfused with terminals projecting from the enteric ganglia to the freshly prepared 4% paraformaldehyde in 0.1 M phos- Proliferative enteropathy (PE)-induced ... 161 phate buffer (pH 7.4). Pigs of A group were nerve terminals containing this substance (Figs 1-4). axotomized one week prior to the perfusion. Ten µm FB-, VAChT-IR neuronal somata, sporadically ob- thick cryostat sections of the IMG were processed for served in control and PE-affected ganglia were also a routine doublelabelling immunofluorescence. Brie- supplied by VAChT-IR nerve terminals. In contrast to fly, sections were incubated in the humid chamber the unchanged number and properties of FB+ norad- with a mixture of two primary antibodies raised in renergic IMG neurons, an increase in the number of different species (overnight, room temperature), in- visible VAChT-IR fibres was found in PE-affected cluding antisera to DβH (1:350; mouse monoclonal, IMG (compare Figs. 1, 3 and 2, 4). Moreover, the Chemicon, USA) or TH (1:80; mouse monoclonal, intensity of immunofluorescence of these terminals Chemicon, USA) and VAChT (1:2400; rabbit poly- was stronger as compared to that observed in the con- clonal, Phoenix, USA). For visualization of complexes trol ganglia (sections from both control and affected of the antigen/primary antibody, a mixture of directly ganglia were stained with the same aliquot of antisera labeled secondary antibodies either conjugated to mixture, as well as were observed and photographed FITC or CY3 (AffiniPure FITC-conjugated donkey with the same camera settings). As revealed by the anti-mouse Fab’ fragment and AffiniPure CY3-con- transsection of caudal colonic nerves (group A), the jugated donkey anti-rabbit Fab’ fragment; both vast majority of VAChT-IR terminals within the por- from Jackson Immunochemicals, USA; used in work- cine IMG originate from enteric ganglia. Thus, results ing dilution 1:1000) were used. Retrogradely labeled/ of the present study suggest that an increase in the /double-immunostained perikarya were then evalu- number of VAChT-IR nerve fibres observed within ated under Olympus BX51 microscope equipped with the porcine IMG reflects sprouting of viscerofugal epi-fluorescence and appropriate filter sets. In order cholinergic nerve terminals or may result from the to estimate the relative number of FB+ cells apposed inhibition of ACh release during Lawsonia intracel- by VAChT-IR terminals, retrogradely labeled neur- lularis infection. ons were counted in each fifth section (only neurons with clearly visible nucleus were included) and presented as mean ± SEM. Pictures were captured by a digital camera connected to a PC, analyzed with Acknowledgments AnalySIS software (version 3.02, Soft Imaging System, FRG) and printed on a wax printer (Phaser 8200, Supported by KBN, grant no. 6P06K 012 20. The Xerox, USA). authors are indebted to Ms. A Piękos and Mr. Z. Liminowicz for their excellent technical help.

Results and Discussion References In the control ganglia, nearly all (90.5 ± 1.8%) retrogradely labeled neurons expressed co-localized β Brown DR, Timmermans JP (2004) Lessons from the por- TH (not shown) and D H (Figs 1, 3). However, IMGs cine enteric nervous system. Neurogastroenterol Motil of animals suffering from PE showed a slight decrease 16: 50-54. in the number of noradrenergic perikarya (80%). In Timmermans JP, Hens J, Adriaensen D (2001) Outer sub- both, control and PE-affected animals, virtually all mucosus plexus: an intrinsic nerve nervork involved in FB+ neurons were VAChT-negative, however, they both secretory and motility processes in the intestine of were surrounded by moderate to dense meshwork of large mammals and humans. 71. Anat Rec 262: 71-78. 162 Wojtkiewicz J. et al. Polish Journal of Veterinary Sciences Vol. 7, No. 3 (2004), Supplement, 163-165

Short original article Potential pathogens in intensive rearing of Wels catfish (Silurus glanis L.)

