Cristiano Boiti · Adriana Ferlazzo Alberto Gaiti · Antonio Pugliese Editors Trends in Veterinary Sciences Current Aspects in Veterinary Morphophysiology, Biochemistry, Animal Production, Food Hygiene and Clinical Sciences Trends in Veterinary Sciences Cristiano Boiti • Adriana Ferlazzo Alberto Gaiti • Antonio Pugliese Editors

Trends in Veterinary Sciences

Current Aspects in Veterinary Morphophysiology, Biochemistry, Animal Production, Food Hygiene and Clinical Sciences

LXV Annual Meeting of The Italian Society for Veterinary Sciences. Tropea-Drapia 2011. Selected Papers

123 Editors Cristiano Boiti Alberto Gaiti Dipartimento di Scienze Biopatologiche Dipartimento di Patologia, Diagnostica Università degli Studi di Perugia Università degli Studi di Perugia Perugia Perugia Italy Italy

Adriana Ferlazzo Antonio Pugliese Dipartimento Scienze veterinarie Dipartimento Scienze veterinarie University of Messina Università degli Studi di Messina Messina Messina Italy Italy

ISBN 978-3-642-36487-7 ISBN 978-3-642-36488-4 (eBook) DOI 10.1007/978-3-642-36488-4 Springer Heidelberg New York Dordrecht London

Library of Congress Control Number: 2013937093

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Part I Biology and Reproduction

1 Seasonal Effect on Hematological and Innate Immune Parameters in Sea Bass (Dicentrarchus labrax) ...... 3 Francesco Pascoli, E. Negrato, C. Poltronieri, G. Radaelli and D. Bertotto

2 Effect of Altitude on Plasma Serotonin Levels in Horses ...... 9 G. Bruschetta, P. Di Pietro, M. Miano, C. Cravana and A. M. Ferlazzo

3 Identification of Aquaporin 1 in Diplodus sargus ...... 15 G. Zanghì, S. Campo, A. D’Ascola, A. Germanà and A. M. Ferlazzo

4 Effect of Dephosphorylation on Donkey Milk Caseins ...... 21 S. Vincenzetti, A. Vita, F. M. Carpi, D. Micozzi and P. Polidori

5 Distribution Pattern and Chemical Coding of Sympathetic Trunk Ganglia Neurons Supplying the Boar Urinary Bladder Trigone ...... 27 F. Gazza, M. Botti, L. Ragionieri, C. Sorteni, D. Russo, P. Clavenzani, R. Chiocchetti, L. Bo Minelli and R. Panu

6 In Vivo Applications of Mesenchymal Stem Cells and Platelet-Rich Plasma to Improve Tendon Regeneration in Sheep ...... 31 M. Patruno, I. Bronzini, L. Maccatrozzo, A. Perazzi, I. Iacopetti, G. M. De Benedictis, S. Testoni, A. Negro, F. Mascarello and T. Martinello

v vi Contents

7 Plasma Fatty Acid Profiles During the First Year in Dogs with and without Hip Dysplasia: Preliminary Results ...... 35 L. Tidu, N. Bacciu, G. Rucco, S. Nardi, M. Santoro and B. Renaville

8 Signaling in Sperm Activation: A Common Strategy for Different Organisms ...... 41 I. Saponaro, N. Bernabò and M. Mattioli

9 Tenogenic Differentiation of Ovine Amniotic Stem Cells Co-Cultured with Tenocytes ...... 45 Valentina Curini, V. Russo, O. Di Giacinto, A. Mauro, E. Galiffa, A. Pomante and B. Barboni

10 Cortisol Changes in Pregnant and Post-Partum Ewes: Effects of Single or Twin Births ...... 51 E. Fazio, M. Manera, S. Mignacca, P. Medica and A. Ferlazzo

Part II Animal Pathology

11 Papillary and Chordoid Meningioma in the Dog: Morphological Findings and Histological Grading ...... 57 S. Pavone and M. T. Mandara

12 Detection of Neutralizing Antibodies in Pigs Inoculated with an Inactivated Vaccine Against Porcine Circovirus Type 2 (PCV2) ...... 63 S. Petrini, M. Paniccià, V. Silenzi, F. Ciuti, M. Bresaola, M. Fortunati, G. M. De Mia, G. Perugini and M. Ferrari

13 Mycobacterium avium subsp. paratuberculosis as an Emergent Pathogen in Raw Ovine Milk Produced in Central Italy ...... 67 A. R. Attili, V. Ngu Ngwa, L. Pacifici, S. Preziuso, A. Domesi and V. Cuteri

14 Canine Filariosis in Sardinia: Epidemiological Findings in the Ogliastra Region...... 73 A. Scala, C. Solinas, A. P. Pipia, G. Sanna, A. Varcasia and G. Tosciri Contents vii

15 Comparison of Serum and Meat Juice for Detection of Anti-Toxoplasma gondii Antibodies in Hunted Wild Boars (Sus scrofa) ...... 79 D. Ranucci, F. Veronesi, I. Di Matteo, R. Branciari, D. Miraglia, C. Marini and D. Piergili Fioretti

16 Eucoleus aerophilus (syn. Capillaria aerophila) and Other Trichinelloid Nematodes in Dogs from Liguria (Northwest Italy) ...... 85 F. Macchioni, L. Guardone, M. C. Prati and M. Magi

17 Helminths in Sheep on Farms of the Basilicata Region of Southern Italy ...... 91 A. Bosco, L. Rinaldi, V. Musella, D. Pintus, M. Santaniello, M. E. Morgoglione, G. Zacometti and G. Cringoli

Part III Pharmacology and Clinical Science

18 Effects of Veterinary Drugs on Swimming Activity in Two Freshwater Organisms ...... 97 M. Dalla Bona, V. Di Leva and M. De Liguoro

19 Interdisciplinary Evaluation of Toxicity in Ostreopsis Ovata: Algal Biotoxins ...... 103 A. Ferrari, I. Schiavetti, C. Bolognesi, D. Pavino and B. Vivaldi

20 Aflatoxin M1 Contamination and Antibacterial Residues in Milk in Kosovo...... 109 G. Gallina, A. Rama, L. Lucatello, C. Benetti, D. Bajraktari, K. Uka and C. Montesissa

21 Heavy Metal Levels in Dog Liver and Kidney in Naples (Campania, Italy) ...... 115 F. P. Serpe, R. Russo, R. De Luna, S. Florio, M. Esposito and L. Severino

22 Ultrasonographic Assessment of Abdominal Lymph Nodes in Normal Puppies: Preliminary Results ...... 119 A. La Pietra and M. De Majo viii Contents

23 Changes in the Metabolic Profile and Performance of Dairy Cows Fed Two Dietary Crude Protein Concentrations ...... 125 D. Bernardini, S. Segato, G. Marchesini, A. L. Stefani, G. Gerardi and I. Andrighetto

24 Impact of Physical Exercise on Release of Cardiac Troponins: Evaluation in Healthy and Cardiopathic Dogs ...... 129 M. Pugliese, A. Seminara, M. De Majo, A. La Pietra and P. P. Niutta

25 Canine Erythrocyte Morphology: Observations of a New Pattern, the ‘‘Quatrefoil’’ Erythrocyte ...... 135 George Lubas, Alessandra Gavazza, Biancaurora Gugliucci, Anna Pasquini and Marianna Ricci

26 Pain Management in Companion Animals: Medical–Legal Aspects ...... 141 V. Quartarone, A. Fazio, G. della Rocca, M. Russo and A. Passantino

Part IV Food Inspection

27 Increase of TVBN and TMA-N in Skin and Gills of Sparus aurata During Storage ...... 149 A. Giuffrida, F. Giarratana, D. Signorino, G. Ziino and A. Panebianco

28 Actin Proteolysis in San Daniele Dry-Cured Ham ...... 153 M. L. Stecchini, A. Fabbro, M. Spaziani, E. Venir and G. Lippe

Part V Husbandry and Zootechnic

29 The Donkey Milk Food Chain: Quality and Certification ...... 159 Stefano Simonella, Cristina Panetta and Biagina Chiofalo

30 Effect of Different Rates of Postmortem pH Decline on the Technological Quality of Calabrian Capocollo...... 165 L. Nanni Costa, F. Tassone, S. Dall’Olio, S. Carpino and V. Russo

31 Preliminary Investigation of the Incidence of Obesity in a Canine Population in the USA ...... 171 G. Biagi, I. Cipollini, M. Grandi, D. Sarti and G. Zaghini Contents ix

32 Administration of Essential Oils Cinnamaldehyde, Eugenol, and Capsicum to Beef Cattle: Effects on Health Status and Growth Performance ...... 177 R. Compiani, C. A. Sgoifo Rossi, A. Pizzi and V. Dell’Orto

33 Extruded Linseed in the Diet of Grazing Goats: Effects on Milk Conjugated Linoleic Acid ...... 181 Raffaella Tudisco, S. Calabrò, M. I. Cutrignelli, M. Grossi, N. Musco, V. Piccolo and F. Infascelli

Index ...... 187 Part I Biology and Reproduction Chapter 1 Seasonal Effect on Hematological and Innate Immune Parameters in Sea Bass (Dicentrarchus labrax)

Francesco Pascoli, E. Negrato, C. Poltronieri, G. Radaelli and D. Bertotto

Abstract The temperate aquatic environment is affected by two primary seasonal components, temperature and photoperiod. Many organisms respond to seasonal change physiologically, behaviorally or both. The aim of this study was to investigate the effect of seasonality on cortisol, hematological, and innate immune parameters in European sea bass (Dicentrarchus labrax) reared under traditional semi-intensive aquaculture. Sea bass were reared in an outdoor pond. Serum cortisol, hematocrit, leucocrit, serum lysozyme activity, and total glutathione (GSH) were monitored bimonthly for 14 months. An effect of sea- sonality was observed for all parameters, with generally higher values in summer and lower values in winter. These results could improve the understanding of the influence of seasonal cues on the immune system and the stress response in fish, to optimize husbandry practices.

Keywords Fish Á Innate immunity Á Cortisol Á Hematology

1.1 Introduction

In the literature, there are numerous studies on the influence of seasonality on fish physiology. The temperate aquatic environment is influenced throughout the year by two main seasonal cues, temperature and photoperiod (Morgan et al. 2008). In fish, seasonality coordinates reproduction, affects body weight and

F. Pascoli (&) Á E. Negrato Á C. Poltronieri Á G. Radaelli Á D. Bertotto Dipartimento di Biomedicina Comparata e Alimentazione, Università degli Studi di Padova, Legnaro (PD), Italy e-mail: [email protected] D. Bertotto e-mail: [email protected]

C. Boiti et al. (eds.), Trends in Veterinary Sciences, 3 DOI: 10.1007/978-3-642-36488-4_1, Ó Springer-Verlag Berlin Heidelberg 2013 4 F. Pascoli et al. physiological status, regulates food intake and locomotion, and is thought also to coordinate the immune response (Bowden et al. 2007). In general, physiological parameters are reduced in winter and raised in summer (Bowden et al. 2007). The purpose of this study was to investigate the effects of seasonality on growth, cortisol, immunological, and hematological parameters in sea bass reared according to conventional semi-intensive method over a period of 14 months.

1.2 Materials and Methods

Juvenile sea bass (Dicentrarchus labrax) were reared in an outdoor tank from May 2009 to July 2010 and monitored every 2 months (initial weight 69 g; final weight 350 g; stocking density 2–12 kg/m3). At each sampling, 20 animals were caught and measured (total and standard length and weight) to observe growth and condition factor (K). Blood samples were collected from the caudal vein. Serum cortisol analysis was carried out by radioimmunoassay (RIA), as described by Simontacchi et al. (2008). Hematocrit and leucocrit were obtained by micro- centrifugation of whole blood (12,500 rpm for 5 min). Serum lysozyme activity was measured by a turbidimetric assay, as described by Parry et al. (1965). Total glutathione (GSH) was determined by an enzymatic assay adapted to microtiter plate (Baker et al., 1990).

1.3 Results

Weight increased from 69.1 ± 3.0 g to 345.5 ± 13.6 g after 14 months. During this period, the condition factor worsened from 1.02 ± 0.03 to 1.21 ± 0.01, with the lowest values in December 2009, January 2010, and March 2010 (0.94 ± 0.02, 0.94 ± 0.02, and 0.95 ± 0.02, respectively) and a significant increase in May and July 2010 (1.22 ± 0.02 and 1.21 ± 0.01, respectively). Serum cortisol was significantly higher in May 2009, May 2010, and July 2010 compared to the other months (p \ 0.05; Fig. 1.1). The lowest levels were recorded in October and December 2009, and January and March 2010 (p \ 0.05). The hematocrit was significantly lower in January and March 2010 than the other samples (p \ 0.05; Fig. 1.2). The leucocrit was significantly lower in December 2009, January 2010, and March 2010 compared to the other months (p \ 0.05; Fig. 1.3). The highest value was recorded in October (p \ 0.01). Serum lysozyme activity increased from May to October 2009, then decreased until January 2010 and increased again after that point (Fig. 1.4). The lowest values were recorded in January (p \ 0.01). Higher values were found in July 2010 than in May, July, and December 2009 and March 2010, but these were not significantly different from those in October 2009 and May 2010. 1 Seasonal Effect on Hematological and Innate Immune Parameters 5

Fig. 1.1 Variations in serum cortisol of sea bass over a 14-month period (mean ± SE). Different letters indicate significant differences (p \ 0.05)

Fig. 1.2 Variations in hematocrit of sea bass over a 14-month period (mean ± SE). Different letters indicate significant differences (p \ 0.05)

The GSH decreased from July to December 2009 and then increased until July 2010 (Fig. 1.5). The lowest values were found in October and December 2009 and January and March 2010, and the highest were in July 2010 (p \ 0.05). 6 F. Pascoli et al.

Fig. 1.3 Variations in leucocrit of sea bass over a 14-month period (mean ± SE). Different letters indicate significant differences (p \ 0.05)

Fig. 1.4 Variations in serum lysozyme activity of sea bass over a 14-month period (mean ± SE). Different letters indicate significant differences (p \ 0.05) 1 Seasonal Effect on Hematological and Innate Immune Parameters 7

Fig. 1.5 Variations in glutathione (GSH) of sea bass over a 12-month period (mean ± SE). Evaluation of GSH in May 2009 was not possible because of a lack of serum. Different letters indicate significant differences (p \ 0.05)

1.4 Discussion

In this study, we investigated the effect of seasonality on some hematological and stress parameters in sea bass reared under conventional semi-intensive aquaculture. The effect of seasonality was observed for all parameters investigated. The growth and condition factors showed a significant reduction during the coldest months (October to March), due to a lower water temperature, which affects food intake, and consequently diet and metabolism (Pastoureaud 1991). Serum cortisol levels showed a seasonal pattern, with lower values during the coldest months and higher values in the warmer months, consistent with the fluctuations of temperature and photoperiod as already reported by Planas et al. (1990). The lowest hematocrit values were recorded in January and March, in corre- lation with the lowest temperatures, which increase the solubility of oxygen in the water and reduce metabolism, requiring a lower number of red blood cells to carry oxygen around the body (Stolen et al. 1984). The highest leucocrit values were recorded in October 2009, whereas the lowest were during the winter months. Seasonal changes in hematological and immunological parameters have been found in other species. It is hypothesized that shorter daylight hours can induce changes in the immune system to prepare for winter. In our study, the highest WBC values, found in October, may also be related to a viral encephalitis that occurred in August 2010 and that has caused the death of 8.31 % of the fish. 8 F. Pascoli et al.

Serum lysozyme activity showed a seasonal trend related to temperature and photoperiod, with lower values during the colder months, consistent with other studies in the literature. The observed levels of GSH suggest a relationship with seasonality, as reported in previous studies. In mammals, fasting and inflammatory processes can affect the levels of GSH, causing a decrease in plasma levels (Malmezat et al. 2000). Consequently, the low levels found in this study during the colder months may be related to winter starvation and to the viral encephalitis that occurred in August and September. In conclusion, all parameters exhibited a seasonal pattern, similar to studies performed in other species. These results may contribute to a better understanding of the mechanisms that regulate the influence of seasonal components on the immune and stress responses in fish, leading to optimized husbandry practices.

References

Baker MA, Cerniglia GJ, Zaman A (1990) Microtiter plate assay for the measurement of glutathione and glutathione disulfide in large number of biological samples. Anal Biochem 190:360–365 Bowden TJ, Thompson KD, Morgan AL, Gratacap RML, Nikoskelainen S (2007) Seasonal variation and the immune response: a fish perspective. Fish Shellfish Immunol 22:695–706 Malmezat T, Breuille D, Capitan P, Minard PP, Obled C (2000) Glutathione turnover is increased during the acute phase of sepsis in rats. J Nutr 130:1239–1246 Morgan AL, Thompson KD, Auchinachie NA, Migaud H (2008) The effect of seasonality on normal haematological and innate immune parameters of rainbow trout Oncorhynchus mykiss L. Fish Shellfish Immunol 25:791–799 Parry RM, Chandan RC, Shahani RM (1965) A rapid sensitive assay of muramidase. Proc Soc Exp Biol Med 119:384–386 Pastoureaud A (1991) Influence of starvation at low temperatures on utilization of energy reserves, appetite recovery and growth character in sea bass, Dicentrarchus labrax. Aquaculture 99:167–178 Planas J, Gutierrez J, Fernandez J, Carrillo M, Canals P (1990) Annual and daily variations of serum cortisol in sea bass Dicentrarchus labrax L. Aquaculture 91:171–178 Simontacchi C, Poltronieri C, Carraro C, Bertotto D, Xiccato G, Trocino A, Radaelli G (2008) Alternative stress indicators in seabass Dicentrarchus labrax L. J Fish Biol 72:747–752 Stolen JS, Gahn T, Kasper V, Nagle JJ (1984) The effect of environmental temperature on the immune response of a marine teleost (Faralichrhys denrufus). Dev Comp Immunol 8:89–98 Chapter 2 Effect of Altitude on Plasma Serotonin Levels in Horses

G. Bruschetta, P. Di Pietro, M. Miano, C. Cravana and A. M. Ferlazzo

Abstract The aim of this work was to carry out a preliminary study about the effect of altitude on plasma serotonin (5-HT) and tryptophan levels in horses. Subjects came from a riding school at sea level and 600 m above sea level. To evaluate animal welfare linked to physiological status, cortisol levels, blood cell count and hematochemical variables (glucose, total proteins, albumin, creat- inine, urea, aspartate transaminase, creatine kinase, lactate dehydrogenase, tri- glycerides and total cholesterol) were measured. Comparison of mean plasma levels of 5-HT, tryptophan, and cortisol in horses coming from different altitudes were not significantly different. Plasma 5-HT levels were affected by altitude in a gender-dependent way, showing an opposing trend between mares and geldings, with the highest levels in plasma of geldings coming from farms at sea level. Both 5-HT and tryptophan were higher in mares than in geldings from 600 m above sea level. Plasma cortisol levels, which were significantly higher in mares than in geldings at sea level, were more affected by gender than altitude.

Keywords Horse Á Plasma serotonin Á Tryptophan Á Biochemical variables

Abbreviations 5-HT Serotonin Try Tryptophan

G. Bruschetta (&) Á P. Di Pietro Á M. Miano Á A. M. Ferlazzo Dipartimento di Morfologia, Biochimica, Fisiologia e Produzioni Animali, Sezione di Biochimica, University of Messina, Messina, Italy e-mail: [email protected] C. Cravana Dipartimento di Morfologia, Biochimica, Fisiologia e Produzioni Animali, Sezione di Fisiologia, University of Messina, Messina, Italy

C. Boiti et al. (eds.), Trends in Veterinary Sciences, 9 DOI: 10.1007/978-3-642-36488-4_2, Ó Springer-Verlag Berlin Heidelberg 2013 10 G. Bruschetta et al.

2.1 Introduction

Serotonin, or 5-hydroxytryptamine (5-HT), is a neurotransmitter produced independently in the central nervous system and peripheral tissues from two distinct isoforms of the enzyme tryptophan hydroxylase (TPH-1 and TPH-2), which catalyses the rate limiting step of the synthesis process. Its many biological functions include pulmonary arterial smooth muscle cell proliferation, smooth muscle bronchial vasoconstriction and local microthrombosis (MacLean et al. 2000). Indeed, high circulating 5-HT levels were associated with the onset of pulmonary arterial hypertension (PAH) (Hervé et al. 1995). Blood 5-HT levels were increased in hypoxic conditions, even in mice (Callebert et al. 2006). 5-HT is synthesised from the amino acid tryptophan (Try) in the brain, in mast cells and in intestinal enterochromaffin cells, where the gene of the classic isoenzyme TPH-1, which controls the peripheral 5-HT production, is mostly expressed (Walther and Bader 2003). Both TPH-1 and peripheral 5-HT play an essential role in the development of hypoxia-induced increased pulmonary pressure and pulmonary vascular remodelling (Morecroft et al. 2007). Platelets do not synthesise 5-HT, but they are its major site of storage and transport in the peripheral blood (Andres et al. 1993). The 5-HT uptake within them keeps plasma 5-HT concentrations low. In the horse, plasma 5-HT values are reported to be higher than in humans (Bailey and Elliott 1998; Di Pietro et al. 2010; Lebelt et al. 1998), and often an increase of circulating 5-HT is linked to common pathological conditions such as laminitis (Bailey et al. 2009). Considering the clinical importance of this variable, the aim of this study was to evaluate, in horses of different breeds, the effect of horse farm altitude on plasma levels of 5-HT and Try, its precursor amino acid. Moreover, some haematochemical and hormonal parameters were determined to signal the animal welfare status and/or the presence of any stress conditions.

2.2 Materials and Methods

Twenty clinically healthy horses (9 geldings and 11 mares) of various breeds (San Fratellana, Sella Italiana and crossbred) were used. Mean age was 10 ± 6 years. Horses came from farms and riding schools in Messina, Catania and their nearby environs at sea level or at an altitude of 600 m above sea level (asl). All horses housed in Messina, in individual boxes, were fed with fresh forages, concentrates and water. Blood samples were collected from the jugular vein into EDTA tubes at 8:30 a.m., at approximately 25 °C, in the month of May. Blood cell count was performed, and after centrifugation at 2,000 g, the following haemato- chemical parameters were detected: glucose (Glu), total proteins (TP), albumin (ALB), creatinine (Crea), urea, aspartate transaminase (AST), creatine kinase (CPK), lactate dehydrogenase (LDH), triglycerides (TG), total cholesterol (TCho), by spectrophotometry and serum cortisol levels (in duplicate, by ELISA kit [Radim, 2 Effect of Altitude on Plasma Serotonin Levels in Horses 11

Pomezia, Italy]). The platelet poor plasma (PPP) fraction was obtained by centrifugation at 4,500 g. Equal volumes of plasma, N-methylserotonin (internal standard) and protein precipitation reagent were vigorously vortex mixed, incubated at 4 °C and centrifuged at 4,500 g. The obtained supernatant was used for the detection of plasma serotonin and tryptophan by reverse phase HPLC (Waters 1,525 binary HPLC pump) with electrochemical detector (ESA). Statistical analysis was done by Student’s unpaired t test and Pearson’s correlation and linear regression tests.

2.3 Results

The blood cell count and haematochemical parameters were in the physiological range for all animals (Kaneko 1989; Ubaldi et al. 1982) and indicated an overall state of wellness. Preliminary data (Table 2.1) did not show any significant differences of plasma 5-HT, tryptophan and cortisol levels with altitude change, nor significant correlations among them. However, a difference in plasma 5-HT levels based on altitude was observed when the horses were divided by gender (Table 2.2). Plasma tryptophan levels (Table 2.2) were similar in mares and geldings at sea level, but slightly higher in mares at 600 m asl, with a trend comparable to 5-HT. In mares, higher concentrations of cortisol were observed in both groups (Table 2.2), which were significant at sea level and at 600 m asl.

2.4 Discussion

Mares seem to respond to higher altitude with greater 5-HT values compared to those housed at sea level (Table 2.2), consistent with observations in cows (Bruschetta et al. 2010) and rats (Awabdy et al. 2003). At higher altitude, platelet function could be downregulated, producing a decrease of platelet serotonin uptake and increased circulating serotonin levels. On the contrary, in geldings, there was a significant increase in 5-HT at sea level. Moreover, a gender-dependent effect on plasma 5-HT levels was detected in horses at sea level, which was slight in horses at 600 m asl. An analysis using a larger sample size could support these results and clarify the existence of an interaction among different variables that may have opposite effects on plasma 5-HT levels. Further, significant findings could come from the comparison of horses coming from farms or riding schools at sea level with horses farmed at altitudes higher than 1,000 m asl.

Table 2.1 Plasma levels of 5-HT, Try and cortisol (Mean ± S.D.) in horses at different altitudes Altitude 5-HT (ng/ml) Try (lg/ml) Cortisol (ng/ml) Sea level (n = 12) 55.4 ± 13.2 7.54 ± 1.37 85 ± 21 600 m (n = 8) 49.9 ± 12.1 7.95 ± 1.78 92 ± 15 12 G. Bruschetta et al.

Table 2.2 Plasma levels of 5-HT, Try and cortisol (Mean ± S.D.) in horses at different altitudes and separated by gender Altitude Sea level Sea level 600 m 600 m Gender Geldings (n. 5) Mares (n. 7) Geldings (n. 4) Mares (n.4) 5-HT (ng/ml) 64.4 ± 6.9 49.0 ± 13.1a 44.3 ± 10.0a 55.5 ± 12.6 Try (lg/ml) 7.52 ± 1.47 7.55 ± 1.42 7.09 ± 1.44 8.80 ± 1.84 Cortisol (ng/ml) 71.0 ± 13.8 95.4 ± 19.8a 84.0 ± 12.2 99.3 ± 14.5a a vs. geldings at sea level: p \ 0.05

The major changes in tryptophan values were observed in mares. These data could suggest the existence of sexual dimorphism, which could be deeper investi- gated using a greater number of horses. The higher concentration of cortisol observed in mares in both groups (Table 2.2) confirms the existence of a regulation of the hypothalamic–pituitary–adrenal axis of horses in a gender-dependent manner, as already observed in humans (Stroud et al. 2011) and rats (Viau et al. 2005). With a larger sample size, this could be of interest for geldings farmed at 600 m asl.

Acknowledgments We thank veterinarians for supplying blood samples. Research work was carried out using PRA 2007 funds—University of Messina.

References

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Lebelt D, Zanella AJ, Unshelm J (1998) Physiological correlates associated with cribbing behaviour in horses: changes in thermal threshold, heart rate, plasma beta-endorphin and serotonin. Equine Vet J Suppl 27:21–27 MacLean MR, Hervé P, Eddahibi S, Adnot S (2000) 5-hydroxytryptamine and the pulmonary circulation: receptors, transporters and relevance to pulmonary arterial hypertension. Br J Pharmacol 131:161–168 Morecroft I, Dempsie Y, Bader M, Walther DJ, Kotnik K, Loughlin L, Nilsen M, MacLean MR (2007) Effect of tryptophan hydroxylase 1 deficiency on the development of hypoxia-induced pulmonary hypertension. Hypertension 49:232–236 Stroud LR, Papandonatos GD, Williamson DE, Dahl RE (2011) Sex differences in cortisol response to corticotropin releasing hormone challenge over puberty: Pittsburgh Pediatric Neurobehavioral Studies. Psychoneuroendocrinology 36:1226–1238 Ubaldi A, Corbella E, Montanari P (1982) Chapter 7. In: Ubaldi A, Corbella E, Montanari P (eds) Diagnostica chimico-clinica veterinaria. Casa editrice Ambrosiana, Milano, p 152 Viau V, Bingham B, Davis J, Lee P, Wong M (2005) Gender and puberty interact on the stress- induced activation of parvocellular neurosecretory neurons and corticotrophin-releasing hormone messenger ribonucleic acid expression in the rat. Endocrinology 146:137–146 Walther DJ, Bader M (2003) A unique central tryptophan hydroxylase isoform. Biochem Pharmacol 66:1673–1680 Chapter 3 Identification of Aquaporin 1 in Diplodus sargus

G. Zanghì, S. Campo, A. D’Ascola, A. Germanà and A. M. Ferlazzo

Abstract Aquaporin 1 (AQP-1) is the first member of the aquaporin family, which includes seven homologs in teleosts, involved in the selective transport of water, small neutral molecules, and ions. AQPs contain six transmembrane helices, five connecting loops, and the amino and carboxyl ends protrude into the cytoplasm. AQPs are important for osmoregulation in fish gills (independent of salinity), kidneys, and intestine. Here, the nucleotide and amino acid sequences of AQP-1 in Diplodus sargus were characterized, and a phylogenetic tree was built to study its evolution. Results showed that AQP-1 mRNA is 1,325 nucleotides in length, and the deduced protein contains two canonical Asn-Pro-Ala (NPA) consensus motifs and all features for water transport. D. sargus AQP-1 is located in a homophyletic branch with Sparus aurata only, inside of a sub-tree in a paraphyletic position with Centropristis striata and Osmerus mordax. Although the structure of the AQP protein in different species is conserved, results showed that the S. aurata and D. sargus AQP-1 proteins have evolved similarly.

Keywords D. sargus Á Aquaporin 1 Á Biochemistry Á Nucleotide and amino acid sequences

G. Zanghì (&) Á A. M. Ferlazzo Dipartimento di Morfologia, Biochimica, Fisiologia e Produzioni Animali, Sezione di Biochimica, Università degli Studi di Messina, Messina, Italy e-mail: [email protected] S. Campo Á A. D’Ascola Dipartimento di Scienze Biochimiche, Fisiologiche e Della Nutrizione, Sezione di Chimica, Università degli Studi di Messina, Messina, Italy A. Germanà Dipartimento di Morfologia, Biochimica, Fisiologia e Produzioni Animali, Sezione di Morfologia, Università degli Studi di Messina, Messina, Italy

C. Boiti et al. (eds.), Trends in Veterinary Sciences, 15 DOI: 10.1007/978-3-642-36488-4_3, Ó Springer-Verlag Berlin Heidelberg 2013 16 G. Zanghì et al.

3.1 Introduction

The survival of fish in the water environment is strictly associated with the correct functional properties of some critical structures, such as ionic channels, which play key roles in electrochemical and osmotic equilibria. Aquaporins (AQPs) facilitate the very fast flux of water molecules to the inside and outside of cells of specific tissues (e.g., proximal tubules, erythrocytes, and membranes of vegetal cell vacuoles). Their presence is necessary for transport of water, which, as a polar molecule, negligibly diffuses through biological membranes. Currently, 13 iso- forms of AQPs have been identified in mammals, variously distributed to various organs (Ablimit et al. 2006; Lee et al. 1997; Lopez et al. 2007), and 17 AQPs have been identified in both fresh and marine teleosts (Cerdà and Finn 2010), particularly in pufferfish and zebrafish (Tingaud-Sequeira et al. 2010). The duplicate isoforms of aquaporin 1 (AQP-1) are involved in intestinal water absorption in marine fish (Tingaud-Sequeira et al. 2008). AQPs comprise a superfamily of intrinsic proteins, with a molecular weight between 36 and 78 kDa, located inside the membrane lipid bilayer, allowing the bidirectional transport of water (Agre 2006). Two families of AQPs were identified (Gonen and Walz 2006): specific aquaporins, allowing only transport of water, and aquaglyceroporins, allowing transport of water, glycerol, and other neutral molecules (Agre et al. 2002; Litman et al. 2009). Both proteins contain transmembrane domains that cross the phospholipid layer (Fu and Lu 2007). AQPs are ethero- and homotetramers. Every protein subunit contains its own pore for transport of one water molecule at a time. Each subunit is characterized by one of six transmembrane domains that cross the lipid bilayer and faces each other: the first three domains (a, b, c) oppose the other three (a1, b1, c1). In the channel pore, the N- and C-terminal ends of the polypeptide chains are oriented toward the intracellular environment. Moreover, the AQP structure contains extracellular loops (A, C, E) and intracellular loops (B, D, F) (Jung et al. 1994). The aqueous channel pore is formed by loops B and E, folded to create the central pore. The loops contain particular Asn-pro-ala (NPA) motifs, allowing an ‘‘hourglass’’ structure (Agre et al. 1999). AQP-1 is the first member of the AQP family, which includes seven homologous proteins in teleosts, that is particularly important in some osmoregulatory fish organs, especially gills, where it is expressed in great amounts, in addition to the kidney and gut (Giffard-Mena et al. 2007). Among marine teleosts of the perciform family, Diplodus sargus (Bargelloni et al. 2005) is found in the Mediterranean Sea, Black Sea, and Eastern Atlantic Ocean. Young specimens live on the surface, and lonely adults live on rocky seabeds at depths of 20–30 m and are 35–45 cm in length. D. sargus reproduces at the beginning of autumn, and its fries remain for some time in brackish waters, then adapt to the changing water salinity. In order to understand the molecular mech- anisms allowing this species to adapt in an extremely good way to different envi- ronmental conditions, the aim of this study was to characterize AQP-1 nucleotide 3 Identification of Aquaporin 1 in Diplodus sargus 17 and amino acid sequences by constructing a phylogenetic tree to evaluate its evolution. The molecular study of this perciform family fish is very interesting, from both a scientific point of view and its commercial consequences.

3.2 Materials and Methods

The research was carried out on 10 specimens of D. sargus, 250 g body weight, farmed for 10 days under normal salinity conditions in an aquarium containing 300 L fresh seawater and gassed with an integrated air pump oxygenator. After anesthesia with MS-222 (40 mg/L ethyl-m-aminobenzoate methanesulfonate), fish were sac- rificed for gill sampling (50–100 mg). After homogenization and total mRNA extraction using the Trizol Reagent kit (Invitrogen, USA) followed by RT-PCR, the AQP-1 nucleotide sequence was identified using the rapid amplification of cDNA ends (RACE) technique. To build the phylogenetic tree, AQP-1 was aligned using ClustalW software with AQPs 1 and 3 of 33 temperate and Antarctic teleosts, a Renibacterium, and other higher vertebrates, representing the outgroup. The tree was calculated by the Neighbor-Joining method with a 1,000 repetitions Bootstrap using the MEGA 5.05 program, whereas the calculus matrix was experimentally evaluated using ProTest software. Phylogenetic trees were drawn to scale, whereas evolutionary distances were calculated using the Poisson correction method. The phylogenetic tree, so drawn, showed a good resolution, given that only one knot had a value lower than 0.5.

3.3 Results

In Fig. 3.1, the nucleotide sequence of D. sargus AQP-1 is reported. The mRNA is 1,325 nucleotides in length; its start codon is located at position 116, the stop codon is located at position 901, and the polyadenylation signals are located at positions 957 and 1,303. In Fig. 3.2, the amino acid sequence of the deduced protein, which is 261 amino acid residues in length and highly homologous to those of other studied fish species is shown. The protein contains two particular NPA motifs and all essential features for water transport. The two NPA domains can be observed at positions 70 and 184, and contain loops B and E, respectively. In addition, it is possible to see amino acid residues of the other four loops. The phylogenetic tree can be divided into four main sub-trees. The first contains AQPs of outgroup species (chicken, Xenopus, and human). Reptile AQP, Vitis vinifer, lies on a distinct branch. On the first sub-tree, in an unusual way, AQPs of two teleosts are included. The fourth sub-tree contains the majority of AQP-3, while the third sub-tree contains the AQP-1 of some teleosts and human AQP-2. Finally, AQP-1 of almost all analyzed teleosts is included in the second sub-tree, which contains different clusters, one formed only from the D. sargus and Sparus aurata AQPs that 18 G. Zanghì et al.

Fig. 3.1 Nucleotide sequence of the AQP-1 gene (GenBank provisory accession number = JN210582)

Fig. 3.2 Amino acid sequence of AQP-1 and consensus loops

obviously are monophyletic, but are in a paraphyletic position with AQP-1 of Centropristis striata and Osmerus mordax. The length of the branches of this cluster also shows that D. sargus and S. aurata AQPs have undergone a very recent and common diversification.

3.4 Discussion

In conclusion, D. sargus mRNA and its deduced protein have been identified. The drawn phylogenetic tree shows that the evolutionary diversification of this gene is similar to that of S. aurata. This study will be strengthened by the characterization and identification of AQP-3 in the same species; AQP-3, in which two duplicated genetic isoforms have been found in zebrafish (Danio rerio), plays a fundamental role in physiological osmoregulatory processes in different teleos- tean organs (Cutler et al. 2007). 3 Identification of Aquaporin 1 in Diplodus sargus 19

Acknowledgments This research work was carried out by PRA 2007 funds at the University of Messina.

References

Ablimit A, Matsuzaki T, Tajika Y, Aoki T, Hagiwara H, Takata K (2006) Immunolocalization of water channel aquaporins in the nasal olfactory mucosa. Arch Histol Cytol 69:1–12 Agre P (2006) The aquaporin water channels. Proc Am Thorac Soc 3:5–13 Agre P, Mathai JC, Smith BL, Preston GM (1999) Functional analyses of aquaporin water channel proteins. Methods Enzymol 294:550–572 Agre P, King LS, Yasui M, Guggino WB, Ottersen OP, Fujiyoshi Y, Engel A, Nielsen S (2002) Aquaporin water channels—from atomic structure to clinical medicine. J Physiol 542:3–16 Bargelloni L, Alarcon JA, Alvarez MC, Penzo E, Magoulas A, Palma J, Patarnello T (2005) The Atlantic-Mediterranean transition: discordant genetic patterns in two seabream species, Diplodus puntazzo (Cetti) and Diplodus sargus Diplodus (L.). Mol Phylogenet Evol 36:523–535 Cerdà J, Finn RN (2010) Piscine aquaporins: an overview of recent advances. J Exp Zool A Ecol Genet Physiol 313:623–650 Cutler CP, Martinez AS, Cramb G (2007) The role of aquaporin 3 in teleost fish. Comp Biochem Physiol A Integr Physiol 148:82–91 Fu D, Lu M (2007) The structural basis of water permeation and proton exclusion in aquaporins. Mol Membr Biol 24:366–374 Giffard-Mena I, Boulo V, Aujoulat F, Fowden H, Castille R, Charmantier G, Cramb G (2007) Aquaporin molecular characterization in the sea-bass (Dicentrarchus labrax): the effect of salinity on AQP1 and AQP3 expression. Comp Biochem Physiol A Integr Physiol 148:430–444 Gonen T, Walz T (2006) The structure of aquaporins. Q Rev Biophys 39:361–396 Jung JS, Preston GM, Smith BL, Guggino WB, Agre P (1994) Molecular structure of the water channel through aquaporin CHIP. The hourglass model. J Biol Chem 269:14648–14654 Lee MD, King LS, Agre P (1997) The aquaporin family of water channel proteins in clinical medicine. Medicine (Baltimore) 76:141–156 Litman T, Søgaard R, Zeuthen T (2009) Ammonia and urea permeability of mammalian aquaporins. In: Beitz E (ed) Aquaporins. Handbook Of Experimental Pharmacology. Springer, Berlin, pp 327–358 Lopez IA, Ishiyama G, Lee M, Baloh RW, Ishiyama A (2007) Immunohistochemical localization of aquaporins in the human inner ear. Cell Tissue Res 328:453–460 Tingaud-Sequeira A, Chauvigné F, Fabra M, Lozano J, Raldúa D, Cerdà J (2008) Structural and functional divergence of two fish aquaporin-1 water channels following teleost-specific gene duplication. BMC Evol Biol 8:259 Tingaud-Sequeira A, Calusinska M, Finn RN, Chauvigné F, Lozano J, Cerdà J (2010) The Zebrafish genome encodes the largest vertebrate repertoire of functional aquaporins with dual paralogy and substrate specificities similar to mammals. Biomed Central BMC Evol Biol 10:38 Chapter 4 Effect of Dephosphorylation on Donkey Milk Caseins

S. Vincenzetti, A. Vita, F. M. Carpi, D. Micozzi and P. Polidori

Abstract Donkey milk caseins display great microheterogeneity by two-dimensional electrophoresis (2-DE), probably because of a variable degree of phosphorylation and/or alternative splicing phenomena. In this work, we have investigated the complexity of the donkey milk caseins: The whole casein fraction was subjected to treatment with calf intestinal alkaline phosphatase to achieve dephosphorylation. The obtained apo forms were analyzed by 2-DE and compared with whole donkey caseins. As a result, donkey milk b-casein and its splicing variants are present as different phosphorylated isoforms, whereas aS1-casein displays several isoforms that are derived from both phosphorylation and glycosylation phenomena.

Keywords Alpha-casein Á Beta-casein Á Dephosphorylation Á Donkey milk Á Two-dimensional electrophoresis

4.1 Introduction

Caseins are a family of acid phosphoproteins (aS1-, aS2-, b-, and j-caseins) that are synthesized in the mammary gland in response to hormonal stimuli and form micellae in milk. In the mare’s milk, the primary structures of the aS1- and

S. Vincenzetti (&) Á A. Vita Scuola di Scienze Mediche Veterinarie, Università di Camerino, Camerino (MC), Italy e-mail: [email protected] F. M. Carpi Á D. Micozzi Scuola di Bioscienze e Biotecnologie, Università di Camerino, Camerino (MC), Italy P. Polidori Scuola di Scienze del Farmaco e dei prodotti della salute, Università di Camerino, Camerino (MC), Italy

C. Boiti et al. (eds.), Trends in Veterinary Sciences, 21 DOI: 10.1007/978-3-642-36488-4_4, Ó Springer-Verlag Berlin Heidelberg 2013 22 S. Vincenzetti et al. b-caseins show several polymorphic patterns due to alternative splicing phe- nomena; furthermore, there are some other isoforms of caseins due to post- translational modifications such as phosphorylation and glycosylation (Miclo et al. 2007). The phosphate group of casein may affect many of its features, such as their digestion and the bioavailability of divalent cations. It is known that in cow’s milk protein allergy, caseins are the proteins widely responsible for this allergic phe- nomenon. In particular, some authors have shown that the aS1-casein in its phosphorylated form plays an important role in the allergenicity of milk and that in general, the aS- and b-caseins that possess serine-phosphorylated residues can be considered immunoreactive and resistant to digestion (Tezcucano et al. 2007). In this work, we investigated the complexity of donkey milk caseins pattern by two- dimensional electrophoresis (2-DE) analyses. For this purpose, the total caseins obtained by isoelectric precipitation were dephosphorylated by alkaline phos- phatase, showing that donkey’s milk b-casein and its splice variants are largely present as phosphorylated isoforms, while donkey’s milk aS1-casein shows dif- ferent isoforms that may result from both phosphorylation and glycosylation phenomena.

4.2 Materials and Methods

Donkey’s milk was collected from 10 Martina Franca breed asses at an interme- diate stage of lactation. The milk was skimmed by centrifugation at 3,000 9 g for 30 min, at a temperature of 15 °C. The casein fraction was obtained from skimmed milk by acidic precipitation at pH 4.6 with 10 % acetic acid, followed by centrifugation at 3,000 g for 10 min. The casein pellet was then resuspended in 50 mM Tris/HCl, pH 7.5, 1 mM DTT, and 8 M urea. The 2-DE was performed as follows: 100 lg casein was treated with the 2D-Clean-Up kit (GE Healthcare) and resuspended in rehydration buffer (8 M urea, 2 % CHAPS, 65 mM DTT, 0.001 % bromophenol blue, and 0.5 % IPG buffer, pH 4–7). Isoelectric focusing was per- formed on an immobilized gradient at pH 4–7 (Immobiline DryStrip gel, 18 cm, GE Healthcare). The IPG strip was rehydrated for 12 h at 15 °C to avoid deam- idation (at the level of a residue of Asn), which may occur when the isoelectric focusing is carried out at temperatures above 20 °C (Matéos et al. 2009). The electrophoretic conditions were 50 A/strip, 1 h at 500 V, 1 h at 1,000 V, and 4 h at 8,000 V. The second dimension consisted of 13 % SDS-PAGE. After electrophoresis, proteins were stained using Coomassie blue or blotted to polyvinylidene difluoride membranes to analyze each spot by N-terminal sequencing. Spot analysis was carried out using PDQuest software (Version 7.1.1; Bio-Rad Laboratories) for spot-intensity calibration, as well as calculation of molecular mass and isoelectric point (pI). In the experiment of casein dephos- phorylation, the casein fraction was resuspended in 10 ml 0.4 % ammonium bicarbonate, pH 8.5, containing 0.5 mM MgCl2. The solution was heated to 80 °C for 5 min to disperse the caseins and then was lyophilized in 5.3-mg aliquots. 4 Effect of Dephosphorylation on Donkey Milk Caseins 23

Lyophilized caseins were resuspended in 50 mM Tris/HCl, 10 mM MgCl2, and 1 mM DTT, pH 7.5, and treated with alkaline phosphatase (20 units) for 3 h at 37 °C. After incubation, the dephosphorylated casein samples were frozen and freeze-dried. Subsequently, dephosphorylated caseins were subjected to 2-DE as described above. The control consisted of caseins treated in the same way as those subjected to dephosphorylation but incubated in the absence of alkaline phosphatase.

4.3 Results

Figure 4.1 shows the electrophoretic pattern of the phosphorylated casein fraction (control, Fig. 4.1a) compared with dephosphorylated caseins (Fig. 4.1b). A shift of dephosphorylated caseins toward more basic pH values with respect to the control is evident, because of the removal of the negative charge of the phosphate groups. The phosphorylated and dephosphorylated caseins were analyzed by PDQuest software and N-terminal sequencing, and the results are summarized in Table 4.1. Spots A–H of the phosphorylated casein fraction are b-caseins with molecular masses (Mr) ranging from 31.15 to 33.80 kDa and pIs ranging from 4.60 to 4.90. Spots I–N are aS1-caseins with lower molecular weights with respect to the b-caseins, ranging from 27.40 to 31.20 kDa and more basic pI values, ranging from 4.90 to 5.40 (Fig. 4.1a and Table 4.1). Spots A1 and B1 observed in the dephosphorylated casein fraction (Fig. 4.1b and Table 4.1) are two b-caseins with very similar molecular masses (33.50 and 32.87 kDa, respectively) but different pI values (5.70 and 5.90, respectively). Spots C1 and I1 are two b-caseins but with slightly lower molecular masses while spots E1, F1, G1, and H1 are aS1-caseins with different pI and molecular mass values. Under our experimental conditions, we have not observed aS2- and j-caseins, which might be present in trace amounts in donkey’s milk, as reported by other authors (Bertino et al. 2010).

Fig. 4.1 Casein dephosphorylation in donkey’s milk: a phosphorylated caseins (control); b dephosphorylated caseins 24 S. Vincenzetti et al.

Table 4.1 Characterization of phosphorylated and dephosphorylated caseins by N-terminal sequencing. Spot analysis was carried out by PDQuest software Version 7.1.1 (Bio-Rad Laboratories)

Spot Mr (kDa) pI Protein identification A 33.74 4.6 b-casein B 33.54 4.7 b-casein C 33.80732.40 4.8 b-casein D 33.54 4.9 b-casein E 31.66 4.7 b-casein F 31.48 4.8 b-casein G 32.15 4.9 b-casein H 31.15 4.9 b-casein

I 31.20 5.1 as1-casein J 31.14 5.2 as1-casein L 31.14 5.4 as1-casein M 28.26 5.0 as1-casein N 27.24 4.9 as1-casein A1 33.50 5.7 b-casein B1 32.87 5.9 b-casein C1 31.90 5.7 b-casein

D1 26.80 5.6 as1-casein E1 28.48 5.6 as1-casein F1 30.60 6.3 as1-casein G1 30.60 6.5 as1-casein H1 30.80 5.4 as1-casein

4.4 Discussion

The phosphorylated casein fraction shows, after 2-DE analysis, the presence of about 14 major spots with molecular masses ranging between 27.24 and 33.74 kDa and pI values between 4.60 and 5.40 (Fig. 4.1a and Table 4.1). N-terminal analysis showed the presence of as1- and b-caseins, suggesting that the heterogeneity observed in the casein fraction could be due to different degrees of phosphorylation or glycosylation (Bertino et al. 2010; Chianese et al. 2010). Through the NetPhos 2.0 and Netglycate 1.0 software (available on the website www.expasy.ch), it is possible to predict phosphorylation and glycosylation on as1- and b-caseins. In donkey milk b-casein, there are 11 serine residues and one threonine residue that can be potentially phosphorylated, and there is one lysine residue that can be potentially glycosylated, whereas in as1-casein, there 10, 2, and 1 potentially phosphorylated sites on serine, threonine, and tyrosine residues, respectively. Furthermore, there are seven potential lysine residues glycosylated. Experiments on casein dephosphorylation have shown that the heterogeneity observed in the donkey’s milk casein fraction is essentially due to post-transla- tional modifications, in particular, to phosphorylation. In fact, comparing the 2-DE 4 Effect of Dephosphorylation on Donkey Milk Caseins 25 patterns obtained using the phosphorylated and dephosphorylated caseins, it can be assumed that spots A, B, C, and D (Fig. 4.1a) assigned the phosphorylated b-caseins, are modified after dephosphorylation of spots A1 and B1 (Fig. 4.1b), where they show similar molecular weights but different pI values. Therefore, spots A1 and B1 could be two isoforms that may be derived from a genetic polymorphism or from the glycosylation of a lysine residue, according to the prediction of glycosylation sites on b-casein. Presumably, spots C1 and I1 (Fig. 4.1b, Table 4.1), characterized by lower molecular masses (31.90 and 32.30 kDa, respectively) than the other two b-casein spots (A1 and B1), may be the dephosphorylated splice variants of b-casein. In fact, Matéos and collaborators (2010) showed the presence of b-casein D5 (30 kDa) characterized as a splice variant of exon 5, in mare’s milk. Therefore, in conclusion, spots A, B, C, and D of donkey’s milk b-casein (Fig. 4.1a) could correspond to different isoforms with different degrees of phosphorylation, while spots E, F, G, and H may be different phosphorylated isoforms of splice variants. Finally, the prediction of glycosylation sites on donkey’s milk as1-caseins highlights the potential for glycosylation of seven lysine residues that may be responsible for the presence of different spots (D1, E1, F1, G1, and H1) observed in donkey’s milk as1-casein even after dephosphorylation.

Acknowledgments This work has been supported by the Italian Ministry of Agriculture; Principal Investigator: Prof. Polidori Paolo.

References

Bertino E, Gastaldi D, Monti G, Baro C, Fortunato D, Perono Garoffo L, Coscia A, Fabris C, Mussap M, Conti A (2010) Detailed proteomic analysis on DM: insight into its hypoallergenicity. Front Biosci 2:526–536 Chianese L, Calabrese MG, Ferranti P, Mauriello R, Garro G, De Simone C, Quarto M, Addeo F, Cosenza G, Ramunno L (2010) Proteomic characterization of donkey milk ‘‘caseome’’. J Chromatogr A 1217:4834–4840 Matéos A, Girardet JM, Mollé D, Dary A, Miclo L, Gaillard JL (2009) Two-dimensional cartography of equine beta-casein variants achieved by isolation of phosphorylation isoforms and control of the deamidation phenomenon. J Dairy Sci 92:2389–2399 Matéos A, Girardet JM, Mollé D, Corbier C, Gaillard JL, Miclo L (2010) Identification of phosphorylation sites of equine beta-casein isoforms. Rapid Commun Mass Spectrom 24:1533–1542 Miclo L, Girardet JM, Egito AS, Mollé D, Martin P, Gaillard JL (2007) The primary structure of a low-Mr multiphosphorylated variant of beta-casein in equine milk. Proteomics 7:1327–1335 Tezcucano Molina AC, Alli I, Konishi Y, Kermasha S (2007) Effect of dephosphorylation on bovine casein. Food Chem 101:1263–1271 Chapter 5 Distribution Pattern and Chemical Coding of Sympathetic Trunk Ganglia Neurons Supplying the Boar Urinary Bladder Trigone

F. Gazza, M. Botti, L. Ragionieri, C. Sorteni, D. Russo, P. Clavenzani, R. Chiocchetti, L. Bo Minelli and R. Panu

Abstract Sympathetic trunk ganglia (STG) neurons projecting to the urinary bladder trigone of the boar were studied by coupling retrograde tracing Fast Blue (FB) and double-labeling immunofluorescence methods. FB-positive neurons were localized in the L1-S3 STG. Immunohistochemical staining revealed the catechol- aminergic (tyrosine hydroxylase-/dopamine beta hydroxylase immunoreactivity) phenotype of the majority of FB-positive neurons, which also preferentially expressed neuropeptide Y. In addition, some of the FB-positive dopaminergic perikarya were immunoreactive for calcitonin gene-related peptide, substance P, vasoactive intestinal peptide, vesicular acetylcholine-transporter, neuronal nitric oxide synthase, somatostatin, and leu-enkephalin. Functional hypotheses have been formulated.

Keywords Boar Á Autonomic neurons Á Retrograde neuronal tracer Á Immunohistochemistry

5.1 Introduction

The urethral bladder trigone (UBT) is a limited area of the urinary bladder where the majority of the bladder’s vessels and nervous fibers enter (Birder et al. 2010) and where intramural neurons are more concentrated (Andersson 2002; Pidsudko 2004). The UBT has remarkable clinical importance, because it is a site for vesical

F. Gazza Á M. Botti Á L. Ragionieri Á L. Bo Minelli (&) Á R. Panu Dipartimento di Scienze Medico-Veterinarie, Università degli Studi di Parma, Parma, Italy e-mail: [email protected] C. Sorteni Á D. Russo Á P. Clavenzani Á R. Chiocchetti Dipartimento di Scienze Mediche-Veterinarie, Università degli Studi di Bologna, Bologna, Italy

C. Boiti et al. (eds.), Trends in Veterinary Sciences, 27 DOI: 10.1007/978-3-642-36488-4_5, Ó Springer-Verlag Berlin Heidelberg 2013 28 F. Gazza et al. cancer origin and proliferation in both human (Shokeir 2004) and veterinary medicine (Saulnier-Troff et al. 2008; Sakai 2011; Wilson et al. 2007). Therefore, a thorough knowledge of the innervation of this area could have important clinical consequences. The aim of this study was to identify and neurochemically characterize the sympathetic trunk neurons involved in the innervation of the boar UBT, combining retrograde neuronal transport Fast Blue (FB) with a double-labeling immunofluorescence method.

5.2 Materials and Methods

The study was carried out on three 40 kg boars. Under general anesthesia, they underwent laparatomy via the ventral midline from the umbilical to pubic regions. The urinary bladder was deflected caudally to expose the UBT. 50 llof2% aqueous solution of FB was injected in 5 random sites (10 ll per site) in the bladder wall of the trigone with a glass Hamilton microsyringe. After an appro- priate survival time (15 days), the animals were deeply anaesthetized and sub- jected to euthanasia by i.v. administration of embutramide, mebenzonium iodide, and tetracaine hydrochloride (Tanax, Intervet, and Italia; 0.3 ml/kg). Bilateral sympathetic trunk ganglia (STG) from T1 to Co1 were collected. Double immu- nolabeling reactions were carried out on 16 lm thick cryosections of the lumbo-sacral ganglia in which FB positive (FB+) neurons were present, to high- light the eventual co-existence of tyrosine hydroxylase (TH) with dopamine b hydroxylase (DbH), vesicular choline acetyl transferase (VChAT), neuronal nitric oxide synthase (n-NOS), calcitonin gene-related peptide (CGRP), leu-enkephaline (LENK), neuropeptide Y (NPY), substance P (SP), vasoactive intestinal polypeptide (VIP), and somatostatin (SOM).

5.3 Results

There were 1845 ± 259 (mean ± S.E.M., n = 3) FB+ neurons distributed in the L1-S3 STG. The vast majority of them (87 ± 5 % of counted FB+ neurons) were counted in the L7-S1 ganglia, primarily in the left ganglion. Morphologically, the FB+ cells were multipolar with an ellipsoidal shape and a major axis of 30 ± 5 lm (200 cells measured for each animal), generally parallel to the longitudinal axis of the ganglion. The labeled cells were isolated or clustered into small groups of two or three neurons scattered in the ganglion. In the most positive ganglia, the labeled cells were localized almost exclusively along one side of the ganglion. Immunohistochemistry showed that 66 ± 10 % of FB+ neurons showed immunoreactivity (IR) for TH and that 92 ± 7 % of these cells co-expressed DbH, revealing the catecholaminergic nature of these neurons. 5 Distribution Pattern and Chemical Coding 29

We observed that 59 ± 8 % of FB+ neurons showed IR to NPY, whereas the percentage of FB+ neurons that co-expressed TH- and NPY-IR was 52 ± 7%. The neurons projecting to the UBT showed positivity also for the other markers tested, in the following percentages: CGRP- (24 ± 3 %), SP- (23 ± 2 %), VIP- (19 ± 2 %), nNOS- (15 ± 2 %), VAChT- (15 ± 2 %), LENK- (14 ± 7 %), and SOM-IR (12 ± 3 %). Moreover, the FB+/TH-IR neurons co-expressed CGRP- (20 ± 2 %), SP- (19 ± 5 %), VIP- (15 ± 2 %), VAChT- (15 ± 2 %), nNOS- (12 ± 3 %), SOM- (11 ± 4 %), and LENK-IR (10 ± 3 %). VAChT-, CGRP-, LENK-IR various fibers, and in minor quantities, nNOS-IR nerve terminals were observed around the FB+ neurons.

5.4 Discussion

The combined use of FB and double immunofluorescence methods carried out on neurons of the STG innervating the boar UBT documented that the contribution of these neurons was quantitatively and qualitatively diversified. The distribution of the FB+ neurons along one side of the ganglia mainly involved in the innervation of the UBT is particular. In the past, a somatotopic organization has been documented in the caudoventral portions of the STG for the neurons projecting to the pig colon (Skobowiat et al. 2010). This localization at the periphery of the ganglia, the small dimensions, the catecholaminergic character, and/or the positivity to NPY or, less frequently to SOM, allow us to hypothesize that the majority of the UBT projecting neurons have a vasoconstrictor activity (Majewski 1999). The adrenergic innervation could also be destined to the musculature of the trigone, where in certain species, the high density of the adrenergic fibers has been proven (Janig and McLachlan 1987; Lakomy et al. 1989, 1990). This could be related to the change of function of the caudal part of the urinary bladder from ‘‘storage’’ to ‘‘sphincter.’’ The FB+ neurons VIP-, NPY-, and VAChT-IR and those TH- and VIP-IR could have a sympathetic vasodilator function (Majewski 1999). The VAChT-IR fibers observed around the FB+ neurons presumably originate from pre-ganglionic cholinergic neurons (Gibbins and Morris 2000). The LENK-IR fibers could originate from neurons located in the STG, whereas the CGRP-IR fibers could originate from primary afferent neurons (personal observations).

References

Andersson KE (2002) Bladder activation: afferent mechanisms. Urology 59(5A):43–50 Birder L, de Groat WC, Mills I, Morrison J, Thor K, Drake M (2010) Neural control of the lower urinary tract: peripheral and spinal mechanisms. Neurourol Urodyn 29:128–139 30 F. Gazza et al.

Gibbins IL, Morris JL (2000) Pathway specific expression of neuropeptides and autonomic control of the vasculature. Regul Pept 93(1–3):93–107 Jänig W, McLachlan EM (1987) Organization of lumbar spinal outflow to distal colon and pelvic organs. Physiol Rev 67(4):1332–1404 Lakomy M, Kaleczyc J, Wasowicz K (1989) Adrenergic innervation of the ureters, urinary bladder, and urethra in pigs. Gegenbaurs Morphol Jahrb 135(2):347–355 Lakomy M, Wasowicz K, Kaleczyc J, Chmielewski S (1990) AChE-positive innervation of the ureters, urinary bladder, and urethra in pigs. Z Mikrosk Anat Forsch 104(2):316–326 Majewski M (1999) Synaptogenesis and structure of the autonomic ganglia. Folia Morphol (Warsz) 58(3 Suppl 2):65–99 (Review) Pidsudko Z (2004) Distribution and chemical coding of neurons in intramural ganglia of the porcine urinary bladder trigone. Folia Histochem Cytobiol 42:3–11 Sakai H, Yonemaru K, Takeda M, Niimi K, Murakami M, Hirata A, Yanai T (2011) Ganglioneuroma in the urinary bladder of a dog. J Vet Med Sci 73(6):801–803 Saulnier-Troff FG, Busoni V, Hamaide A (2008) A technique for resection of invasive tumors involving the trigone area of the bladder in dogs: preliminary results in two dogs. Vet Surg 37:427–437 Shokeir AA (2004) Squamous cell carcinoma of the bladder: pathology, diagnosis and treatment. BJU Int 93:216–220 Skobowiat C, Calka J, Wasowicz K, Majewski M (2010) Distribution pattern and chemical coding of neurons of the sympathetic chain ganglia supplying the descending colon in the pig. Acta Vet Hung 58(2):189–198 Wilson HM, Chun R, Larson VS, Kurzman ID, Vail DM (2007) Clinical signs, treatments, and outcome in cats with transitional cell carcinoma of the urinary bladder: 20 cases (1990–2004). J Am Vet Med Assoc 231:101–106 Chapter 6 In Vivo Applications of Mesenchymal Stem Cells and Platelet-Rich Plasma to Improve Tendon Regeneration in Sheep

M. Patruno, I. Bronzini, L. Maccatrozzo, A. Perazzi, I. Iacopetti, G. M. De Benedictis, S. Testoni, A. Negro, F. Mascarello and T. Martinello

Abstract The ‘‘restitutio ad integrum’’ pursue in the treatment of tenodesmic lesions might represents a tangible target thanks to the increased number of novel cellular-based therapies. In this work, we evaluated the efficacy of the application of platelet-rich plasma (PRP), mesenchymal stem cells (MSCs), and PRP ? MSCs to experimentally injured sheep deep digital flexor tendon (DM n° 97/2010-B). Our results indicate that the in vivo integration of injected MSCs was successful as verified by the presence of green fluorescent protein GFP-positive cells. Tissue architecture and the tendon linear fiber pattern were significantly improved on histologic sections, especially after the use of MSCs, and the clinic evaluation was also satisfactory after the use of PRP.

Keywords Adult stem cells Á Platelet-rich plasma Á Tendon regeneration

6.1 Introduction

In veterinary medicine, the tenodesmic pathologies are clinically relevant because: (1) they occur very often; (2) it is difficult to obtain a complete functional recovery; and (3) they cause long periods of inactivity. The commonly used therapeutical protocols so far does not involve achieving a ‘‘restitutio ad

M. Patruno (&) Á I. Bronzini Á L. Maccatrozzo Á F. Mascarello Á T. Martinello Department of Comparative Biomedicine and Food Science, University of Padova, Legnaro, Italy e-mail: [email protected] A. Perazzi Á I. Iacopetti Á G. M. De Benedictis Á S. Testoni Department of Department of Animal Medicine, Production and Health, University of Padova, Legnaro, Italy A. Negro Department of Biomedical Science, University of Padova, Padua, Italy

C. Boiti et al. (eds.), Trends in Veterinary Sciences, 31 DOI: 10.1007/978-3-642-36488-4_6, Ó Springer-Verlag Berlin Heidelberg 2013 32 M. Patruno et al. integrum’’, and therefore the research community continues to look for new therapies that are able to increase the regeneration of damaged tissue. In particular, the advantages of using mesenchymal stem cells (MSCs) isolated from adult tissues (Martinello et al. 2010) and biomaterials such as platelet-rich plasma (PRP) are being investigated. The therapeutical potential based on the regenerative capability of stem cells is a real possibility, even in veterinary medicine (Ferrari et al. 2007; Crovace et al. 2010; Watts et al. 2011), and the beneficial effects of growth factors found in PRP are already known (Cenni et al. 2010). Moreover, the association of MSCs and PRP likely will allow for improved in vivo growth of implanted cells; therefore, their combined use represents an intriguing option in the treatment of tenodesmic pathologies in the veterinary orthopedic fields. Our research aims to study the efficacy of MSCs, together with the use of PRP, implanted in experimentally injured tendons of sheep.

6.2 Materials and Methods

All protocols used in this research involving live animals were approved by the University Ethics Committee for Animal Experimentation and by the Italian Ministry of Health on 17 May 2010 (DM n° 97/2010-B). The project was divided into three phases: (1) bilateral induction of experimental tendon lesions in three groups of sheep; (2) monolateral application of PRP in one group, MSCs in the second group, and both PRP ? MSCs in the third group, with a clinical follow-up for all groups; and (3) post mortem histological analysis of tendons. For each sheep, the left hind limb received one of the three treatments, and the right hind limb was injected with the same volume of saline solution phosphate-buffered saline (PBS) and served as the internal control (placebo). Nine female Bergamasca sheep homogeneous for size and age were used in this study. Parasitological and biochemistry exams were carried out to ensure the good health of the subjects. Sheep were sedated by intravenous administration of 0.2 mg/kg Metadone (Eptadone) and 10 lg/kg Medetomidine (SedatorÒ) and positioned in lateral recumbency. Into each deep digital flexor tendon (DDFT), 500 IU filter sterilized bacterial Collagenase type 1A (C-9891, Sigma, Milan, Italy) was injected bilaterally (left and right hind legs) under ultrasonographic guidance. The injection was performed using a 23 gage needle positioned 15 cm at the proximal–distal direction from the calcaneal bone. Seven days after creation of the lesions, the animals were treated with one of the three methods described above. 1 ml of PRP (972 9 106 platelets) was prepared following standard procedures and inoculated as a liquid, taking advantage of the intrinsic coagulation factors that induce its gelification in situ once injected. One hundred milliliters of peripheral blood were collected from the jugular vein of sheep 8 weeks before treatment. To isolate MSCs, a Ficoll-paque solution (Amersham Biosciences) was used according to a protocol described previously for horse but adaptable for sheep 6 In Vivo Applications of Mesenchymal Stem Cells and Platelet-Rich Plasma 33

(Martinello et al. 2010). Isolated cells were also transfected with a plasmid expressing the green fluorescent protein (GFP). A total of 15 9 106 MSCs were used for each inoculation in 1 ml hyaluronic acid (Hyalgan, Fidia, Padova, Italy) solution as a vector solution for the second group of sheep and with 1 ml PRP for the third group. The efficacy of each treatment was monitored through clinical and ultrasound examinations conducted regularly until sacrifice of the animal 1 month after treatment. Histological and immunohistochemical analyses were carried out to evaluate different parameters involved in tendon regeneration: the alignment of the collagen fibers, the amount, and morphology of new cells, the grade of vascularization, the presence of inflammatory elements, and the expression of collagen types I and III together with matrix fibrillar proteins.

6.3 Results

The clinical and ultrasound examinations revealed that in all subjects, a local reaction to the collagenase injection was evident as well as the formation of a peritendinous edema associated with loss of ecogenity of the tissue. The clinical follow-up results were similar in all three groups of treated animals, and the treated limbs showed a quicker reduction of clinical signs due to the inflammatory response respect to control limbs. The ecographic controls at 7, 14, 21, and 28 days from the treatment indicated a slow but progressive filling of the anechoic area in the treated tendons. Regarding the histological profile, we observed complete disorganization of the control tendons (untreated lesions, placebo), whereas in the subjects treated with PRP, better tissue organization was seen, although tenoblasts appeared still round in shape. By contrast, in subjects treated with MSCs, better alignment of collagen fibers was observed, tenoblasts appeared similar to proper tenocytes, and a higher expression of collagen I was detected. The expression of collagen III was observed in the placebo and weakly in tendons treated with PRP but not in tendons treated with MSCs. The expression of matrix proteins cartilage oligomeric matrix protein (COMP) and biglycan was increased in all treated tendons. The presence and viability of injected cells was confirmed using an antibody against GFP in tendons treated with MSCs alone and PRP ? MSCs.

6.4 Discussion

Our data allow for speculation about the ability of adult stem cells to improve regenerative potential of tendon, because treatment with these cells stimulated mechanisms that resulted in histological features similar to native tendons. It is always very difficult to evaluate the quality of matrix produced using clinical and ultrasound investigations, and our results did not show significant differences in 34 M. Patruno et al. outcomes after the use different treatments (PRP, MSCs, and PRP ? MSCs). Histological examinations indicated that all treatments reduced the vascularization and inflammatory response. Treatment with PRP improved the general organiza- tion of injured tendons. However, the use of MSCs seemed to increase the quality of the repaired tendons according to histological features and because of a lack of expression of collagen III; the latter is a collagen type that is present in natural healing tendons but does not allow full recovery of the mechanical characteristics of tendons. Moreover, only tendons treated with MSCs exhibited an elongated morphology that is similar to that of quiescent tenocytes.

References

Cenni E, Savarino L, Perut F, Fotia C, Avnet S, Sabbioni G (2010) Background and rationale of platelet gel in orthopaedic surgery. Musculoskelet Surg 94:1–8 Crovace A, Lacitignola L, Rossi G, Francioso E (2010) Histological and immunohistochemical evaluation of autologous cultured bone marrow mesenchymal stem cells and bone marrow mononucleated cells in collagenase-induced tendinitis of equine superficial digital flexor tendon. Vet Med Int. doi:10.4061/2010/250978 Ferrari M, Corradi A, Lazzaretti M, De’Cillà M, Losi CG, Villa R, Lanfranchi A (2007) Adult stem cells: perspectives for therapeutic applications. Vet Res Commun 31(Suppl 1):1–8 Martinello T, Bronzini I, Maccatrozzo L, Iacopetti I, Sampaolesi M, Mascarello F, Patruno M (2010) Cryopreservation does not affect the stem characteristics of multipotent cells isolated from equine peripheral blood. Tissue Eng Part C 16:771–781 Watts AE, Yeager AE, Kopyov OV, Nixon AJ (2011) Fetal derived embryonic-like stem cells improve healing in a large animal flexor tendonitis model. Stem Cell Res Ther 2:4 doi:10.1186/scrt45 Chapter 7 Plasma Fatty Acid Profiles During the First Year in Dogs with and without Hip Dysplasia: Preliminary Results

L. Tidu, N. Bacciu, G. Rucco, S. Nardi, M. Santoro and B. Renaville

Abstract At the Military Veterinary Center of Grossetto, where operative dogs from the Italian Army are raised and trained, more than 70 % of the discharges for unfitness are due to articular pathologies like hip and elbow dysplasia. The aim of this study was to investigate fatty acid metabolism of dogs during the growth phase, and its modulation by a fish-based diet. Only 2 out of the 32 subjects were affected by articular pathologies during the study. Still, both subjects had lower levels of arachidonic acid and higher levels of docosahexaenoic acid. Moreover, we observed that the ratio of eicosapentaenoic to docosahexaenoic acids, an indicator of delta-6 desaturase activity, drops dramatically during the first year in the German shepherd.

Keywords Dog Á Dysplasia Á Fatty acids

L. Tidu Centro Militare Veterinario, Grosseto, Italy N. Bacciu agn Genetics GmbH, Davos, Switzerland G. Rucco Comando Logistico dell’Esercito, Roma, Italy S. Nardi Ospedale Militare Veterinario, Montelibretti, Italy M. Santoro Corso Addestramento e Allevamento Cani, Castiglione del Lago, Italy B. Renaville (&) Dipartimento Scienze degli Alimenti, Udine, Italy e-mail: [email protected]

C. Boiti et al. (eds.), Trends in Veterinary Sciences, 35 DOI: 10.1007/978-3-642-36488-4_7, Ó Springer-Verlag Berlin Heidelberg 2013 36 L. Tidu et al.

7.1 Introduction

Hip dysplasia is one of the most common inherited joint diseases affecting large breed dogs. It can lead to secondary hip osteoarthritis, resulting in pain and lameness. In dogs with hip dysplasia, the head of the femur does not fit well into the acetabulum, resulting in painful friction (Lust 1997). This pathology has a genetic basis but is also influenced by the environment. Animals with a similar genetic background may show different levels of dysplasia, and animals with a genetic background that predisposes them to develop the disease may remain clinically normal (Willis 1989). Nutrition is one of the main environmental factors. Indeed, a diet too rich in energy is a predisposing factor for dysplasia. Lipid metabolism can interact with bone metabolism through various mechanisms, such as through the balance between adipocytes and osteoblasts. Osteoblasts and marrow adipocytes have the same bone marrow progenitor, and bone loss is often associated with an expansion of marrow adipose tissue. Another mechanism is through the de novo synthesis of fat. Melhus et al. (2008) demonstrated that the fracture risk in elderly men is strictly correlated with the activity of stearoyl-CoA desaturase (SCD), which is a key enzyme of fat synthesis. Moreover, a diet rich in palmitic acid leads to a reduction of the calcium concentration in bones. Also involved is the synthesis of eicosanoids, hormones that generally act at a local level and are also known for their role in inflammatory processes. Eicosa- noids are synthesized from arachidonic acid, an omega-6 fatty acid. Among these, the prostaglandin (PG) E2 is known as an important mediator of bone remodu- lation by inducing osteoclast formation and increasing the synthesis of collagen and fibronectin from osteoblasts. At the Centro Militare Veterinario in Grosseto, Italy, where operative dogs from the Italian Army are raised and trained, more than 70 % of the discharges for unfitness are due to articular pathologies such as hip and elbow dysplasia. As this pathology has a high incidence and no treatment is proved to be effective, this study aimed to investigate fatty acid profiles and the effect of dietary poly-unsaturated fatty acids as a preventive solution.

7.2 Materials and Methods

The study was conducted on 32 puppies from the Italian Army that were born and raised at the Centro Militare Veterinario in Grosseto, Italy. The subjects included one litter of seven Belgian shepherds, three litters of German shepherds (six puppies each), and one litter of seven crossbreed dogs. Half of the puppies from each litter received a standard diet (Premium, Eukanuba) and the other half received a fish-based diet (SANYpet S.p.A.). Each diet was fed from weaning until the end of the experiment. Both hips and elbows were radiographed (MAXIVETÒ H.F. High Frequency, Multimage) at 5 and 12 months of age. For fatty acid analyses, blood samples were collected in EDTA tubes from the brachial 7 Plasma Fatty Acid Profiles 37 vein. Immediately after centrifugation (3,000 g, 10 min), plasma was stored at -20 °C until analysis. For each sample, fatty acids were extracted using the Bligh and Dyer method (1959), methylated (Renaville et al. 2006), and characterized using gas chromatography. A multivariate repeated measures analysis of variance (ANOVA) was carried out on the proportion of each fatty acid using Proc MIXED (SAS Institute Inc. 2010). This procedure allowed the obtainment of the variance– covariance matrix by specifying the direct (Kronecker) product structures of a first covariance matrix (across multivariate fatty acid observations) with an additional covariance matrix (repeated measures of a specific fatty acid across time or another factor). Ultimately, this procedure was used because it can efficiently deal with missing records.

7.3 Results

The concentrations of the following fatty acids were measured: palmitate (C16:0), palmitoleate (C16:1x9), stearate (C18:0), oleate (C18:1x9), vaccenate (C18:1x11), linoleate (C18:2x6), arachidonate (C20:4x6), eicosapentanoate (C20:5x3, EPA), docosaepentanoate(C22:5x3,DPA),anddocohexaenoate(C22:6x3,DHA). The profile of the plasma fatty acid concentrations in dogs fed a standard diet or a fish-based diet did not significantly differ. Unfortunately, in this study, only two dogs presented signs of articular pathologies, both of which showed hip dysplasia with signs of osteoarthrosis on radiography at 12 months of age. It is noteworthy that both affected animals presented lower levels of arachidonic acid and higher levels of both EPA and DHA (Fig. 7.1). An interesting observation from this study is that the ratio of EPA and DHA varies with age (P \ 0.01), and this was especially observed in German shepherd dogs (Fig. 7.2).

Fig. 7.1 Plasma fatty acid profile in littermate dogs affected by hip dysplasia from litter 1 and 2 38 L. Tidu et al.

Fig. 7.2 Ratio between the fatty acids DHA and EPA with age in German shepherds (n = 6)

7.4 Discussion

In this study, the onset of articular pathologies causing osteoarthritis was not related to the diet, as only two animals were affected by hip dysplasia. In the dysplastic subjects, both German shepherds, we observed lower levels of arachidonic acid. This may be due to local utilization of this fatty acid in the area of inflammation as a precursor of pro-inflammatory molecules, although the small number of affected dogs did not allow a valid statistical analysis. In the small number of studies performed in dogs (Roush et al. 2010; Fritsch 2010), other authors only used animals affected with osteoarthritis, in which supplementation of the diet with long chain polyunsaturated fatty acids (LC-PUFAs) improved the symptoms of affected dogs and increased the plasma concentrations of LC-PUFAs in affected and control group dogs. The fact that we did not observe significant differences between the two diets might have resulted from the fact that the healthy dog is able to synthesize LC-PUFAs from the other fatty acids present in the diet, because normal dogs have normal elongase and desaturase enzymatic activity. Therefore, what we still need to answer is whether healthy dogs should be supplemented or not. According to Walters et al. (2010), PUFA supplementation of normal dogs for 21 days leads to an increase of lipid peroxidation, revealed by a reduction of GSH and an increase of isoprostane in the urine. Our results suggest that a different metabolic mode for using LC-PUFAs is probably linked to genetic factors, for example, that the activity of D6-desaturase in German shepherds is different from that of other breeds studied, and decreases with age. It would be of interest to investigate the genetic role that D6-desaturase, or other enzymes implicated in lipid metabolism, has on the incidence of osteo-articular pathologies. This might reveal the implication of LC-PUFAs in the turn-over of anti-inflam- matory molecules and clarify if a specific role does exist in articular metabolism. We can conclude from this preliminary study, that the enzymatic activity of D6- desaturase gets reduced during the first year of life in German shepherds and that dysplastic dogs seems to have lower plasma concentrations of arachidonic acid. 7 Plasma Fatty Acid Profiles 39

Acknowledgments This research was supported by the Italian ‘‘Ministero della difesa,’’ grant name: ‘‘Ricerca sull’incidenza dell’artrosi nel cane militare e strategia di prevenzione e controllo’’.

References

Bligh E, Dyer W (1959) A rapid method of total lipid extraction and purification. Can J Biochem Physiol 37:911–917 Fritsch DA, Allen TA, Dodd CE, Jewell DE, Sixby KA, Leventhal PS, Brejda J, Hahn KA (2010) A multicenter study of the effect of dietary supplementation with fish oil omega-3 fatty acids on carprofen dosage in dogs with osteoarthritis. J Am Vet Med Assoc 236:535–539 Lust G (1997) An overview of the pathogenesis of canine hip dysplasia. J Am Vet Med Assoc 210:1443–1445 Melhus H, Risérus U, Warensjö E, Wernroth L, Jensevik K, Berglund L, Vessby B, Michaëlsson K (2008) A high activity index of stearoyl-CoA desaturase is associated with increased risk of fracture in men. Osteoporos Int 19:929–934 Renaville B, Mullen A, Moloney F, Larondelle Y, Schneider YJ, Roche HM (2006) Eicosapentaenoic acid and 3, 10 dithia stearic acid inhibit the desaturation of trans-vaccenic acid into cis-9, trans-11-conjugated linoleic acid through different pathways in Caco-2 and T84 cells. Br J Nutr 95:688–695 Roush JK, Cross AR, Renberg WC, Dodd CE, Sixby KA, Fritsch DA, Allen TA, Jewell DE, Richardson DC, Leventhal PS, Hahn KA (2010) Evaluation of the effects of dietary supplementation with fish oil omega-3 fatty acids on weight bearing in dogs with osteoarthritis. J Am Vet Med Assoc 236:67–73 SAS Institute Inc. (2010) SAS/IML Ò 9.22 User’s Guide. Cary, NC: SAS Institute Inc.Walters J, Hockett T, Ogilvie G, Fettman M (2010) Polyunsaturated Fatty Acid dietary supplementation induces lipid peroxidation in normal dogs. Vet Med Int, 2010 Article ID 619083, 4 pages. doi:10.4061/2010/619083 Willis M (1989) Hip dysplasia. In: Willis MB (ed) Genetics of the dog. Howell Book House, New York, pp 144–179 Chapter 8 Signaling in Sperm Activation: A Common Strategy for Different Organisms

I. Saponaro, N. Bernabò and M. Mattioli

Abstract The molecular events occurring during sperm activation were studied and compared in three species with very different biological features (sea urchin, C. elegans, and human) to verify if a common signaling strategy has evolved. The comparison was carried out by representing the molecular events involved in sperm activation as biological networks of nodes, (the molecules) connected by links (interactions). Analysis of the main topological parameters of the three networks was carried out and the results were compared. In all the cases, the sperm activation network displayed a scale-free topology and the most linked node was 2+ [Ca ]i. We suppose that this common architecture has evolved to fulfill important evolutionary advantages, ensuring robustness against random failure and efficiency and speed of signaling.

Keywords Spermatozoa Á Sperm activation Á Sea urchin Á Caenorhabditis elegans Á Human Á Biological networks

8.1 Introduction

Spermatozoa have unique characteristics that make them different from all other cell types: they are transcriptionally silent cells, with a peculiar organization of DNA, and autonomous motility. In addition, they are produced at the end of spermatogenesis in an inactive form, needing a complex series of chemical and physical changes collectively known as ‘‘activation’’ before acquiring functional competence. This process has long been a research focus because of its important

I. Saponaro Á N. Bernabò Á M. Mattioli (&) Department of Comparative Biomedical Sciences, Teramo, Italy e-mail: [email protected]

C. Boiti et al. (eds.), Trends in Veterinary Sciences, 41 DOI: 10.1007/978-3-642-36488-4_8, Ó Springer-Verlag Berlin Heidelberg 2013 42 I. Saponaro et al. implications in both basic sciences (developmental biology, endocrinology, and biochemistry), and in applied sciences (andrology, male infertility, and con- traception). Thanks to the available data, it was possible to verify that the spermatozoa of organisms with different reproductive strategies and sexual behaviors, such as sea urchins, Caenorhabditis elegans, and humans, are all required to complete activation before they can successfully fertilize oocytes. The sea urchin belongs to the phylum Echinodermata, lives in a marine envi- ronment, and has both female and male subjects. The sperm of the urchin, initially not motile within the male genital tract, is activated and begin to move only when released into the water where, attracted by chemotactic substances, they move toward the oocyte and fertilize it (Barnes 1982). C. elegans is a nematode worm, approximately 1 mm in length, that lives in the soil of tem- perate regions. There are two sexually different types of adults, hermaphrodites (99.95 % of individuals) and males (0.05 % of individuals), which provide the genetic variability. The eggs pass through four larval stages (L1-L4). During the L4 stage, the hermaphrodites produce sperm and eggs, whereas males produce only sperm. Thus, hermaphrodites can reproduce by self-fertilization, generating individuals identical to themselves (clones), or they can mate with the males (crossbreeding) (Fraire-Zamora and Cardullo 2010). In humans, reproduction takes place through a process of internal fertilization, whereby the erect male penis is inserted into the female’s vagina and the ejaculation of semen takes place. The sperm then pass through the female genital tract, until they reach the oviduct, where a complex series of events promotes the acquisition of fertilizing ability (capacitation) and fertilization. Starting from this premise, the purpose of this work was to verify if the acti- vation process of sperm has evolved as a species-specific signaling system or if there is a common strategy of behavior. To this aim, the biochemical events in question were modeled as biological networks consisting of nodes (molecules) connected by links (interactions between molecules). The identification of com- mon structural determinants in sperm activation was examined by comparing the statistical and topological properties of these networks.