I. Zmysłowska, J. Guziur1, J. Szarek2, J. Krause

Chair of Environmental Microbiology, University of Warmia and Mazury in Olsztyn, Prawocheńskiego St. 1, 10-957 Olsztyn, Poland 1 Chair of Biology of Fish and Pisciculture, University of Warmia and Mazury in Olsztyn, Poland 2 Department of Forensic and Administration of Veterinary Medicine, University of Warmia and Mazury in Olsztyn, Poland

Abstract

Seasonal occurrence of bacteria Enterobacteriaceae, Aeromonas hydrophila, Pseudomonas fluorescens, Staphylococcus spp. and fungi in post-cooling water, skin mucus and the alimentary tract contents in Wels catfish (Silurus glanis L.) of intensive fish cage breeding was determined. The microorganisms were found in the smallest amounts in mucus, whereas their largest amounts were found in the fish alimentary tract contents. The bacteria Enterobacteriaceae and fungi were the dominant microorganisms in water and alimentary tract contents, whereas in mucus fungi dominated. The microorganisms in question were usually the most numerous in summer and the least numerous in spring.

Key words: microorganism, post-cooling water, intensive fish rearing

Introduction alimentary tract contents of Wels catfish (Silurus glanis L.) during intensive fish cage rearing in Fish are exposed to various microorganisms which post-cooling waters. are either saprophytic microflora of aquatic environment or get there from soil, waste or from air. Some of the microoragnisms are pathogenic to fish, others Materials and Methods are conditionally pathogenic, yet other are non-pathogenic. However, not always they can be The measurements were done with samples of classified as strictly saprophytic and pathogenic to fish water and fish (Silurus glanis L.) taken in the Centre (Kozińska et al. 2002). Quick development of piscicul- of Fish Rearing in Ostrołęka where intensive rearing ture associated to intensive fish rearing has been was carried out in fish cages placed in a canal, to noted for some recent years. In such conditions fish which post-cooling water from a heat and power sta- are much more vulnerable to diseases and the symp- tion was drained off. toms are much more intense than in fish living in the Samples of water and fish were taken in three per- wild. The aim of the study was to determine potential iods of 2003 – in spring (01 June), in summer (24 July) pathogens present in water, in skin mucus and in the and in autumn (23 September). Samples of water

Correspondence to: I. Zmysłowska, tel.: +48-89 523-45-32, e-mail: [email protected] 164 Zmysłowska I. et al. were taken at two depths of the fish cage (0.3 m and The obtained, characteristic colonies on selective 1.3 m), and the samples of fish, three individuals each media were subjected to confirmation testing, ident- time, were taken from the same place. Bacteria from ifying them with a microscope (shape and position of the family Enterobacteriaceae, Aeromonas hydrophila, cells, movements, Gram staining), enzymatic and bio- Pseudomonas fluorescens, Staphylococcus spp. and chemical tests (Api 20 NE, bioMe`rieux). fungi were determined quantitatively and qualitatively in all the samples of water and skin mucus, as well as Results in the fish alimentary tract contents. Quantitative determinations were done by a culture method, on selec- The smallest numbers of potential pathogens in tive culture media (Table 1), in three parallel replica- question were found in summer, and the highest tions. The results were converted into colony forming – usually in spring. The skin mucus was contaminated units (cfu) in 1 cm3 of water, in 1 cm2 of skin mucus with the microorganisms to the lowest extent, whereas and in 1 g of fish alimentary tract contents. Physiologi- the highest numbers of microorganisms were found in cal saline was used for diluting the samples (0.85% the fish alimentary tract contents. Fungi dominated in NaCl). all environments and the bacteria Enterobacteriaceae – in water and in the fish alimentary tract contents (Table 2). The highest average numbers of microorganisms from the whole study period were found in the ali- Table 1. Rearing conditions of microorganisms determined mentary tract contents for the bacteria Enterobac- in water and in fish. teriaceae (1300 cfu/g) and for fungi (1420 cfu/g). These microorganisms also dominated in water – 380 Incubation Microorganisms Medium cfu/cm3 and 230 cfu/cm3, respectively. These bacteria time were not found in the mucus and fungi were found in o Enterobacteriaceae Endo 37 C/24h the amount of 270 cfu/cm2. The highest average Aeromonas hydrophila mA 37oC/24h o number of bacteria Aeromonas hydrophila was found Pseudomonas fluorescens Kinga B 26 C/48h 3 o in water (40 cfu/cm ), Pseudomonas fluorescens in mu- Staphylococcus spp. Chapmana 37 C/48h 2 Fungi Saboraunda 25oC/5 days cus (110 cfu/cm ), and Staphylococcus spp. in the fish alimentary tract contents (190 cfu/g) (Fig. 1).