8.2 Materials and Methods

Data were obtained from the international literature using PubMed (www.ncbi.nlm.nih.gov/pubmed/) and were organized into three databases (Microsoft Office Excel 2003, Redmond, WA) as previously described (Bernabò et al. 2010, 2011). From these databases, Cytoscape 2.6.3 software (www.cytoscape.org; Institute for System Biology, San Francisco, California) was used to identify networks, represented using the Cytoscape Spring-embedded Layout. Statistical and topological analyses were conducted using the Cytoscape plugin Network Analyzer (http://med.bioinf.mpi-inf.mpg.de/netanalyzer/help/ 2.6.1/index.html). 8 Signaling in Sperm Activation 43

Table 8.1 Main topological parameters of the sperm activation networks of sea urchins, C. elegans, and humans Sea urchin C. elegans Human N° nodes 127 100 151 N° links 175 132 202 Clustering coefficient 0.023 0.032 0.028 Diameter 23 23 20 Average n° neighbors 2.740 2.620 2.662 Characteristic path length 8.128 7.816 6.546 2+ 2+ 2+ Most linked node [Ca ]i [Ca ]i [Ca ]i N° links of most linked node 19 (14.6 %) 10 (10 %) 25 (17.1 %) The number of nodes represents the total number of molecules; the number of bonds represents the total number of interactions; clustering coefficient is calculated as CI = 2nI/k (k-1), where nI is the number of links that connect kI nodes to the I node; the diameter of the network is the greatest distance between two nodes; the average n° neighbors represents the average number of connections for each node; the characteristic path length indicates the distance between two nodes connected to each other

8.3 Results

In all three cases, the distribution of the number of links per node was in accor- dance with the equation y = ax-b (scale-free topology). The topological and statistical parameters that characterized the networks are summarized in Table 8.1. For all species, the clustering coefficient was low (0.023–0.032) and did not depend on the number of links per node (R2 always less than 0.250). The char- acteristic path length was always between 6.6 and 7.6. In all networks, the most 2+ linked node is the intracellular concentration of calcium [Ca ]i. Glycolysis and mitochondrial oxidative phosphorylation were common to all tested organisms (Fig. 8.1).

Fig. 8.1 Biological networks representing sperm activation process in a sea urchin, b C. elegans, and c humans. The size of each node is proportional to the number of connections, and the color gradient depends on the closeness centrality. This parameter, calculated as Cc (n) = 1/avg (L (n, m)), where L (n, m) is the length of the shortest path between two nodes n and m, may vary between 0 and 1, measuring the speed with which information passes from one node to other nodes 44 I. Saponaro et al.

8.4 Discussion

All analyzed networks had the typical topology of scale-free networks: a relatively small number of highly connected nodes (hubs), with the remaining majority of the nodes being poorly linked. This particular structure gives them a great robustness against accidental damage. These damages will have no significant effect on the overall system, because they will most likely involve poorly connected nodes, which represent more than 95 % of the nodes. In addition, calcium, the most linked node in all networks, emerges as a key molecule in signaling mechanisms of sperm activation. This ion, acting as a second messenger, converts extracellular stimuli into chemical responses in several pathways, including that of protein kinase C, protein kinase A, and actin, fundamental in the activation process (Darszon et al. 2005). The other common nodes are those involved in the energetic metabolism of sperm. In all cases, the energy is supplied in the form of ATP, through glycolysis and mitochondrial oxidative phosphorylation (Storey 2008). In conclusion, the use of biological networks has allowed us to describe and analyze the biological organization and functions of various cellular components of sperm, representing an important tool for understanding the principles that underlie the evolutionary strategies of living organisms. Indeed, it is evident that sea urchins, C. elegans, and humans have all evolved a common strategy for signaling and a similar arrangement in the metabolic processes of sperm activa- tion, as indicated by similar network topology. This strategy provides important evolutionary advantages such as robustness against accidental damage, efficiency, and speed of signal transduction.

References

Barnes RD (1982) Invertebrate zoology. Holt-Saunders International, Philadelphia, pp 961–981 Bernabò N, Mattioli M, Barboni B (2010) The spermatozoa caught in the net: the biological networks to study the male gametes post-ejaculatory life. BMC Syst Biol 4:87–98 Bernabò N, Berardinelli P, Mauro A, Russo V, Lucidi P, Mattioli M, Barboni B (2011) The role of actin in capacitation-related signaling: an in silico and in vitro study. BMC Syst Biol 5:47–59 Darszon A, Nishigaki T, Wood C, Treviño CL, Felix R, Beltrán C (2005) Calcium channels and Ca2+ fluctuations in sperm physiology. Int Rev Cytol 243:79–172 Fraire-Zamora JJ, Cardullo RA, (2010) The physiological acquisition of amoeboid motility in nematode sperm: is the tail the only thing the sperm lost? Mol Reprod Dev 77:739–750 Storey BT (2008) Mammalian sperm metabolism: oxygen and sugar, friend and foe. Int J Dev Biol 52:427–437 Chapter 9 Tenogenic Differentiation of Ovine Amniotic Stem Cells Co-Cultured with Tenocytes

Valentina Curini, V. Russo, O. Di Giacinto, A. Mauro, E. Galiffa, A. Pomante and B. Barboni

Abstract Tendons are constantly exposed to mechanical loads, particularly to injuries in sportsmen, workers, and elderly people. Spontaneous healing, slow and incomplete, often results in scar formation. An effective treatment that is able to stimulate complete tendon healing remains to be developed. The introduction of stem cells that are able to differentiate into mature tenocytes represents a prom- ising strategy for tendon repair. The aim of the present research was to assess whether co-culture with ovine or equine tenocytes can stimulate well-characterized ovine amniotic epithelial stem cells (AESCs) to differentiate into tenocytes. Both co-cultures reprogrammed ovine AESCs toward a tenogenic lineage as shown by the acquisition of a fusiform shape with a flat nucleus, by the secretion of collagen type I, and by the expression of tendon—related genes.

Keywords Ovine amniotic epithelial stem cells Á In vitro co-culture Á Tenogenic differentiation Á Ovine tenocytes Á Equine tenocytes

9.1 Introduction

Tendons must bear stresses that, especially among athletes and the elderly, can occasionally be excessive and cause serious tendon injuries. Currently, pharma- cological (anti-inflammatory) and nonpharmacologic (physical therapy and sur- gery) therapies are essentially aimed at providing symptomatic relief, but an

O. Di Giacinto Á E. Galiffa Á A. Pomante Department of Biomedical Comparative Sciences, University of Teramo, Teramo, Italy V. Curini (&) Á V. Russo Á A. Mauro Á B. Barboni Department of Biomedical Comparative Sciences, University of Teramo, StemTeCh Group, Teramo, Italy e-mail: [email protected]

C. Boiti et al. (eds.), Trends in Veterinary Sciences, 45 DOI: 10.1007/978-3-642-36488-4_9, Ó Springer-Verlag Berlin Heidelberg 2013 46 V. Curini et al. effective treatment that is able to stimulate a complete healing of the tendon has not yet been identified. Tenocytes are highly differentiated cells that have a limited replication potential, and low and slow repair capability for damaged tissue. For these reasons, instead of reconstruction of the tendon tissue, in most cases, scar tissue is formed, characterized by hypocellularity, which compromises the bio- mechanical properties of the tendon and causes significant dysfunction and dis- ability. Recently, to overcome the low number of cells present in tissues during the process of regeneration, attention has been paid to the use of regenerative cell therapies, aimed at stimulating cellular activity and extracellular matrix production at the site of the lesion (Longo et al. 2010). These new therapeutic approaches are conditioned by the availability of alternative cell sources to tenocytes, of appro- priate media in which to introduce these cells into injured tissues (scaffolds), and of appropriate protocols of in vitro tenogenic differentiation. For tendon repair, dermal fibroblasts (Liu et al. 2006), which have a reduced differentiation potential, embryonic stem cells, and mesenchymal progenitor cells, present in bone marrow, adipose tissue, or umbilical cord, were used (Harris et al. 2004). During osteogenic differentiation where cells are cultured in osteo-inducing medium, it is difficult to trigger tenogenic differentiation in the same cells. In fact, in vitro cultures have been tested with different cocktails of growth factors, such as BMP-12, FGF-2, and GDF-5, or transcription factors, such as Six1 and Smad8, but the composition of the medium needed to stimulate efficient differentiation has not yet been identified (Farng et al. 2008). Recently, the ability to program the differentiation of stem cells by co-culture with differentiated cells, such as osteoblasts and chondrocytes, was verified, which appeared to recreate the microenvironment of the tissues from which the cells were isolated (Luo et al. 2009). In this context, the influences exerted by differentiated tenocytes regarding re-programming of stem cells form the foundation upon which co-culture systems aimed at producing tenocytes for tissue engineering of tendons have been devel- oped; at the same time, these systems provide valuable information about changes that the same cells undergo if they are inserted into tendon tissues in need of repair. On this basis, the aim of the research was to determine whether the presence of tenocytes, isolated from sheep or horse tendon in co-culture, was able to promote the tenogenic differentiation of stem cells isolated from ovine amniotic membranes (amniotic epithelial stem cells, AESCs). The results obtained demonstrate that the co-culture system is able to induce the rapid transition of AESC morphology, accompanied by the activation of gene and protein expression within the tenogenic cell line.

9.2 Materials and Methods

Co-culture. Biological samples were collected from animals slaughtered at the abattoir. In particular, AESCs were isolated from fetuses, 25–35 cm in length, at 3 months of development. Calcaneal tendons were obtained from 1-year-old adult 9 Tenogenic Differentiation of Ovine Amniotic Stem Cells Co-Cultured 47 sheep. To isolate primary tenocytes, the tendons were dissected with a scalpel, and the central portion was divided into pieces approximately 1 mm in size. Each tendon piece was mechanically disaggregated under a stereomicroscope and then placed in a Petri dish with 2 ml medium consisting of a-MEM supplemented with 10 % FCS, 1 % penicillin/streptomycin, and 1 % L-glutamine. The plates were incubated at 38.5 °Cat5%CO2. After 10 days, the first colonies of tenocytes were found to have migrated from the tendon. At confluence, the cells were trypsinized with 0.05 % trypsin/EDTA, counted in a Burker chamber, and used for co-culture. The co-culture system was constructed by inserting ‘‘transwell cham- bers’’ (Corning), with 0.4 lm pores, into each well of a 12-well plate (Luo et al. 2009). On the bottom of each well, 3 9 103 AESCs were plated; the same number of tenocytes was placed on the membrane of the transwell. To each well was added 2 ml medium. In this system, the two cell types develop in the same medium but without coming into contact with each other. The plates were cultured at 38.5 °C at 5 % CO2 for 28 days. In parallel, for the same period of time and with the same soil, an additional plate containing only 3 9 103 AESCs in each well was cultured as a negative control for differentiation. Morphological evaluation. The AESCs in co-culture were observed under a stereomicroscope to assess possible morphological changes. Assessment of differentiation. Differentiation was analyzed by immunocyto- chemistry and RT-PCR after 14 and 28 days of culture. For immunocytochemis- try, cells were fixed in 4 % paraformaldehyde/PBS for 15 min at room temperature and subjected to antibodies against an epithelial factor (cytokeratin), a mesen- chymal factor (a-SMA), and collagen type 1 (COL I). For RT-PCR, total RNA was extracted using TriReagent (Sigma), reverse transcribed into cDNA, and used to amplify the genes for COL I and COL III and osteocalcin (OCN). Xeno co-culture. Ovine AESCs (3 9 103) were placed in co-culture, as pre- viously described, with equine tenocytes obtained from in vitro culture as described for ovine. Cell morphology, expression of cytokeratin, a-SMA, and COL I by immunocytochemistry, and expression of specific tenocyte genes by RT-PCR were assessed.

9.3 Results

The AESCs co-cultured with ovine primary tenocytes acquired a fusiform appearance similar to that of tendon cells and formed circular cell aggregates (spheroids) after 15 days in culture. At 28 days, the spheroids organized them- selves into elongated, tendon-like, three-dimensional (3D) structures. The char- acterization of the AESCs in co-culture, performed by immunocytochemistry, showed the presence of cytokeratin only in cells of the monolayer but not in cells organized within the 3D structures. In contrast, a-SMA was detected only in cells of the tendon-like structures. Finally, immunocytochemical analyses showed, in the first 2 weeks of culture, the synthesis of intracellular COL I and its deposition in the extracellular matrix, with abundant collagen fibers arranged within the 3D 48 V. Curini et al. structures. Additionally, RT-PCR analysis confirmed tenogenic differentiation of the AESCs, showing typical expression patterns of the cells of tendon tissues: an increase in the expression levels of COL I and OCN and reduced expression levels of COL III. In control cultures, no signs of differentiation by AESCs were observed. AESCs co-cultured with equine tenocytes underwent morphological transformation into spindle cells and aggregated into spheroids during the first week of culture. After 28 days, in contrast to ovine co-cultures, the AESC-equine tenocyte co-cultures did not develop 3D structures. The AESCs produced COL I, did not deposit it outside the cell, and did not form organized fibers. In addition, RT-PCR results reveal lower expression levels of the genes of tenogenic differ- entiation than those found in AESCs co-cultured with ovine tenocytes. In both cases, the influences exerted by the tenocytes do not require direct contact with the cells but are mediated by soluble factors.

9.4 Discussion

Ovine AESCs have characteristics that are ideal for extended use in regenerative therapies for damaged organs and tissues, including plasticity, the ease with which they can be retrieved, high proliferative potential, and low immunogenicity (Insausti et al. 2010). The results of this research confirm these properties and extend their differentiation capabilities to tenogenic cell lines when cultured in the presence of primary tenocytes. AESCs in co-culture, in fact, rapidly acquire the typical fusiform appearance of tenocytes and are organized into cellular bundles with 3D structures similar to a tendon. The presence of cytokeratin in the cells of the monolayer and of a-SMA in cells of the tendon-like structures demonstrates the transition of AESCs from an initial epithelial state to a mesenchymal state. In addition, during culture, there is an increase in collagen, which is then deposited outside the cells to form well-organized fibrils that are oriented along the longi- tudinal axis of the 3D structures. Finally, tenogenic differentiation was confirmed by the study of the expression of the COL I, COL III, and OCN genes. At the end of the culture period, AESCs display a pattern of expression similar to that seen in tenocytes themselves. Even equine tenocytes are able to support the tenogenic reprogramming of ovine stem cells, although this process is slower than that induced by sheep tenocytes. This observation suggests that ovine AESCs can regenerate after xenotransplantation into injured tendons of other species, like horse. In conclusion, co-culture is an efficient method for differentiation, which is based on the stimulatory effects of paracrine factors released by differentiated cells that recreate a tendon-like microenvironment. The co-culture system allows us to bypass normal operating limits that control tenogenic differentiation, and it represents a promising strategy to produce teno- cytes and/or engineered tendons that are increasingly required for the regeneration of injured tendons by cell therapies. 9 Tenogenic Differentiation of Ovine Amniotic Stem Cells Co-Cultured 49

Acknowledgments This work was supported by a Tercas Foundation grant.

References

Farng E, Urdaneta AR, Barba D, Esmende S, McAllister DR (2008) The effects of GDF-5 and uniaxial strain on mesenchymal stem cells in 3-D culture. Clin Orthop Relat Res 466:1930–1937 Harris MT, Butler DL, Boivin GP, Florer JB, Schantz EJ, Wenstrup RJ (2004) Mesenchymal stem cells used for rabbit tendon repair can form ectopic bone and express alkaline phosphatase activity in constructs. J Orthop Res 22:998–1003 Insausti CL, Blanquer M, Bleda P, Iniesta P, Majado MJ, Castellanos G, Moraleda JM (2010) The amniotic membrane as a source of stem cells. Histol Histopathol 25:91–98 Liu W, Chen B, Deng D, Xu F, Cui L, Cao Y (2006) Repair of tendon defect with dermal fibroblast engineered tendon in a porcine model. Tissue Eng 12:775–788 Longo UG, Lamberti A, Maffulli N, Denaro V (2010) Tissue engineered biological augmentation for tendon healing: a systematic review. Brit Med Bull 98:31–59 Luo Q, Song G, Song Y, Xu B, Qin J, Shi Y (2009) Indirect co-culture with tenocytes promotes proliferation and mRNA expression of tendon/ligament related genes in rat bone marrow mesenchymal stem cells. Cytotechnology 61:1–10 Chapter 10 Cortisol Changes in Pregnant and Post-Partum Ewes: Effects of Single or Twin Births

E. Fazio, M. Manera, S. Mignacca, P. Medica and A. Ferlazzo

Abstract Cortisol is a reliable physiological index of stress in sheep. Pregnancy and post-partum conditions, the influence of a single or a twin birth, and simultaneous milking may represent significant sources of stress for the mother and lambs. We characterized the adrenal response in ewes during late pregnancy and post-partum by accounting for the possible influence of single or twin birth on changes in circulating cortisol levels. Cortisol levels were in agreement with normal physiological ranges reported for sheep and with previous data obtained from lactating ewes. There was a significant increase in cortisol levels in post- partum compared with pregnancy, and there was a comparable trend between Comisana and Pinzirita crossbreds. With regard to the influence of single or twin birth, post-partum cortisol levels were higher in ewes with both single and twin birth. In addition, post-partum cortisol levels were increased in ewes with male births and both female and male births.

Keywords Ewe Cortisol Pregnancy Single birth Twin birth Post-partum

10.1 Introduction

Reproductive function involves significant endocrine changes and intense meta- bolic activity. Adrenocortical function, pregnancy, and post-partum period are physiologically correlated. In sheep, there is also evolutionary evidence to support the interaction of the mother-fetus relationship, the effects of single or twin birth, and the hypothalamus–pituitary–adrenal axis responses (Smith and Dobson 2002; Phogat et al. 1999). The productive utility and performance can be modified by

E. Fazio (&) M. Manera S. Mignacca P. Medica A. Ferlazzo Dipartimento di Morfologia, Biochimica, Fisiologia e Produzioni Animali, Università di Messina, Messina, Italy e-mail: [email protected]

C. Boiti et al. (eds.), Trends in Veterinary Sciences, 51 DOI: 10.1007/978-3-642-36488-4_10, Ó Springer-Verlag Berlin Heidelberg 2013 52 E. Fazio et al. endocrine changes occurring in lactating animals. In addition, the amplitude of adrenocortical response may correlate with high or low milk yield (Fazio et al. 1994). The aim of this study was to characterize mechanisms involved in ovine adrenocortical response to psychophysical stress during the pre- and post-partum period. We also compared our results with values previously obtained from ewes in different conditions of pregnancy and post-partum periods (Fazio et al. 1994; Medica et al. 2009).

10.2 Materials and Methods

The study design consisted of 20 healthy ewes (10 Comisana and 10 Pinzirita crossbreds) that were 2–3 years old. These ewes were at the late stage of pregnancy (approximately 14 day pre-partum) or approximately 9 days post-partum. All animals had access to free range pasture and were provided with hay once per day. The animals originated from a flock of 200 ewes at a farm in Enna, Sicily (37°340000N. latitude; 14°160000E. longitude) that was located 200 m above sea level. Pregnant ewes that were near parturition were separated from the flock and confined to a pen with 80 adult animals. Because they did not have free access to pasture they were fed hay ad libitum, 100 g of chard beet pulp, and concentrated mix (corn and broad bean). The amount fed was increased as pregnancy progressed, until they were fed 600 g as lactating ewes during the post-partum period. Blood samples were taken once per week from the jugular vein (10:00–12:30 a.m.), from October (at the late stage of pregnancy) to November (birth period: 13 single births and 7 twin births), and in the first four post-partum weeks. Serum cortisol concentrations were analyzed in duplicate using a commercially available immunoenzyme kit (RADIM Diagnostics, Pomezia, Roma, Italy). A one-way repeated measures analysis of variance (RM-ANOVA) was performed to deter- mine whether there was a significant effect of pregnancy state on cortisol levels. The Student’s unpaired t test was used to determine whether there were significant differences between Comisana and Pinzirita crossbreds and between single and twin births. The Student’s paired t test was used to compare pre- and post-partum cortisol values.

10.3 Results

Cortisol levels were higher in the post-partum than in the pre-partum period (Table 10.1;(P \ 0.001). This trend was also observed in the Comisana (P \ 0.001) and Pinzirita (P \ 0.02) breeds. When comparing different breeds, there were no significant differences between pre- and post-partum values. Therefore, the analysis for the influence of single and twin birth was performed using all subjects combined, irrespective of breed. 10 Cortisol Changes in Pregnant and Post-Partum Ewes 53

Table 10.1 Serum cortisol levels (mean ± SD) in pre- and post-partum Comisana and Pinzirita crossbred ewes and in ewes with single or twin birth lambs Cortisol (nmol/L) Pre-partum Post-partum ANOVA F= P Total 87.96 ± 44.15 131.39 ± 56.68*** 1.566 n.s. Comisana crossbred 84.12 ± 40.73 131.91 ± 58.98*** 3.615 \0.001 Pinzerita crossbred 91.81 ± 47.57 130.87 ± 54.39* 2.650 \0.02 Single birth 90.93 ± 47.25 125.20 ± 41.68** 1.285 n.s. Twin birth 86.86 ± 41.86 144.64 ± 79.77*** 3.631 \0.001 Vs. pre-partum: *P \ 0.02; **P \ 0.01; ***P \ 0.001

Table 10.2 Lamb gender comparisons of serum cortisol levels (mean ± SD) in pre- and post- partum Comisana and Pinzirita crossbred ewes Cortisol (nmol/L) Pre-partum Post-partum ANOVA FP Females 92.80 ± 53.80 113.54 ± 51.44 1.094 n.s. Males 87.09 ± 38.20 139.77 ± 55.13** 2.082 n.s. Males and females 88.33 ± 44.25 178.33 ± 75.29* 2.895 n.s. Vs. pre-partum: *P \ 0.05; **P \ 0.001

Compared to pre-partum values, both ewes with single birth (P \ 0.01) and those with twin birth (P \ 0.001) had higher cortisol levels in post-partum (Table 10.2). One-way RM-ANOVA showed a significant effect of only twin birth (F = 3.631; P \ 0.001) on changes in cortisol levels. Cortisol levels were sig- nificantly higher in post-partum with male birth (P \ 0.001) or male and female births (P \ 0.05) than the pre-partum values (Table 10.2). However, there were no significant effects of gender on cortisol changes.

10.4 Discussion

Ewe cortisol values were in agreement with the physiological range reported for sheep (Kaneko 1989; Phogat et al. 1999) and with previous data obtained from lactating ewes (Fazio et al. 2000; Medica et al. 2005). Our results confirmed the observation that ovine species maintain lower cortisol levels during the pre-partum compared with the post-partum period (Fazio et al. 1994). Lower cortisol levels in the mother during pre-partum could be due to a secondary negative feedback effect stimulated by high fetal cortisol levels that are even higher in ewes with twin pregnancy. Hence, the highest mean cortisol levels observed in post-partum could be related to the negative correlation between cortisol and circulating estrogen levels that significantly increase in ewes during late pregnancy, with a decrease of circulating cortisol levels that occur at the beginning of post-partum (Smith and 54 E. Fazio et al.

Dobson 2002). Moreover, the highest cortisol levels in post-partum could also be an expression of greater metabolic stress on mothers with twin birth compared with mothers of one lamb. However, male only or male and female fetuses induced significant maternal cortisol increases in post-partum. In addition, it is possible that the increase of cortisol levels in post-partum could be due to confinement of animals near parturition and to the start of lactation, irrespective of breed and number of offspring. Cortisol changes were within the wide range reported in sheep for various physiological conditions. Nevertheless, the blood sampling time, the different breeding for meat or milk production and nutritional factors may also be relevant considerations.

References

Fazio E, Murania C, Piccione G, Criscione G (1994) Iodotironine totali e libere e cortisolo del siero in pecore a differente produzione lattea. Atti SIPAOC XI:53–56 Fazio E, Medica P, Aronica V, Alberghina D, Ferlazzo A (2000) Adrenocortical response to confinement in the presence of co-specifics after weaning of lambs. Proc Internat Congr FeMeSPRum 8:88–92 Kaneko JJ (1989) Clinical biochemistry of domestic animals. In: Kaneko JJ (ed) Academic, San Diego pp 886–891 Medica P, Cravana C, Aronica V, Fazio E, Ferlazzo A (2005) Effetto differenziato degli stress da contenimento e da tosatura e del loro grado di novità sulla risposta dell’asse ipofisi- corticosurrene nella Pecora. Atti Soc it Sci vet 59:49–50 Medica P, Mignacca S, Fazio E, Cravana C, Ferlazzo A (2009) Risposta corticosurrenalica di pecore vuote e gravide allo stress da trasporto di media durata. Atti Soc it Sci vet 63:50–52 Phogat JB, Smith RF, Dobson H (1999) Effect of transport on pituitary responsiveness to exogenous pulsatile GnRH and oestradiol-induced LH release in intact ewes. J Reprod Fertil 116:9–18 Smith RF, Dobson H (2002) Hormonal interactions within the hypothalamus and pituitary with respect to stress and reproduction in sheep. Domest Anim Endocrinol 23:75–85 Part II Animal Pathology Chapter 11 Papillary and Chordoid Meningioma in the Dog: Morphological Findings and Histological Grading

S. Pavone and M. T. Mandara

Abstract Based on histomorphology, biological behavior, and outcome predic- tion, human papillary meningioma (PM) and chordoid meningioma (CM) are considered as grades III and II, respectively. In dogs, they are rare histotypes of which only PM is recognized in the domestic animal World Health Organization (WHO) classification system as a benign tumor. This study describes four cases of CM and seven cases of PM in dogs, providing contributions to their histomor- phological features in this species. Applying the current human WHO histological classification system, we identified PM of grades I and II, and CM of grades I and III. To verify a correlation between the histological grade and the biological behavior, it is necessary to collect sufficient data on post-surgery recurrence rate and survival time in dogs.

Keywords Dog Á Meningioma Á Papillary Á Chordoid Á Grading

11.1 Introduction

Meningiomas are common central nervous system (CNS) tumors in both humans (Perry et al. 2007) and domestic animals (Summers et al. 1995; Kostner et al. 1999). In humans, meningiomas are generally considered benign, corresponding to grade I in the World Health Organization (WHO) histological classification system (Perry et al. 2007). They include meningothelial, fibrous, transitional, psammomatous, angiomatous, microcystic, secretory, lymphoplasmacyte-rich, and metaplastic histological variants. On the other hand, specific histological variants of human

S. Pavone (&) Á M. T. Mandara Dipartimento di Scienze Biopatologiche ed Igiene delle Produzioni Animali e Alimentari, Facoltà di Medicina Veterinaria, Università degli Studi di Perugia, Via S. Costanzo 4 06126 Perugia, Italy e-mail: [email protected]

C. Boiti et al. (eds.), Trends in Veterinary Sciences, 57 DOI: 10.1007/978-3-642-36488-4_11, Ó Springer-Verlag Berlin Heidelberg 2013 58 S. Pavone and M. T. Mandara meningioma characterized by particular combinations of morphological parameters are associated with a less favorable prognosis. In these cases, meningiomas corre- spond to grades II (atypical, chordoid, and clear cell meningioma) and III (anaplastic or malignant meningioma, papillary, and rhabdoid meningioma) (Perry et al. 2007). Conversely, based on traditional histological parameters of malignancy, the current domestic animal WHO histological classification system of meningiomas catego- rized these tumors into two major groups: benign (meningothelial, fibroblastic, transitional, psammomatous, angiomatous, papillary, granular cell, and myxoid meningioma) and anaplastic meningiomas (Koestner et al. 1999).

11.2 Materials and Methods

Seven papillary meningiomas (PM) and four chordoid meningiomas (CM) diag- nosed in the last 3 years were selected for this study. The definitive diagnosis of PM and CM was performed by routine histological examination. PMs were observed in dogs aged 7–14 years. CM were diagnosed in dogs aged 4–16 years. PMs were intracranial, except for a case occurring at the C1–C2 spinal segment level. On the other hand, all CM developed in the spinal cord. The selected tumors were graded by applying the morphological criteria used in the human WHO histological classification (Perry et al. 2007). Therefore, meningiomas showing low mitotic activity [B4 mitosis/10 high-power fields (HPF)] were classified grade I (Perry et al. 2007). Diagnostic criteria used for atypical meningiomas (grade II) were mitotic index (C4 mitoses/10 HPF) or at least three of the following criteria: patternless sheets, increased cellularity, high nucleus/cytoplasm (N/C) ratio, ma- cronucleoli, spontaneous necrosis, or brain invasion (Perry et al. 2007). Finally, meningiomas showing features of frank anaplasia or at least 20 mitoses/10 HPF were considered grade III (Perry et al. 2007). In addition, immunolabeling was carried out by the avidin–biotin complex (ABC) method using antibodies against Ki-67 antigen (clone MIB-1), to evaluate the proliferative index of the tumors.

11.3 Results

Histological examination of the seven tumors classified as PM showed typical papillary figures containing a central core of connective tissue and small and medium-sized vessels (Fig. 11.1). The neoplastic cells had abundant eosinophilic cytoplasm and round-to-oval nuclei, and were arranged perpendicularly to the vascular structures. Anisocytosis and anisokaryosis were moderate in all cases; mitotic figures were rare (\4 mitoses/10 HPF), except for a case with 10 mitoses/ 10 HPF. In some cases, papillary growth pattern was mingled with areas of meningothelial, microcystic, and fibroblastic type. However, in all cases papillary growth was predominant. Spontaneous foci of necrosis were detected in three Papillary and Chordoid Meningioma in the Dog 59

Fig. 11.1 Twelve-year-old female Siberian Husky dog (left panel). Retrobulbar papillary meningioma. a The neoplastic tissue is characterized by a perivascular papillary pattern (H&E, Bar = 100 lm). Sixteen-year-old female mixed-breed dog (right panel). Spinal chordoid meningioma (C2). b The neoplastic cells are typically arranged in cords enmeshed in an abundant mucinous matrix (H&E, Bar = 20 lm) cases. Based on the criteria applied in human histological grading, five PM were considered grade I. Two meningiomas were classified as grade II according to the infiltration of adjacent brain tissue observed histologically and reported by surgical history. Neutrophilic infiltration was observed in all cases, except one. The pro- liferative index ranged from 9 to 24 mitoses/10 HPF in grade I PM, and from 5 to 59 mitoses/10 HPF in grade II papillary tumors. Histologically, CM consisted of polyhedral to spindle-shaped bipolar cells, and multipolar cells, arranged in cords and nests enmeshed in a weakly basophilic mucinous matrix (Fig. 11.1). Sometimes, the neoplastic cells showed abundant cytoplasmic vacuolizations. Anisokaryosis and anisocytosis were moderate to marked. Two cases showed severe cellular atypia (high N/C ratio, irregular or vesicular nuclei, and macronucleoli). The mitotic index was low in two cases (\4 mitoses/10 HPF) and high in the remaining two cases (25–30 mitoses/10 HPF). In two cases, the prevalent chordoid pattern was associated with meningothelial and microcystic areas. Foci of spontaneous necrosis were detected in three cases. Based on the criteria applied in human histological grading, two CM were considered grade I, and the remaining two cases were grade III. Infiltration of the spinal cord was not detected in any case. The proliferative index ranged from 19 to 28 mitoses/10 HPF in grade I CM and from 200 to 381 mitoses/10 HPF grade III tumors.

11.4 Discussion

PM is a rare histological variant of human beings and dogs. In humans, this tumor represents 1.0–2.5 % of all meningiomas and tends to affect younger patients, including children (Russel et al. 1989). Because of its high invasiveness of the brain, recurrence, and metastasis rate, PM is considered grade III in man (Perry et al. 2007). PM has been reported in dogs by Schulman et al. (1992), Summers 60 S. Pavone and M. T. Mandara et al. (1995), and Kaldrymidou et al. (2008), and it is currently included in the group of benign meningiomas in the histological classification of CNS tumors of domestic animals (WHO) (Koestner et al. 1999). In the present study, canine PM presented histological features reported in the human form. However, the tumor did not affect young patients. Dogs older than 7 years were the most affected, in line with the average age of onset for canine meningiomas (Patnaik et al. 1986). Applying the morphological parameters used in grading of human meningiomas (WHO), in our study, PMs were characterized by moderate histological malig- nancy consistent with grades I and II. Moreover, they occurred mainly in the brain, occasionally associated with brain invasion, as documented in two cases. This tumor showed foci of spontaneous necrosis and neutrophilic inflammation. The proliferation index ranged from low to medium in line with the mitotic index detected by routine histological examination. CM is a rare histological variant in humans and represents 0.5 % of all menin- giomas (Couce et al. 2000). In dogs, it has been reported only in two cases (Sturges et al. 2008). However, the morphological and immunophenotypic features, and the biological behavior of this variant are not available in dogs. As in humans, in this study canine CM showed histological features suggestive of chordoma, consisting in polymorphic, often vacuolated cells, arranged in small nests or cords. In humans, since the recurrence rate of CM and its correlation with hemato- logical disorders (e.g., Castleman’s disease) (Perry et al. 2007), this tumor is considered grade II. Conversely, in the present study, two cases of CM were classified as grade I and two cases were classified as grade III. Brain invasion was not detected in any case. Indeed, all CM consisted of a well-circumscribed neo- plasia with an easy surgically smooth cleavage plane. This variant seemed to frequently develop spontaneous foci of necrosis and was associated with mild inflammatory infiltration. The proliferative index, which was medium for menin- giomas classified as grade I and very high in those classified as grade III, appeared in line with the mitotic index detected by routine histological examination and correlated to grade of tumor. It appeared to exponentially increase in grade III CM. In conclusion, this study has reported the morphological features of two rare histological variants of canine meningioma, attempting to provide histological grading according to the morphological criteria used in the human classification of meningiomas. Indeed, in the last several years, veterinary scientific community has been calling for a grading scheme that is able to recognize a further form of meningioma intermediate between benign and frankly anaplastic form in dogs. Contrary to the current domestic animal classification system (WHO) (Koestner et al. 1999), in the present study, we identified a variable range of malignancy morphological features for canine PM, including brain invasiveness, supporting a potential high rate of recurrence for this tumor, as seen in humans. Furthermore, this study confirmed the presence of CM in dogs as a distinct rare histological variant. Also this histotype this tumor is characterized by highly variable malig- nancy morphological features, resulting in both grades I and III. Papillary and Chordoid Meningioma in the Dog 61

These preliminary results call for an assessment of the real biological behavior of canine meningioma. The high histological variability expressed by the same histotype. For this aim, further studies examining post-surgery recurrence rate and survival time must be assessed for canine PM and CM.

References

Couce ME, Aker FV, Scheithauer BW (2000) Chordoid meningioma: a clinicopathologic study of 42 cases. Am J Surg Pathol 24:899–905 Kaldrymidou E, Polizopoulou ZS, Papaioannou N, Koutinas AF, Poutahidis T, Papadopoulos T (2008) Papillary meningioma in the dog: a clinicopathological study of two cases. J Comp Path 124(3):227–230 Koestner A, Bilzer T, Fatzer R, Schulman FY, Summers BA, Van Winkle TJ (1999) Histological classification of tumors of the nervous system of domestic animals. Schulman FY (ed) WHO, Armed Forces Institute of Pathology, American Registry of Pathology, Washington DC Patnaik AK, Kay WJ, Hurvitz AI (1986) Intracranial meningioma: a comparative pathologic study of 28 dogs. Vet Path 23:369–373 Perry A, Louis DN, Scheithauer BW, Budka H, von Diemling A (2007) Meningiomas. In: Louis DN, Cavenee WK, Ohgaki H, Wiestler OD (eds) World Health Organization classification of tumours of the central nervous system, 4th edn. World Health Organization, Lyon, pp 164–172 Russel DS, Rubinstein LJ (1989) Tumours of the meninges and related tissues. In: Russel DS, Rubinstein LJ (eds) Pathology of tumours of the nervous system, 5th edn. Edward Arnold, London, pp 481–483 Schulman FY, Carpenter JL, Ribas JL, Brum DE (1992) Cystic papillary meningioma in the sella turcica of a dog. JAVMA 200(1):67–69 Sturges BK, Dickinson PJ, Bollen AW, Koblick PD, Kass PH, Vernau KM, Knipe MF, LeCouteur RA, Higgins RJ (2008) Magnetic resonance imaging and histological classification of intracranial meningiomas in 112 dogs. J Vet Intern Med 22:586–595 Summers BA, Cummings JF, DeLahunta A (1995) Tumours of the central nervous system. In: Summers BA, Cummings JF, DeLahunta A (eds) Veterinary neuropathology. Mosby, St. Louis, pp 351–401 Chapter 12 Detection of Neutralizing Antibodies in Pigs Inoculated with an Inactivated Vaccine Against Porcine Circovirus Type 2 (PCV2)

S. Petrini, M. Paniccià, V. Silenzi, F. Ciuti, M. Bresaola, M. Fortunati, G. M. De Mia, G. Perugini and M. Ferrari

Abstract Currently, few information is available on the stimulation of neutralizing antibodies (NA) to porcine circovirus type 2 (PCV2). The aim of the present study was to characterize the humoral response, particularly the production of NA in pigs inoculated with an inactivated vaccine against PCV2. Twenty-five conventional pigs, seronegative to PCV2, were divided into two groups. Group 1 was composed of 14 animals that were injected by intramuscular route with 1 ml of inactivated vaccine. Group 2 was composed of 11 pigs and was used as a negative control. ELISA antibodies and NA were evaluated and a virological investigation was performed. The vaccine did not induce any clinical signs in immunized pigs. The animals in Group 1 developed IgG antibodies, detected by ELISA tests, between 14 and 42 post-vaccination days (PVD). In the same group, NA were produced between 35 and 42 PVD. In Group 2, neither ELISA antibodies nor NA were produced. No virus was isolated from any of the pigs during the experimental period.

Keywords Pigs Á PCV2 Á Vaccination Á Virus neutralization test

12.1 Introduction

Porcine circovirus type 2 (PCV2) infection is associated to several diseases including: (1) postweaning multisystemic wasting syndrome (PMWS); (2) porcine dermatitis and nephropathy syndrome (PDNS); (3) proliferative and necrotizing pneumonia (PNP); (4) porcine respiratory disease complex (PRDC); (5) enteritis;

S. Petrini (&) Á M. Paniccià Á V. Silenzi Á F. Ciuti Á M.Fortunati Á G. M. De Mia Á G. Perugini Istituto Zooprofilattico Sperimentale Umbria-Marche, 06126 Perugia, Italy e-mail: [email protected] M. Bresaola Á M. Ferrari Istituto Zooprofilattico Sperimentale Lombardia and Emilia-Romagna, 25124 Brescia, Italy

C. Boiti et al. (eds.), Trends in Veterinary Sciences, 63 DOI: 10.1007/978-3-642-36488-4_12, Ó Springer-Verlag Berlin Heidelberg 2013 64 S. Petrini et al. and (6) reproductive disorders. Such diseases (Petrini et al. 2011) are currently designated as porcine circovirus diseases (PCVD). Among them, PMWS is the most studied to date. The immune response of the host organism is crucial in the path- ogenesis of the disease. The role played by neutralizing antibodies (NA) in the course of PMWS or after vaccination against PCV2 infection is not yet clearly understood. Previous studies (Meerts et al. 2005, 2006) reported that NA against PCV2 are produced during the course of experimental or natural PCV2 infection, but are not evident during viraemia or when PCV2 is localized in target tissues (lymphoid tissue, lung, etc.). In Europe, three commercial vaccines are available for the control of PCV2 infection. Two of them are formulated with a subunit vaccine containing PCV2 capsid protein while the third consists of inactivated PCV2 vac- cine with a paraffin oil adjuvant. So far, immunity induced by the above-mentioned vaccines has been studied only by evaluating immunoglobulin isotype M (IgM) or immunoglobulin isotype G (IgG) production by enzyme-linked immunosorbent assay (ELISA) test. Moreover, as already highlighted, NA production after vacci- nation has not been well studied. Thus, the present study aimed to develop a virus neutralization test (VNT) against PCV2 for the evaluation of NA in conventional pigs previously vaccinated with an inactivated PCV2 vaccine.

12.2 Materials and Methods

Twenty-five conventional pigs of 2–3 months of age, seronegative for PCV2, were used. The pigs were tested for PCV2 twice in 1 month at their respective farms before being transported to our animal facilities. Pigs were divided into two groups in separate boxes. Group 1 was composed of 14 pigs injected with 1 ml of inac- tivated PCV2 vaccine into the neck muscle. Group 2 was composed of 11 pigs that received no treatment and served as a negative control. The animals in Group 1 were observed daily for local or general adverse reactions. Skin response at the inoculation site was evaluated and rectal temper- ature was taken. Nasal swabs and serum samples were collected from each pig on post-vaccination days (PVD) 0, 7, 14, 21, 28, 35, and 42. Nasal swabs were used for virus isolation on porcine kidney clone 28 (PK15-28) cell line. For the viral identification, an anti-PCV2 polyclonal antiserum conjugated to fluorescein isothiocyanate was used (WMRD, Pullman, USA). Serum IgM and IgG levels were determined by a commercial ELISA test Ò Ò (Ingezim PCV IgG ; Ingezim PCV IgM , Ingenasa, Spain) according to manu- facturer recommendations. For the optimization of VNT conditions, inactivated (56 °C, 30 min) as well as non-inactivated serums were used. Several concentrations of PCV2 (100 TCID50/ 0.05 ml, 200 TCID50/0.05 ml, 400 TCID50/0.05 ml) were seeded in each well; different incubation times (36, 48, and 72 h) were determined for different mix- tures of serum samples, PCV2, and cell cultures. Briefly, 50 ll of each tested serum (inactivated or non-inactivated) were serially diluted from 1:2 to 1:4096 in Earle-minimum essential medium (E-MEM), and 12 Detection of Neutralizing Antibodies in Pigs Inoculated with an Inactivated Vaccine 65 supplemented with 5 % fetal bovine serum (FBS) and antibiotics (10,000 IU/ml penicillin, 5,000 IU/ml streptomycin, and 5 lg/ml amphotericin B). Later, different concentrations of a virulent PCV2 62297/08 LN strain previ- 6,50 ously produced on PK15-28 cell lines, with a titer of 10 TCID50/0.1 ml, were inoculated into each well (Chamber Slide, Nunc, DK) for 1 h at 37 °C in the presence of 5 % CO2. After incubation, PK15-28 cell lines were added at a concentration of 2 9 104/ well and incubated for 36, 42, or 72 h. Then, the cell lines were fixed in absolute acetone for 30 min at room tem- perature and then left in an acetone/methanol solution (75/25) at room temperature for 20 min. Successively, they were dried for 10 min at 37 °C. Finally, the cell lines were incubated for 30 min at 37 °C with an anti-PCV2 antiserum conjugated to fluorescein isothiocyanate (WMRD, Pullman, USA), then rinsed five times with phosphate buffered saline (PBS) solution, and examined for fluorescence by ultraviolet (UV) microscopy after placing a cover slip with mounting buffer (glycerol/PBS, 50/50). Serum samples with a known antibody titer were added as a positive control, and E-MEM was used as a negative control. Cells with nuclear and/or cytoplasmic fluorescence were considered infected.

12.3 Results

The animals injected with the inactivated PCV2 vaccine did not induce any clinical symptoms or adverse reactions during the evaluation period. The viro- logical investigations were consistently negative for the presence of PCV2 in all animals tested. In Group 1, IgM was detected from 14 to 42 PVD, whereas IgG was detected from 21 PVD up to the end of the experiment. Animals of Group 2 remained IgM- and IgG-negative during the entire experiment. No significant differences were observed between inactivated and non-inactivated serum samples when perform- ing VNT. As to viral inoculum, the best results were obtained using a virus dose of 200–400 TCID50/0.05 ml. An incubation time of 48 or 72 h yielded the same optimal results, while a low reproducibility was observed after a 36 h incubation. VNT was optimized using 3 -1 inactivated serum samples, 200 TCID50/0.05 ml (4 9 10 TCID50 ml ,50ll) concentration of the test virus and a 72 h incubation period. Using such parame- ters, NA were detected from 35 PVD on in Group 1, with a mean titer of 0.60 log2. No NA were observed in Group 2.

12.4 Discussion

Vaccination against PCV2 appears to be an effective strategy for the control of PCVD (Petrini et al. 2011). Neutralizing antibodies play an important role against viral infections, because they are able to neutralize the viral antigen and prevent 66 S. Petrini et al. virus diffusion by inhibiting its ability to enter receptive cells. NA produced against several viral infections can be detected early in the blood, whereas in PCV2 infection they can only be detected 4 weeks after experimental infection (Meerts et al. 2005). During the first stage of disease, NA are detected at lower titers, which do not interfere with PCV2 replication and diffusion in the host organism, as the viral epitopes are not recognized by the immune system (Meerts et al. 2006). The neutralizing activity of such antibodies appears to be mainly supported by IgG rather than IgM, as demonstrated by the respective specific isotypes detected by ELISA tests. Furthermore, IgM produced against PCV2 seems to have a shorter persistence in the blood (2–3 weeks), compared to what was previously described by Meerts et al. (2006), who detected IgM up to 6 weeks after experimental infection. Nevertheless, such a discrepancy might be due to a different sensitivity of the ELISA test employed. With regard to the results obtained by VNT in this study, they are similar to those published by Meerts et al. (2006).

Acknowledgments This research was financially supported by the Italian Ministry of Health— Research Project IZSUM 2007/13 (D.L. 502/92, art. 12).

References

Meerts P, Misinzo G, Nauwynck HJ (2005) Enhancement of porcine circovirus 2 replication in porcine cell lines by IFN-gamma before and after treatment and by IFN-alpha after treatment. J Int Cytok Res 25:684–693 Meerts P, Misinzo G, Lefebvre D, Nielsen J, Botner A, Kristensen CS, Nauwynck HJ (2006) Correlation between the presence of the neutralizing antibodies against porcine circovirus 2 (PCV2) and protection against replication of the virus and development of PCV2-associated disease. BMC Vet Res 2:6 Petrini S, Paniccià M, Gavaudan S, Simone E, Sensi M, Filipponi G, Rigotti L, Fortunati M, Ferrari M, De Mia GM (2011) Porcine circovirus type 2 (PCV2) associated diseases. Large Anim Rev 17:89–98 Chapter 13 Mycobacterium avium subsp. paratuberculosis as an Emergent Pathogen in Raw Ovine Milk Produced in Central Italy

A. R. Attili, V. Ngu Ngwa, L. Pacifici, S. Preziuso, A. Domesi and V. Cuteri

Abstract This study was performed to verify the safety of raw ovine milk and its role as a vehicle for Mycobacterium avium subsp. paratuberculosis (MAP) transmission. Milk samples from 697 randomly chosen adult sheep, reared in 17 dairy flocks in central Italy, were examined using Ziehl Neelsen (ZN) staining, bacteriological culture (BC), and indirect ELISA tests. Using ELISA, we identi- fied 70 % of MAP-infected ovine dairy farms. MAP infection was confirmed in 24.3 and 21.6 % of ELISA-positive milk samples (n = 37) by ZN staining and BC, respectively. A fair (k = 0.43) and slight (k = 0.33) agreement was observed between ELISA–ZN and ELISA–BC, respectively. The presence of MAP in raw ovine milk produced in central Italy confirmed a risk of potential transmission to humans through consumption of milk and cheese manufactured from unpas- teurized milk.

A. R. Attili (&) Á S. Preziuso Á V. Cuteri School of Veterinary Medical Sciences, University of Camerino, Via Circonvallazione 93/95 62024 Matelica, Italy e-mail: [email protected] S. Preziuso e-mail: [email protected] V. Cuteri e-mail: [email protected] V. Ngu Ngwa School of Veterinary Medicine and Sciences, University of Ngaoundere, 254 Ngaoundere, Cameroon e-mail: [email protected] L. Pacifici Á A. Domesi ASUR 10, 62032 Camerino, Marche Region, Italy e-mail: pacifi[email protected] A. Domesi e-mail: [email protected]

C. Boiti et al. (eds.), Trends in Veterinary Sciences, 67 DOI: 10.1007/978-3-642-36488-4_13, Ó Springer-Verlag Berlin Heidelberg 2013 68 A. R. Attili et al.

Keywords Ovine Á Milk Á Mycobacterium avium subsp. paratuberculosis Á Public Health

13.1 Introduction

Paratuberculosis, otherwise known as Johne’s disease (1895), is a contagious, chronic, and incurable granulomatous disease caused by Mycobacterium avium subsp. paratuberculosis (MAP). MAP can infect a wide range of domestic and wild ruminants and a series of reared animals (Pavlik et al. 2000). Transmission is achieved by congenital and fecal-oral routes, including by food intake and/or contaminated water. Infected colostrum and milk can be a source of infection for young animals. In sheep, the three described forms are paucibacillary, multibac- illary, and asymptomatic. Paratuberculosis causes chronic enteritis, with animals showing lower ponderal index values, ruffled hairs, dehydration, decrease in milk production, hypofertility, and in the late stages, intermittent diarrhea and progressive weight loss. Paratu- berculosis causes significant economic losses due to decreased production and premature culling of infected animals (Smith et al. 2009). A potential role of MAP in the etiology of Crohn’s disease in humans has been suggested. In 1913, Dalziel reported the clinical and histopathological similarities among animal paratuber- culosis, intestinal tuberculosis, and chronic granulomatous enteritis in humans. Recent studies have also suggested the involvement of MAP in the pathogenesis of human type I diabetes: 70 % positivity was observed in Sardinia, Italy, and in England, while 40 % positive correlation was recorded in Lombardia, Italy (Rosu et al. 2009). MAP can survive pasteurization treatments if present in milk at a high concentration, with consequent risk to public health (Ellingson et al. 2005). Food safety problems and the risk of transmission of zoonotic agents have increased with the growing use of automated raw milk systems, mainly in cattle, as authorized by Regulation (EC) 853/2004, together with increased consumption of cheeses made from raw milk. The aim of this work was to estimate the prevalence of MAP infection and the degree of safety of raw ovine milk and its role as a source of MAP infection for humans in the Marche Region (Italy), where the production of raw milk cheeses is high.

13.2 Materials and Methods

The survey was carried out between October 2009 and July 2010. A total of 697 ewes, older than 2 years, were randomly selected from 17 farms located in the Marche region which produced raw milk cheese. The animals were Comisana, Massese, Fabriano, Sarda, Sopravvissana, and mixed breed ewes at the beginning, 13 Mycobacterium avium subsp. paratuberculosis as an Emergent Pathogen 69 end, or peak of lactation. From each animal, after teat disinfection with chlorh- exidine, approximately 100 ml of milk were collected. Samples were kept at 4 °C during transport to the laboratory and frozen at -20 °C until microbiological investigations. All milk samples were tested for the detection of anti-MAP antibodies using a commercial immunosorbent assay kit (ELISA Paratuberculosis screening kit; Institut PourquierÒ, France) following the protocol that the manufacturer recom- mends for bovine milk. Briefly, serum was diluted 1:2 and pre-incubated with Mycobacterium phlei, limiting the possibility of cross-reactions with heterologous mycobacteria and significantly improving the specificity of the ELISA. The test was validated when the optical density (OD450 nm) of the positive control was C0.350 and the ratio of OD means of the positive and negative controls was C3. The milk samples were recorded as positive if they scored C40 %, questionable if between 30 and 40 % and negative if\30 %. All samples were subjected to BC and to ZN staining. The isolation of MAP was performed on Herrold’s egg yolk medium (HEYM) containing mycobactin J (2 lg/ml) following the protocol previously developed by Gao et al. (2005). A 50-ml aliquot of milk was centrifuged at 3,000 rpm for 30 min, and the serum was removed. After the addition of 20 ml of benzalkonium chloride (0.3 %), samples were left at room temperature for 2 h with stirring at 30-min intervals and then subjected to a final centrifugation at 2,500 rpm for 10 min. The supernatant was removed, and the sediment resuspended in 0.5 ml of antibiotic solution (50 mg amphotericin B, 100 mg nalidixic acid, 100 mg vancomycin, and BHI broth 18.5 g/L, Sigma-Aldrich, Italy). After incubation at 37 °C for 24 h, 0.2 ml of the suspension was spread on HEYM and placed at 37 °C for 16 weeks. Periodic inspections allowed for the identification of suspected colonies and ZN staining highlighted the typical clusters of acid-fast bacilli. Sta- tistical analysis was conducted using STATA software version 5 (STATA Cor- poration, College Station, TX, USA) and differences assessed using the Chi-square test. P values\0.05 were considered significant. The degree of agreement between tests was estimated using the kappa coefficient (k) statistic, and the k-value inter- preted as reported by Holton et al. (1998).

13.3 Results

ELISA was able to identify 12/17 (70 %) MAP-infected ovine dairy farms. MAP antibodies were evidenced in 37 (5.3 %) milk samples with a mean optical density of 1.5 ± 0.8 (SD). In particular, 7.8 % (n = 270) and 3.7 % (n = 427, P = 0.021) of ewes tested positive at early/late and peak lactation, respectively. MAP infection was confirmed in 24.3 and 21.6 % of ELISA-positive milk samples (n = 37) by ZN staining and BC, respectively (Table 13.1). A fair (k = 0.43) and slight (k = 0.33) agreement was observed between ELISA–ZN and ELISA–BC, respectively. When the higher antibody-level milk 70 A. R. Attili et al.

Table 13.1 Mycobacterium avium subsp. paratuberculosis in the milk of ovine dairy flocks reared in the Marche region (Central Italy), using three diagnostic tests Positive/Total Percentage ELISA 37/697 5.3 ZN staining 9/37 24.3 BC 8/37 21.6 samples were considered, an almost perfect agreement (k = 0.90) was attained between milk ELISA–BC or –ZN.

13.4 Discussion

In Italy, the normative (Intesa Stato Regioni 25/1/2007) defines the criteria of acceptability of raw milk sold directly to consumers or by automated distributors. It individualizes parameters that must be respected for the most important bacterial agents of food-borne illness, but these do not include emergent pathogens of public health significance such as MAP (Intesa Governo, Regioni e Provincie Autonome di Trento e Bolzano N.5/CSR 2007; Autori Vari 2006). Many authors have suggested the transmission of various pathogens to the consumer through the consumption of raw milk. Those that play a particularly important role are E. coli O157: H7, thermophilic Campylobacter, Salmonella spp., Listeria monocytogenes, Staphylococcus aureus, and Yersinia enterocolitica. The danger of these pathogens, MAP and other zoonotic agents should lead to an attitude of caution in consuming raw milk. Our results indicate that almost two-thirds of the sampled farms were infected and that MAP is excreted in the milk. Eight (47.0 %) and twelve (70.6 %) of the 17 dairy sheep farms in the Marche region were positive using BC or ZN staining combined with ELISA, respectively. The indirect ELISA, performed on milk, was significantly more sensitive when carried out on sheep at the beginning/end of lactation than when performed on animals at the peak of lactation (P \ 0.021). This is in agreement with previous works of Williams and Millar (1979) and Nielsen et al. (2002), and could be attributed by the decrease in IgG concentration during peak lactation, causing a dilution effect due to increased milk production. The 21.6 % of raw milk samples that were positive for MAP on ELISA is lower than that reported by Gao et al. (2009), who isolated the organism in 34.6 % (44/133) of samples, but is higher than that obtained in other studies, which recorded rates of isolation of 10.0 % (Ayele et al. 2005), 8.3 % (Streeter et al. 1995) and 11.7 % (Singh et al. 2007). This study also demonstrated that an immunoenzymatic technique validated from bovine milk can also be applied to the sheep (Ngu Ngwa et al. 2010). In conclusion, the data obtained from this study suggest that ovine paratubercu- losis is widespread in the Marche region and that MAP, eliminated through the milk of infected sheep, represents a potential risk of infection to humans via 13 Mycobacterium avium subsp. paratuberculosis as an Emergent Pathogen 71 consumption of raw milk and its products. A precautionary approach to legislation should therefore be taken by the competent authority for the protection of public health.

References

Ayele WY, Svastova P, Roubal P, Bartos M, Pavlik I (2005) Mycobacterium avium subspecies paratuberculosis cultured from locally and commercially pasteurized cow’s milk in the Czech Republic. Appl Environ Microbiol 71:1210–1214 Ellingson JL, Anderson JL, Koziczkowski JJ, Radcliff RP, Sloan SJ, Allen SE, Sullivan MN (2005) Detection of viable Mycobacterium avium subsp. paratuberculosis in retail pasteurized whole milk by two culture methods and PCR. J Food Prot 68(5):966–972 Gao A, Odumeru J, Raymond M, Mutharia L (2005) Development of improved method for isolation of Mycobacterium avium subsp. paratuberculosis from bulk tank milk: effect of age of milk, centrifugation, and decontamination. Can J Vet Res 69:81–87 Gao A, Odumeru J, Raymond M, Hendrick S, Duffield T, Mutharia L (2009) Comparison of milk culture, direct and nested polymerase chain reaction (PCR) with fecal culture based on samples from dairy herds infected with Mycobacterium avium subsp. paratuberculosis. Can J Vet Res 73(1):58–64 Holton LL, Scott EM, Nolan AM, Reid J, Welsh E, Flaherty D (1998) Comparison of three methods used for assessment of pain in dogs. J Am Vet Med Assoc 212(1):61–66 Intesa Governo, Regioni e Provincie Autonome di Trento e Bolzano in materia di vendita di latte crudo per l’alimentazione umana. Repertorio Atti N.5/CSR del 25/01/2007 Ngu Ngwa V, Attili AR, Preziuso P, Valente C, Cuteri V (2010). A comparative study of serum and milk ELISAs for the diagnosis of Ovine Paratuberculosis (Ovine Johne’s Disease: OJD). The Acts of 18th International Congress of FeMeSPRum: 32–37 Nielsen SS, Grohn YT, Enevoldsen C (2002) Variation of the milk antibody response to paratuberculosis in naturally infected dairy cows. J Dairy Sci 85:2795–2802 Pavlik I, Bartl J, Dvorska L, Svastova P, Du Maine R, Machackova M, Ayele WY, Horvathova A (2000) Epidemiology of paratuberculosis in wild ruminants studied by restriction fragment length polymorphism in the Czech Republic during the period 1995–1998. Vet Microbiol 77:231–251 Rosu V, Ahmed N, Paccagnini D, Gerlach G, Fadda G, Hasnain SE, Zanetti S, Sechi LA (2009) Specific immunoassays confirm association of Mycobacterium avium subsp. paratuberculosis with type-1 but not type-2 diabetes mellitus. PLoS One 4(2):e4386 Singh SV, Singh AV, Singh R, Sandhu KS, Singh PK, Sohal JS, Gupta VK, Vihan VS (2007) Evaluation of highly sensitive indigenous milk ELISA kit with fecal culture, milk culture and fecal-PCR for the diagnosis of bovine Johne’s disease (BJD) in India. Comp Immunol Microbiol Infect Dis 30:175–186 Smith RL, Grohn YT, Pradhan AK, Whitlock RH, Van kessel JS, Smith JM, Wolfgang DR, Schukken YH (2009) A longitudinal study on the impact of Johne’s disease status on milk production in individual cows. J Dairy Sci 92(6):2653–2661 Streeter RN, Hoffsis GF, Bechnielsen S, Shulaw WP, Rings M (1995) Isolation of Mycobac- terium paratuberculosis from colostrum and milk of subclinically infected cows. Am J Vet Res 56:1322–1324 Vari A (2006) Parere in materia di Rischi per la Salute degli animali derivanti da un’alimentazione con prodotti lattiero caseari pronti all’uso senza ulteriore trattamento. EFSA J 347:1–21 Williams MR, Millar P (1979) Changes in serum immunoglobulin levels in Jerseys and Friesians near calving. Res Vet Sci 26:81–84 Chapter 14 Canine Filariosis in Sardinia: Epidemiological Findings in the Ogliastra Region

A. Scala, C. Solinas, A. P. Pipia, G. Sanna, A. Varcasia and G. Tosciri

Abstract From May 2010 to April 2011, 242 family dogs from 14 municipalities of the Ogliastra region (Sardinia) were examined for microfilaremia using the modified knott method. Overall, 32.6 % microfilaremia were recorded. The spe- cies found were Acanthocheilonema reconditum (28.1 %), Dirofilaria immitis (11.6 %), and Dirofilaria repens (11.2 %). The most important risk factors for microfilaremia were the age of dogs ([6 years) and only for D. repens, the dif- ferences in the use of the dog (pet versus hunting and guard). A negative corre- lation was also found between the altitude and the prevalence of D. immitis and D. repens. In light of these epidemiological results, it would therefore be desirable to implement pharmacological prophylaxis to prevent filariosis in dogs in the monitored region.

Keywords Dog Á Filariosis Á Modified knott method Á Ogliastra (Sardinia)

14.1 Introduction

Dog heartworms are currently one of the most important parasitological diseases, due to the health impact in animals (i.e., Dirofilaria immitis), the possible zoonotic implications (especially Dirofilaria repens), and recent epidemiological changes. In particular, concerning the latter point, several authors have recently shown an expansion of the potential vectors of Dirofilaria spp., such as Aedes albopictus (the tiger mosquito) (Pietrobelli 2010). Moreover, climatic and environmental changes, as well as the increase in the possible handling of dogs for activities related to tourism, hunting, etc. (Pietrobelli

A. Scala Á C. Solinas Á A. P. Pipia Á G. Sanna Á A. Varcasia (&) Á G. Tosciri Dipartimento di Medicina Veterinaria, Sassari, Italy e-mail: [email protected]

C. Boiti et al. (eds.), Trends in Veterinary Sciences, 73 DOI: 10.1007/978-3-642-36488-4_14, Ó Springer-Verlag Berlin Heidelberg 2013 74 A. Scala et al.

2010), make the epidemiological picture of canine heartworm extremely variable. For all of these reasons, it should be monitored continuously. In this context, we have carried out an investigation to provide adequate answers to veterinarians on prophylactic and therapeutic measures against these parasites. The first survey carried out in Sardinia on canine heartworm by Arru et al. (1968) showed the presence of microfilariae in 11.2 % of examined hosts. Diro- filaria repens was then the most widespread species (7.1 %), followed by Diro- filaria immitis (1.6 %), and Acanthocheilonema (syn. Dipetalonema) reconditum (1.2 %), and 1.2 % were mixed infections. Subsequently, other investigations were carried out by Tarantini et al. (1983) and Garippa et al. (1993), and more recently by Scala et al. (2004). The latter study confirmed some data, such as the rate of microfilaremia (12.3 %) and the spreading of D. immitis (3.8 %), but also an increase in A. reconditum (7.4 %) and a decrease in the prevalence of D. repens (Scala et al. 2004). This latest survey also enabled the identification of some areas of the island with a higher risk of infection from D. immitis, such as the Oristano and Sulcis-Iglesiente regions, where the preva- lence of microfilariae of this species was 17.1 and 7.4 %, respectively. Unfortunately, these regions have been recently confirmed to be at particular risk for the occurrence of filariasis by D. immitis (Genchi et al. 2010); in the district of Oristano, the first infection from D. immitis in cats in Sardinia was also reported (Scala et al. 2009). In the same Oristano region, the seroprevalence of 14 % for D. immitis in a cat population (Scala et al. 2010) was highlighted. The presence of filariosis in cats therefore confirms the high parasitic pressure for D. immitis in this area (Dillon 1986). In this survey, to complete the epidemiological picture of the different areas of Sardinia, we report the results of an epidemiological survey carried out on canine heartworm in dogs from Ogliastra, the central-eastern region of Sardinia.

14.2 Materials and Methods

From May 2010 to April 2011, 242 blood samples collected from dogs (129 males and 113 females) were examined with the modified knott technique. Examined dogs, ranging in age from 6 months to 19 years (mean = 3.9 years, SD = 2.98), were from 14 municipalities of the Ogliastra region. The sampling was carried out with the help of the owners by collecting data on age, sex, type of hair (shaved, short, or long), and the use of the dog. A clinical examination to ascertain the presence or absence of symptoms of canine filariosis by D. immitis was also performed. The identification of microfilariae was performed according to the morphometric keys as described by Euzeby (1981). Data were stratified by sex, age, living environment, season, and dog category, and then processed statistically using the software EPI INFO version 6.04. 14 Canine Filariosis in Sardinia: Epidemiological Findings in the Ogliastra Region 75

14.3 Results

Microfilariae were found in 32.6 % of the examined dogs (79), belonging to the species A. reconditum, D. immitis, and D. repens, identified in 28.1 % (68), 11.6 % (28), and 11.2 % (27) of examined animals, respectively. Multiple infections were observed in 24 dogs (9.9 %) [5 D. immitis ? D. repens (2.1 %); 8 D. imm- itis ? A. reconditum (3.3 %); and 11 D. immitis ? D. repens ? A. reconditum (4.5 %)]. Microfilaremia was detected in 36.4 % of males (47/129) and in 28.3 % of females (32/113), with odds ratio (OR) values of 1.45 (0.81 \ OR \ 2.59). Results of prevalence were processed by stratifying the animals by gender for each Filariidae and are reported in Table 14.1. No significant differences were found for the prevalence of Filariidae species in either sex (v2 = 1.804; P = 0.179), although there was a slight tendency to significance for A. reconditum, where higher prevalence was found in females (v2 = 2.72; P = 0.099) with OR values of 1.62 (0.88 \ OR \ 2.98). Prevalence of microfilaremia stratified by host age is shown in Table 14.2. OR values for the various age classes of dogs with microfilaremia and for each Filariidae are reported in Table 14.3. The prevalence in dogs stratified according to category is shown in Table 14.4. The stratification of the data into three categories according to the type of fur did not show statistically significant differences (P [ 0.05). Prevalence of

Table 14.1 Filariidae Males (129) Females (113) prevalence stratified by n (%) n (%) animal gender A. reconditum 42 (32.6) 26 (23.0) D. immitis 16 (12.4) 12 (10.6) D. repens 16 (12.4) 11 (9.7)

Table 14.2 Microfilaremia prevalence in dogs stratified by age Classes of age Number Microfilariae + D. immitis + D. repens + A. reconditum examined n (%) n (%) n (%) + n (%) \1 year 6 2 (33.3 %) 0 0 2 (33.3 %) 1 year 38 9 (23.7 %) 4 (10.5 %) 4 (10.5 %) 9 (23.7 %) 2 years 61 18 (29.5 %) 9 (14.8 %) 7 (11.5 %) 16 (26.2 %) 3–5 years 87 31 (35.6 %) 8 (9.2 %) 10 (11.5 %) 24 (27.6 %) 6–10 years 42 15 (35.7 %) 6 (14.3 %) 6 (14.3 %) 13 (31 %) [10 years 8 4 (50 %) 1 (12.5 %) 0 4 (50 %) v2 trend / 5.768 0.121 1.388 1.725 Level of significance / P = 0.016 P = 0.727 P = 0.239 P = 0.189 76 A. Scala et al.

Table 14.3 Odds ratio values for each age class of dogs and for each Filariidae Classes of age (Years) Microfilariae D. immitis D. repens A. reconditum OR OR OR OR \1 1.00 / / 1.00 1 1.24 1.00 1.00 1.24 2 0.74 1.47 1.10 1.42 3–5 2.21 0.86 1.10 1.52 6–10 2.22 1.42 1.42 1.79 [10 4.00 1.21 / 4.00

Table 14.4 Prevalence stratified according to dog category Category Number Microfilariae D. immitis D. repens A. reconditum examined +n (%) + n (%) + n (%) + n (%) Pet 18 7 (38.9 %) 5 (27.8 %) 5 (27.8 %) 3 (16.7 %) Hunting 206 67 (32.5 %) 20 (9.7 %) 19 (9.2 %) 61 (29.6 %) Guard 18 5 (27.8 %) 3 (16.7 %) 3 (16.7 %) 4 (22.2 %) v2 / 0.51 5.78 6.34 1.71 Level of / P = 0.773 P = 0.056 P = 0.042 P = 0.427 significance

D. immitis and D. repens found in 14 municipalities was negatively correlated with the altitude of the same municipalities (D. immitis -0.542, P = 0.045; D. repens -0.593, P = 0.026) (Pearson correlation).

14.4 Conclusions

Our results show the presence of the three Filariidae previously described in Sardinia in the Ogliastra region: A. reconditum, D. immitis, and D. repens. Additionally, in this region, A. reconditum plays a predominant role. This species differs from the others of the genus Filaria for its intermediate hosts, and therefore for its mode of transmission. This finding could explain the lack of a negative correlation between the altitude of the municipalities of the positive dogs and prevalence rates. The presence of A. reconditum requires the implementation of an accurate differential diagnosis with D. immitis microfilariae that have the same length. The prevalence of Filariidae found in Ogliastra, especially for D. immitis, requires the implementation of chemoprophylaxis. It is obvious that, as for other coastal areas of Sardinia, such as Oristano and Sulcis-Iglesiente, which are already reported as risk areas for D. immitis, the Ogliastra region should be considered an area in which this parasite is an important health problem for dogs. 14 Canine Filariosis in Sardinia: Epidemiological Findings in the Ogliastra Region 77

References

Arru E, Nuvole A, Mann P (1968) La filariosi del cane in Sardegna. Riv Parasitol 30:49–58 Dillon AR (1986) Feline heartworm disease. In: Otto GF (ed) Proceeding Heartworm Symposium American Heartworm Society, Washington, DC, p 149–154 Euzeby J (1981) Diagnostic Experimental des Helminthoses animals. Livre 1 Edition Information Tecniques des services Vétérinaires, Paris Garippa G, Sanna ML, Delogu E, Arru E (1993) Ulteriori indagini sulla filariosi del cane in Sardegna. Atti Soc It Sci Vet 48:1417–1420 Genchi M, Scala A, Piazza C, Melis R, Viglietti A (2010) Can San Pietro island (Sardinia) be a possible cause of filarial infection throuhghout Europe? Parassitol 52(1–2):318 Pietrobelli M (2010) Dirofilariosi canina: un esempio di federalismo parassitario? La Sett Vet 698(suppl 5–6):7–18 Scala A, Atzori F, Varcasia A, Garippa G, Genchi C (2004) Le filariosi canine in Sardegna: aggiornamenti epidemiologici (1998–2003). Atti Soc It Sci Vet 58:120–121 Scala A, Pazzola M, Giobbe M, Sanna G, Briguglio P, Lutzu M, Carta A, Genchi M (2009) Is a heartworm preventive treatment also advisable in Sardinia? 2hd Eur Dir Days 197 Scala A, Briguglio P, Sanna G, Pipia AP, Varcasia A, Garippa G, Genchi M, Genchi C (2010) Feline hearthworm (Dirofilaria immitis) infection in Sardinia Parassitol 52(1-2):257 Tarantini SM, Garippa G, Leoni A (1983) Attuale incidenza della filariosi del cane in Sardegna. Parasitol 25:361–364 Chapter 15 Comparison of Serum and Meat Juice for Detection of Anti-Toxoplasma gondii Antibodies in Hunted Wild Boars (Sus scrofa)

D. Ranucci, F. Veronesi, I. Di Matteo, R. Branciari, D. Miraglia, C. Marini and D. Piergili Fioretti

Abstract Samples of serum and diaphragm meat juice obtained from wild boars were tested for anti-Toxoplasma gondii antibodies by means of an indirect immunofluorescent antibody test (IFAT). The results were compared to evaluate the concordance between the two matrices. A total of 160 blood and muscle juice samples were tested. Antibodies against T. gondii were detected in 28 (17.5 %) blood samples and in 22 meat juice samples (13.75 %). The results showed an ‘‘almost perfect’’ concordance between serum and meat juice analyses (K value = 0.86). These preliminary data show that diaphragm meat juice could be an alternative to serum when using IFAT for T. gondii monitoring purposes in wild boars.

Keywords Wild boar Á Toxoplasma gondii Á Meat juice Á IFAT

15.1 Introduction

Wild and farmed game meat consumption is considered as an emerging risk factor for Toxoplasma gondii infection in humans (Kijlstra and Jongert 2008). The most recent estimates provided by the European Food Safety Authority (EFSA 2007) reported that approximately 50 % of wild game is seropositive for T. gondii.In wild boar (Sus scrofa, Linnaeus, 1758), epidemiological surveys conducted in Europe revealed seropositivity rates up to 38.4 % (Gauss et al. 2005). Parasitized

D. Ranucci (&) Á F. Veronesi Á I. Di Matteo Á R. Branciari Á D. Miraglia Á D. Piergili Fioretti Dipartimento Scienze Biopatologiche ed Igiene delle Produzioni Animali ed Alimentari, Università degli Studi di Perugia, Perugia, Italy e-mail: [email protected] C. Marini Istituto Zooprofilattico Sperimentale Umbria e Marche, Perugia, Italy

C. Boiti et al. (eds.), Trends in Veterinary Sciences, 79 DOI: 10.1007/978-3-642-36488-4_15, Ó Springer-Verlag Berlin Heidelberg 2013 80 D. Ranucci et al. wild boar meat can be a source of infection for hunters during evisceration pro- cedures, and for consumers (Choi et al. 1997; Cook et al. 2000). This makes the implementation of monitoring plans beginning at the hunt, as provided by Euro- pean legislation (Directive EC/99/2003), of utmost importance. Serologic testing is not always practical in the hunted boar because of difficulties in sample collection. Recently, an alternative approach based on antibody screening performed on meat juice has been suggested in swine (Dubey et al. 2005; Berger-Schoch et al. 2011). Encouraging results for the determination of antibodies to T. gondii in meat juice from domestic pigs were obtained by applying an indirect immunofluorescent antibody test (IFAT) method (Ranucci et al. 2010). The aim of this study was to evaluate the applicability of an IFAT technique conducted on meat juice obtained from the diaphragm muscle of hunted wild boar and compare it to the traditional serum antibody research.

15.2 Materials and Methods

Serum and meat juice samples obtained from 160 wild boar hunted in 2009 and 2010 were analyzed. The blood of each animal was collected via intracardiac puncture immediately after killing. Samples were centrifuged at 4,000 rpm for 15 min, and the separated serum was stored at -20 °C until analysis. A fragment (2.5 9 2.5 cm) of the diaphragm muscle was taken for meat juice extraction. Muscle samples were placed in containers (Sardstedt, Numbrecht, Germany), maintained at -20 °C for 12 h, and then thawed at 4 °C for 8–12 h. Meat juice was collected in test tubes and quantified. Blood and meat juice samples were subjected to screening by IFAT for the determination of anti-T. gondii Immuno- globulin G (IgG) using commercial slides (Mega Cor Diagnostik, Horbranz, Osterreich) obtained from T. gondii tachyzoites cultured on Vero cells and fluo- rescein isothiocyanate conjugated anti-swine IgG (anti-swine IgG FITC conjugate; Sigma Immunochemicals, St. Louis, MO) diluted 1/32. Sera were tested at a screening dilution of 1/40 (cut off) (Bartová et al. 2006), while for meat juice, in the absence of accurate bibliographic references, a cut-off value of 1/5 was adopted on the basis of previous evaluations. All positive samples were subjected to 2-fold serial dilutions to determine the final titer (endpoint). The concordance between serum and meat juice overall results was calculated by McNemar’s Chi-square test, using 2 9 2 contingency tables where serum was considered as the comparative test and meat juice as the alternative test. The overall agree- ment between test and the one relative to two distinct serum antibody level titers (1/40, C 1/40) was evaluated by calculating the K value, interpreted according to the following guidelines: \0.2 = slight agreement, 0.2–0.4 = fair, 0.4–0.6 = moderate, 0.6–0.8 = substantial, [0.8 = almost perfect (Thrusfield 2007). All statistical analyses were carried out using EpiInfo 6.01 free software (CDC, Atlanta, GA) and the statistical significance level was set at p \ 0.05. 15 Comparison of Serum and Meat Juice 81

Table 15.1 Distribution of positive serum and meat juice samples according to different titer levels Meat juice Total Titer (IGg) 1/5 1/10 C1/20 Number 15 6 1 22 Serum Total Titer (IGg) 1/40 1/80 C1/160 Number 16 8 4 28

Table 15.2 Comparison of IFAT assay results of the determination of anti-T.gondii antibodies (IGg) in serum and meat juice samples IFAT Meat juice p value K (95 % CI) + 2 Serum total + 22 6 0.007 0.86 (0.75–0.97) 2 0 132 Serum 1/40 + 12 4 0.023 0.84 (0.63–0.93) 2 0 132 Serum [ 1/40 + 10 2 0.079 0.90 (0.77–1.00) 2 0 132

15.3 Results

Of the 160 animals examined, 28 serum samples (17.5, 95 % Confidence Interval (CI): 12–24.3 %) and 22 meat juice samples (13.75, 95 % CI: 8.8–20.1 %) were positive for IgG antibodies to T. gondii. The distribution of the results on the basis of antibody titers is shown in Table 15.1. With regard to serum samples, 16 positive samples (57.1 %) had titers of 1/40, eight samples (28.6 %) had titers of 1/80 and four samples (14.3 %) had titers C1/160. Relative to meat juice, most of the positive samples (15/22, 68.2 %) had titers of 1/5. The results obtained using the two different matrices are compared in Table 15.2. The percentage of positive serum samples was significantly different from meat juice samples (p \ 0.01). The overall agreement between tests was ‘‘almost perfect’’ (K = 0.86, 95 % CI: 0.75–0.97) despite the six negative discrepancies observed. This correlation also showed optimal values (K = 0.9, 95 % CI: 0.63–0.94) on meat juice samples obtained from animals with serum antibody titers C1/40, while it was slightly reduced (K = 0.84, 95 % CI: 0.77–1) on samples with lower titers.

15.4 Discussion

The agreement found between the two matrices allows the consideration of meat juice as an alternative to serum for epidemiological screening in hunted wild boar. The feasibility of muscle sampling during hunting makes this result relevant and 82 D. Ranucci et al. important, as wild boar is fundamental for the persistence of the biological cycle of T. gondii in the wild. Although an optimal agreement was obtained, a statistically significant difference between the percentages of positive samples in the two matrices was found. The use of meat juice, therefore, cannot be considered comparable to serum when the seronegative state is taken as a discriminant for the infection (Berger-Schoch et al. 2011). Using the IFAT method allows us to compare the data with those obtained in domestic pigs (Ranucci et al. 2010). A higher agreement was detected in wild boar (0.86 versus 0.72) due to a higher antibody concentration in meat juice. This could be due to the amount of blood in the muscles, as meat juice reactivity depends on the blood concentration in the muscle (Mecca et al. 2011), and to the higher antigenic stimulation due to sub- sequent re-infection, sometimes with different protozoan strains, common in the wild. The choice of the diaphragmatic muscle was dictated both by the previous positive observation in pigs, compared to other muscle, and by the routine sam- pling of this muscle for the detection of Trichinella spp. in meat (Regulation EC/ 2075/2005). Taking advantage of the public health aspect, it would be possible to carry out large epidemiological surveys on the territory for T. gondii. However, further studies have to be performed beforehand for the definition of all the parameters of accuracy (sensitivity, specificity, positive predictive value, and negative predictive value) necessary for the diagnostic applicability of the tech- nique on meat juice.

References

Bartová E, Sedlák B, Literák I (2006) Prevalence of toxoplasma gondii and neospora caninum antibodies in wild boars in the Czech Republic. Vet Parasitol 142:150–153 Berger-Schoch AE, Bernet D, Doherr MG, Gottstein B, Frey CF (2011) Toxoplasma gondii in Switzerland: a serosurvey based on meat juice analysis of slaughtered pigs, wild boar, sheep and cattle. Zoonoses Public Health 58:472–478 Choi WY, Nam HO, Kwak NH, Huh W, Kim YR, Kang MW, Cho SY, Dubey JP (1997) Foodborne outbreaks of human toxoplasmosis. J Infect Dis 175:1280–1282 Cook AJ, Gilbert RE, Buffolano W, Zufferey J, Petersen E, Jenum PA, Foulon W, Semprini AE, Dunn DT (2000) Sources of Toxoplasma infection in pregnant women: European multicentre case-control study. European research network on congenital toxoplasmosis. Br Med J 321:142–147 Directive EC/99/2003. Official Journal of the European Union L 325/31 Dubey JP, Hill DE, Jones JL et al (2005) Prevalence of viable Toxoplasma gondii in beef, chicken, and pork from retail meat stores in the United States; risk assessment to consumers. J Parasitol 91(5):1082–1093 European Food Safety Authority (2007) Monitoring of Toxoplasma in humans, food and animals. EFSA Journal 583:1–64 Gauss CB, Dubey JR, Vidal D, Ruiz F, Vicente J, Marco I, Lavin S, Gortazar C, Almeria S (2005) Seroprevalence of Toxoplasma gondii in wild pigs (Sus scrofa) from Spain. Vet Parasitol 131:151–156 Kijlstra A, Jongert E (2008) Control of the risk of human toxoplasmosis transmitted by meat. Int J Parasitol 38:1359–1370 15 Comparison of Serum and Meat Juice 83

Mecca JN, Meireles LR, de Andrade HFJ (2011) Quality control of toxoplasma gondii in meat packages: standardization of an ELISA test and its use for detection in rabbit meat cuts. Meat Sci 88(3):584–589 Ranucci D, Veronesi F, Branciari R, Miraglia D, Mammoli R, Piegili Fioretti D (2010) Comparison of an IFA assay for the detection of Toxoplasma gondii antibodies in serum and meat juice of slaughtered pigs. Parassitologia 330 Regulation EC/2075/2005. Official Journal of the European Union L 338/60 Thrusfield M (2007) Veterinary Epidemiology, 3rd edn. Blackwell Science Ltd Oxford, UK Chapter 16 Eucoleus aerophilus (syn. Capillaria aerophila) and Other Trichinelloid Nematodes in Dogs from Liguria (Northwest Italy)

F. Macchioni, L. Guardone, M. C. Prati and M. Magi

Abstract As a part of a wider study that examined endoparasites of carnivores in northwest Italy, 270 canine fecal samples were analyzed with flotation techniques. Overall prevalence was 7.4 % for Capillaria spp. and 12.2 % for Trichuris vulpis. This article stresses the importance of an accurate morphological and morpho- metrical analysis to distinguish eggs of T. vulpis from eggs of the genus Capillaria. We also suggest that different Capillaria spp. are present in the Italian dog population.