Table 2. Means and the ranges of microorganism numbers in post-cooling waters (cfu/cm3), skin mucus (cfu/cm2) and alimentary tract contents (cfu/g) of Wels catfish (Silurus glanis L.) during intensive fish cage breeding.

Aeromonas Pseudomonas Staphylococcus Enterobacteriaceae hyrophila fluorescens spp. Fungi

W 740 180 10 50 300 210-1270 110-240 9-13 50-50 20-580 Water L 390 50 4 4 100 360-420 40-60 2-5 3-5 90-110 J10 70 10 30 180 10-10 40-100 10-10 25-35 250-310

W 0 0 680 5 500 380-750 2-9 230-630 Mucus L 30 150 0 00 10-40 130-170 J0 405 100 20-50 1-8 0 80-160

W 1990 140 200 500 0 Alimentary 1200-2880 80-190 160-240 420-610 contents L 230 130 80 130 50 120-270 90-170 70-110 90-150 30-80 J 1680 450 3710 00 1150-1950 380-510 1900-4100

Explanation: W – spring, L – summer, J – autumn Potential pathogens ... 165

10000 cate organic contamination of the allochtonic type. Bacteria Aeromonas hydrophila, Pseudomonas fluorescens and Staphylococcus spp. were also found. 1000 These results confirm and supplement the research conducted by the authors who dealt with these issues (Zmysłowska 2000a, b, c, Lewandowska et al. 2001). 100 Aquatic bacteria are highly changeable. In certain cfu conditions saprophytic bacteria can become virulent and dangerous to fish. It is related to the factors, 10 which make fish vulnerable to infections caused by relatively pathogenic, or even saprophytic bacteria; it is especially true about fish of reduced immunity. 1 water mucus alimentary tract References Enterobacteriaceae Staphylococcus ssp. Aeromonas hydrophila Fungi Esteve C, Garray E (1991) Heterotrophic bacterial flora as- Pseudomonas fluorescens sociated with Europen eel Angilla angilla reared in fresh water. Nippan Suisan Gakkaiski 57: 1369-1375. Fig. 1. Mean numbers of potentially pathogenic microorgan- Kozińska A, Guz L, Pękala A (2002) Diagnostyka wyb- 3 isms in post-cooling water p (cfu/cm ), skin surface mucus ranych patogenów bakteryjnych w ichtiopatologii. 2 (cfu/cm ) and alimentary tract contents (cfu/g) in Wels cat- Państwowy Instytut Weterynaryjny, Puławy. fish (Silurus glanis L.) during intensive fish cage breeding. Lewandowska D, Zmysłowska I, Gołaś I (2001) Comparison of the microflora present in the aquatic environment, fish, and fish feeds. Fol Univ Agric Stetin 218 Piscaria Discussion 28: 77-88. Zmysłowska I, Lewandowska D, Guziur J (2000a) Microbi- Data from literature suggest that because of a large ological study of ide (Leuciscus idus L.) from ponds of amount of organic matter which gets to water during different trophy. Arch Pol Fish 8: 259-269. the intensive fish rearing an increased numbers of the Zmysłowska I, Lewandowska D, Pimpicka E (2000b) Micro- microorganisms are found. They are mainly Aero- biological evaluation of water and digestive tract contents monas, Pseudomonas, Plesiomonas, Acinetobacter and of tench (Tinca tinca L.) during tank rearing. Arch Pol Fish 8: 95-105. the family Enterobacteriaceae (Esteve 1991, Lewan- Zmysłowska I, Lewandowska D, Pimpicka E (2000c) Micro- dowska et al. 2001, Zmysłowska et al. 2001). Contami- biological studies of tench (Tinca tinca L) and water of nation of the aquatic environment is reflected in the Dgał Wielki Lake. Arch Pol Fish 8: 107-117. microbiological condition of fish. The results of own Zmysłowska I, Harnisz M, Lewandowska D (2001) Sanitary research have shown that bacteria Enterobacteriaceae and bacteriological evaluation of water quality during and fungi dominated in the post-cooling water and in cage culture of wels (Silurus glanis L.) in cooling water. the fish alimentary tract contents. Both groups indi- Arch Pol Fish 9: 85-93. 166 Zmysłowska I. et al. Polish Journal of Veterinary Sciences Vol. 7, No. 3 (2004), Supplement, 167-169