Keywords Capillaria spp. Eucoleus aerophilus Dog Italy

16.1 Introduction

Dogs can be infected by several parasitic species of the family Trichuridae that can be difficult to specifically identify via copromicroscopic examination. The eggs of these nematode species are characterized by a particular barrel shape and polar plugs, with only minor morphological differences (Campbell 1991). This family includes two subfamilies, Capillarinae and Trichurinae, which contain the genus Capillaria and the genus Trichuris, respectively.

F. Macchioni (&) L. Guardone M. Magi Dipartimento di Patologia Animale Profilassi e Igiene degli Alimenti, Università di Pisa, Viale delle Piagge, 2, 56127 Pisa, Italy e-mail: [email protected] M. C. Prati Scuola Normale Superiore di Pisa, Pisa, Italy M. C. Prati INFN Sezione di Pisa, Pisa, Italy

C. Boiti et al. (eds.), Trends in Veterinary Sciences, 85 DOI: 10.1007/978-3-642-36488-4_16, Ó Springer-Verlag Berlin Heidelberg 2013 86 F. Macchioni et al.

The genus Capillaria has been revised several times. It is currently divided into Eucoleus spp. for parasites found in the airways, Aonchotheca spp. for those living in the gastrointestinal tract, and Pearsonema spp. for parasites of the urinary tract (Moravec 1982; Burgess et al. 2008). Eucoleus aerophilus (syn. Capillaria aerophila) lives in the trachea, bronchi, and bronchioles of domestic and wild carnivores. Infection with this parasite can cause verminous tracheo-bronchitis. It is occasionally reported in humans (Laloševic et al. 2008). It is present in Europe, and North and South America (Conboy 2009). In Italy E. aerophilus has been reported in wild animals (Rossi et al. 1983; Balestrieri et al. 2006; Magi et al. 2009) and in dogs and cats (Traversa et al. 2009; Di Cesare 2010). The adult females lay eggs after mating. The eggs reach the pharynx, are swallowed, and then eliminated through the feces. The eggs have an elliptical shape and asymmetrical polar plugs and show a reticular surface with anastomosed ridges. They are 60–83 lm long and 26–40 lm wide (Campbell 1991). Outside of the host, they mature in 30–45 days. Infecting larvae can reach the definitive host directly or via an earthworm, the intermediate host. Infesting larvae migrate in 7–10 days from the enteric wall of the host toward the site of choice (Euzeby 1961). E. boehmi (syn. C. boehmi) lives in the nasal passages and sinuses of wild and domestic canids. This species has been reported in America and Europe (Conboy 2009). The eggs measure 54–60 9 30–35 lm. They exhibit a characteristic space between the embryo and the shell and have a smoother surface with small wells (Campbell 1991; Campbell and Little 1991). E. boehmi was identified for the first time in Germany in the silver fox (Supperer 1953). A. putorii (syn. C. putorii) inhabits the stomach and small intestine of domestic and wild carnivores. It has been reported in North America and Europe (Campbell 1991). In Italy it has been reported only in foxes (Iori et al. 1990; Di Cerbo et al. 2008; Guardone et al. 2010). The eggs measure 53–70 9 20–30 lm and have a reticular surface organized in a longitudinal pattern (Campbell 1991). P. plica (syn. C. plica) is localized in the urinary bladder of dogs, cats, and wild carnivores. It has been reported in North America and Europe (Campbell 1991; Whitehead 2009; Senior et al. 1980). In Italy it has been reported in foxes and in a dog (Iori et al. 1990; Callegari et al. 2010). The eggs measure 58–74 9 23–31 lm, and have a rough surface (Campbell 1991). T. vulpis is a cosmopolitan parasite of dogs and wild canids and has occasionally been reported in humans (Dunn et al. 2002; Traversa 2011). It inhabits the large intestine, especially the cecum. The eggs, which are similar in size to those of the genus Capillaria, measure 70–84 9 30–40 lm. They have a typical symmetrical barrel shape, are yellowish-brown in color, and have a very smooth surface (Campbell 1991). The aim of this study was to confirm the presence of Capillaria spp. and T. vulpis in dogs in an area where these parasites are known to infect wild animals, especially foxes (Guardone et al. 2010). 16 Eucoleus aerophilus (syn. Capillaria aerophila)87

16.2 Materials and Methods

Between January 2010 and March 2011, we examined fecal samples from 270 dogs (163 from hunting dogs and 107 from dogs in kennels) in the province of Imperia (Liguria, Northern Italy). Fresh fecal samples were examined using flotation in zinc sulphate (specific gravity = 1.350), combined with centrifugation. Using an optical microscope at 1009 and 4009 magnification, trichurid eggs were identified according to a morphological and morphometric analysis following in the reported descriptions (Campbell 1991).

16.3 Results

Out of a total of 270 samples of fresh dog feces, 48 were positive for Trichuridae eggs. Fifteen dogs were positive for Capillaria spp., and 28 were positive for T. vulpis. Five dogs were infested with two species. The overall prevalence of infection with Capillaria spp. was 7.4 % (95 % confidence interval [CI], 4.3–10.5 %). The prevalence of infection with T. vulpis was 12.2 % (95 % CI, 8.3–16.1 %). Among the 20 dogs infected with Capillaria spp., 12 were positive for eggs of E. aerophilus (prevalence, 4.4 %; 95 % CI, 2.0–6.8 %). In nine cases, we found eggs belonging to the genus Capillaria that had different morphological characteristics (one dog had a double infestation of E. aerophilus and Capillaria spp.). In six dogs (2.2 %), the eggs had characteristics similar to eggs of E. boehmi. Most of them were partially embryonated and a few contained a living embryo. In three dogs (1.1 %), the eggs had characteristics similar to those of A. putorii. Samples collected during this study are currently being examined with biomolecular tools (Traversa et al., Deplazes et al., unpublished data) to confirm these identifications. With the exception of three dogs, which had respiratory symptoms with cough and nasal discharge, most of the dogs infected with Capillaria spp. were asymptomatic. The prevalence of Capillaria spp. was 8.6 % in the hunting dogs and 5.6 % in the kennel dogs. This difference was statistically significant. The prevalence of T. vulpis in the hunting dogs was 2.4 and was 27.1 % in the kennel dogs. These differences were highly significant. However, dogs from a heavily infested kennel were present in the kennel dog samples. Some of the 48 dogs were coinfested by Toxocara canis, Toxascaris leonina, Ancylostomatidae, Angiostrongylus vasorum, Crenosoma vulpis,orDipilydium caninum. 88 F. Macchioni et al.

16.4 Discussion

In Italy, the prevalence of infestation by E. aerophilus is likely underestimated. Reports are more common in wild animals, in which the coprological diagnosis is confirmed by finding adult worms in the trachea or lungs during necropsy examinations. In the same geographic area of this study, we detected E. aerophilus in the airways and A. putorii and T. vulpis in the intestines of red foxes. The sharing of habitat between wild and domestic animals can lead to an increased risk of infestation for domestic species. The prevalence of E. aerophilus in dogs (4.4 %) found in this study does not differ significantly from that reported by Traversa et al. (2009) and Di Cesare et al. (2010). The prevalence of T. vulpis (12.2 %) found in dogs in the Liguria region contributes to the data reported by other authors in other Italian regions (Poglayen et al. 2000; Perrucci et al. 2001; Rinaldi et al. 2006; Zanzani et al. 2010). In conclusion, the various capillarid eggs display very similar morphological and morphometric characteristics. Therefore, new biomolecular methods should be developed to aid in the identification of these parasites.

References

Balestrieri A, Remonti L, Ferrari N, Ferrari A, Lo Valvo T, Robetto S, Orusa R (2006) Sarcoptic mange in wild carnivores and its co-occurence with intestinal helminthes in the Western Italian Alps. Eur J Wild Res 52:196–201 Burgess H, Ruotsalo K, Peregrine AS, Hanselman B, Abrams-Ogg A (2008) Eucoleus aerophilus respiratory infection in a dog with Addison’s disease. Can Vet J 49:389–392 Callegari D, Kramer L, Cantoni AM, Di Lecce R, Dodi PL, Grandi G (2010) Canine bladderworm (Capillaria plica) infection associated with glomerular amyloidosis. Vet Parasitol 168:338–341 Campbell BG (1991) Trichuris and other Trichinelloid nematodes of dogs and cats in the United States. Compend Contin Educ Vet 13(5):769–778 Campbell BG, Little MD (1991) Identification of the eggs of a nematode (Eucoleus boehmi) from the nasal mucosa of North American dogs. J Am Vet Med Assoc 198:1520–1523 Conboy G (2009) Helminth parasites of the canine and feline respiratory tract. Vet Clin North Am Small Anim Pract 39:1109–1126 Di Cerbo AR, Manfredi MT, Bregoli M, Ferro Milone N, Cova M (2008) Wild carnivores as source of zoonotic helminths in north-eastern Italy. Helmintologia 45(1):13–19 Di Cesare A, Meloni S, Milillo P, Castagna G, Otranto D, Paoletti B, Bartolini R, Avolio S, Traversa D (2010) Feline and canine cardio-pulmonary nematodes in central and southern Italy. Parassitologia 52(1–2):307 Dunn J, Columbus S, Aldeen W, Davis M, Carroll K (2002) Trichuris vulpis recovered from a patient with chronic diarrhea and five dogs. J Clin Microbiol 40(7):2703–2704 Euzeby J (1961) Trichuroidea In: Les maladies vermineuses des animaux domestique, Tome I, Maladies dues aux nemathelminthes. Vigot Frères Editeurs, Paris, France, p. 54 Guardone L, Macchioni F, Prati MC, Mignone W, Torracca B, Magi M (2010) Common parasites of dogs (Canis familiaris) and foxes (Vulpes vulpes) in an area of Maritime Alps (north- western Italy). LXIV Convegno Nazionale S.I.S.Vet Asti, Italy, 8–10 Sep 2010 16 Eucoleus aerophilus (syn. Capillaria aerophila)89

Iori A, Costantini R, Cancrini G (1990) Parassiti in volpi provenienti da alcune regioni italiane. Parassitologia 32:153–154 Laloševic D, Laloševic V, Klem I, Stanojev-Jovanovic0 D, Pozio E (2008) Pulmonary capillariasis miming bronchial carcinoma. Am J Trop Med Hyg 78(1):14–16 Magi M, Macchioni F, Dell’Omodarme M, Prati MC, Calderini P, Gabrielli S, Iori Cancrini G (2009) Endoparasites of Vulpes vulpes in central Italy. J Wildl Dis 45(3):881–886 Moravec F (1982) Proposal of a new systematic arrangement for nematodes of the family Capillaridae. Folia Parasitol 29:119–132 Perrucci S, Glorioso A, Tarantino C (2001) Parassitosi nei canili e nei gattili. Ob Doc Vet 22(5):37–40 Poglayen G, Giannetto S, Macrì B, Garippa G, Scala A, Cambosu C, Giangaspero A, Paoletti B, Montauti AE, Traldi G, Habluetzel A (2000) Canine zoonoses by environmental faecalization. Parassitologia 42:220 Rinaldi L, Biggeri A, Carbone S, Musella V, Catelan D, Veneziano V, Cringoli G (2006) Canine faecal contamination and parasitic risk in the city of Naples (southern Italy). BMC Vet Res 2:29 Rossi L, Iori A, Cancrini G (1983) Osservazioni sulla fauna parassitaria della popolazione di volpi presente nel parco regionale La Mandria. Parassitologia 25:340–343 Senior DF, Solomon GB, Goldschmidt MH, Joyce T, Bovee KC (1980) Capillaria plica infection in dogs. J Am Vet Med Assoc 176:901–905 Supperer R (1953) Capillaria boehmi Spec. Nov., Eine neue Haarwurm-Art aus den Stirnhoehlen des Fuchses Z Parasitenk 16:51–55 Traversa D (2011) Are we paying too much attention to cardio-pulmonary nematodes and neglecting old-fashioned worms like Trichuris vulpis? Parasit Vectors 4:32 Traversa D, Di Cesare A, Milillo P, Iorio R, Otranto D (2009) Infection by Eucoleus aerophilus in dogs and cats: is another extra-intestinal parasitic nematode of pets emerging in Italy? Res Vet Sci 87:270–272 Whitehead M (2009) Urinary capillariosis in a cat in the UK. Vet Rec 165:757 Zanzani S, Bonassi L, Maffi S, Mandredi MT (2010) Prevalence of intestinal parasites in dogs and cats from two provinces of Lombardy region and perception of related risks by the pet owners. Parassitologia 52(1–2):341 Chapter 17 Helminths in Sheep on Farms of the Basilicata Region of Southern Italy

A. Bosco, L. Rinaldi, V. Musella, D. Pintus, M. Santaniello, M. E. Morgoglione, G. Zacometti and G. Cringoli

Abstract The aim of this study was to obtain up-to-date information on the presence and distribution of helminths in grazing sheep from the Basilicata region of southern Italy. A cross-sectional coprological survey was conducted on 98 sheep farms and the FLOTAC dual technique was used to detect and count par- asitic elements. The most frequent nematodes found were gastrointestinal (GI) strongyles (91.8 %), in particular Haemonchus (76.9 %), Trichostrongylus (91.8 %), Teladorsagia (88.8 %), Cooperia (77.6 %), and Oesophagostomum (72.5 %). They were followed by lungworms (50.0 %), including Muellerius (37.8 %), Neostrongylus (13.3 %), Protostrongylus (4.4 %), Cystocaulus (2 %), and Dictyocaulus (2 %). We also found Trichuris (39.8 %), Nematodirus (24.5 %), Strongyloides (4.1 %), and Skrjabinema (2.0 %). Trematoda were also found with the following prevalence: Calicophoron daubneyi (10.2 %), Dicro- coelium dendriticum (61.2 %), and Fasciola hepatica (1.0 %). Moniezia was found on 35.7 % of farms. Interestingly, the spatial analysis showed clusters for C. daubneyi and D. dendriticum.

Keywords Helminths Á Sheep Á GIS Á FLOTAC Á Epidemiology Á Southern Italy

A. Bosco (&) Á L. Rinaldi Á D. Pintus Á M. Santaniello Á M. E. Morgoglione Á G. Cringoli Department of Pathology and Animal Health, University of Naples Federico II, Cremopar, Italy e-mail: [email protected] V. Musella Department of Clinical and Experimental Medicine, University of Catanzaro Magna Graecia, Catanzaro, Italy G. Zacometti Provincial Association of Farmers, Potenza, Basilicata, Italy

C. Boiti et al. (eds.), Trends in Veterinary Sciences, 91 DOI: 10.1007/978-3-642-36488-4_17, Ó Springer-Verlag Berlin Heidelberg 2013 92 A. Bosco et al.

17.1 Introduction

Sheep farming in the Basilicata region of southern Italy (378,966 head; ISTAT, 2010) is an important part of the region’s rural economy. Information on the distribution of helminths on sheep farms in this region, and on the health aspects related to these parasitic infections, are scant and out of date. Therefore, the aim of this study was to update the data on the presence and distribution of helminth infections on sheep farms in the Basilicata region. A cross-sectional coprological survey was conducted using geographical information systems (GIS) for territorial sampling and for the representation of results with parasitological maps. In addition, a spatial analysis was conducted to detect possible clusters of positive farms.

17.2 Materials and Methods

A GIS of the Basilicata region was constructed using administrative boundaries at the provincial and municipal levels as data layers. The region was divided into ninety-eight 10 9 10 km quadrants. The centroid of each quadrant was identified and the farm closest to each centroid was sampled. A total of 98 sheep farms were sampled, one for each quadrant. On each farm, 20 sheep (15 adults and 5 juve- niles) were subjected to parasitological investigation, for a total of 1,960 subjects. In the laboratory, individual samples were grouped into four composites (each consisting of equal parts of five individual samples), one for juveniles and three for adult animals. Copromicroscopic analyses were performed using the FLOTAC dual technique (Cringoli et al. 2010) with an analytic sensitivity of two eggs/larvae per gram (EPG/LPG) of feces. Two flotation solutions were used, the first consisting of sodium chloride [specific gravity (SG) = 1.200] to detect and count eggs of the following genera/groups of nematoda: gastrointestinal (GI) strongyles, Nemato- dirus, Strongyloides, and Trichuris. A second solution consisting of zinc sulfate (SG = 1.350) was used to detect and count eggs/larvae of Skrjabinema, Fasciola, Calicophoron, Dicrocoelium, Moniezia, and lungworms. Coprocultures were performed for each farm to identify the genera of GI strongyles (van Wyk et al. 2004). The GIS software ArcGIS 9.3 GIS (ESRI, Redlands, CA, USA) was used for spatial analysis to detect cluster distribution of each parasite.

17.3 Results

The results showed the presence of GI strongyles on 91.8 % (90/98) of farms, including Haemonchus 76.9 % (78/98), Trichostrongylus 91.8 % (90/98), Tela- dorsagia 88.8 % (87/98), Cooperia 77.6 % (76/98), and Oesophagostomum 17 Helminths in Sheep on Farms of the Basilicata Region of Southern Italy 93

Fig. 17.1 Distribution of C. daubneyi and D. dendriticum in the Basilicata region. Parasitolog- ical maps with proportional circles and clusters detected by spatial analysis

72.5 % (71/98). The following genera of nematoda were also found: Trichuris 39.8 % (39/98), Nematodirus 24.5 % (24/98), Strongyloides 4.1 % (4/98), and Skrjabinema 2 % (2/98). The most frequent lungworms were Muellerius, found on 37.8 % (37/98) of farms, followed by Neostrongylus 13.3 % (13/98), Proto- strongylus 4.4 % (4/98), Cystocaulus 2 % (2/98), and Dictyocaulus 2 % (2/98). Cestoda of the genus Moniezia were found on 35.7 % (35/98) of farms. Among trematoda, D. dendriticum was found on 61.2 % (60/98) of the farms, followed by C. daubneyi on 10.2 % (10/98), and F. hepatica on 1 % of the farms (1/98). The two parasitological maps with proportional circles shown in Fig. 17.1 show the distribution and intensities of D. dendriticum and C. daubneyi. Spatial analyses showed a cluster distribution pattern for both D. dendriticum (P = 0.05) and C. daubneyi (P = 0.01).

17.4 Discussion

This survey showed a noteworthy distribution of helminths on sheep farms of the Basilicata region. The most prevalent helminths were GI strongyles, detected on most of the farms (91.8 %), and comprised the genera Haemonchus (76.9 %), Trichostrongylus (91.8 %), Teladorsagia (88.8 %), Cooperia (77.6 %), and Oesophagostomum (72.5 %). Lungworms were also widely distributed (50 %) and Muellerius was most commonly detected genus (37.8 %). These findings are consistent with similar studies performed in other Apenninic areas of southern Italy (Cringoli et al. 2002; Biggeri et al. 2007). The prevalence values for Trichuris (39.8 %), Nematodirus (24.5 %), Strongyloides (4.1 %), and Skrjabinema (2 %) are also consistent with those recently reported on sheep farms in other Italian regions (Bosco et al. 2011). D. dendriticum was the most prevalent liver fluke 94 A. Bosco et al.

(61.2 %), whereas F. hepatica was reported on only 1 % of the farms. Param- phistomes (C. daubneyi) were reported on 10.2 % of farms. Spatial analysis detected an interesting cluster distribution of D. dendriticum and C. daubneyi in the study area, likely due to soil and environmental charac- teristics on the biology of the intermediate hosts. Further studies are needed to clarify this finding. In conclusion, considering that the health status of livestock is fundamental for the productive and reproductive performances of these animals, the economic impact of this parasitological situation is likely very significant, although not quantified in this study (Cringoli et al. 2004, 2007, 2008). Local knowledge about the presence and distribution of parasitic infections in sheep is a basic requirement to better plan and implement appropriate control protocols and targeted strategies.

Acknowledgments Part of this research was funded by the European Union Seventh Framework Programme FP7 –KBBE-2011-5 under grant agreement n° 288975. The authors would like to express sincere appreciation to Giovanna Cappelli, Ida Guariglia, Davide Ianniello, and Mario Parrilla for the laboratory analyses.

References

Biggeri A, Catelan D, Dreassi E, Rinaldi L, Musella V, Veneziano V, Cringoli G (2007) Multivariate spatially-structured variability of ovine helminth infections. Geospat Health 2(1):97–104 Bosco A, Rinaldi L, Musella V, Pintus D, Morgoglione ME, Santaniello M, Prestera G, Zacometti I (2011) Mappe parassitologiche degli elminti nei bovini semibradi in Basilicata. J Italian Assoc Buiatrics 6:43–47 Cringoli G, Rinaldi L, Veneziano V, Capelli G, Malone JB (2002) A cross-sectional coprological survey of liver flukes in cattle and sheep from an area of the southern Italian Apennines. Vet Pararasitol 108:137–143 Cringoli G, Taddei R, Rinaldi L, Veneziano V, Musella V, Cascone C, Sibilio G, Malone JB (2004) Use of remote sensing and geographical information systems to identify environmental features that influence the distribution of paramphistomosis in sheep from the southern Italian Apennines. Vet Pararasitol 122(1):15–26 Cringoli G, Veneziano V, Rinaldi L, Sauve C, Rubino R, Fedele V, Cabaret J (2007) Resistance of trichostrongyles to benzimidazoles in Italy: a first report in a goat farm with multiple and repeated introductions. Parasitol Res 101:577–581 Cringoli G, Veneziano V, Jackson F, Vercruysse J, Greer AW, Fedele V, Mezzino L, Rinaldi L (2008) Effects of strategic anthelmintic treatments on the milk production of dairy sheep naturally infected by gastrointestinal strongyles. Vet Parasitol 156:340–345 Cringoli G, Rinaldi L, Maurelli MP, Utzinger J (2010) FLOTAC: new multivalent technique for qualitative and quantitative copromicroscopic diagnosis of parasites in animals and humans. Nat Prot 5:503–515 van Wyk JA, Cabaret J, Michael LM (2004) Morphological identification of nematode larvae of small ruminants and cattle simplified. Vet Parasitol 119:277–306 Part III Pharmacology and Clinical Science Chapter 18 Effects of Veterinary Drugs on Swimming Activity in Two Freshwater Organisms

M. Dalla Bona, V. Di Leva and M. De Liguoro

Abstract Alterations in swimming activity may influence ecologically relevant performance, such as predation avoidance, prey capture, growth, stress resistance, mating, and longevity. The evaluation of swimming activity supports toxicological investigation with endpoints other than traditional LC50s values and may aid in investigating the environmental relevance of low-level exposures and determining ‘‘no observable effect concentration’’ (NOEC) and ‘‘lowest observable effect concentration’’ (LOEC). In this chapter, some veterinary antibacterial compounds that may contaminate the aquatic environment due to their use in livestock and/or mass aquaculture treatments are evaluated for their effects on swimming activity of Daphnia magna (primary consumer) and Poecilia reticulata (secondary con- sumer). Results show that the chosen endpoint may call to the attention of eco- toxicology some compounds that are otherwise negligible, based on lethality tests.

Keywords Swimming activity Á Daphnia magna Á Poecilia reticulata Á Toxicity test

18.1 Introduction

Behavioral changes represent integrated responses of the whole organism. These altered responses may be associated with reduced fitness and survival, resulting in adverse consequences at the population level (Bridges 1997). Until recently, behavioral endpoints have been slow to be integrated into aquatic toxicology, not only because of the poor understanding of their conse- quences on ecologically relevant activities, such as predation avoidance, prey

M. Dalla Bona (&) Á V. Di Leva Á M. De Liguoro Department of Biomedicine and Food Safety, University of Padua, Viale dell’Università 16 35020 Legnaro, PD, Italy e-mail: [email protected]

C. Boiti et al. (eds.), Trends in Veterinary Sciences, 97 DOI: 10.1007/978-3-642-36488-4_18, Ó Springer-Verlag Berlin Heidelberg 2013 98 M. Dalla Bona et al. capture, growth, and reproduction, but also because it is difficult to obtain quan- tifiable and reproducible data (Kane et al. 2005). Recent improvements in computer and video automation have made significant progress in the ease, utility, and affordability of obtaining, interpreting, and applying behavioral endpoints in a variety of applications, including aquatic toxicity tests, possible. Currently, after exposing an aquatic organism to a sub- stance, it is possible to acquire a film by means of video tracking software and analyze it to accurately evaluate the swimming activity through graphical and statistical elaboration of data. Acute toxicity tests, while demanding little time and labor, are generally less sensitive than chronic toxicity tests. Their sensitivities can be improved by eval- uating sublethal effects such as the behavioral consequences. Generally, the acute toxicity (lethality) of antibacterial toward nontarget organisms such as crustaceans and fishes occurs only at concentrations higher than 100 mg/L; substances with EC50 values higher than this threshold are considered safe for the aquatic envi- ronment (Regulation 1272/2008/EEC). In this chapter, some methodologies are described that can be used for swim- ming activity evaluation of fishes and crustaceans after exposure to antibacterial. It is shown that significant effects of these drugs on swimming activity may be detected at concentrations that are unable to cause any lethality of the test organisms.

18.2 Materials and Methods

In accordance with the fish acute toxicity test (OECD 203 1992), the following compounds were tested at a concentration of 100 mg/L (limit test) on Poecilia reticulata: enrofloxacin (EFX), its metabolite ciprofloxacin (CFX), trimethoprim (TMP), sulphamethazine (SMZ), sulphaguanidine (SGD), and sulphaquinoxaline (SQO). As no lethality was recorded after 96 h, the test was extended to 14 days (OECD 204 1984). At the end of this prolonged toxicity test and before sacrificing the animals with an overdose of MS-222, each group of seven fish was allocated in a small, round tank (20 cm in diameter) under standard conditions of light and temperature and filmed from above for 12 min using a digital video camera (JVC EVERIO GZ-MS100E); the middle 2 min of each video sequence was analyzed on a PC AcerAspire M3201, using Swistrack 4.0 (Lochmatter et al. 2008). The spatial coordinates (in two dimensions) of the movements were then exported to Excel (MicrosoftÒ) software to perform graphical and statistical elaborations. Based on the results of this test, the following compounds were selected to be tested on Daphnia magna: EFX, TMP, SGD, and SQO. Ten daphnids (6 days of age) were allocated in Roux flasks (75 cm2)at20± 1 °C and with a 16 h pho- toperiod (200 lux), and exposed for 24 h to a single compound dissolved in ADaM medium (Klüttgen et al. 1994) at 100 mg/L concentration. At the end of the test, each daphnid was frontally filmed for 5 min (see above), and the middle 2 min of 18 Effects of Veterinary Drugs on Swimming Activity in Two Freshwater Organisms 99 the video sequence was analyzed on a PC Acer Aspire M3201 using Kinovea software (freeware). For all tests, statistical analysis was performed using ANOVA followed by Dunnett’s test with the SPSS StatisticsÒ 17.0 application. This experimentation was authorized by the Italian Ministry of Health (decree n. 175/ 2010-B).

18.3 Results

No lethality was observed in any of the tests. In Fig. 18.1, box plots of swimming activity of fish (a) and daphnids (b) exposed to the various compounds are shown. With the exception of SMZ (in fish) and SGD (in daphnids), all the drugs tested elicited a statistically significant reduction of the traveled distance. In Fig. 18.2,as an example, video recordings of the controls and subjects exposed to TMP are compared, for both P. reticulata (a) and D. magna (b).

18.4 Discussion

Based on the results from the algal toxicity tests (EC50 \ 100 mg/L), TMP is already classified as ‘harmful to aquatic organisms’ and ‘possibly able to cause long-term adverse effects in the aquatic environment’ (Phrases R42 and R43). However, as frequently happens, an acute lethality test on Brachydanio rerio (Halling-Sørensen et al. 2000) yielded negative results, pointing to a no observed effect concentration (NOEC) for fish of 100 mg/L (Chemwatch 2009). For P.

Fig. 18.1 Box plots showing the distance traveled in 2 min by seven specimens of Poecilia reticulata (a) and by 10 specimens of Daphnia magna (b), for control group (k) and for groups exposed to the antibacterial compounds (100 mg/L). Bowties are outliers, and asterisks indicate significant difference from k (P \ 0.01) 100 M. Dalla Bona et al.

Fig. 18.2 a Two-minute paths of seven specimens of Poecilia reticulata (filmed from above): control group and group exposed for 14 days to 100 mg/L TMP are compared. b Two-minute paths of 10 specimens of Daphnia magna (frontally filmed): control group and group exposed for 24 h to 100 mg/L TMP are compared reticulata, these data would have been confirmed by our test, but referring to the sublethal endpoint (swimming activity inhibition), exposure to 100 mg/L is not devoid of effects. In fact, the protocol for the acute lethality test (OECD 203 1992) recommends the importance of recording any sublethal effect. Thus, it will be important, in the future, to evaluate the same endpoint with scaled concentrations of TMP to determine both the NOEC and the EC50 with confidence limits. Swimming activity inhibition was also recorded with the other five compounds; more detailed study is therefore recommended, although with less urgency than with TMP. It is worth noting that in this study, the evaluation of more refined components of swimming that could increase the sensitivity of the tests has not been considered. These actions include acceleration, freezing, angles of turns, horizontal and vertical distribution, predator avoidance, etc. In Fig. 18.2, for example, one notes that the control group swims more peripherally (P. reticulata) or has a higher tendency to move in the water column (D. magna) compared to the TMP-exposed groups. OECD protocols for both acute (immobilization) and chronic (reproduction) tests on D. magna require the use of newborn specimens. However, in our novel approach to the evaluation of the effects of acute exposure to drugs on swimming activity, we have chosen to use 6-day-old daphnids. This age was chosen, because the organisms are sufficiently developed to be filmed with a conventional video camera but not yet ready for reproduction. Indeed, reproduction in D. magna causes a physiological fluctuation of swimming activity that would unavoidably interfere with the test results. The test was quick and easy to perform, and showed, at least with TMP, to be more sensitive than the traditional immobilization test (OECD 202 2004) in which the calculated EC50 was 149 mg/L (De Liguoro et al. 2009). Taken together, our results confirm that swimming activity is a valuable end- point in aquatic toxicology and indicate the need for more in-depth studies on TMP toxicity. It is not clear whether the strong effect on swimming activity is simply the consequence of the metabolic disturbance of folate synthesis caused by the drug or whether the effect might be related to direct impacts on the nervous system of aquatic organisms. It should be noted, in this regard, that TMP is largely used in both human and veterinary medicine. Furthermore, its use has been 18 Effects of Veterinary Drugs on Swimming Activity in Two Freshwater Organisms 101 extended to aquaculture facilities, where nonnegligible inputs of the compound can be directly released into the aquatic environment when metaphylactic treat- ments are in place. Moreover, TMP is poorly biodegradable, having a long ([22 days) environmental half-life for primary degradation (Chemwatch 2009), and its effects in the aquatic environment may add to those of other antifolic compounds (sulphonamides) that have been frequently detected in surface waters and are as persistent in the environment (De Liguoro et al. 2010).

Acknowledgments This investigation was funded by grants from the University of Padua.

References

Bridges CM (1997) Tadpole swimming performance and activity affected by acute exposure to sublethal levels of carbaryl. Environ Toxicol Chem 16:1935–1939 Chemwatch (2009) Trimethoprim. MSDS no.sc203302 [Online]. Chemwatch: Victoria. Austra- lia, 7 April, 2009. http://datasheets.scbt.com/sc-203302.pdfS. Accessed 22 Feb 2011 De Liguoro M, Fioretto B, Poltronieri C, Gallina G (2009) The toxicity of sulfamethazine to D. magna and its additivity to other veterinary sulfonamides and trimethoprim. Chemosphere 75(1519):1524 De Liguoro M, Di Leva V, Gallina G, Faccio E, Pinto G, Pollio A (2010) Evaluation of the aquatic toxicity of two veterinary sulfonamides using five test organisms. Chemosphere 81:788–793 Halling-Sørensen B, Holten-Lützhøft HC, Andersen HR, Ingerslev F (2000) Environmental risk assessment of antibiotics. Comparison of mecillinam, trimethoprim and ciprofloxacin. J Antimicrob Chemother 46:53–58 Kane AS, Salierno JD, Brewer SK (2005) Fish models in behavioral toxicology: automated techniques, updates and perspectives. In: Ostrander GK (ed) Methods in aquatic toxicology, vol 2. Lewis Publishers, Boca Raton, pp 559–590 Klüttgen B, Dülmer U, Engels M, Ratte HT (1994) ADaM, an artificial fresh-water for the culture of zooplankton. Water Res 28:743–746 Lochmatter P, Roduit P, Cianci C, Correl N, Jacot J, Martinoli A (2008) SwisTrack—a flexible open source tracking software for multi-agent systems. In: Proceedings of the 2008 IEEE/RSJ international conference on intelligent robots and systems, pp. 4004–4010 OECD (1984) OECD guidelines for the testing of chemicals. Guideline 204: fish, prolonged toxicity test. Organization for Economic Co-operation and Development, Paris OECD (1992) OECD guidelines for the testing of chemicals. Guideline 203: fish, acute toxicity test. Organization for Economic Co-operation and Development, Paris OECD (2004) OECD guidelines for testing of chemicals. Guideline 202. Daphnia sp. acute immobilisation test. Organization for Economic Co-operation and Development, Paris Regulation 1272/2008/EEC of the European Parliament and of the Council of 16 December 2008 on classification, labelling and packaging of substances and mixtures, amending and repealing Directives 67/548/EEC and 1999/45/EC, and amending Regulation (EC) No 1907/2006 Chapter 19 Interdisciplinary Evaluation of Toxicity in Ostreopsis Ovata: Algal Biotoxins

A. Ferrari, I. Schiavetti, C. Bolognesi, D. Pavino and B. Vivaldi

Abstract An interdisciplinary study was performed to characterize the biotoxins produced by Ostreopsis ovata and their consequences for health and food safety. O. ovata, a benthic dinoflagellate widespread in tropical seas, has recently also colonized several areas of the Italian coast. O. ovata causes respiratory symptoms in exposed humans, and microalgae have the ability to release ostreocina during flowering, which accumulates in many aquatic organisms. The results show a significant correlation between time of death in mice subjected to chemical analysis and quantification of biotoxins performed by mass spectrometry, indi- cating its validity in detecting the toxicity profile of O. ovata. The potential mutagenicity of toxins detected by genotoxic analysis has yet to be determined.

Keywords Algal biotoxins Ostreopsis ovata Toxicity

19.1 Introduction

Ostreopsis ovata is a benthic dinoflagellate that is most commonly found in tropical seas but has recently also colonized several portions of the Italian coast, due to peculiar climatic and environmental conditions. Marked evidence of its presence was found between 2005 and 2006 in Liguria and Sicily. Today, O. ovata is believed to cause respiratory symptoms in exposed people, but the specific cause-effect relationship has not yet been identified. In addition,

A. Ferrari (&) I. Schiavetti D. Pavino B. Vivaldi Istituto Zooprofilattico Sperimentale del Piemonte, Liguria e Valle d’Aosta, Genoa, Italy e-mail: [email protected] C. Bolognesi Istituto Nazionale per la Ricerca sul Cancro (IST), S.S. Cancerogenesi Ambientale, Genoa, Italy

C. Boiti et al. (eds.), Trends in Veterinary Sciences, 103 DOI: 10.1007/978-3-642-36488-4_19, Springer-Verlag Berlin Heidelberg 2013 104 A. Ferrari et al. potential environmental risks have been identified, associated with the capacity of the microalgae to release ostreocina during flowering. Ostreocina is a palytoxin- like substance that accumulates in many aquatic organisms and concentrates along the food chain. Within the framework of a research project funded by the Ministry of Health, the Experimental Zooprophylactic Institute of Piedmont, Liguria and Aosta Valley (IZSPLV)—Genoa Section, in cooperation with the Liguria Regional Agency for Environmental Protection (ARPAL), National Cancer Research Centre—Genoa (IST), Liguria Regional Government, and Marine Research Centre of Cesenatico (CRM), has conducted an interdisciplinary (biotoxicological, chemical, and genotoxic) study aimed at characterizing the biotoxins produced by O. ovata and their consequences on human health and food safety.

19.2 Materials and Methods

Based on the literature available and on experience, the sampled area included the first few meters of submerged beach to approximately 1 m of depth in the water column. Protected and sheltered coast sections, characterized by both natural and artificial substrates, are the most exposed to O. ovata flowering. Furthermore, based on risk signals collected during previous bathing seasons, as well as on some peculiar past events, some ‘‘sensitive sites’’ have been identified to be at risk of algal flowering. A report was filed for each sampling point, prepared in cooperation with ARPAL, including all information and details regarding the sampling site and environmental physical–chemical parameters. The sites selected for the study were: • For the Gulf of Genoa: Pontetto, Bagnara, Cinque Maggio • For the Province of La Spezia: Falconara, Fiascherino, Tellaro • Sites dedicated to mussel farming in the Gulf of La Spezia: Diga Levante, Diga Ponente, Diga Centro, Portovenere, Palmaria. Biotoxicity tests were performed on 400 mussel samples and 40 echinoderm samples. The investigation was conducted on mice (mouse test), in compliance with the annex of DM 16/5/2002 (Maximum contents and test methods of algal biotoxins in live bivalve molluscs, echinoderms, tunicates, and marine gastropods) as the official reference method for detecting the presence of all classes of toxins that may be associated with paralytic (PSP) and diarrhea (DSP) molecules (DM 16/5/2002). The mouse test evaluates the acute toxicity of an extract of the edible body of molluscs injected into the peritoneum of adult mice (Swiss albino mice of 18–20 days of age). Homogenized mussels were extracted with 1:1 acetone and methanol according to the indications given in protocol 2. Subsequently, the extracts were combined and a further extraction was performed with 1:3 dichlo- romethane and methanol to isolate two groups of toxins: (1) okadaic acid toxins 19 Interdisciplinary Evaluation of Toxicity in Ostreopsis Ovata: Algal Biotoxins 105 and their analogs dinophysistoxin, pectenotoxins, and DSP lipo-soluble toxins, identified as azaspiracid, in a dichloromethane extract; (2) yessotoxins and paly- toxins in a methanol extract. The lipidic residues obtained from the evaporation of solvents were resuspended in a Tween 60 solution. Intraperitoneal injections into three mice were then performed (Mouse Test). Toxicity was assessed based on the death times of the mice, and the class of toxins responsible for the death was able to be recognized. High performance liquid chromatography-mass spectrometry (HPLC-MS) tests (Riobò et al. 2006) were performed with a Varian 1,200 L Triple Quadrupole type device (Varian Inc., Walnut Creek, CA, USA) on the digestive gland of the mussels, where the toxins are most concentrated. Specifically, 22.5 g of hepato- pancreas were extracted in Ultra Turrax three times with 50 mL of 90 % MeOH. The extract was brought to a final volume of 160 mL with methanol. Seven millilitres, corresponding to 0.98 g of tissue, were evaporated and reconstituted in 2 mL of 50 % MeOH. The samples then underwent a fluid–fluid partitioning with CH2Cl2 to remove lipids from the extract and then purification with an OASIS MAX cartridge (Waters). The eluate was further concentrated 10 times before injecting 20 lL into the HPLC-MS. The palytoxin was separated by chromatography using a Polaris C18, 3 lm, 50 9 2.00 mm column (Varian Inc.) and gradient elution with water and aceto- nitrile/water (95:5), both containing 30 mM 1 % acetic acid. The determination was performed considering the transitions with a more intense response, i.e., m/z 327 [ 75 and m/z 1,278 [ 327, and by comparison with the peaks obtained from different dilutions of a palytoxin standard. Genotoxicity was tested with the Comet assay and DNA damage induction. In particular, the first assay, simple and sensitive, was performed in the alkaline version proposed by Singh et al. (1988). The slides were prepared with cells sandwiched between two layers of agarose gel for 24 h and then immersed in a lysis solution. Electrophoresis was then conducted for 25 min with the slides in an alkaline environment at 25 V and 300 mA, to be later incubated in a neutralizing solution, colored with ethidium bromide, and dehydrated. After observing the slides under a fluorescence microscope, the comets were classified into four distinct categories and results were reported in terms of DNA fragmentation index, based on the evaluation of 200 comets per slide.

19.3 Results

Sampling was performed throughout the year, with an emphasis on the bathing season (May–October). The positive results of biotoxicology tests obtained over 3 years are given below (Table 19.1). The mice showed respiratory failure, posterior limb paralysis, and sometimes post-mortem eye opacity. 106 A. Ferrari et al.

Table 19.1 Biotoxicological Samples received Positive samples % of positives test results 2008 250 50 20 2009 100 10 10 2010 400 2 0.5

The chromatograms of the HPLC-MS tests are given in Fig. 19.1, and the results and comparisons between chemical and biotoxicological test results are given in Table 19.2.