Short original article Antibiotic-resistant bacteria isolated from fish and from water during intensive fattening

I. Zmysłowska, M. Harnisz, J. Guziur1, J. Szarek2

Chair of Environmental Microbiology, University of Warmia and Mazury in Olsztyn, Prawocheńskiego St. 1, 10-957 Olsztyn, Poland 1 Chair of Fish Biology and Pisciculture, University of Warmia and Mazury in Olsztyn, Poland 2 Department of Forensic and Administration of Veterinary Medicine, University of Warmia and Mazury in Olsztyn, Poland

Abstract

The resistance of bacteria Aeromonas hydrophila, Pseudomonas fluorescens and Enterobacteriaceae isolated from water, the alimentary tract contents and skin mucus of catfish (Silurus glanis L.) to flumequine, oxytetracycline, novobiocin, trimethoprim, enrofloxacin, canamycin, oxolinic acid and neomycin was examined. The bacteria were the most resistant to novobiocin and the least resistant to canamycin. No difference was found in the resistance to medicine among the strains from various environments.

Key words: antibiotic-resistant bacteria, catfish (Silurus glanis L.), intensive fattening of fish

Introduction Materials and Methods

Progressive intensification of fish production and The samples of water, alimentary tract contents an increase in the number of diseases caused by bac- and skin mucus of catfish (Silurus glanis L.), which teria in fish are accompanied by the problem of the were taken in February, March and April 2004 in the increasing resistance of bacteria to antibiotics, both in Fish Breeding Centre in Ostrołęka were the material pathogens and in saprophytes. The issue is particular- used in the experiment. In the course of the study the ly important because of the transfer of the genes of bacteria Aeromonas hydrophila, Pseudomonas fluor- resistance, both among pathogens and from sap- escens and those of the family Enterobacteriaceae were rophytes to pathogens. isolated. They were growing in the form of character- The aim of the study was to determine the sensitiv- istic colonies on selective media (mA, King B and ity of bacteria Aeromonas hydrophila, Pseudomonas Endo, respectively) and their sensitivity to antibiotics fluorescens and those of the family Enterobacteriaceae was determined. The taxonomic identity was con- isolated from water, alimentary tract contents and firmed microscopically and biochemically. skin mucus of catfish (Silurus glanis L.) to antibiotics The sensitivity of the isolates to antibiotics was de- commonly used in fish diseases in Poland. termined by the method of diffusion disks on Muel-

Correspondence to: I. Zmysłowska, tel.: +48 89 523-45-32, e-mail: [email protected] 168 Zmysłowska I. et al. ler-Hinton broth. The disks with the following anti- 100 biotic media were used in the experiment: flumequine 90 water (UB, 30 µg), oxytetracycline (OT, 30 µg), novobiocin 80 alimentary tract 70 of catfish (NV, 5 µg), thrimetoprim (W, 5 µg), enrofloxacin skin mucus of µ µ 60 catfish (ENR, 5 g), canamycin (K, 30 g), oxolinic acid (OA, 50 30 µg), neomycin (N, 30 µg). 40 30 20

% of resistant10 isolates Results 0 UB OT NV W ENR K OA N During the whole experiment 114 strains of bac- antibiotic teria were isolated. More than a half (60.53%) came Fig. 1. Frequency of resistance to antibiotics (UB from the aquatic environment and the others were – flumequine, OT – oxytetracycline, NV – novobiocin, isolated from the fish alimentary tract contents and W – trimetoprim, ENR – enrofloxacin, K – canamycin, OA from skin mucus. Aeromonas hydrophila accounted – oxolinic, N – neomycin) for bacterial strains. for the majority of the isolates (Table 1).

Table 1. Number of isolates resistant to particular antibiotics.

Number UB OT NV W ENR K OA N of isolates

water 1 3 22 8 1 1 2 4 22 Aeromonas hydrophila alimentary tract* 3 5 11 5 1 0 4 4 11 skin mucus* 5 2950340 10

water 2 3 27 26 1 3 16 4 29 Pseudomonas fluorescens alimentary tract 0 2330010 3 skin mucus 2 1662161 6 water 3 0 17 10 2 2 2 2 18 Enterobacteriaceae alimentary tract 1 3440001 7 skin mucus 3 2551152 8

Explanation: UB – flumequine, OT – oxytetracycline, NV – novobiocin, W – trimetoprim, ENR – enrofloxacin, K – canamycin, OA – oxolinic, N – neomycin, * – catfish (Silurus glanis L.)

Table 2. Number of antibiotics to which strain resistance was found.

Number of antibiotics to which Source strain resistance was found water alimentary tract of catfish skin mucus of catfish 01–– 11422 22593 31868 4923 5227 6––– 7––– 8––– Number of isolates 69 21 24 % resistant isolates 98.55% 100% 100% Antibiotic-resistant bacteria ... 169

Novobiocin was the antibiotic the resistance to range action, a relative safety of use and low cost of which was found to occur the most frequently. The production and sales. The resistance to oxytetracyc- resistance to trimethoprim was also frequent. line was as high as 52.36% among the strains isolated Canamycin and enrofloxacin were the antibiotics to from the alimentary tract contents. The absence of which the absence of sensitivity was found the least sensitivity to this antibiotic was most often found in frequently (Fig. 1). No difference was found in the the rods Aeromonas hydrophila. These bacteria cause resistance to the medicine among the strains from motile aeromonas septicemia, although those strains various environments. isolated from water, alimentary tract contents and Among 114 strains there was only one, isolated from mucus are not necessarily virulent (McPhearson from water, which was sensitive to all the antibiotics et al. 1991). Magee and Quinn (1991) found the resis- used. Usually a resistance to one, two or three anti- tance to oxytetracycline in the bacteria isolated from biotics was found. No strain was found to be resistant the environments where no antibiotics had been used, to more than five antibiotics (Table 2). and Miranda and Zamelman (2001) – in bacteria isolated from the fish living in the wild. This indicates a possibility of transfer of the genes of resistance to Discussion this antibiotic.

The study revealed that the resistance to novobiocin and to trimethoprim existed in more iso- References lates in comparison with other antibiotics (85.9% Magee AM, Quinn JP (1991) Antibiotic bacteria in the re- – 95.7% and 57.12% – 70.08% of isolates, respective- mote upland river catchment. Lett Appl Microbiol 13: ly). The mechanism of resistance to these two anti- 145-149. biotics and to oxytetracycline may be the result of ac- Markiewicz Z, Kwiatkowski ZA (2001) Bakterie, an- tive removal of antibiotics with a protein acting as tybiotyki, lekooporność. Wydawnictwo Naukowe PWN, a pump (Markiewicz and Kwiatkowski 2001). A high Warszawa. frequency of resistance to antibiotics in the bacteria McPhearson RM, DePaola A, Zywno SR, Motes ML Jr., isolated from fish during intensive fattening indicates Guarino AM (1991) Antibiotic resistance in Gram-nega- a role which may be played by fish breeding farms as tive bacteria from cultured catfish and aquaculture ponds. Aquaculture 99: 203-211. reservoirs of genes of resistance, which may be trans- Miranda CD, Zamelman R (2001) Antibiotic resistant bac- ferred to other animals and to humans. teria in fish from the Concepcion Bay, Chile. Marine Pol- Like in the studies conducted by other authors lution Bulletin 11: 1096-1102. (McPhearson et al. 1991, Miranda and Zamelman Miranda CD, Zamelman R (2002) Antimicrobial multiresis- 2001) the bacterial strains were the most sensitive to tance in bacteria isolated from freshwater Chilean canamycin and enrofloxacin. salmon farms. Sci Total Environ 293(1-3): 207-218. Oxytetracycline is the most frequently used anti- Miranda CD, Zamelman R (2002) Bacterial resistance to biotic in fish diseases in Poland. It is caused by its vast oxytetracycline in Chilean salmon farming. Aquaculture 212: 31-47. 170 Zmysłowska I. et al. Polish Journal of Veterinary Sciences Vol. 7, No. 3 (2004), Supplement, 171-172

Short original article Telomerase activity in squamous cell carcinoma in dogs

M. Żmudzka, E. Malicka, M. Sobczak-Filipiak, R. Lechowski

Department of Clinical Sciences, Faculty of Veterinary Medicine, Warsaw Agricultural University, Nowoursynowska St. 159, 02-776 Warsaw, Poland

Abstract

The eight cases of sqamous cell carcinoma in dog were investigated for telomerase activity. The main value was 398.8 ± 98.9 U/µg protein. This is the first report about reference value of telomerase activity in canine squamous cell carcinoma.