19.4 Discussion

Biotoxicological tests showed the highest incidence of positive results in July and August, times consistent with stable marine meteorological conditions, mild wave motion, and high sun radiation with water temperatures ranging from 25 to 26 C. The death times of the Echinoderms suggest that these organisms are more

Fig. 19.1 Examples of HPLC-MS test chromatograms: a Standard palytoxin (WAKO chemicals GmbH, Neuss, Germany); b Mussel samples collected offshore in La Spezia 19 Interdisciplinary Evaluation of Toxicity in Ostreopsis Ovata: Algal Biotoxins 107

Table 19.2 List of some samples tested for comparison with HPLC-MS and Mouse tests CRM code Material LC-MS Mouse test pPlTX OVTX-a Death times (84100/6) G.SP. Met extract mussels. \LOD \LOQ 2 h–3 h–3 h (84100/7) G.SP. Met extract mussels. \LOD \LOQ 2 h–3 h–3 h (84100/3) G.SP. Met extract mussels. \LOD \LOQ 2 h–2 h 300–2 h 300 2859/09 (84474) G.SP. Met extract mussels. \LOD \LOQ 1 h–2 h–2 h 2860/09 (Fiascherino) Met extract echinoderms \LOD 34 400–400–2 h 450 2861/09 (Falconara) Met extract echinoderms \LOD 37 300–300–300 2862/09 (Tellaro) Met extract echinoderms \LOD 27 380–380–380 2863/09 (Fiascherino) Homogenated echinoderms \LOD 111 400–400–2 h 450 2864/09 (Falconara) Homogenated echinoderms \LOD 180 300–300–300 2865/09 (Tellaro) Homogenated echinoderms \LOD 55 380–380–380 2866/09 (84100) G.SP. Homogenated mussels \LOD 39 2 h–3 h–3 h LOD limit of detection, LOQ limit of quantification sensitive to this class of biotoxins (Wiles et al. 1974). This assumption suggests that further environmental monitoring trials for palytoxins are needed using sea urchins. Our results show an appreciable correlation between the death times of the mice and the chemical quantitation performed with a mass spectrometer, indicating the validity of this device in detecting the toxic profile of O. ovata. The potential mutagenicity of the toxins detected in genotoxic tests has yet to be confirmed. In the light of these results, we consider it necessary to refine the investigation methods proposed and to adopt an interdisciplinary approach that may allow researchers to obtain a global picture of the different classes of algal biotoxins.

References

DM 16/05/2002 Tenori massimi e metodiche di analisi delle biotossine algali nei molluschi bivalvi vivi, echinodermi, tunicati e gasteropodi marini. GU N. 165 del 16/07/2002 Riobò P, Paz B, Franco JM (2006) Analysis of palytoxin-like in Ostreopsis cultures by liquid chromatography with precolumn derivatization and fluorescence detection. Anal Chim Acta 566:217–223 Singh NP, McCoy MT, Tice RR, Schneider EL (1988) A simple technique for quantitation of low levels of DNA damage in individual cells. Exp Cell Res 175:184–191 Wiles JS, Vick JA, Christensen MK (1974) Toxicological evaluation of palytoxin in several animal species. Toxicon 12:427–433 Chapter 20 Aflatoxin M1 Contamination and Antibacterial Residues in Milk in Kosovo

G. Gallina, A. Rama, L. Lucatello, C. Benetti, D. Bajraktari, K. Uka and C. Montesissa

Abstract A survey was performed to assess the occurrence of aflatoxin M1 (AM1) and antibacterial residues in milk produced in Kosovo in 2009. AM1 was detected by enzyme-linked immunosorbent assay (ELISA) in 3 % of the tested samples, but the contamination was consistently below the limit (50 ng/kg) set by the European Union (EU). Antibacterial residues were detected in 5 % of milk samples tested by an inhibition assay (Delvotest SP). The results were confirmed by specific receptor assays (SNAP tests, IDEXX), and 40 of 52 samples were positive for beta-lactam antibiotics. The presence of residues of ampicillin, amoxicillin, penicillin G, and cloxacillin (12/25 at concentration [ Maximum Residue Limits) was confirmed by liquid chromatography-tandem mass spectrometry.

Keywords Milk Á Aflatoxin M1 Á Antibacterial residues Á b-Lactam antibiotics

20.1 Introduction

Dairy farming is a key sector agriculture in Kosovo. The presence of veterinary drug residues in milk is becoming important to Kosovo consumers. Antibacterial drugs, such as beta-lactams, tetracyclines, and sulphonamides, are routinely used

G. Gallina Á L. Lucatello Á C. Montesissa (&) Department of Comparative Biomedicine and Food Science, University of Padua, Viale dell’Università 16, 35020 Legnaro, PD, Italy e-mail: [email protected] A. Rama Faculty of Agriculture and Veterinary, Pristina University, Pristina, Kosovo C. Benetti Istituto Zooprofilattico Sperimentale delle Venezie, Legnaro, Italy D. Bajraktari Á K. Uka Food and Veterinary Agency, Pristina, Kosovo

C. Boiti et al. (eds.), Trends in Veterinary Sciences, 109 DOI: 10.1007/978-3-642-36488-4_20, Ó Springer-Verlag Berlin Heidelberg 2013 110 G. Gallina et al. for prevention and therapy of diseases in milking cows in Kosovo, but limited data are available regarding the presence of antibacterial residues in milk (Rama et al. 2010). Aflatoxins are mycotoxins of major concern to the dairy industry. The presence of aflatoxin M1 (AM1) in milk and milk products is considered unde- sirable. The risk of AM1 should not be underestimated, due to the unpredictability of weather and ambient conditions and the inability of certain agricultural systems, characterized by poor economic conditions and/or lack of knowledge, to face and manage mycotoxin prevention and contamination (Prandini et al. 2009). This study was performed to assess the occurrence of AM1 and antibacterial residues in milk produced in Kosovo in 2009. The aim of the project was to support the development of a monitoring plan and to underline the importance of continuous surveillance for AM1 and antibacterial residues in milk to ensure consumer health.

20.2 Materials and Methods

Milk samples were collected over the whole of 2009, in six regions of Kosovo (Prishtina, Gjilan, Mitrovica, Peja, Gjakova, and Prizren). The sampling of raw milk was territorially programmed in such a way that the results could describe the level of milk contamination in the whole territory of Kosovo. Evaluation for AM1 was performed on 675 milk samples: 524 samples came from milk collection points (MCPs), 129 from individual farms, and 42 samples were ultra high temperature (UHT) milk from retailers. A total of 1,015 samples were collected to be analyzed for antibacterial contamination: 826 came from MCPs, 150 from individual cows, and 39 samples of UHT milk from retailers. The screening tests were performed at the Kosovo Food and Veterinary Agency. Concentrations of AM1 were determined by an enzyme-linked immu- nosorbent assay (ELISA) commercial kit (Tecna, Trieste, Italy). All milk samples were screened within 24 h for the presence of antimicrobials by Delvotest SP (DSM Food Specialties, Dairy Ingredients, Delft, The Netherlands). All Delvotest SP-positive samples were checked with a SNAP test (IDEXX Laboratories, Westbrook, ME, USA) specific for penicillins, tetracyclines, and sulphonamides. Only the samples that were confirmed positive by the second screening test underwent the confirmation analysis by liquid chromatography-tandem mass spectrometry (Rama et al. 2011).

20.3 Results

The detection capacity of the ELISA test was lower than the maximum permitted level of AM1. The good performance of this method was confirmed by the sat- isfactory results obtained in the proficiency test, ‘‘Progetto Trieste’’ (z-score of 20 Aflatoxin M1 Contamination and Antibacterial Residues in Milk in Kosovo 111

0.32 and 0.65) (Progetto Trieste—Final Report Mycotoxins). In this study, all milk samples had values below the maximum level (50 ng/kg) determined by the European Union (EU) and Kosovo legislation. The level of AM1 was below the detection capacity of the test in 97 % of samples analyzed. The mean concen- tration of AM1 detected in the 20 contaminated samples was 12 ng/kg. Table 20.1 shows the results of the antibacterial residue analyses. The overall positivity rate on the Delvotest SP was near 5 %. The positivity rates for farms, MCPs, and UHTs were 4.9, 6.6, and 2.5 %, respectively. The samples from the region of Peja showed the highest rate of contamination. When the 52 samples that reacted positively on the Delvotest SP were analyzed further with the SNAP test for penicillins, tetracyclines, and sulphonamides, only 40 samples (77 %) were positive for beta-lactams. Twenty-five of these samples were randomly selected to be confirmed by liquid chromatography-tandem mass spectrometry. Sixteen samples were positive for residues of amoxicillin, penicillin G, or cloxacillin (12 at a concentration higher than the Maximum Residual Limits).

20.4 Discussion

The data presented in this chapter, obtained from the first monitoring plan per- formed in Kosovo to investigate the presence of AM1 in milk, are encouraging. The results pointed out that the content of AM1 in the contaminated samples was always below that allowed by EU legislation. Comparison of data from neigh- boring Western Balkan countries showed similar results. AM1 was detected in 30 % of Serbian pasteurized and UHT milk but none of the samples exceeded the concentrations of 50 ng/kg (Polovinski-Horvatovic´ et al. 2009). In Croatia, 98.4 % of the analyzed milk samples had AM1 values below the EU maximum level (Bilandzˇic et al. 2010). The level of AM1 milk contamination in Kosovo is dif- ferent from that in countries with similar agricultural practices but different cli- mates, like Iran and Turkey, where AM1 in milk currently appears to be a serious public health problem (Unasan 2006; Kamkar 2008). Due to the unpredictability of climatic and environmental conditions, it is essential that the program of moni- toring of AM1 in milk in Kosovo will continue, to avoid an undetected problem, like occurred in Italy in 2003.

Table 20.1 (a) Delvotest SP screening analysis; (b) beta-lactam detection by LC/MS-MS (a) Delvotest Analyzed Positive (b) LC/MS- MRL (lg/ Range detected SP samples (n) samples (n) MS kg) (lg/kg) Farms 150 10 Penicillin G 4 0.7–1973.4 Collection 826 41 Cloxacillin 30 6.4–542.0 points UHT milk 39 1 Amoxicillin 4 0.2–42.9 Total 1,015 52 Ampicillin 4 1.4–784.2 112 G. Gallina et al.

The results of antibacterial residue analyses showed that milk samples collected from different parts of Kosovo were often contaminated, most significantly with beta-lactams. The results confirmed that penicillin residues were represented mainly by penicillin G, amoxicillin, and ampicillin, often occurring at concen- trations higher than the MRL set by the EU (Ghidini et al. 2002; Holstage et al. 2002). These findings were in agreement with reports of several researchers (McEwen et al. 1991; Mitchell et al. 1998) and reflect the frequent use of intra- mammary infusions containing beta-lactams for the treatment of mastitis. The results from UHT milk reported in this study revealed levels higher than those reported in The Netherlands and Costa Rica (Wit et al. 1996; Bayenes et al. 1999). Results from Kosovo were in agreement with those reported by Fonseca et al. (2009), confirming the presence of antibiotic residue in UHT milk produced and sold in Brazil. Given the considerable levels of residues detected in raw milk and the worri- some consequences for consumers, competent authorities should apply continuous monitoring programs to produce safe milk products which pose no health risk to Kosovo consumers. In addition, more research should be carried out to accurately focus the problem and implement effective corrective actions to reduce milk contaminants. Further studies will be conducted to evaluate the performance of several screening tests for milk residues, to identify methods that are easy to use, suitable for a broad spectrum of antimicrobials, and inexpensive.

Acknowledgments Research partially funded by the University of Padua (Fondo PVS 2009).

References

Bayenes R, Lyman R, Anderson KL, Brownie CF (1999) A preliminary survey of antibiotic residues and viable bacteria in milk from the Caribbean basic countries. J Food Prot 62:177–180 Bilandzˇic´ N, Varenina I, Solomun B (2010) Aflatoxin M1 in raw milk in Croatia. Food Control 21:1279–1281 Fonseca GP, Cruzi AG, Faria AF, Silvia R, Moura MRL, Carvalho LMJ (2009) Antibiotic residues in Brazilian UHT milk: a screening study. Ciência e Tecnologia de Alimentos 29:451–453 Ghidini SM, Zanardi E, Varisco G, Chizzolini R (2002) Prevalence of molecules of ß-lactam antibiotics in bovine milk in Lombardia and Emilia Romagna (Italy). Ann Fac Medi Vet di Parma 22:245–252 Holstage DM, Punchner B, Whitehead G, Galey FD (2002) Screening and mass spectral confirmation of ß-lactam antibiotic residues in milk using LC-MS/MS. J Agric Food Chem 50:406–411 Kamkar A (2008) The study of aflatoxin M1 in UHT milk samples by ELISA. J Vet Res 63:7–12 McEwen SA, Meek AH, Black WD (1991) A dairy farm survey of antibiotic treatment practices, residue control methods and associations with inhibitors in milk. J Food Prot 54:454–459 Mitchell JM, Griffiths MW, McEwen SA, McNab WB, Yee AJ (1998) Antimicrobial drug residues in milk and meat: causes, concerns, prevalence, regulations, tests, and test performance. J Food Prot 61:742–756 20 Aflatoxin M1 Contamination and Antibacterial Residues in Milk in Kosovo 113

Polovinski-Horvatovic´ M, Juric´ V, Glamocˇic´ D (2009) Two year study of incidence of aflatoxin M1 in milk in the region of Serbia. Biotechnol Anim Husbandry 25:713–718 Prandini A, Tansini G, Sigolos S (2009) On the occurrence of aflatoxin M 1 in milk and dairy products. Food Chem Toxicol 47:984–991 Rama A, Lucatello L, Benetti C, Bajraktari D, Uka K, Montesissa C (2010) Recent surveillance activity of veterinary drugs in Kosovo: a two year survey of antibacterial residues in milk. In: Proceedings of the 6th International Symposium on Hormone and Veterinary Drug Residue Analysis (Ghent), 190 Rama A, Lucatello L, Montesissa C, Benetti C, Bajraktari D, Uka K (2011) Veterinary drug use in Kosovo: results from two year surveillance of antibacterial residues in milk. J Int Environ Appl Sci 6:401–411 Unusan N (2006) Occurence of aflatoxin M1 in UHT milk in Turkey. Food Chem Toxicol 44:1897–1900 Wit B, Van Buuren RD, Van Ooy-Landman AN, Van Hattum E, Van Ramshorst E (1996) Detection of antibiotic substances in milk using capacitance measurement. Neth Milk Dairy J 50:573–580 Chapter 21 Heavy Metal Levels in Dog Liver and Kidney in Naples (Campania, Italy)

F. P. Serpe, R. Russo, R. De Luna, S. Florio, M. Esposito and L. Severino

Abstract The aim of the current study was to carry out a retrospective analysis of heavy metal (Pb, Cd, and Hg) levels in liver and kidney of 38 dogs living in an urban habitat (city of Naples). Tissues were homogenized, digested in a microwave digestion system, and analyzed by atomic absorption spectrometry. The results of this study showed generally low levels of heavy metals in tissues of all examined dogs; only mercury concentrations in kidneys of pet dogs were higher than in stray dogs, and no significant age-dependent differences in metal levels were shown between the two groups. In conclusion, these results suggest the involvement of ad hoc-formulated pet food exposure to heavy metals in domestic animals.

Keywords Dog Á Heavy metals Á Environment

21.1 Introduction

Environmental contamination by heavy metals is almost ubiquitous. Their presence is partly attributable to the natural abundance of heavy metals in the earth’s crust in addition to human activities. Some of these metals, besides not being essential for living organisms, are significantly toxic. Given the paucity of the scientific literature on the exposure of domestic species to heavy metals (Sakai et al. 1995; Balagangatharathilagar et al. 2006; Lopez-Alonso et al. 2007), the purpose of this study was to undertake a retrospective analysis of the levels of lead,

F. P. Serpe (&) Á M. Esposito Istituto Zooprofilattico Sperimentale del Mezzogiorno, Dipartimento di Chimica, Portici, NA, Italy e-mail: [email protected] R. Russo Á R. De Luna Á S. Florio Á L. Severino Università degli Studi di Napoli Federico II, Facoltà di Medicina Veterinaria, Naples, Italy

C. Boiti et al. (eds.), Trends in Veterinary Sciences, 115 DOI: 10.1007/978-3-642-36488-4_21, Ó Springer-Verlag Berlin Heidelberg 2013 116 F. P. Serpe et al. cadmium, and mercury in tissues of dogs (stray and domestic) that died from various causes, all living in the urban area of Naples.

21.2 Materials and Methods

As part of the necropsy of the 38 dogs from the Department of Pathology and Animal Health, University of Naples ‘‘Federico II,’’ livers and kidneys were collected and stored at -20 °C until analysis. Aliquots of homogenized bodies were taken, after thawing, equal to 0.50 ± 0.01 g; wet mineralization of the samples was performed by treating each aliquot with 4.0 mL of 70 % HNO3 (Carlo Erba) and 1.5 mL of 30 % H2O2 (Baker Analyzed) in a total volume of 8.0 mL (difference made with deionized water) for atomic absorption spectroscopy (Best Chemicals) in a microwave oven (Milestone, FKW) at a temperature of 190 °C. The sample was analyzed for lead and cadmium levels using an atom- ization technique in a graphite furnace (AAS 800, Perkin Elmer) and for total mercury levels using the hydride generation technique (AAS 3110, Perkin Elmer). Quantification was carried out after external standardization, correcting for recovery percentage. The animals, divided into domestic (n = 19) and stray (n = 19) groups, were further divided into groups according to age (Group I: 2–9 years, Group II: 10–17 years). The results obtained were statistically pro- cessed using the Student’s t test.

21.3 Results

The results of this work are summarized in Table 21.1. With regard to lead, which was present in all examined samples, levels between 0.074 and 0.949 mg/kg (Xm = 0.321) in the liver and between 0.049 and 1.001 ppm in the kidney

Table 21.1 Levels of lead, cadmium, and mercury in liver and kidney (mg/kg f.w.) of dogs from the urban area of Naples (n = 38) Pb Cd Hg Liver Average 0.321 0.093 0.054 SD 0.198 0.079 0.044 Min 0.074 ND ND Max 0.949 0.352 0.418 Kidney Average 0.293 0.259 0.040 SD 0.231 0.238 0.021 Min 0.049 0.011 ND Max 1.001 0.984 0.328 21 Heavy Metal Levels in Dog Liver and Kidney in Naples 117

(Xm = 0.293) were detected. For cadmium, found in all kidney samples and 95 % of all liver samples, the concentrations ranged from ND to 0.352 mg/kg (Xm = 0.093) in the liver and between 0.011 and 0.984 mg/kg (Xm = 0.259) in the kidney. Finally, for mercury, present in 83 % of all samples, values between ND and 0.418 mg/kg (Xm = 0.054) were found in liver samples, whereas in the kidney, the levels of this metal ranged between ND and 0.328 mg/kg (Xm = 0.040). The higher concentrations of mercury were found in domestic subjects.

21.4 Discussion

The results of this study showed that all subjects examined have been exposure to at least one heavy metal, although hepatic and renal concentrations of all metals were not generally considered likely to give rise to clinical toxicity (Hansmann et al. 2009). In particular, with regard to lead, measured concentrations were higher than the other metals but lower than values considered ‘‘normal’’ in the kidney of the canine species (Beretta 1984). For cadmium, the highest levels were found in the kidney (Xm = 0.25 mg/kg), which represent, as well-known, the organ in which this metal has a particular tropism. Even mercury concentrations detected provide signs of a generally low exposure to this metal, but its concentrations in the kidney were higher in domestic than in stray dogs (P \ 0.01). With regard to age, no statistically significant differences were detected between the two groups. Despite the limited number of subjects and the different variables inherent in such a retrospective study, the results of this work, which we consider preliminary, provide guidance to continue a wider study of different nutritional statuses and living environments. In particular, the major concentrations of mercury found in domestic dogs might be considered as a starting point to investigate the role of diet in exposure of pets to contaminants that may end in inadequately controlled commercial food.

References

Balagangatharathilagar M, Swarup D, Patra RC, Dwivedi SK (2006) Blood lead level in dogs from urban and rural areas of India and its relation to animal and environmental variables. Sci Tot Environ 359:130–134 Beretta C (1984) Piombo. In: Beretta C (ed) Tossicologia Veterinaria. Editoriale Grasso, Bologna, pp 153–166 Hansmann F, Stephan I, Wirtz A, Gruber AD, Wohlsein P (2009) Mercury poisoning in a German shepherd dog. Vet Rec 165:447–448 Lopez-Alonso M, Miranda M, García-Partida P, Cantero F, Hernández J, Benedito JL (2007) Use of dogs as indicators of metal exposure in rural and urban habitats in NW Spain. Sci Tot Environ 372:668–675 Sakai T, Ito M, Aoki H, Aimi K, Nitaya R (1995) Hair mercury concentrations in cats and dogs in central Japan. Br Vet J 151:215 Chapter 22 Ultrasonographic Assessment of Abdominal Lymph Nodes in Normal Puppies: Preliminary Results

A. La Pietra and M. De Majo

Abstract Data on the ultrasonographic size of normal abdominal lymph nodes in the puppy are lacking. Length, thickness, and width were measured on serial evaluation of 12 puppies every 15 days from 6 to 12 weeks of age. Some mea- surements showed no or negative correlation with increasing body weight and were comparable to published data for adult dogs with very large ranges of age and body weight. Higher immunological activity in young dogs may be a possible explanation for these results.

Keywords Dog Á Puppy Á Ultrasonography Á Abdominal lymph nodes

22.1 Introduction

Reports of ultrasound examination of the abdominal lymph nodes and their normal size ranges in healthy adult dogs have recently been published (Agthe et al. 2009; Llabres-Diaz Francisco 2004; Mayer et al. 2010). This work was possible due to the availability of probes with higher resolution and ability to identify very small anatomical structures. Abdominal and pelvic lymph nodes are classified as visceral or parietal depending on lymph drainage from the viscera or parietal regions of the abdomen. Among the former, the jejunal lymph nodes are the most frequently evaluated; among the latter, the hypogastric and the medial iliac are the most frequently evaluated. The jejunal lymph nodes, usually found as a pair, are the largest of the abdominal lymph nodes and lie at the root of the jejunal and ilial mesentery, in

A. La Pietra (&) Á M. De Majo Dipartimento di Sanità Pubblica Veterinaria, Sezione di Medicina e Farmacologia, Università degli studi di Messina, Messina, Italy e-mail: [email protected]

C. Boiti et al. (eds.), Trends in Veterinary Sciences, 119 DOI: 10.1007/978-3-642-36488-4_22, Ó Springer-Verlag Berlin Heidelberg 2013 120 A. La Pietra and M. De Majo close proximity with the cranial mesenteric vessels. They are also referred to as the cranial mesenteric lymph nodes, and drain the jejunum, ileum, and pancreas (Bezuidenhout 1993). The medial iliac lymph nodes are parietal lymph nodes, the largest in the iliosacral lymphocenter, and are found in variable numbers stretching cranially to the deep circumflex iliac arteries and aorta and caudally to the origin of the external iliac arteries. The left node is often located in a position more ventral than the right one. They drain the genital organs, peritoneum, anus, rectum, colon, skin, subcutaneous tissue, and muscles of the pelvic region, including the tail (Bezuidenhout 1993). The hypogastric lymph nodes are located in the corner formed by the internal iliac vessels and the median sacral artery and belong to the same lymphocenter. They are smaller than the medial iliac nodes and have a spherical shape. These lymph nodes drain almost the same areas as the medial iliac nodes (Bezuidenhout 1993). The abdominal lymph nodes are usually readily detectable in young subjects (Pugh 1994), but the size of these lymph nodes has not yet been determined in healthy puppies. Serial ultrasound evaluations of the jejunal, medial iliac, and hypogastric lymph nodes in clinically healthy subjects aged between 6 and 12 weeks were conducted to identify a size reference range and to define what constitutes enlarged abdominal lymph nodes in puppies.

22.2 Materials and Methods

Ultrasound assessment of abdominal lymph nodes was conducted every 15 days in 12 puppies (2 boxers, 8 American Staffordshire terriers, and 2 Maremma sheep- dogs; seven females, five males), from 6 until 12 weeks of age. All of the puppies were healthy on the basis of clinical examination, blood count, and fecal parasi- tological examination. The puppies were fasted overnight with free access to water prior to each ultrasound and were weighed on a digital pediatric scale. Consent was provided by the owners of all puppies. Mylab Five Vet echograph (Esaote, Italia), set for shallow abdominal evalua- tion, was used with an 8-MHz microconvex transducer at 5 cm working depth and 60 % power emission. The subjects were examined by a single operator. They were placed in left lateral recumbency with right paramedian retrocostal scanning for the jejunal lymph nodes and prepubic paramedian scanning for the right medial iliac and hypogastric nodes. The puppies were then placed in right lateral recumbency, and the medial iliac and ipsilateral hypogastric nodes were viewed with the same scans. The scanning plane was adjusted to obtain the best view of the lymph node’s long axis, and then the scan axis was rotated 90° for evaluations of the short axis. After a careful evaluation of lymph node echogenicity and optimization of the widest section, measurements of length, thickness, and width (in mm) were made; the width/length ratio (w/l) was then calculated. For jejunal lymph nodes, measurements were performed only on the largest lymph nodes. 22 Ultrasonographic Assessment of Abdominal Lymph Nodes 121

Student’s t test was performed between the parameters measured in the right and left lymph nodes and between the first and the second measurements. Linear regression on body weight was performed after Student’s t test on consecutive measurements of body weight. Statistical analysis was performed by specific software GraphPad InStat (GraphPad Software, La Jolla, CA, USA) with signifi- cance limit of P \ 0.05.

22.3 Results

The data reported here include only the first and second evaluations, which were all that were completed at the time of writing. The medial iliac lymph nodes appeared iso-to hypo-echoic compared to mesenteric fat and were always easily identifiable. They had a homogeneous appearance or a cortical-medullar structure. Visualization was aided by their location on the abdominal aorta at the lumbar vertebrae, which served as a dorsal landmark. They appeared with larger dimen- sions at the cranial and caudal ends and narrower in the center. On cross-section, they appeared ovoid (Fig. 22.1). In longitudinal section, the hypogastric lymph nodes had an elongated oval shape, and had close communication with the surrounding vessels, which served as major landmarks. In cross-section, they appeared spherical and in line with the iliac arteries. They were hypoechoic compared to mesenteric fat and appeared generally homogeneous. The jejunal lymph nodes appeared very clearly in the right paramedian scan- ning, and were bigger than both other abdominal lymph nodes on both longitudinal and transverse scanning. The vessels located in the middle of the same lympho- center or the cranial artery and mesenteric vein were important landmarks. Two very large lymph node-like objects were also easily identifiable. On cross-section, they appeared oval shaped, nearly circular, iso-to hypoechoic compared to the mesentery, and generally homogeneous. Descriptive statistics related to lymph node size is shown in Table 22.1. At the first evaluation, body weight was 3.48 ± 0.4 kg (mean ± S.D.); at the second evaluation, body weight was 4.48 ± 0.7 kg. This difference in weight was

Fig. 22.1 Lymph nodes: a Medial iliac; b Hypogastric; c Jejunal 122 A. La Pietra and M. De Majo

Table 22.1 Measurements of length, thickness, and width of abdominal lymph nodes and the width/length ratio (w/l) 1st Control 2nd Control Average S.D. Median Range Media S.D. Mean Range Jejunal Length (mm) 37.6 3.8 39.2 31.0–42.9 40.1 5.3 38.7 32.8–48.2 Thickness (mm) 6.8 0.8 6.7 5.6–8.4 7.7 1.5 7.3 5.4–10.2 Width (mm) 9.4 2.1 9.3 4.8–12.8 9.6 3.3 8.9 5.6–18.4 w/l 0.25 0.06 0.25 0.17–0.41 0.24 0.05 0.22 0.17–0.38

Iliac medial Right Average S.D. Median Range Average S.D. Mean Range Length (mm) 21.0 6.0 21.2 10.6–30.4 23.6 3.7 24.0 19.8–33.0 Thickness (mm) 4.3 1.5 3.9 2.3–7.4 4.7 1.0 4.8 3.1–6.4 Width(mm) 7.0 1.3 7.3 4.3–9.1 6.5 1.2 6.4 4.4–8.6 w/l 0.34 0.08 0.35 0.22–0.53 0.27* 0.05 0.26 0.21–0.34

Left Media S.D. Median Range Average S.D. Mean Range Length (mm) 22.9 6.5 26.0 11.4–29.8 26.3 4.6 25.2 20.4–37.6 Thickness (mm) 4.1 1.0 4.2 2.2–5.4 4.5 0.5 4.5 3.5–5.4 Width (mm) 7.2 2.2 7.3 4.8–10.8 5.2* 1.3 5.6 2.6–7.5 w/l 0.32 0.10 0.29 0.17–0.52 0.35 0.05 0.20 0.14–0.32

HYPOGASTRIC Right Average S.D Median Range Average S.D. Mean Range Length (mm) 10.1 2.0 10.0 6.4–12.8 10.7 1.8 10.2 7.8–13.8 Thickness (mm) 3.1 0.7 2.9 2.2–4.8 2.3* 0.3 2.4 1.8–2.8 Width (mm) 4.1 0.5 4.1 3.5–5.4 3.8 0.8 3.8 2.7–5.4 w/l 0.40 0.07 0.41 0.22–0.49 0.36 0.08 0.37 0.23–0.52

Left Average DS Median Range Average S.D. Mean Range Length (mm) 11.1 2.3 11.2 6.9–14.6 10.3 1.9 10.3 6.4–12.7 Thickness (mm) 2.6 0.5 2.8 1.8–3.5 2.3 0.3 2.2 1.9–2.5 Width (mm) 4.2 0.5 4.1 3.4–5.2 3.5* 0.8 3.6 2.5–5.5 w/l 0.38 0.08 0.39 0.24–0.55 0.35 0.09 0.33 0.23–0.53 Data that were significantly different between the first and second evaluations are marked by asterisks significant (P \ 0.01). None of the remaining measured parameters showed any difference between the left and right lymph nodes or a correlation with weight gain.

22.4 Discussion

Ultrasonography of abdominal lymph nodes is particularly important to make a distinction between inflammatory and neoplastic reactivity, by evaluating the size, shape, and margins of the internal structure, as well as the vascular markings. 22 Ultrasonographic Assessment of Abdominal Lymph Nodes 123

Ultrasound also provides a fundamental aid to needle aspiration of abdominal lymph nodes (Nyman and O’Brien 2007). The sizes of the medial iliac and jejunal nodes were similar to the bibliographic data obtained from adult subjects with variable weights (Agthe et al. 2009; Mayer et al. 2010); this may be due to the increased activity of the lymphatic stations in puppies. The statistically significant decrease of some of the measured parameters between the evaluations suggests that the size of the lymph nodes may be uncorrelated or inversely correlated with the weight of the animal. We believe that the results of this survey can be definitive only following a third evaluation. Limitations of the study are the low number of subjects, even though they were of average constitution and serially assessing puppies is difficult. At this time, the authors have not reported data for subjects of small size, because the number is still too small for statistical consideration.

References

Agthe P, Caine AR, Posch B, Herrtage EM (2009) Ultrasonographic apparance of jejunal lymph nodes in dogs without clinical signs of gastrointestinal desease. Vet Radiol Ultrasound 50:195–200 Bezuidenhout AJ (1993) The lymphatic system. In: Evans HE (ed) Miller’s anatomy of the dog, 3rd edn. WB Saunders, Philadelphia, pp 717–757 Llabres-Diaz Francisco J (2004) Ultrasonography of the medial iliac lymph nodes in the dog. Vet Radiol Ultrasound 45:156–165 Mayer MN, Lawson AJ, Silver I (2010) Sonographic characteristic of presumptively normal canine medial iliac and superficial inguinal lymph nodes. Vet Radiol Ultrasound 51:638–641 Nyman HT, O’Brien RT (2007) The sonographic evaluation lymph nodes. Clin Tech Small Anim Pract 22:128–137 Pugh CR (1994) Ultrasonographic examination of abdominal lymph nodes in the dog. Vet Radiol Ultrasound 35:110–115 Chapter 23 Changes in the Metabolic Profile and Performance of Dairy Cows Fed Two Dietary Crude Protein Concentrations

D. Bernardini, S. Segato, G. Marchesini, A. L. Stefani, G. Gerardi and I. Andrighetto

Abstract According to a 2 9 2 cross-over design, 14 lactating Holstein–Friesian cows were fed two isofibrous and isoenergetic diets based on either 14.5 % of crude protein (LOW CP diet) or 16.5 % (HIGH CP diet). Milk yield and quality and metabolic profile were detected during the third week of each experimental period. Dietary treatment had no effect on dry matter (DM) intake (21.0 kg/d on average), milk yield (34.7 kg/d on average), and milk protein content (3.48 % on average). LOW CP fed cows showed a lower milk urea content (19.4 vs. 29.7 mg/ dl; P \ 0.01). In LOW CP treatment, it was observed a significant increase of plasma globulins, glucose, and creatinine meanwhile the urea level was significant lower. The efficiency of CP utilization was higher (0.39 vs. 0.34; P \ 0.05) for the LOW CP group.

Keywords Dairy cows Á Metabolic profile Á Dietary crude protein

23.1 Introduction

High dietary protein content can increase milk yield and protein concentration in dairy cows. When the nitrogen supply exceeds the cow’s requirement, a reduction in efficiency may occur both in the digestive process and in absorbed amino acid

D. Bernardini (&) Á G. Gerardi Dipartimento di Scienze Cliniche Veterinarie, Università degli Studi di Padova, Viale dell’Università 16, 35020 Legnaro, PD, Italy e-mail: [email protected] S. Segato Á G. Marchesini Dipartimento di Scienze Animali, Università degli Studi di Padova, Viale dell’Università 16, 35020 Legnaro, PD, Italy A. L. Stefani Á I. Andrighetto Istituto Zooprofilattico Sperimentale delle Venezie, Viale dell’Università 10, 35020 Legnaro, PD, Italy

C. Boiti et al. (eds.), Trends in Veterinary Sciences, 125 DOI: 10.1007/978-3-642-36488-4_23, Ó Springer-Verlag Berlin Heidelberg 2013 126 D. Bernardini et al. utilization during the anabolic phase. Along with the environmental risk of nitrogen pollution, excessive CP can increase milk urea concentration and have negative consequences on the coagulation of milk (Segato et al. 2007). In dairy cows with a milk yield up to 35 kg/day, the nitrogen content should be maintained within 26 g/kg of dry matter (DM) (equal to 16.3 % of CP). Achieving lower nitrogen levels is possible by feeding highly digestible forages (De Campaneneere et al. 2006) and/or free amino acids (Cozzi et al. 2003). Along with measuring nitrogen input and output, analysis of the metabolic profile could explain anabolic (protein synthesis) and catabolic (urea excretion) processes involving nitrogen (Bernardini et al. 2005), which would allow the assessment of nitrogen efficiency in dairy cows. The aim of this study was to assess the effect of dietary protein reduction on the productive performance and metabolic profile of dairy cows fed with a maize silage-based total mixed ration (TMR).

23.2 Materials and Methods

According to a 2 9 2 cross-over design (2 diets 9 2 periods), 14 lactating Holstein–Friesian cows (142 ± 79 days in milk; 35.1 ± 5.3 kg/day milk pro- duction) were fed two isoenergetic (0.95 UFL/kg DM) and isofibrous (31.5 % DM) diets characterized by different crude protein levels: LOW CP (14.5 % DM) and HIGH CP (16.5 % DM). In the LOW CP diet, a part of the soybean by-products was replaced with fine bran and maize meal supplemented with L-Lysine and DL-methionine. For each experimental period, the metabolic profile and milk yield and quality were analyzed after 3 weeks of adaptation. DM intake was continuously and automatically measured (Biocontrol A/S, Rakkestad, Norway) and blood samples were collected into heparinized tubes after the morning milking. All metabolic parameters were determined using an automatic analyzer (Hitachi 911, Roche Diagnostics GmBH Mannheim), with the exception of nonesterified fatty acids (NEFA), which were analyzed using a commercial kit (Novatech Diagnostics, Randox). The assumption of normality of all variables was tested before carrying out the ANOVA (Proc Mixed) according to a linear model, which considered diet and period as main effects and cow as random effect (SAS 2002).

23.3 Results

Dietary treatment did not affect DM intake (20.9 vs. 21.1 kg/day; P [ 0.10), milk yield (35.0 vs. 34.4 kg/day; P [ 0.10), or milk protein content (3.48 vs. 3.47 %; P [ 0.10). The CP ingestion was significantly lower in LOW CP-fed cows (3.02 vs. 3.42 kg/day; P \ 0.05), which also resulted in more effective nitrogen effi- ciency, expressed as the ratio between milk nitrogen and ingested nitrogen (0.39 23 Changes in the metabolic profile and performance 127

Table 23.1 Effect of dietary treatment on metabolic profile of lactating dairy cows Diet P SEM Low CP High CP 14.5 (%) 16.5 (%) Total protein (g/L) 84.0 81.9 1.6 Albumin (g/L) 35.0 35.1 ns 0.7 Globulins (g/L) 49.0 46.8 * 2.1 Urea (mmol/L) 3.80 5.89 ** 0.24 Glucose (mmol/L) 3.49 3.38 * 0.05 Total cholesterol (mmol/L) 5.36 5.14 ns 0.25 NEFA (mEq/L) 0.12 0.17 ns 0.04 Direct bilirubin (lmol/L) 1.72 1.67 ns 0.18 Total bilirubin (lmol/L) 3.92 3.89 ns 0.19 Creatinine (lmol/L) 63.6 60.3 ** 2.7 AST (U/L) 75.0 75.9 ns 2.8 cGT (U/L) 26.5 26.8 ns 1.7 CK (U/L) 157 159 ns 9 Low CP diet with low CP content (14.5 % DM); High CP diet with high CP content (16.5 % DM). ** P \ 0.01; * P \ 0.05; P \ 0.10; ns not significant vs. 0.34; P \ 0.05). Furthermore, the LOW CP-fed cows’ milk was characterized by a lower urea concentration (19.4 vs. 29.7 mg/dL; P \ 0.01). The results of the hematological profile are reported in Table 23.1. The LOW CP diet led to a tendency toward a higher amount of total protein, likely as a result of increased globulins. Furthermore, LOW CP-fed cows had significantly higher glucose and creatinine values, while the urea level was significantly lower.

23.4 Discussion

In diets for lactating dairy cows in which protein sources are mainly soybean by- products, lysine, and methionine are the limiting amino acids. The supplementing of these amino acids in the LOW CP diet likely improved its amino acid profile, leading to an increase in nitrogen efficiency, calculated as the ratio between milk and dietary CP percentage (Cozzi et al. 2003). However, a lower nitrogen supply, even when supported by a lysine and methionine supplementation, caused greater muscular protein mobilization to support milk protein synthesis. This hypothesis was confirmed by the higher blood creatinine level, which is an indicator of higher endogenous protein catabolism. The higher glucose levels in the LOW CP-fed cows may be due to the lower urea content in plasma and milk resulting in less liver fatigue needed to support urea conversion from ammonia. Even though cows were in a mid-stage of lactation, the LOW CP diet showed greater dietary nitrogen efficiency (Mäntysaari et al. 2005). This finding is of noticeable importance for the application of the EU Nitrates Directive, especially in vulnerable areas where there 128 D. Bernardini et al. are more intensive dairy farms (Segato et al. 2009). In conclusion, the results of this study confirm the possibility of reducing the amount of dietary nitrogen fed to high producing dairy cows without reduction in performance, at least after 4 months from parturition. The use of lower dietary CP is also suitable to reduce blood and milk urea levels, leading to better cheese-making traits. It should therefore be appropriate to further reduce dietary CP content by using high bio- logical value protein sources and/or supplying the diet with free essential amino acids.

Acknowledgment Research financially supported by Fondazione Cariverona (call 2003).

References

Bernardini D, Segato S, Gerardi G, Berzaghi P, Elia CA, Ferrari V, Poser H, Dorigo M (2005) Valutazione del metabolismo azotato in bovine da latte alimentate con dieta a base di insilato di mais o fieno. In: Proceeding of 13th Fe.Me.S.P.Rum, pp 78–79 Cozzi G, Berzaghi P, Segato S, Andrighetto I (2003) Productive response of dairy cows to a supplementation with methionine hydroxy analog. Ital J Anim Sci 2(Suppl 1):222–224 De Campaneneere S, De Brabander DL, Vanacher JM (2006) Milk urea concentration as affected by the roughage type offered to dairy cattle. Livestock Sci 103:30–39 Mäntysaari P, Huhtanen P, Nousianinen J, Virkki M (2005) The effect of protein strategy during lactation on performance of primiparous dairy cows fed total mixed ration. Livestock Prod Sci 94:189–198 SAS (2002) User’s guide: statistic. SAS Inst. Inc., Cary, NC (USA) Segato S, Balzan S, Elia CA, Lignitto L, Granata A, Magro L, Contiero B, Andrighetto I, Novelli E (2007) Effect of period of milk production and ripening on quality traits of Asiago cheese. Ital J Anim Sci 6(Suppl 1):469–471 Segato S, Marchesini G, Andrighetto I (2009) A comparison of nitrogen use efficiency and surplus in two dairy farms typologies. Ital J Anim Sci 8(Suppl 3):178–180 Chapter 24 Impact of Physical Exercise on Release of Cardiac Troponins: Evaluation in Healthy and Cardiopathic Dogs

M. Pugliese, A. Seminara, M. De Majo, A. La Pietra and P. P. Niutta

Abstract The purpose of this study was to evaluate the impact of physical exercise on the release of cardiac troponins (cTn). Sixteen dogs were evaluated: 8 clinically healthy (group A) and 8 (group B) with valve disease in the asymp- tomatic stage (class I, New York Heart Association). All dogs, after a complete cardiac evaluation, were forced to exercise on a motorized treadmill at three different speeds: 1.8 km/h (low speed), 3.6 km/h (medium speed), and 5.4 km/h (high speed) lasting 6 min. Each test was performed on different days. Plasma samples were refrigerated at -20°. cTnI and cTnT were measured just before the running test, after a recovery period of 20 min, and then 2, 4, and 6 h later. cTnI concentrations increased in group A only at the highest speed, while cTnT did not change. These results suggest that exercise may induce myocardial suffering.