Key words: telomerase, skin neoplasia, sqamous cell carcinoma

Introduction diagnoses of SCC were based on histopathological examination of haematoxylin and eosin stained sec- Telomeres are the regions of linear chromosomes tions. The samples for telomerase activity determina- essential for their stability. In the majority of normal tion were frozen in liquid nitrogen. When several somatic cells telomeres tend to successive cell divi- samples were collected they were homogenized and sion. Telomerase is a ribonucleoprotein enzyme which examined of protein concentration. Telomerase activ- maintains the lengths of telomeres. It is present in ity was measured by the telomeric amplification pro- fetal tissues, and shortly after birth only in immortal tocol (TRAP) using TAGGG Telomerase PCR and germ line cells (Biller et al. 1998, Argyla et al. ELISA plus from Roche. As a control five samples of 2003). Telomerase activity can also be found in most regular skin from dogs were used. malignant tumours in human and other mammals (Biller et al. 1998, Argyla et al. 2003). Information about values of telomerase activity in variety canine Results skin neoplasm in literature is very fragmentary. The aim of the study was to investigate telomerase activity The results of telomerase activity in SCC are in neoplastic tissue in dogs with squamous cell car- shown in the Table 1. The activity of telomerase in cinoma (SCC). samples of regular skin was less than 1 U/µg protein in all cases.

Materials and Methods Discussion The samples of skin tumours were obtained from dogs that were surgically treated. Immediately after From 1994 when Kim et al. (1994) announced re- surgical resection neoplastic tissues were divided in sults of their work about the method of measurement two parts: one for histopathological examination and of telomerase activity in tissue samples (TRAP assay) other for telomerase activity determination. The many scientists have undertaken researches on this

Correspondence to: R. Lechowski, tel.: +48 22 843-90-41, e-mail: [email protected] 172 Żmudzka M. et al.

Table 1. Telomerase activity in neoplastic tissue of SCC. samples. It is difficult to compare this data with the results concerning skin cancer in dogs obtained by Telomerase activity other authors because they are very few. In all Dog µ (U/ g protein) samples of regular skin telomerase activity did not ex- 8 years old, female, mix 560 ceed 1 U/µg protein. Our results show that telomerase 11 years old, female, mix 354 activity may be useful indicator of malignancy in ca- 9 years old, female, mix 527 nine skin tumours. Its utility in differential diagnosis 6 years old, female, mix 343 of sqamous cell carcinoma needs further investiga- 8 years old, female, mix 278 tions. 10 years old, male, mix 339 7 years old, male, mix 379 References 7 years old, male, mix 410

Mean ± SD 398.8 ± 98.9 Argyle DJ, Nasir L (2003) Telomerase: A Potential Diagnos- tic and Therapeutic Tool in Canine Oncology. Vet Pathol 40: 1-7. Biller BJ, Kitchell BE (1998) Evaluation of an assay for de- tecting telomerase activity in neoplastic tissues of dogs. topic. Many studies on telomerase activity in various A.J.V.R. 59: 1526-1529. human tissues have been published. Taylor at al. Kim NW, Piatyszek MA, Prowse KR, Harley CB, West MD, (1996) analysed telomerase activity in various types of Ho PL, Coviello GM, Wright WE, Weinrich SL, Shay JW skin diseases. They found it in 15 of 18 nonmetastatic (1994) Specific association of human telomerase activity cutaneous sqamous cell carcinoma (SCC). Ueda with immortal cells and cancer. Science 266: 2011-2015. (2000) showed telomerase activity in 6 of 6 SCC. In Parris CN, Jezzard S, Silver A, MacKie R, McGregor JM, contrary Paris et al. (1999) reported telomerase activ- Newbold RF (1999) Telomerase activity in melanoma ity only in 3 of 12 SCC. In conclusion, this studies and non-melanoma skin cancer. Br J Cancer 79: 47-53. show that although discrepancies exist, telomerase is Taylor RS, Ramirez RD, Ogoshi M, Chaffins M, Piatyszek frequently activated in sqamous cell carcinoma in hu- MA, Shay JW (1996) Detection of telomerase activity in malignant and nonmalignant skin conditions. J Invest man. Similar results of the researches of Ueda (2000) Dermatol 106: 759-65. in human were obtained in our investigations in dogs Ueda M (2000) Telomerase in cutaneous carcinogenesis. where telomerase activity was found in all SCC J Dermatol Sci 23 (Suppl) 1: 37-40. Polish Journal of Veterinary Sciences Vol. 7, No. 3 (2004), Supplement