Keywords Exercise test Á Cardiac troponins Á Heart failure

24.1 Introduction

Numerous studies have observed that prolonged strenuous exercise promotes the elevation of cardiac-specific biomarkers [cardiac Troponin I (cTnI) and T (cTnT)] (Hermann et al. 2003; Vidotto et al. 2005; Shave et al. 2007; La Gerche et al. 2008), as a physiological response of the body to physical exertion (Scharhag et al. 2005; Scharhag et al. 2008). The knowledge and clinical interpretation of exercise-related changes in cardiac-specific biomarkers is complicated by high inter-individual variation in the available field-based competitive data that may be

M. Pugliese (&) Á A. Seminara Á M. De Majo Á A. La Pietra Á P. P. Niutta Unit of Medicine and Pharmacology Department of Veterinary Public Health, University Messina, Messina, Italy e-mail: [email protected]

C. Boiti et al. (eds.), Trends in Veterinary Sciences, 129 DOI: 10.1007/978-3-642-36488-4_24, Ó Springer-Verlag Berlin Heidelberg 2013 130 M. Pugliese et al. partially explained by large variations in exercise parameters such as duration and intensity (Vidotto et al. 2005) or evidence of underlying heart disease not clinically manifest. The purpose of this study was to evaluate the impact of physical exercise on the release of cardiac troponins (cTn) in healthy and card- iopathic dogs with valve disease in the asymptomatic stage (class I, New York Heart Association).

24.2 Materials and Methods

Eight healthy dogs (group A) and eight with valve disease in the asymptomatic stage (group B) were recruited for this study and written informed consent was obtained from each patient’s owner. The group A was represented by five American Stafford Shire Terrier (two males and three females), two Cross-Breed males and one Boxer female. The average age was 5.2 years. The group B (two females and six males) included four Cross-Breed, two English Setter, one Boxer, and one English Bulldog. The average age in group B was 8.4 years. All dogs underwent a full cardiac investigation (history, physical examination, arterial blood pressure measurement, full hematology, routine serum biochemistry, elec- trocardiography, survey radiographs, and color Doppler echocardiography). Diagnosis of valve disease and clinical staging was based on clinical findings, on echocardiography and on radiographic examination of cardiac enlargement by vertebral heart score (VHS) (Buchanan and Bucheler 1995). Patients were exer- cised on a motorized treadmill (GrilloÒ, Italy) graded at three different speeds: 1.8 km/h (low speed), 3.6 km/h (moderate speed), and 5.4 km/h (high speed) with a prior heating phase of 3 min. The duration of each test was 6 min. A period of 7 days between the tests was allowed. A session of pre-trained was carried out as the subjects were not familiar with the treadmill and the equipment. It was esti- mated a resting (recovery time) of about 20 min. The test was planned to terminate at an earlier stage (end point) if a patient showed signs of fatigue, pain, or distress or other symptoms of intolerance. Blood samples were collected 20 min before the test, 20 min afterwards (recovery period), and 2, 4, and 6 h after the exercise; they were centrifuged, immediately after collection and stored at -20 °C. Serum car- diac troponin-I was measured using a immunochemiluminescence test (AccessÒ— AccuTnIÒ). Cardiac troponin-T was measured by enzyme immunoassay test (ElecsysÒ—Roche). Levels of cTnI were below of 0.30 ng/ml (Spratt et al. 2005) and of cTnT below 0.04 ng/ml were considered normal. Samples of plasma were used for the assay of lactate, considered the most sensitive parameter for the evaluation of sub-maximal physical exercise (Larsen et al. 2001). Statistical analysis performed using Student’s t-tests included: comparison of the cTnI and cTnT concentrations on pre and post exercise; the values of cTn at the end of exercise at different speeds. Data are expressed as means with the corresponding standard deviations. 24 Impact of Physical Exercise on Release of Cardiac Troponins 131

24.3 Results

Before exercise, lactate levels (mean ± S.D.) were within the reference ranges for both groups (0.87 ± 0.32; references range 0.40–1.40 mmol/L); at the higher speeds (3.6 and 5.4 km/h) recovery levels were 2.8 ± 0.44 mmol/L. The concen- trations of cTnI (mean ± S.D.) in the pre-exercise were within the reference ranges in both groups (1.8 ± 0.006 and 2.4 ± 0.007 ng/ml, in groups A and B, respec- tively). Already 2 h after the end of the exercise, it showed a statistically significant increase (p = 0.011) of cTnI at higher speeds (mean 0.093 ± 0.02 ng/ml at 3.6 km/h; mean 0.1 ± 0.02 ng/ml at 5.4 km/h) in group B. No significant increase (p = 0.174) was found at the slowest speed (walk, 1.8 km/h, mean of cTnI 0.035 ± 0.01 ng/ml) (Fig. 24.1). Increased values of cTnI showed at 2 h after end of test remained higher at 4 and 6 h. The increase of plasma concentrations of troponins post-exercise compared between the two groups at speed 3.6 and 5.4 km/ h was significant (p = 0.022; p = 0.014). Changes of cTnT (mean pre-exercise 0.014 ± 0.07 ng/ml; mean post-exercise 0.012 ± 0.06 ng/ml) were not statistical significant.

24.4 Discussion

In human medicine, exercise testing (ET) is considered as a practical method, which provides to give important informations about the stage of heart failure. It is useful to formulate a accurate prognosis. The test is based on the response to physical effort by the subjective perception of fatigue, electrocardiographic monitoring, and blood pressure measure (Lainchbury and Richards 2002). The feasibility of ET in patients with congestive heart failure has been investigated in dogs by Kittleson et al. (1996), using submaximal test and measurements of lactate threshold and venous oxygen tension. More recently, Boddy et al. (2004) evaluated the suitability of the 6 min walk test in dogs. However, although the 6 min walk

0.12

0.1

0.08 1.8 Km/h 0.06 3.6 Km/h

0.04 5.4 Km/h

0.02

0 Pre Post Post 2h Post 4h

Fig. 24.1 Trends in mean concentrations of cTnI in group B in relation to different types of physical activity 132 M. Pugliese et al. test is simple, it is not based on a full objective evaluation. Indeed, several ele- ments may affect the distance walked in 6 min by a dog, such as the pace of the person walking the dog or the presence of distracting stimuli such as people, animals, or noise in the surrounding environment. One of the primary problems encountered by Kittleson and collegues was the unwillingness of approximately 50 % of the dogs to walk on a treadmill. In our study, none of the patients appeared reluctant undergo the test, probably because the owners were allowed to walk on the treadmill with their dogs. The increased level of lactate above the physiological ranges suggested that all dogs were subjected to a submaximal physical effort. The failure to increase levels of cTn in subjects of the control group is partially in agreement with those reported in humans and in the horse: cTnI does not undergo significant changes during exercise in healthy subjects (Eijsvogels et al. 2010; Nostell and Häggström 2008). Unlike the significant increase of cTnI in patients of group B (asymptomatic subjects with valve disease) occurred at higher speeds where suggested that exercise may induce myocardial suffering, evidenced by the increase of these values. The significant increase of cTnI if not exceeding the references range for the dog may indicate the presence of a myocardial insult mild and of short duration, such as not to exceed the physiological threshold of cTnI. The release of cTnI could be related to the increase in myocardial work and to higher demand of oxygen or as consequence of oxidative stress triggered in patients by with valve dysfunction by various factors, such as wall stress, spasm coronary artery, and ischemia. Although in our case, the increase in cTnI during treadmill exercise in subjects on class I New York Heart Association (NYHA) was not associated with the signs of exercise intolerance. Then, this parameter can be defined a good indicator of exercise tolerability. In human patients, it has been demonstrated that troponin-I reaches the peak plasma concentration approximately 6 h after acute myocardial damage (Schober et al. 2002). A similar peak was observed with serum troponin-I in rats after a prolonged and intense. Our study has shown that concentrations of cTnI were raised 2 h after the end of exercise test and remained increased in hours later. The lack of variation of cTnT in control group would seem to be connected to a diagnostic sensitivity of cTnI greater than the cTnT, which seems to have a greater prognostic value (Schober et al. 2002). In conclusion, our study suggests that intensity and duration of exercise can lead to myocardial suffering, undetectable except through objective parameters such as the measurement of cTnI.

References

Boddy KN, Roche BM, Schwartz DS, Nakayama T, Hamlin RL (2004) Evaluation of the six- minute walk test in dogs. Am J Vet Res 65(3):311–313 Buchanan JW, Bücheler J (1995) Vertebral scale system to measure canine heart size in radiographs. J Am Vet Med Assoc 15; 206(2):194–199 24 Impact of Physical Exercise on Release of Cardiac Troponins 133

Eijsvogels T, George K, Shave R, Gaze D, Levine BD, Hopman MT, Thijssen DH (2010) Effect of prolonged walking on cardiac troponin levels. Am J Cardiol 15;105(2):267–272 Herrmann M, Scharhag J, Miclea M (2003) Post-race kinetics of cardiac troponin. Clin Chem 49:831–834 Kittleson MD, Johnson LE, Pion PD (1996) Submaximal exercise testing using lactate threshold and venous oxygen tension as endpoints in normal dogs and in dogs with heart failure. J Vet Intern Med 10(1):21–27 La Gerche A, Connelly KA, Mooney DJ, MacIsaac AI, Prior DL (2008) Biochemical and functional abnormalities of left and right ventricular function after ultra-endurance exercise. Heart 94(7):860–866 Lainchbury JG, Richards AM (2002) Exercise testing in the assessment of chronic congestive heart failure. Heart 88(5):538–543 Larsen AI, Aarsland T, Kristiansen M, Haugland A, Dickstein K (2001) Assessing the effect of exercise training in men with heart failure; comparison of maximal, submaximal and endurance exercise protocols. Eur Heart J 22(8):684–692 Nostell K, Häggström J (2008) Resting concentrations of cardiac troponin I in fit horses and effect of racing. J Vet Cardiol 10(2):105–109 Scharhag J, Herrmann M, Urhausen A, Haschke M, Herrmann W, Kindermann W (2005) Independent elevations of N-terminal pro-brain natriuretic peptide and cardiac troponins in endurance athletes after prolonged strenuous exercise. Am Heart J 150(6):1128–1134 Scharhag J, Meyer T, Auracher M, Müller M, Herrmann M, Gabriel H, Herrmann W, Kindermann W (2008) Exercise-induced increases in NT-proBNP are not related to the exercise-induced immune response. Br J Sports Med 42(5):383–385 Schober KE, Oechtering G, Aupperle H (2002) Diagnosis of myocardial cell injury in cats. Tierärztl Prax 30:290–294 Shave R, George KP, Atkinson G (2007) Exercise-induced cardiac troponin T release: a meta- analysis. Med Sci Sports Exerc 39(12):2099–2106 Spratt DP, Mellanby RJ, Drury N, Archer J (2005) Cardiac troponin I: evaluation I of a biomarker for the diagnosis of heart disease in the dog. J Small Anim Pract 46(3):139–145 Vidotto C, Tschan H, Atamaniuk J, Pokan R, Bachl N, Müller MM (2005) Responses of N- Terminal Pro-Brain Natriuretic Peptide (NT-proBNP) and Cardiac Troponin I (cTnI) to competitive endurance exercise in recreational athletes. Int J Sports Med 26:645–650 Chapter 25 Canine Erythrocyte Morphology: Observations of a New Pattern, the ‘‘Quatrefoil’’ Erythrocyte

George Lubas, Alessandra Gavazza, Biancaurora Gugliucci, Anna Pasquini and Marianna Ricci

Abstract The ‘‘quatrefoil’’ erythrocyte pattern may be observed during erythro- cyte morphology evaluation in canine complete blood counts (CBCs). ‘‘Quatre- foil’’ red blood cells (RBCs) have been documented in blood smears using both optical and scanning electron microscopy. In our examination of 3,958 CBCs, we found a prevalence of 3.89 % ‘‘quatrefoil’’ RBCs. These RBCs mostly occurred between the body and feathered edge of the blood smears. This pattern was not statistically related to the breed or sex, but to the age of the dog (older dogs; p \ 0.0001). ‘‘Quatrefoil’’ RBCs are not significantly associated with other mor- phological erythrocyte changes. However, compared with populations of dogs without ‘‘quatrefoil’’ RBCs, dogs with this RBC pattern are significantly more likely to exhibit lower total leukocyte and neutrophil counts. Hypotheses describing the origin of ‘‘quatrefoil’’ RBCs include artifact effects, dacryocyte overlapping, and the effect of adhesion forces between two circulating RBCs in the blood stream (i.e., tank-trading and/or tumbling).

Keywords Erythrocyte Á Morphology Á Poikilocytosis Á Dog Á Quatrefoil erythrocyte

25.1 Introduction

Microscopic evaluation of blood smears is an essential part of the complete blood count (CBC) and an important component of validation of data generated by hematology analyzers. The most sophisticated analyzers do not detect many blood abnormalities. Furthermore, many of the analyzed parameters that provide an

G. Lubas (&) Á A. Gavazza Á B. Gugliucci Á A. Pasquini Á M. Ricci Dipartimento di Scienze Veterinarie, Università di Pisa, Via Livornese, San Piero a Grado, Pisa, Italy e-mail: [email protected]

C. Boiti et al. (eds.), Trends in Veterinary Sciences, 135 DOI: 10.1007/978-3-642-36488-4_25, Ó Springer-Verlag Berlin Heidelberg 2013 136 G. Lubas et al. objective characterization of erythrocyte morphology, e.g., anisocytosis, represent only an average of all the erythrocytes in the sample. Erythrocyte morphologic evaluation of a well-prepared blood smear performed by an expert microscopist at the optical microscope (OM) provides insight into the etiologies of anemia, the presence of underlying metabolic processes, the possibility of underlying specific organ dysfunction, and identification of infectious agents (Harvey 2001; De Nicola 2008; Walker 2008). The aim of this study was to describe the presence of a new erythrocyte alteration (‘‘quatrefoil’’ or ‘‘Pisa cross’’ erythrocytes) observed in canine blood smears examined with a scanning electron microscope (SEM). To understand the meaning of this morphological alteration, the possible correlation with signalment and other hemato-biochemical parameters was also investigated.

25.2 Materials and Methods

We evaluated 3,958 canine CBCs, which were selected between May 2009 and October 2010. All of the blood samples were collected in ethylenediaminetetra- acetic acid (EDTA) tubes containing the correct anticoagulant-blood ratio. The samples were analyzed with a veterinary impedance cell counter HeCoVet (SEAC, Calenzano, FI, Italia) within 3 h after collection. Blood smears, which were stained with Diff-QuickÒ, were also prepared within 3 h after collection. Smears were viewed with an OM at 209 magnification to evaluate cell distribution and to perform the platelet count. A magnification of 1009 was used to evaluate eryth- rocyte morphology (e.g., anisocytosis, polychromasia, poikilocytosis, and rouleaux formation), leukocyte and platelet morphology and to perform the differential leukocyte count (Paltrinieri et al. 2010). The CBCs in which ‘‘quatrefoil’’ eryth- rocytes were observed were statistically compared with a control population [CBCs not showing ‘‘quatrefoil’’ red blood cells (RBCs) in blood smears]. Blood smears examined with the SEM were initially fixed in methanol and viewed with the OM to locate areas that contained the ‘‘quatrefoil’’ RBC pattern. Suitable smears were prepared, without dehydration, for the SEM (JEOL JSM-5410) by the Inter-Departmental Microscopy Centre, and were processed with gold staining. The following tests were used for the statistical analysis: Fisher’s Exact test was used for the breed analysis, the Chi-squared test was used for the sex and age analysis, and the ANOVA test was used to compare CBC parameters, biochemical profiles, and serum protein electrophoresis results.

25.3 Results

The ‘‘quatrefoil’’ erythrocyte pattern was found during blood smear evaluation, generally in the central portion of the smear, between the body and feathered edge. These erythrocytes were isolated or occurred with other RBC modifications. In the 25 Canine Erythrocyte Morphology 137 central portion of the smear, there were minimal distortions and slide preparation artifacts. Approximately 50 % of the RBCs were barely touching each other (Figs. 25.1 and 25.2). The prevalence of the ‘‘quatrefoil’’ RBC morphology was 3.89 % (154/3,958). With regard to breed, the ‘‘quatrefoil’’ RBC pattern was observed in mixed breeds (n = 40; 25.9 %), Labrador Retrievers (n = 18; 11.8 %), German Shep- herds (n = 11; 7.2 %), Golden Retrievers (n = 8; 5.2 %), Poodles, Rottweilers, Setters each (n = 5; 3.2 %), Boxers (n = 4; 2.6 %), and others (n = 58; 37.7 %) (Fisher’s Exact test; p [ 0.05). The Chi-squared analysis was not statistically significant with regard to sex (p [ 0.05) (n = 81 males, 52.6 % and n = 73 females, 47.4 %). Dogs with ‘‘quatrefoil’’ RBCs were split in three age groups: puppy (0–12 months, n = 27 dogs), adults (13 months–6 years, n = 42 dogs), and old (C7 years old, n = 85 dogs). Compared with the control population, the Chi- square analysis revealed an increased frequency of ‘‘quatrefoil’’ RBCs in older dogs (p\0.0001). In dogs with ‘‘quatrefoil’’ RBCs, the following alterations were also observed (prevalence in the control population in brackets): anisocytosis (+1/ +3), 70.7 % (61.3 %); polychromasia (±/+2), 35.1 % (25.1 %); echinocytes, 22.7 % (20.1 %); rouleaux formation, 9 % (14.5 %); dacryocytes, 9 % (6.4 %); elliptocytes, 7.8 % (6.1 %); acanthocytes, 3.9 % (3.9 %); schistocytes, 3.2 % (4.5 %); eccentrocytes, 2.3 % (1.2 %); keratocytes, 2.3 % (2.1 %); and target cells and spherocytes, 1.3 % (0.7 %), Howell–Jolly bodies, 19.4 % (11.6 %). None of these changes were significantly associated (ANOVA test) with the appearance of ‘‘quatrefoil’’ RBCs. The ANOVA test performed on CBCs, biochemical profiles and serum protein electrophoresis parameters revealed that leukocyte counts (absolute) and neutrophil counts (absolute and percentage) were significantly (p \ 0.0001) lower in ‘‘qua- trefoil’’ RBC populations than in the control population.

25.4 Discussion

This study is the first to describe the prevalence of these various erythrocyte alter- ations. The occurrence of ‘‘quatrefoil’’ RBCs is quite rare, about 4 % (154/3,958 dogs). They have been observed more frequently in older dogs, with no differences between breed or sex. These dogs also exhibited more RBC changes (i.e., higher

Fig. 25.1 ‘‘Quatrefoil’’ erythrocytes viewed optical microscopy (black arrows) (91,000) 138 G. Lubas et al.

Fig. 25.2 ‘‘Quatrefoil’’ erythrocytes viewed with scanning electron microscopy (white arrows)(91,500)

grade of anisocytosis and polychromasia), even though they were not statistically significant (p [ 0.05). Compared with the control population, ‘‘quatrefoil’’ RBCs were more frequently found in dogs with lower leukocyte and neutrophil counts. One hypothesis that explains the formation of ‘‘quatrefoil’’ RBCs describes them as EDTA-induced artifacts. Another hypothesis is that dacryocytes overlap with other erythrocytes at the feathered edge of the blood smear. Upon further observation, this hypothesis was rejected because ‘‘quatrefoil’’ RBCs were found with higher fre- quency in the area between the body of the smear and the feathered edge. In addition, intermediate forms often exhibited round cell characteristics, which were different from dacryocytes. Rouleaux formation was less frequent in dogs with ‘‘quatrefoil’’ RBCs, and thus does not explain the cause of this cell pattern. SEM examination of cell morphology changes confirmed the presence of the ‘‘quatrefoil’’pattern in blood smears, which disproves the hypothesis of an optical illusion or accidental finding. Furthermore, the SEM analysis provided a basis for a 3-D model. The ‘‘quatrefoil’’ erythrocytes, examined with higher magnification compared to the optical micros- copy, displayed an ‘‘embraced’’ cell formation (Fig. 25.3). This pattern is different from the ‘‘coin-pile’’ formation seen in rouleaux formation or the chaotic arrange- ment seen with agglutinated RBCs. A second hypothesis was that this pattern was an unusual circulating cell. Cellular adhesion is a fascinating and difficult to investigate process. Blood cir- culation in vessels is accompanied with cell-to-cell and cell-to-wall collisions that could lead to adhesion. These interactions can be influenced by electrostatic, steric, hydration, Van der Waals, and specific chemical bonding forces. Adhesion is inversely related to the surface tension of erythrocytes’ suspension, but it is directly related to viscosity. The surface tension is related to the number of adhesion molecules, most of which are proteins attached to the cytoskeleton (Barshtein et al. 2000; Elblbesy et al. 2009). The extent of this aggregation is

Fig. 25.3 Hypothesis of ‘‘quatrefoil’’ erythrocyte formation 25 Canine Erythrocyte Morphology 139 determined by opposing forces; from one side the presence of macromolecules and from the other side superficial negative charge and shear stress. The RBC consists, from a physical point of view, of an outer flexible cell membrane and inner viscous fluid and its deformation derives from the membrane elastic properties together with a high surface-volume ratio. Experimental observations of erythrocytes in shear flows demonstrate two types of movement. One type is ‘‘tank-treading’’, a rotation of the membrane around the viscous core of the erythrocyte that maintains a constant shape. The other type is ‘‘tumbling’’ an overall rotation of the entire cell as a rigid body. If fluid external force is greater than membrane elastic resistance force, the membrane maintains its tank-treading movement. Otherwise the motion changes to tumbling. When the maximum elastic resistance force is comparable to the external fluid force there is a transition between the two kinds of movement, that is, an oscillation of the RBC accompanied by membrane rotation (Tsubota et al. 2010). The ‘‘quatrefoil’’ pattern could be a particular type of adhesion between two RBCs into a relatively small area of contact, together with the influence of shear flow forces and tank-treading motions which may lead to a slipping effect between two RBC membranes, and consequently cell deformation through the loss of biconcavity. The end result is an ‘‘embraced shape’’. Causes of adhesion could be related to the production of molecules from the immune system (inflammation and immune-mediated phenomena) and increased turbulence in damaged vascular beds. Alternatively, ineffective erythrocatheresis and a longer life span of circulating erythrocytes (e.g., in older animals) may contribute to increased adhesion. More data are required to understand the mechanism of this RBC morphological alteration. A large number of dogs with more detailed clinical data about health status may produce the data needed to correlate ‘‘quatrefoil’’ RBCs with a specific disorder or a group of diseases (e.g., inflammatory, neoplastic, or degenerative).

References

Barshtein G, Wajnblum D, Yedgar S (2000) Kinetics of linear rouleaux formation studied by visual monitoring of red cell dynamic organization. Biophys J 78:2470–2474 DeNicola DB (2008) Blood film cytology—Red blood cell morphologic changes; Clue to the cause of anemia and diagnostic direction to possible organ dysfunction. In: Proceedings 59° Intl Congr SCIVAC, Rimini, pp 109–110 Elblbesy M, Hereba A (2009) Effect of viscosity and surface tension on erythrocyte adhesion. Curr Appl Phys 9:872–874 Harvey JW (2001) Atlas of veterinary hematology: blood and bone marrow of domestic animals. Saunders, Philadelphia Paltrinieri S, Bertazzolo W, Giordano A (2010) Patologia clinica del cane e del gatto (Clinical Pathology of the dog and cat). Elsevier-Masson, Milano, pp 22–64 Tsubota K, Wada S (2010) Elastic force of red blood cell membrane during tank-treading motion: consideration of the membrane’s natural state. Int J Mech Sci 52:356–364 Walker D (2008) Peripheral blood smears. In: Cowell RL, Tyler RD, Meinkoth JH, DeNicola DB (eds) Diagnostic cytology and hematology of the dog and cat. Mosby Elsevier, Missouri, pp 390–421 Chapter 26 Pain Management in Companion Animals: Medical–Legal Aspects

V. Quartarone, A. Fazio, G. della Rocca, M. Russo and A. Passantino

Abstract Preventing and managing pain has become a fundamental part of quality and compassionate patient care in veterinary medicine. In fact, animal pain and suffering are clinically important conditions that adversely affect an animal’s quality of life. Methods to prevent and control pain must be tailored to the indi- vidual animal. As advocates for their patients, the veterinary team has the responsibility to recognize, assess, prevent, and treat pain. Considering that many dogs and cats still receive little or not analgesia following surgery, trauma, or disease, we suggest the development of guidelines on pain management to assist the veterinarian in optimizing comfort care.

Keywords Pain Á Therapy Á Dog Á Cat Á Legislation

V. Quartarone Á A. Fazio Dottorato di Ricerca in Scienze Mediche Veterinarie, Curriculum ‘‘Normative dei Paesi della UE relative al benessere e protezione animale’’, Università degli Studi di Messina, Messina, Italy G. della Rocca Dipartimento di Patologia, Diagnostica e Clinica Veterinaria, Università degli Studi di Perugia, Perugia, Italy M. Russo Avvocato e Dottore di Ricerca in ‘‘Normative dei Paesi della UE relative al benessere e protezione animale’’, Università degli Studi di Messina, Messina, Italy A. Passantino (&) Dipartimento di Sanità Pubblica Veterinaria, Università degli Studi di Messina, Messina, Italy e-mail: [email protected]

C. Boiti et al. (eds.), Trends in Veterinary Sciences, 141 DOI: 10.1007/978-3-642-36488-4_26, Ó Springer-Verlag Berlin Heidelberg 2013 142 V. Quartarone et al.

26.1 Introduction

Pain control is an important issue, which in recent years has been given particular attention. The Faculty of Veterinary Medicine of Perugia University established the Study Center on Animal Pain (CeSDA) to ‘‘homogenize and coordinate sci- entific, cultural, and educational initiatives in the field of problems related to animal pain’’ (https://centri.unipg.it/cesda/). Successful diagnostic and therapeutic interventions aimed at detecting the presence of pain and implementing appropriate analgesic therapies require excellent knowledge of the pathophysiology of pain, the clinical consequences of inadequately treated pain, the kinetics and dynamics of analgesic drugs available, and of additional therapeutic options. The veterinarian must recognize the presence of pain and its location, intensity, type, and pathogenesis, and implement a treatment plan specifically adapted for each patient. Choice and conduct of the treatment are the sole responsibility of the vet- erinarian, who makes decisions according to the clinical evaluation of the patient. Pain therapy is one of the branches of medicine in which biological phenomena can also be examined from the aspect of ethics (res medica sub specie iuris). In veterinary medicine, there are no legal references on the issue, so the veterinarian is forced to refer to human regulations. A variety of practical and legal limits often lead to an underestimation by the veterinarian of the problem of pain and of its treatment. Consequently, we need guidelines that allow veterinarians to evaluate the different scientifically validated treatment options available, taking into account all aspects of patient care, quality of animal life, and compliance, with a proper veterinary-client-patient relationship.

26.2 Limits of Treatment of Pain in Animals

After recent scientific advances, pain is no longer considered just a symptom. It is a symptom of tissue damage, where it plays a biological role of protection (acute, physiological, and inflammatory pain), but it is also a pathology itself where, once the initial damage has been repaired, or in the absence of a lesion, it is still present (chronic and pathological pain). The transition from acute to chronic pain is due to the establishment in the nervous system of a series of morphological and functional changes that explain the pain, even in the absence of afferent stimulation. A characteristic of chronic pain is a lesser or complete lack of response to analgesic drugs normally used in acute pain, though it may eventually respond to other classes of drugs. It becomes necessary to go back to the pathogenesis of the pain occurring at the time of the diagnosis, thus defining the type. This is not always easy, especially given the fact that pain is a dynamic event that tends to change over time, with different and complex pathogenic mechanisms. 26 Pain Management in Companion Animals 143

It can be difficult to recognize the presence of pain in animals. They cannot verbalize, and at present there are no objective methods to demonstrate and quantify the presence of pain. Changes in physiological, metabolic, and neuro- endocrine parameters are not just indicative of pain but are also associated with anxiety and stress. In veterinary medicine, it is preferable to assess behavioral responses to pain. It requires a good knowledge of the behavioral repertoire of the species, as well as the individual, in response to pain. In particular, the relief of abnormal behavior patterns is considered particularly useful and meaningful in this respect. It can also be useful to estimate the potential level of pain that an animal may have, following a surgery or a disease, and to apply ‘‘pain scales.’’ These systems all have limits and it is often necessary to apply various diagnostic methods in combination, as part of a careful clinical evaluation of the patient. Analgesic therapy cannot be predetermined, but must be adapted to the type of pain, the animal species, the individual response to the drug, and to the health status of the patient. In addition to the classes of analgesics commonly used to combat acute pain (opioids, NSAIDs, local anesthetics, b2-agonists, and trama- dol), we must also consider other molecules (ketamine, anticonvulsants, antidepressants, GABA-mimetics, etc.), and nonpharmacological approaches (e.g., technical and physical rehabilitation, acupuncture, etc.) which can be applied and combined as part of a multimodal analgesic protocol. Medication side effects and drug interactions have to be taken into account, while remembering that the clinical consequences of pain, whether acute or chronic, are still often more severe than complications due to treatments.

26.3 Medical–Legal Considerations

In recent years, animals have been assigned important emotional, social, and supportive roles and this has allowed the conception of an animal as ‘‘res’’ (Article 812 of Italian Civil Code) (Passantino 2008) and the recognition, even from a legal point of view, of its nature as alive, sentient, and able to experience suffering and pain (EC 2007). For this reason, many researchers studied animals’ sensitivity and ability to feel pain (Bianchi et al. 2003; della Rocca and Di Salvo 2008; della Rocca et al. 2009). Such studies have shown that animals feel pain, as they have all of the anatomic and functional components necessary for the perception of painful stimuli as both reflex stimuli and conscious stimuli. It was also shown that fish are sentient and are capable of feeling pain (Algers et al. 2009). In some fish species, two types of nociceptors (A-delta and C) were identified to be responsible for the transduction and conduction of painful stimuli (Braithwaite and Boulcott 2007; Broom 2007; Hastein 2008; Sneddon 2002; Sneddon et al. 2003). The sensitivity of pet owners to the suffering of their animals has inspired progress in this area, putting pain control at the forefront of ‘‘compassionate care’’ (della Rocca and Di Salvo 2008; Ogilvie 2004). The treatment of pain should be an integral and inalienable part of 144 V. Quartarone et al. the therapeutic process in the animal patient as it is in the human patient, and must take into consideration the value and the dignity of the animal. There is no community or national legislation defining proper conduct of the veterinarian in the treatment of pain in animals or providing guidelines regarding the diagnosis or treatment of pain. At present, the only legal regulations related to pain therapy, concerning nar- cotic drugs and their storage, are in reference to humans (Anon 1990, 2001, 2006). Moreover, our country has recently issued a regulation (Anon 2010) relating to palliative care and pain therapy in the human field, providing an obligation to report pain level (Article 7 of Law no. 38/2010), the applied analgesic technique, drugs used, dosages, and the obtained results in the patient’s medical records. This regulation also considers the simplification of prescribing drugs for pain relief (Article 10 of Law no. 38/2010). The same has not been determined for veterinary medicine. These deficiencies are reflected in clinical practice, where pain management is often superficial or lacking. It is, therefore, necessary for the legislation to at least propose a measure aimed at regulating the use of analgesic drugs in veterinary medicine. It would also be desirable to formulate guidelines on the minimum requirements in the practice of pain management, which would aim to standardize medical conduct. These rec- ommendations could be met, modified, or rejected depending on the clinical or surgical needs of the animal patient, but each case would be supported by an updated tool, utilizing data from the current literature, and the synthesis of expert opinion and clinical practice. In the absence of a legal act, the veterinarian must always make choices respecting the life and health of the patient, and evaluating the relationship between costs and benefits, as determined by the code of ethics. It is, therefore, important to keep the animal’s life free of pain and stress as much as possible. This objective can be achieved only by the veterinary surgeon who possesses the appropriate knowledge to prevent and stop suffering.

References

Algers B, Blokhuis HJ, Bøtner A, Broom DM et al (2009) General approach to fish welfare and to the concept of sentience in fish. EFSA J 954:1–27 Anon (1990). D.P.R. 9 ottobre 1990, n. 309. G.U. 31 ottobre 1990, n. 255—S.O. n. 67 Anon (2001). Legge 8 febbraio 2001, n.12. G.U. 19 febbraio 2001, n 41 Anon (2006). Legge 21 febbraio 2006, n. 49. G.U. 27 febbraio 2006, S.O. n 45 Anon (2010). Legge 15 marzo 2010, n. 38. G.U. 19 marzo 2010, Serie Generale n 65 Bianchi E, Leonardi L, Breghi G, Melanie P (2003). Le scale del dolore come ausilio nell’interpretazione dello stato algico nel cane. Annali Della Facoltà di Medicina Veterinaria di, vol LVI. Pisa, pp 267–277 Braithwaite VA, Boulcott P (2007) Pain perception, aversion and fear in fish. Dis Aquat Organisms 75:131–138 26 Pain Management in Companion Animals 145

Broom DM (2007) Cognitive ability and sentience: which aquatic animals should be protected. Dis Aquat Organisms 75(2):99–108 Della Rocca G, Di Salvo A (2008) Il dolore negli animali: perché è importante trattarlo Patogenesi e conseguenze cliniche del dolore patologico. Parte 2. Bollettino AIVPA 1:29–35 Della Rocca G, Olivieri E, Di Salvo A, Gogny M (2009) Studio epidemiologico sulla attitudine dei medici veterinari alla gestione del dolore negli animali da compagnia. Bollettino AIVPA 2:15–21 European Community (2007) Treaty of lisbon amending the treaty on european union and the treaty establishing the european community, signed at lisbon. Offi J C 306, pp. 1–271 Hastein T (2008) Welfare of fish in aquaculture. Bulletin OIE 2:8–10 Ogilvie GK (2004). Fulfilling the first commandment: Providing analgesia and compassionate care. In: Proceedings of the 29th World Small Animal Veterinary Congress-WSAVA, October 6–9, Rhodes, pp 30–37 Passantino A (2008) Non-domesticated animals kept for companionship: an overview of the regulatory requirements in Italy to address animal welfare and human safety concerns. Eur J Companion Anim Pract 18(2):119–126 Sneddon LU (2002) Anatomical and electrophysiological analysis of the trigeminal nerve in a teleost fish, Oncorhynchus mykiss. Neurosci Lett 319:167–171 Sneddon LU, Braithwaite VA, Gentle MJ (2003) Do fish have nociceptors? Evidence for the evolution of a vertebrate sensory system. Proc R Soc London 270:1115–1121 Part IV Food Inspection Chapter 27 Increase of TVBN and TMA-N in Skin and Gills of Sparus aurata During Storage

A. Giuffrida, F. Giarratana, D. Signorino, G. Ziino and A. Panebianco

Abstract The aim of this work was to assess the increase of total volatile basic nitrogen (TVBN) and trimethylamine nitrogen (TMA-N) in the skin, gills and muscle of gilthead seabream (Sparus aurata) during refrigerated storage and to relate these increases to sensorial scores and spoilage bacteria growth. TVBN and TMA-N increases in skin and gills were more correlated to the sensorial scores obtained by the quality index method and to bacterial growth, in comparison to TVBN and TMA-N of muscle. Since the bacterial load of muscle is very low until the 168th hour of storage, according to the obtained results, measurement of TVBN and TMA-N of skin and gills could prove useful for the assessment of the shelf life of gilthead seabream.

Keywords Gilthead seabream Á Total volatile basic nitrogen (TVBN) Á Trimethylamine nitrogen (TMA-N) Á Skin Á Gills Á Shelf life

27.1 Introduction

The shelf life of fish is due to several intrinsic and extrinsic factors that can influence enzyme activity and the behaviour of spoilage bacteria. Some authors (Gram and Dalgaard 2002) have emphasised the importance of certain bacteria called specific spoilage organisms (SSO), including Pseudomonas spp., Shewa- nella putrefaciens and Photobacterium phosphoreum, which are able to exploit specific metabolites of fish muscle for their growth. Particularly, these bacteria are able to reduce trimethylamine oxide (TMA-O) into trimethylamine nitrogen (TMA-N), which causes the specific odour of spoiled fish. The increase of total

A. Giuffrida (&) Á F. Giarratana Á D. Signorino Á G. Ziino Á A. Panebianco Dipartimento Sanità Pubblica Veterinaria, Università di Messina-Polo Universitario Annunziata, Messina, Italy e-mail: [email protected]

C. Boiti et al. (eds.), Trends in Veterinary Sciences, 149 DOI: 10.1007/978-3-642-36488-4_27, Ó Springer-Verlag Berlin Heidelberg 2013 150 A. Giuffrida et al. volatile basic nitrogen (TVBN) during fish storage is related to the activity of SSO against muscle proteins and for this reason TVBN is considered as a good indicator of fish spoilage, especially for certain species. Several studies have been carried out to assess the relationships between SSO behaviour, sensorial parameters and chemical indicators such as TMA-N and TVBN, especially for species widely exploited in aquaculture as Sparus aurata (Giuffrida et al. 2005; Huidobro et al. 2000; Koutsoumanis and Nychas 2000; Lougovois et al. 2003; Marrone et al. 2007). In this species, however, the increase of TVBN and TMA-N during refrigerated storage did not always correlate to sensorial decay (quality index method, QIM and score) or to the growth of SSO. It is well-known that the bacterial load of fish muscle can be very low for several days of refrigerated storage, especially in fish with thick skin like gilthead sea- bream. Conversely, skin and gills normally have a high bacterial load that affects sensorial parameters early. These aspects could explain the low correlation between muscle chemical indicators and sensorial or microbiological parameters. Thus, the aim of this work was to study the increase of TVBN and TMA-N in the skin and gills of specimens of Sparus aurata during refrigerated storage, and at the same time to evaluate the relationship between these chemical indicators, spoilage flora and the QIM scores.

27.2 Materials and Methods

This study was carried out using 21 specimens of Sparus aurata obtained from a Sicilian aquaculture plant. Fish, after harvest, were freighted to the laboratory within 2 h and submitted to analytical and sensorial evaluation. The former was carried out after 0, 120, 168, 216 and 288 h of storage at 6 °C and included the sterile sampling of gills, skin and muscle from three specimens. These samples were cultured onto Levine Iron Agar at 25 °C for 72 h, after 10-fold dilutions in peptone water. An aliquot of each subsample was used for the determination of TVBN and TMA-N by the microdiffusion method in Conway cells, according to Mahmud et al. (2007). The sensorial evaluation was carried out at the same time intervals using the quality index method (Huidobro et al. 2000). Microbial, chemical and sensorial data were statistically evaluated by linear regression tests.

27.3 Results

Figure 27.1a shows the spoilage bacteria trends on skin, gills and muscle during storage. Of note is the quick growth on skin and gills and the slight growth in muscle until the end of storage, when the fish were considered completely spoiled. The partial (QIMskin-eye, QIMgills and QIMmuscle) and total scores of QIM (Fig. 27.1b) appear in agreement to the bacterial trends, showing a rejection sensorial point when the concentration of the spoilage flora reaches values [ Log 27 Increase of TVBN and TMA-N in Skin and Gills of Sparus aurata During Storage 151

Fig. 27.1 Trends of SSO (a), QIM (b), TVBN (c) and TMA-N (d) in skin (filled circle), gills (filled squre) and muscle (filled triangle) during storage. The QIM total scores (b) are indicated by the star symbol

7 cfu/g. Figure 27.1c and d shows TVBN and TMA-N trends for skin, gills and muscle during storage. The concentration of muscle TVBN almost remained constant until the 166th hour, while the values for skin and gills progressively increased along the entire storage period. Concerning TMA-N values (Fig. 27.1d), only in the gills did the increase appear to correlate with prolonged storage period; on the contrary, the muscle values had a fluctuating trend, and the skin values showed a moderate increase. All the above-mentioned aspects are confirmed by regression tests which are summarised in Table 27.1.

Table 27.1 Coefficient of determination R2 for TVBN and TMA-N versus QIM scores and SSO counts TVBN TVBN TVBN TMA-N TMA-N TMA-N gills skin muscle gills skin muscle QIM 0.8404 0.8797 0.6985 0.9479 0.8714 0.6439 SSO gills 0.6564 – – 0.8452 – – SSO skin – 0.6462 – – 0.9297 – SSO – – 0.4686 – – 0.5221 muscle 152 A. Giuffrida et al.

27.4 Discussion

These results confirm the low significance of muscle TVBN and TMA-N values to assess the shelf life of gilthead seabream, according to Koutsoumanis and Nychas (2000) and Marrone et al. (2007). Conversely, these chemical indicators on gills and skin could be useful as their increase appeared correlated to the storage duration. These aspects could be due to the high level of bacterial contamination in these sites, and at the same time to the accumulation on skin and gills of several mucoproteins and excretion compounds. It is known that, for some species, the potential accumulation of TMA-O on skin and gills is to regulate hydro-osmotic homeostasis (Wood 1993). Therefore, our results appear worthy of further inves- tigations, especially in order to utilise a more correct application of Regulations 853/2003 and 2074/2005 EC concerning the use of these chemical indicators.