Authors index

Andrzejewska A. 135, 139, 147 Lipińska J. 69, 135, 139, 143, 147 Apoznański J. 9 Lorek M.O. 143 Aupperle H. 5 Łakomy M. 115 Babińska I. 69, 135, 139, 143, 147 Majewski M. 13, 17, 21, 37, 45, 49, 53, 73, 77, 97, Bakuła T. 9 155, 159 Bigoszewski M. 85 Malicka E. 171 Bossowska A. 13, 17, 21, 45, 49, 53, 73, 77, 155, 159 Małaczewska J. 81 Bownik A. 25 Mikulska-Skupień E. 85 Morand M. 123 Całka J. 13, 29, 41, 45, 155 Obrembski K. 9 Cendrowska I. 131 Osińska B. 65, 89 Chorostowska-Wynimko J. 101, 131 Pavlidou E. 93 Chrószcz A. 57 Pidsudko Z. 17, 21, 73, 97 Chrystowska D. 131 Podlasz P. 41 Czerski A. 57 Prevost G. 25 Dang X. 33 Procajło Z. 85 Dhein S. 5 Rogala E. 101 Dziedzic-Gocławska A. 131 Rotkiewicz Z. 81 Dzienis A. 37 Rymuszka A. 105 Fabczak J. 69 Rzewuska M. 151 Felsmann M.Z. 139, 143 Sadło J. 131 Filewska M. 101 Saurek D. 109 Franke-Radowiecka A. 41 Schneider K. 5 Gajęcki M. 9 Schoon H.A. 5 Garbade J. 5 Sender-Janeczek A. 57 Gonkowski S. 13, 17, 21, 45, 49, 53, 73, 77, 155, 159 Sienkiewicz W. 49, 53, 77, 115 Gugołek A. 143 Sierosławska A. 119 Guziur J. 163, 167 Siwicki A.K. 25, 81, 101, 105, 119, 123, 127, 131, 147 Harnisz M. 167 Skobowiat C. 13 Henklewski R. 57 Skopińska-Różewska E. 101, 131 Huang Z. 33 Skopiński P. 131 Jakubowski K. 61 Sobczak-Filipiak M. 151, 171 Jana B. 37 Sommer E. 101, 131 Janeczek M. 57 Steiger K. 5 Janeczek W. 57 Szarek J. 69, 127, 135, 139, 143, 147, 163, 167 Jakubowski J. 135 Szweda W. 85 Jedlińska-Krakowska M. 61 Terech-Majewska E. 127, 147 Józefowicz A. 17, 21 Tian D. 33 Kaba J. 151 Tian S. 33 Kaleczyc J. 13, 17, 21, 29, 41, 45, 49, 53, 73, 77, 115, Trapkowska S. 123, 127 155, 159 Truszczyńska M. 143, 147 Kamiński A. 131 Wąsowicz K. 29, 41 Katkiewicz M. 65, 89, 109 Wiese I. 9 Kazuń B. 123, 127 Witkowski L. 151 Kita J. 151 Wojtasik E. 101 Koncicki A. 135 Wojtkiewicz J. 13, 17, 37, 45, 49, 53, 73, 77, 155, 159 Kowalski A. 61 Wolf G. 29 Kowalski I.M. 69, 139 Zduńczyk Z. 135 Krause J. 163 Zmysłowska I. 163, 167 Lechowski R. 171 Żmudzka M. 171