References

Giuffrida A, Ziino G, Pennisi L, Panebianco A, Donato G (2005) Valutazioni comparative sulla conservabilità di Sparidi allevati. Ind Alim 44:381–386 Gram L, Dalgaard P (2002) Fish spoilage bacteria-problems and solution. Current Op in Biotec 13:262–266 Huidobro A, Pastor A, Tejada M (2000) Quality index method developed for Raw Gilthead Seabream (Sparus aurata). J Food Sci 65:1202–1205 Koutsoumanis K, Nychas GJE (2000) Application of a systematic experimental procedure to develop a microbial model for rapid fish shelf life predictions. Int J of Food Microbiol 60:171–184 Lougovois VP, Kyranas ER, Kyrana VR (2003) Comparison of selected methods of assessing freshness quality and remaining storage life of iced gilthead sea bream (Sparus aurata). Food Res Int 36:551–560 Mahmud MM, Hossain MA, Jahan I, Banerjee P, Rahaman MA (2007) Effect of delayed icing on the quality characteristics of Bagda (Penaeus monodon fabricius, 1798). Int J Sustain Crop Prod 2:24–30 Marrone R, Pennisi L, Colarusso G, Ghedini M, Ianieri A, Anastasio A (2007) Shelf-life di orate (Sparus aurata) provenienti da allevamenti off-shore e confezionate in atmosfera protettiva. Atti AIVI 17:194–198 Wood CM (1993) Ammonia and urea metabolism and excretion. In: Evans DH (ed) The rhysiology of fishes. CRC Press, Florida, pp 379–425 Chapter 28 Actin Proteolysis in San Daniele Dry-Cured Ham

M. L. Stecchini, A. Fabbro, M. Spaziani, E. Venir and G. Lippe

Abstract The aim of this work was to define the actin degradation pattern during the production of dry-cured San Daniele ham, as a factor that could influence its ripening and sensory characteristics. Biceps femoris muscle samples from San Daniele hams were subjected to denaturing and reducing conditions and one- dimensional sodium dodecyl sulphate–polyacrylamide gel electrophoresis followed by immuno-detection analysis. The degradation of actin was not extensive and was evident only after the salting stage, which could have labilized protein interactions in the myofibrillar structure. This limited proteolysis may be associated with the inaccessibility of the actin molecule to proteolytic enzymes.

Keywords Actin Á Dry-cured ham Á Proteolysis

28.1 Introduction

The production of dry-cured ham requires a long processing time and is associated with intense proteolytic activity on the myofibrillar as well as on the sarcoplasmic proteins, resulting in their progressive degradation. Due to their low stability, the cytosolic endopeptidases, calpains, poorly contribute to protein degradation. In contrast, cathepsins significantly sustain proteolysis, but their activities gradu- ally decrease throughout processing (Toldrà and Etherington 1988). A residual activity of only 5–10 % for cathepsins B, H, and L has been reported at 15 months of processing (Toldrà et al. 1993). The initial breakdown of muscle proteins by endopeptidases is followed by the action of exopeptidases, giving rise to small peptides and free amino acids. These final products can contribute directly or

M. L.Stecchini (&) Á A. Fabbro Á M. Spaziani Á E. Venir Á G. Lippe Department of Food Science, University of Udine, Udine, Italy e-mail: [email protected]

C. Boiti et al. (eds.), Trends in Veterinary Sciences, 153 DOI: 10.1007/978-3-642-36488-4_28, Ó Springer-Verlag Berlin Heidelberg 2013 154 M. L. Stecchini et al. indirectly, as precursors of other compounds (keto acids, amines, aldehydes, methyl ketones, etc.), to flavor development in dry-cured ham (Hinrichsen and Pedersen 1994). On the other hand, excessive proteolysis could be responsible for the appearance of defects (softness and bitter flavor) and might be avoided by removing raw materials with high proteolytic activity, as suggested for Parma ham (Schivazappa et al. 2002). Structural modifications also associated with proteolysis are essential in determining the typical texture of dry-cured ham and contributing to flavor perception (Larrea et al. 2007). A number of studies have been published on the use of nonprotein nitrogen fraction as an indicator of dry-cured ham proteolysis. More recently, proteomics has been used to study the evolution of myofibrillar and sarcoplasmic protein hydrolysis (Di Luccia et al. 2005; Picariello et al. 2006). This latter approach is useful for identifying molecular markers to predict and discriminate quality characteristics. In a recent proteomic study on Slovenian dry-cured hams (Kraški pršut) (Škrlep et al. 2011), the sensory defects found were related to the degra- dation patterns of proteins, including actin. This study aimed to investigate the degradation of actin during processing of San Daniele dry-cured ham, providing a characterization of the process with respect to myofibrillar protein fragmentation, which could influence the final sensory characteristics.

28.2 Materials and Methods

Hams were obtained from Large White 9 Landrace pigs, suitable for the protected designation of origin (PDO) Prosciutto di San Daniele. The internal Biceps femoris muscles, characterized by low salt penetration, were excised from the hams and analyzed at crucial steps of the ham-curing process (T1 = out of salting, 14–18 days; T2 = introduction to resting, 35 days; T3 = after washing and dry- ing, 117 days; T4 = after greasing, 211–221 days; T5 = end of curing, 413 days). The protein extraction was carried out in a sodium dodecyl sulfate (SDS) solution containing 100 mM dithiothreitol (DTT) to optimize protein recovery. Extracts were analyzed using one-dimensional denaturing electrophoresis (SDS- PAGE) for the separation of proteins between 10 and 100 kDa. Western blotting was then run with anti-actin antibody (Sigma-Aldrich) and immunoreactive bands were detected by chemiluminescence.

28.3 Results

In all samples, the presence of intact actin with an apparent molecular weight of 42 kDa was observed. Dry-cured ham processing gave rise to a limited degrada- tion of this myofibrillar protein. From the drying phase, fragments around 29 and 28 Actin Proteolysis in San Daniele Dry-Cured Ham 155

22 kDa were detected using anti-actin antibody. After 210–220 days, a new fragment of 38 kDa appeared and remained intact to the end of the curing, along with the 29- and 22-kDa fragments.

28.4 Discussion

Despite the number of papers published on dry-cured ham proteolysis, the research has only recently focused on actin proteolysis. The presence of intact actin was restricted to the first 6 months of the Italian dry-cured ham process. Actin then seemed to disappear after 10 months of processing (Di Luccia et al. 2005). Such a complete hydrolysis of actin was not observed in Spanish dry-cured hams (Teruel), where actin appeared to decrease less in the Semimembranosus muscle than in the Biceps femoris (Larrea et al. 2006). Small (1502–1971 Da) peptide fragments of actin have been identified at the end of Serrano dry-cured ham processing, sup- porting the relevance of action of cathepsin D (Sentandreu et al. 2007). Cathepsin D may remain active during a large part of the processing period, although a reduction is expected with increasing NaCl concentrations (Sarraga et al. 1993). We hypothesize that actin degradation in San Daniele ham, which seems to be similar to the degradation of myosin previously reported (Spaziani et al. 2009), may occur because of weaker myofibrillar-protein electrostatic interactions induced by modifications of ionic strength due to salt penetration during pro- cessing. This would explain the lag between the salting step and the appearance of proteolytic fragments, which are probably generated by cathepsin activities.

Acknowledgments This research was supported by a grant from ‘‘Legge regionale L.R. 26/2005 articolo 23: Innovazione e Ottimizzazione nella Filiera del Prosciutto Crudo Tipico’’.

References

Di Luccia A, Picariello G, Cacace G, Scaloni A, Faccia M, Liuzzi V, Alviti G, Spagna Musso S (2005) Proteomic analysis of water soluble and myofibrillar protein changes occurring in dry- cured hams. Meat Sci 69:479–491 Hinrichsen LL, Pedersen SB (1994) Relationship among flavor, volatile compounds, chemical changes and microflora in Italian-type dry-cured ham during processing. J Sci Food Agric 43:2932–2940 Larrea V, Hernando I, Quiles A, Lluch MA, Pérez-Munuera I (2006) Changes in proteins during Teruel dry-cured ham processing. Meat Sci 74:586–593 Larrea V, Pérez-Munuera I, Hernando I, Quiles A, Llorca E, Lluch MA (2007) Microstructural changes in Teruel dry-cured ham during processing. Meat Sci 76:574–582 Picariello G, De Martino A, Mamone G, Ferranti P, Addeo F, Faccia M, Spagna Musso S, Di Luccia A (2006) Proteomic study of muscle sarcoplasmic proteins using AUT-PAGE/SDS- PAGE as two-dimensional gel electrophoresis. J Chromatogr B 833:101–108 156 M. L. Stecchini et al.

Sàrraga C, Gil M, Garcìa-Regueiro JA (1993) Comparison of calpain and cathepsin (B, L, and D) activities during dry-cured ham processing from heavy and light large white pigs. J Sci Food Agric 62:71–75 Schivazappa C, Degni M, Nanni Costa L, Russo V, Buttazzoni L, Virgili R (2002) Analysis of raw meat to predict proteolysis in Parma ham. Meat Sci 60:77–83 Sentandreu MA, Armenteros M, Calvete JJ, Ouali A, Aristoy MC, Toldrà F (2007) Proteomic identification of actin-derived oligopeptides in dry-cured ham. J Agric Food Chem 55:3613–3619 Škrlep M, Cˇ andek-Potokar M, Mandelc S, Javornik B, Gou P, Chambon C, Santé-Lhoutellier V (2011) Proteomic profile of dry-cured ham relative to PRKAG3 or CAST genotype, level of salt and pastiness. Meat Sci 88:657–667 Spaziani M, Bisetto E, Simula MP, Fabbro A, Stecchini ML, Lippe G (2009) Myosin degradation products in dry-cured San Daniele ham. In: 55th International Congress of Meat Science and Technology, Copenhagen, Denmark Toldrá F, Etherington DJ (1988) Examination of cathepsins B, D, H and L activities in dry-cured hams. Meat Sci 23:1–7 Toldrá F, Rico E, Flores J (1993) Cathepsin B, D, H and L activity in processing of dry-cured ham. J Sci Food Agric 62:157–161 Part V Husbandry and Zootechnic Chapter 29 The Donkey Milk Food Chain: Quality and Certification

Stefano Simonella, Cristina Panetta and Biagina Chiofalo

Abstract Donkey milk, which is used for its nutritional and sensory character- istics and for its high digestibility, is very similar to human milk, representing a good substitute for infants affected by cow milk protein allergy. The aim of this study was to support a farm oriented in the production of donkey milk to develop a technical discipline (TD) for the certification of conformity (CC). The achieve- ment of the CC, guaranteeing that any procedures related to the food in each link of the supply chain are in full compliance with the requirements of the TD, was an important goal to protect consumers that, in this case, are infants. This study has permission to obtain, for the first time in Italy, certified donkey raw milk with technical, scientific, economic, and social implications. The CC is viewed as necessary for the effective regulation of food quality and safety throughout the contemporary agrifood system.

Keywords Donkey milk Á Quality Á Voluntary certification of product Á Food safety

29.1 Introduction

Donkey milk, which is used for its nutritional and sensory characteristics (Chiofalo and Panetta 2011) and for its high digestibility (Dugo et al. 2005), is very similar to human milk, so it represents a good substitute for infants affected by cow milk

S. Simonella (&) Consorzio Ricerca Filiera Carni, Università degli Studi di Messina, Messina, Italy e-mail: [email protected] C. Panetta Á B. Chiofalo Dipartimento di Morfologia, Biochimica, Fisiologia e Produzioni Animali, Università degli Studi di Messina, Messina, Italy e-mail: [email protected] B. Chiofalo e-mail: [email protected]

C. Boiti et al. (eds.), Trends in Veterinary Sciences, 159 DOI: 10.1007/978-3-642-36488-4_29, Ó Springer-Verlag Berlin Heidelberg 2013 160 S. Simonella et al. protein allergy. Nevertheless, the use of donkey milk for therapeutic purposes is hampered by several factors: difficulties in food security, ensuring supply conti- nuity, and the medical and legal risks associated with the administration of a nonconventional diet therapy. EC Regulation 853/2004b does not integrate gap legislation for feeding use and marketing of donkey milk, although it can be considered a good starting point. In this legislative situation, the adoption of voluntary certification systems for product guarantees of food safety and quality throughout the global supply chain is important. In recent years, the issue of food security has assumed a significant central role, even in the Italian food system, a crucial sector for the economy, to direct the efforts of farmers and agribusiness toward achieving higher standards. Consequently, food security is configured as a requirement to be pursued, rather than a prerequisite; for this reason, consumer protection should be guaranteed, and therefore should be ensured not only through compliance with applicable law but also through voluntary, accredited certifica- tion. Certification of the food system offers several advantages, including increasing the value of the special characteristics of the product or of the process through which it is obtained; guarantee of sensory and nutritional characteristics of the product, and its quality and uniqueness (traceability in food chains); greater attention to sanitary aspects of food than is required by law to assure food safety; and promotion of the image of the company and product so as to yield a com- petitive advantage for the producer in the markets. In this context, the aim of the present study was to support a farm oriented in the production of donkey milk to develop a technical discipline (TD) that specifies the requirements for the regu- lation of food safety and quality practices. The implementation of good practices is an essential part of the agrifood system to obtain a certification of conformity (CC) for the food product.

29.2 Materials and Methods

The study was carried out at the ASILAT Srl farm in Milo (Catania province), which covers approximately 4 ha, with a herd consisting of about 80 donkeys belonging to the Ragusana, Siciliana, and Pantesca populations. The farm obtained in 2002 permission to sell ‘‘raw donkey milk’’; in 2008, production was adapted to EC Regulation 852/2004a. Breeding is based on the principles of animal welfare assurance (Passantino 2007) and the hygienic production of milk. Feeding rations differ in relation to their physiological phases: lactation, dry, and foals (Chiofalo 2008). Foodstuffs are given three times per day in a manger; animals have access to fresh water ad libitum. Housing allows the movement of animals in a non- traumatic way and without the use of electrical or other systems. The foals are with the dams and fed directly by the udder, and are not moved from the dams for the first 30 days of life; consequently, during this period, the dams are never milked. The dams are milked by machine, and the foals are physically separated from the dams for 3–5 h before each milking, even if they remain in direct visual contact 29 The Donkey Milk Food Chain 161

(D’Alessandro and Martemucci 2007). The pilot milking machine consists of a type of wheeled trolley with a sheep cluster; according to a study on dairy don- keys, operative parameters were established at 35 kPa vacuum and 50 % pulsation ratio, with a pulsation rate at 60 cycles/min (Salimei et al. 2006; Fantuz et al. 2007). The farm also has a milking parlor with six workstations. The product of ASILAT is raw milk, which is obtained no more than once a day, kept at a temperature not higher than +6 °C and sold directly to the consumer. In this study, the first step was to develop a TD based on the specific characteristics of the farm by setting certifiable requirements for the quality practices of the breeding system and for the food safety of the product. Subsequently, the farm was accompanied and supported in the application of the TD. To establish certifiable criteria and limits of the composition and hygienic (sanitary) characteristics of the product, and to develop standards for food safety management systems to obtain raw donkey milk of high quality, the principal aspects for drafting of the TD for the CC were as follows: characterization of the product; identification and traceability of animals and animal products; control plans; and staff training. For this purpose, monthly (following the TD and self-assessments at the farm), bulk samples (approximately 50 mL) were taken directly from the cooling tank (T = +4 °C) and placed in sterile containers. Samples were then transported, under isothermal conditions, to laboratories for microbiological, physical, and chemical analyses.

29.3 Results

Based on the results of the analyzed samples and those reported in the literature on raw milk (Chiofalo et al. 2004; Conte and Passantino 2008; Salimei et al. 2004), the ranges of the physical, chemical, and microbiological parameters were defined and reported in the TD to obtain the CC. When a draft of the TD was completed, the ASILAT farm was supported in the application of disciplinary procedures, helping the quality manager of the farm in the preparation of the required docu- mentation. Therefore, the farm was assisted in the achievement of the parameters indicated in the TD. Among the results of the physical, chemical, and microbio- logical parameters recorded in the analyzed samples, the total bacteria count (TBC) of milk (at +30 °C) was higher (500,000 CFU/mL) than the value fixed in the TD (\100,000 CFU/mL). Indeed, the value recorded in the samples appears much lower than that (\1,500,000 CFU/mL) fixed by the EC legislation on raw milk (EC Regulation 853/2004b). Nevertheless, several studies (Conte 2008) showed that this value is acceptable in milk of other animal species, but it seems very high and not suitable for donkey milk, especially when this milk is used for highly sensitive patients such as infants. In fact, the value set out in the TD was fixed by taking into account both the positive activity of lysozyme on the microbial status of donkey milk (Conte et al. 2006) and the people to whom the milk is recommended (Chiofalo et al. 2006). To reduce the TBC, particular attention was focused on all operations prior to the milking (rinsing, cleaning, and disinfection of 162 S. Simonella et al. the milking machine and storage tank) and during the milking. After corrective actions were taken, the results concerning the TBC were in line with those reported in the TD, confirming the importance of the adoption of routine operations for milk hygiene.

29.4 Discussion

To obtain the CC for raw donkey milk, the CoRFilCarni-GCC, the certification body recognized by the Italian Ministry of Agriculture and Forestry that is accredited by the Italian National Accreditation Body, was instructed to verify the compliance of the milk with the specifications in the TD. The certification body, after conducting pre-assessments, a documentation review of the supplier’s facilities and production operations, field audits, and verification of the confor- mity, issued the CC, allowing the supplier to label its product as certified. The label shows the mark of conformity and the logo of the third-party certifier. The achievement of the CC, guarantying that any procedures related to the food in each link of the supply chain are in full compliance with the requirements of the TD, was an important goal to protect consumers that, in this case, are infants. In conclusion, this study has permitted a farm to obtain, for the first time in Italy, certified raw donkey milk, with technical, scientific, economic, and social impli- cations. The CC is viewed as necessary for the effective regulation of food quality and safety throughout the contemporary agrifood system. Producers that are cer- tified may stand to gain economic advantages in the marketplace.

References

Chiofalo B (2008) Strategie nutrizionali per l’ottimizzazione dell’effetto probiotico del latte di asina. In: Conte F L’asino all’attenzione della comunità scientifica e del territorio. Chiriotti Editori, pp 27–31 Chiofalo B, Azzara V, Liotta L, Chiofalo L (2004) The chemical and physical parameters of the Ragusana ass’s milk during lactation. In: Proceedings of 6th national meeting: new findings in equine practice, pp 77–84 Chiofalo B, Drogoul C, Salimei E (2006) Other utilisation of mare’s and ass’s milk. In: Miraglia N, Martin-Rosset W (eds) Nutrition and feeding of the broodmare, vol 120. Wageningen Academic Publishers, Wageningen, pp 133–147 Chiofalo B, Panetta C (2011) Gestione nutrizionale dell’asina per l’ottimizzazione dell’effetto probiotico del latte quale alimento funzionale. In: Milonis E e Polidori P Latte di asina produzione, caratteristiche e gestione dell’azienda asinina. Fondazione Iniziative Zooprof- ilattiche e Zootecniche pp 151–160 Conte F (2008) Definizione dei principali criteri per il controllo del latte d’asina. In: Conte F L’asino all’attenzione della comunità scientifica e del territorio. Chiriotti Editori, pp 57–64 Conte F, Passantino A (2008) Isolation of Enterobacter sakazakii from ass’milk in Sicily: case report, safety and legal issues. Travel Med Infect Dis 6:250–252 29 The Donkey Milk Food Chain 163

Conte F, Piccinini R, Dapra’ V, Gagliano V (2006) Valutazione dell’attività del lisozima nel latte di asina. Atti LX Convegno Nazionale Società Italiana delle Scienze Veterinarie, pp 453–454 D’Alessandro AG, Martemucci G (2007) Influence of milking number and frequency on milk production in Martina Franca breed asses. Ital J Anim Sci 6:643–645 Dugo P, Kumm T, Lo Presti M, Chiofalo B, Salimei E, Fazio A, Cotroneo A, Mondello L (2005) Determination of triacylglycerols in donkey milk by using high performance liquid chromatography coupled with atmospheric pressure chemical ionization mass spectrometry. J Sep Sci 28:1023–1030 Fantuz F, Maglieri C, Casamassima M, Palazzo M, Chiofalo B, Salimei E (2007) Nutritional status of dairy asses managed with different machine milking strategies. Ital J Anim Sci 6:647–649 Passantino A (2007) Protezione del benessere degli asini nell’allevamento: attualità e prospettive. Lar Anim Rev 3:103–107 EC REGULATION No 852/2004 of the European Parliament and of the Council 29 April 2004 ‘‘On the hygiene of foodstuffs’’ EC REGULATION No 853/2004 of the European Parliament and of the Council of 29 April 2004 ‘‘Laying down specific hygiene rules for food of animal origin’’ Salimei E, Fantuz F, Coppola R, Chiofalo B, Polidori P, Varisco G (2004) Composition and characteristics of ass’s milk. Anim Res 53:67–78 Salimei E, Fantuz F, Polidori P, Coppola R, Chiofalo B, Varisco G (2006) Ass’s milk: nutritional and functional characteristics. In: Rubino R, Sepe L, Dimitriadou A, Gibon A Livestock farming system: product quality based on local resources leading to improved sustainability, vol 118. Wageningen Academic Publishers, Wageningen, pp 93–98 Chapter 30 Effect of Different Rates of Postmortem pH Decline on the Technological Quality of Calabrian Capocollo

L. Nanni Costa, F. Tassone, S. Dall’Olio, S. Carpino and V. Russo

Abstract Ninety carcasses from cross-bred pigs [Duroc 9 (Landrace 9 Large White)] were examined to study the effect of postmortem acidification rate on weight loss during the processing of protected designation of origin Calabrian Capocollo. The pH was measured in the loin at the 6th rib at 1, 3, and 6 h postmortem. Thirty-four carcasses were selected on the basis of pH decline at 3 h: slow (pH3 [ 6.20, n = 11), intermediate (5.91 [ pH3 B 6.20, n = 11), and fast (pH3 B 5.90, n = 12). Before and after curing and ripening, the Capocollo weights were recorded and the respective losses (%) were calculated. At 1, 3, and 7 days postmortem, measurements of pH, color, and water holding capacity (WHC) were recorded from adjacent loin samples. Fast acidification was associ- ated with a lighter color and lower WHC of fresh meat but did not affect weight loss after curing and ripening, which were very similar between the three groups.

Keywords Pig Á Pork Á pH Á Technological losses Á Calabrian Capocollo

30.1 Introduction

‘‘Capocollo of Calabria’’ is one of four types of Calabrian salami with protected designation of origin (PDO). As required by the regulation, it is prepared from the cranial part of the loin of pigs raised exclusively in the Calabria region. After deboning, the cut is dry salted for 4–8 days. After salting the Capocollo is washed

S. Carpino Associazione Provinciale Allevatori, Cosenza, Italy L. Nanni Costa (&) Á F. Tassone Á S. Dall’Olio Á V. Russo Dipartimento di Protezione e Valorizzazione Agro-alimentare, University of Bologna, Via Fanin, 46 40127 Bologna, Italy e-mail: [email protected]

C. Boiti et al. (eds.), Trends in Veterinary Sciences, 165 DOI: 10.1007/978-3-642-36488-4_30, Ó Springer-Verlag Berlin Heidelberg 2013 166 L. Nanni Costa et al. with water, wet with wine vinegar, and subjected to ‘‘massaging’’ and ‘‘pressing’’ treatments. Black pepper grains are then added, and it is wrapped in pig diaphragm wall. The cut is then tied with natural twine, and the wrapping is perforated. To limit the development of microbial flora and to facilitate slow ripening, aging takes place in temperature and humidity controlled rooms for not fewer than 100 days after salting. The Calabrian Capocollo consists of a single whole piece of meat. Therefore, the quality of the raw material can influence weight loss during pro- cessing. The aim of this study was to evaluate the influence of the rate of post- mortem pH decline on fresh meat quality and on weight loss during Capocollo processing in order to characterize the raw meat for the attitude to the production of this PDO salami.

30.2 Materials and Methods

The carcasses of 90 cross-bred pigs [Duroc 9 (Landrace 9 Large White)], which were reared in the province of Reggio Calabria, were examined. Pigs were slaughtered after 38 h of rest in a slaughterhouse located in the province of Cosenza. After electrical stunning (220 V, 0.9 Amp), pigs were eviscerated and hot carcass weight was recorded. At 1, 3, and 6 h postmortem, the pH of the longissimus thoracis muscle at the level of the sixth thoracic vertebra was mea- sured on left half-carcasses. On the basis of pH measured at 3 h postmortem, 36 half carcasses were selected. At dissection, the number of carcasses was reduced to 34 due to blood splashing in the loin. The 34 half carcasses were divided into three groups; slow (pH3 [ 6.20, n = 11), intermediate (5.91 [ pH3 B 6.20, n = 11), and fast (n = 12, pH3 B 5.90) pH decline. Capo- collo was isolated from the half carcasses, then immediately deboned and trim- med. An adjacent sample was collected from between the 6th and the 10th thoracic vertebrae for the color measurement (Minolta CRC300, L*, a*, b* system) at 6 h postmortem and for subsequent analyses. The muscle samples were then trans- ported by air to a Department laboratory. The pH, color, and cooking losses (Honikel 1998) were measured at 1, 3, and 7 days postmortem. Drip losses (Honikel 1998) were assessed at 3 days postmortem. After weighing, the Capocolli were subjected to two dry saltings, each 4 days in length. A mixture of salt, flavorings, and additives equal to 2 % and to 1 % of the batch weight were added at the first and second saltings, respectively. The Capocolli samples were weighed at the end of the second salting and after 98 days ripening. Weight losses after salting and ripening were calculated on Capocolli weight recorded before the first salting. Data were analyzed using the SAS mixed procedure (SAS 1996). For the analysis, pH decline was a fixed factor and subject was a random factor. The mixed repeated measures analysis procedure was used to analyze the parameters at different times postmortem. 30 Effect of Different Rates of Postmortem pH Decline 167

30.3 Results

Because of the high carcass weights (mean ± SD, 148.8 ± 16.1 kg), the Capo- colli raw weight was higher than normal, but within the 3.5–5.5 kg range required by the original regulation and subsequent amendments. The average weight (mean ± SD) was 5.08 ± 0.40 kg after trimming, 4.98 ± 0.39 kg after salting, and 2.83 ± 0.35 kg after drying. Table 30.1 lists the postmortem change in pH and the L* color coordinate. The weights recorded in these processing steps were not significantly different between the groups with different rates of pH decline (P [ 0.05). At 3 h postmortem, the pH was significantly different between the three groups. However, for subsequent measurements, only the differences between slow and fast pH decline groups reached the threshold of statistical sig- nificance. In addition, the pH values of groups with fast and slow pH decline were far from the thresholds which identify pale, soft and exudative (PSE; pH1 \ 6.00) and dark, firm, and dry (DFD; pH24 [ 6.00) defects, respectively. The color L*coordinate, a measure of the meat lightness, was significantly lower in groups with slow and intermediate pH decline. This change was with respect to the fast pH decline group, which had a lighter color at all measurement times. Weight losses observed in the fresh meat after packing and cooking, and in Capocolli at the end of salting and ripening, are reported in Table 30.2. Drip losses were significantly higher in the group with a fast rate of pH decline. A similar trend was observed for cooking losses. However, the differences between groups with fast pH decline and the other two groups did not become significantly

Table 30.1 Effect of postmortem pH decline rate on pH and on color coordinate L* measured on m. longissimus thoracis at the 6th thoracic vertebra Time Slow pH decline Intermediate pH decline Fast pH decline postmortem (pH3 [ 6.20) (5.91 [ pH3 B 6.20) (pH3 B 5.90) pH 1 h 6.48x 6.36x 6.27x 3 h 6.27a, y 5.93b, y 5.78c, y 6 h 5.85a, z 5.78ab, z 5.62b, z 24 h 5.80a, z 5.71ab, z 5.58 b, z 72 h 5.83a, z 5.70ab, z 5.63b, z SEM 0.058 0.058 0.056 Color, L* coordinate 6 h 45.20a, x 46.22a, x 52.88b 24 h 47.34a, x 47.92a, x 53.94b 72 h 49.29a,y 50.88ab, y 52.82b 168 h 49.41a, y 51.16a, y 54.35b SEM 1.098 1.098 1.051 a, b within rows, mean values without a common superscript differ (P \ 0.05) x, y within columns, mean values without a common superscript differ (P \ 0.05) SEM Standard error of the mean 168 L. Nanni Costa et al.

Table 30.2 Effect of postmortem pH decline rate on weight losses of fresh meat and on Ca- pocollo during processing Slow pH decline Intermediate pH decline Fast pH decline (pH3 [ 6.20) (5.91 [ pH3 B 6.20) (pH3 B 5.90) Drip loss (%) 1.68a 2.04a 3.36b SEM 0.233 0.233 0.223 Cooking loss (%) 1 day 15.03x 16.84x 17.29x 3 days 10.37a, y 11.52ab, y 14.17b, y 7 days 12.34y 11.44y 14.33y SEM 1.023 1.073 0.979 After salting 0.80 0.82 0.83 (%) SEM 0.023 0.023 0.022 After 44.18 44.60 44.85 ripening (%) SEM 0.727 0.727 0.696 a, b within rows, mean values without a common superscript differ (P \ 0.05) x, y within columns, mean values without a common superscript differ (P \ 0.05) SEM Standard error of the mean different until 3 days postmortem (P \ 0.05). Even though there was a weak tendency to show slightly higher losses in the fast pH decline group, there were no significant differences in weight loss between the three groups of Capocolli after salting and ripening.

30.4 Discussion

In this study, there were no large variations in pH or signs of PSE or DFD defects. However, the results confirmed the effect of the rate of pH decline on relevant traits of fresh pork such as color and WHC (Huff-Lonergan et al. 2002). Fast pH decline was associated with lighter pork color, which was recorded up to 7 days after slaughter. Water holding capacity was also reduced by the fast rate of pH decline, even though the largest differences with slow or normal rate of pH decline were observed at 3 days postmortem. The rate of pH decline did not affect the technological weight losses of Capocollo salami. There were no differences between Capocolli produced by pork with different rates of pH decline after a second salting and curing. This result may be due to several factors, such as a normal final pH in the all groups or the inclusion of additives during salting. It may also be due to the simultaneous presence in the cut of the m. longissimus muscle, with a rapid acidification rate, and other neck muscles such as the m. semispinalis capitis, with a slow pH decline. Thus, the different rates of pH decline affected 30 Effect of Different Rates of Postmortem pH Decline 169 only one portion of the lean part of the Capocollo. In general, the results show that the variation in the rate of pH decline detected in the longissimus thoracis portion did not cause significant changes in technological losses during Calabrian Capollo processing.

Acknowledgments This research was supported by the Ministry of Agricultural, Food and Forestry Policies (Contratto di Filiera Cozac). We would like to convey special thanks to the staff of the Cooperativa Agricola Calabrese di Piano Lago (CS) for their collaboration.

References

Honikel KO (1998) Reference methods for the assessment of physical characteristics of meat. Meat Sci 49:447–457 Huff-Lonergan E, Baas TJ, Malek M, Dekkers JCM, Prusa K, Rothschild MF (2002) Correlations among selected pork quality traits. J Anim Sci 80:617–627 SAS (1996) SAS/STAT User’s guide, 4th edN. SAS Institute Inc., Cary, NC Chapter 31 Preliminary Investigation of the Incidence of Obesity in a Canine Population in the USA

G. Biagi, I. Cipollini, M. Grandi, D. Sarti and G. Zaghini

Abstract Obesity represents the most common nutritional pathology in com- panion animals. At least one-third of the canine population is estimated to be overweight or obese. The aim of the present study was to evaluate the nutritional status of a canine population in the USA and assess its correlation with some specific individual factors. During a period of approximately 2 months, data concerning nutritional status, dietary management, and clinical situation were collected for 158 adult dogs. Half of the animals were overweight or obese with an average body condition score (BCS) of 3.5/5. Factors such as age, neutering, hormone and chronic arthritic disorders, and breed were significantly positively correlated with BCS. On the contrary, gender, the presence of other animals and type of food provided did not correlate with BCS. Despite the relatively low number of dogs involved, these results confirm that overweight status affects a large portion of the canine population in the USA and is influenced mainly by endogenous factors.

Keywords Dog Á Obesity Á Neutering Á Hormone disorders Á Chronic arthritis

31.1 Introduction

Obesity is currently the most common nutritional pathology among dogs and cats living in Western countries (Courcier et al. 2010). Regarding dogs, recent studies have shown that at least one-third of the canine population is obese, although this statistic is subject to considerable variation depending on geographical area and diagnostic criteria used (McGreevy et al. 2005; Colliard et al. 2006; Lund et al.

G. Biagi (&) Á I. Cipollini Á M. Grandi Á D. Sarti Á G. Zaghini Dipartimento di Scienze Mediche Veterinarie, Università di Bologna, Ozzano Emilia, Italy e-mail: [email protected]

C. Boiti et al. (eds.), Trends in Veterinary Sciences, 171 DOI: 10.1007/978-3-642-36488-4_31, Ó Springer-Verlag Berlin Heidelberg 2013 172 G. Biagi et al.

2006; Courcier et al. 2010). This pathological condition is secondary to a long- term imbalance between energy intake and energy expenditure, which causes an increase in body weight and change in body composition. Obesity is classified as a multi-factorial pathology, because it is associated with many risk factors that can be classified into two main categories: endogenous (breed, gender and reproduc- tive status, age, and hormonal disorders) and exogenous (diet, exercise, and other environmental factors) (Case et al. 2000). Moreover, severe obesity can make animals prone to numerous pathologies which may cause a substantial decline in their quality of life and life expectancy. The aim of the present study was to evaluate the nutritional status of a canine population in the USA and to assess its correlation with factors that may predispose dogs to obesity and other pathologies.

31.2 Materials and Methods

Data were collected for 158 dogs at Canton Veterinary Hospital in Massachusetts (USA) in 2009 over a period of 2 months. A form was created to record data regarding individual factors (age, gender, breed, weight, and reproductive status), diet (quantity and type of food provided, dry vs. wet food, and commercial vs. home-prepared food, number of meals per day, and whether snacks and/or left- overs are offered between meals), physical activity, and the state of health of each dog. Moreover, nutritional state of each dog was assessed using a 5-point body condition score (BCS) scale (1 = cachexia, 3 = normal and 5 = obese). The collected data were analyzed by one-way ANOVA, and the differences among groups were assessed using Student’s t test. Finally, the incidence of certain pathological conditions in relation to BCS (B3 vs. [3) was determined using a Chi-square test. Differences were considered significant if P \ 0.05.

31.3 Results

The canine population investigated included male and female subjects aged from 1 to 15 years which were mostly castrated (87 %). The most prevalent breeds were Labrador Retriever (n = 20), Golden Retriever (n = 10), Cocker Spaniel (n = 9), and Shih-tzu (n = 9). Mean BCS was 3.5. 50 % of the dogs examined were overweight (76 subjects with a BCS of 4 and three subjects with a BCS of 5). Only three dogs were underweight (BCS of 2), and the remaining 76 were of ideal weight (BCS of 3). At the time the data were processed, it was decided to exclude information about the amount of food given and the level of physical activity, given the low reliability of this information when provided by dog owners. Whereas gender proved not to have any influence on BCS, a significantly higher BCS was observed in neutered dogs (3.6 vs. 3.2; P = 0.003). With respect to age, the BCS observed in subjects between 1 and 3 years old (BCS 3.3) was lower than 31 Preliminary Investigation 173

Table 31.1 BCS of dogs (mean ± SD) according to age Age (yrs) 1–3 3–6 6–9 9–12 12–15 P ANOVA BCS 3.3 ± 0.5 3.5 ± 0.6 3.8 ± 0.4 3.6 ± 0.5 3.8 ± 0.5 0.019 No. 58 53 23 19 5

Table 31.2 BCS of dogs (mean ± SD) according to breed BREED No. BCS P ANOVA Labrador Retriever 20 3.9 ± 0.6 0.013 Golden Retriever 10 3.8 ± 0.6 Cocker Spaniel 9 3.7 ± 0.4 Shih-tzu 9 3.3 ± 0.5 Crossbred 25 3.4 ± 0.5 Other breeds (\9 subjects) 85 3.4 ± 0.6

Table 31.3 Incidence of other pathologies in relation to the BCS of dogs Pathology BCS B 3 BCS [ 3 P ANOVA No. 79 79 Hypothyroidism or hyperadrenocorticism 1 (1.3 %) 8 (10.1 %) 0.040 Chronic arthropathies 0 14 (17.7 %) \0.001 Traumatic lesions of the musculoskeletal system 7 (8.9 %) 11 (13.9 %) 0.453 that of dogs over 3 years (P = 0.019; Table 31.1). Of breeds represented by at least nine subjects, Labrador Retrievers (BCS 3.9), Golden Retrievers (BCS 3.8), and Cocker Spaniels (BCS 3.7) had a significantly higher BCS than the rest of the population (P = 0.013); the mean BCS of crossbred subjects, was slightly lower than the rest of the population (BCS 3.4; Table 31.2). Type of food provided and the consumption of snacks or leftovers did not appear to be factors influencing the BCS. A lower BCS (BCS 3.1) was observed in subjects fed ad libitum compared to the others (P = 0.020). Living together with other animals did not appear to have any influence on mean BCS. Overweight dogs did show a higher incidence of several pathologies, such as hormonal disorders (hypothyroidism and hyperad- renocorticism, P = 0.040) and chronic arthropathies (P \ 0.001; Table 31.3).

31.4 Discussion

The high percentage of dogs characterized by a state of overnutrition in the present study confirms what has recently been reported by other authors, who found the prevalence of overweight dogs equal to approximately 60 % of the canine popu- lation (Courcier et al. 2010). It should be remembered that, in both humans and dogs, obesity represents a problem that is closely tied to social context and its 174 G. Biagi et al. incidence may vary according to geographical area. Still, if we compare our data with previous studies, we observe a recent increase in overweight dogs (McGreevy et al. 2005; Colliard et al. 2006; German et al. 2006). The significant correlations found in this study among obesity and age, reproductive status, and breed were previously highlighted by other authors, who observed a significant increase in the incidence of overweight dogs over 6 years old, in neutered subjects irrespective of gender, and in certain breeds like Labrador Retrievers, Golden Retrievers, Cocker Spaniels, Dalmatians, and Rottweilers (Lund et al. 2006). However, the slightly lower BCS found in crossbred subjects in the present study is in disagreement with other studies, in which crossbred dogs had a greater predisposition to becoming overweight (Lund et al. 2006; Courcier et al. 2010). In relation to dietary factors, the finding of a significantly lower BCS in dogs fed ad libitum contradicts what was observed in another study, in which a positive correlation was found between obesity and the number of meals provided daily (Kienzle et al. 1998). However, although it cannot be ruled out that consuming small meals during the day might help some subjects to maintain an ideal weight, this kind of dietary regime is generally adopted by owners of dogs who do not have a voracious appetite and show little tendency toward gaining weight. Contrary to what one might logically expect, the present study revealed the lack of any influence of the type of diet or the habit of giving snacks or leftovers. This is in disagreement with what has been reported by other authors, who report that snacks and leftovers are among the factors predisposing dogs to an overweight condition (Kienzle et al. 1998). The nearly constant finding of a higher BCS in subjects affected by hypothyroidism or hyperadrenocorticism confirms what was previously observed in a number of studies (Rijnberk et al. 1968; Daminet et al. 2003) and supports the assumption that these pathologies play a role in predisposing to obesity. Finally, the significant correlation between overnutrition and the presence of chronic arthropathies sug- gests that, consistent with numerous studies (Impellizzeri et al. 2000; Smith et al. 2006), the latter are likely to be worsened by an overweight condition. Likewise, the reduction in physical activity of an arthritic dog may contribute to the onset of an overweight condition. In conclusion, despite the limited number of cases, the present study has served to confirm that a substantial part of the canine population is in an overweight condition and that there are numerous factors, particularly endogenous ones, which may favor the onset of this condition.

References

Case LP, Carey DP, Hirakawa DA, Daristotle L (2000) Obesity. In: Canine and feline nutrition: a resource for companion animal professional, 2nd edn. Mosby ed, St. Louis, USA, pp 303–312 Colliard L, Ancel J, Benet JJ, Paragon BM, Blanchard G (2006) Risk factors for obesity in dogs in France. J Nutr 163:1951S–1954S Courcier EA, Thomson RM, Mellor DJ, Yam PS (2010) An epidemiological study of environmental factors associated with canine obesity. J Small Anim Pract 51:362–367 31 Preliminary Investigation 175

Daminet S, Jeusette I, Duchateau L, Diez M, Van de Maele I, De Rick A (2003) Evaluation of thyroid function in obese dogs and in dogs undergoing a weight loss protocol. J Vet Med Assoc 50:213–218 German AJ, Holden SL, Bissot T, Hackett RM, Biourge V (2006) Dietary energy restriction and successful weight loss in obese client-owned dogs. J Nutr 136:2031S–2033S Impellizzeri JA, Tetrick MA, Muir P (2000) Effect of weight reduction on clinical signs of lameness in dogs with hip osteoarthritis. J Am Vet Med Assoc 216:1089–1091 Kienzle E, Bergler R, Mandernach A (1998) A comparison of the feeding behavior and the human–animal relationship in owners of normal and obese dogs. J Nutr 128:2779S–2782S Lund EM, Armstrong PJ, Kirk CA, Klausner JS (2006) Prevalence and risk factors for obesity in adult dogs from private US veterinary practices. Intern J Appl Res Vet Med 4:177–186 McGreevy PD, Thomson C, Pride C, Fawcett A, Grassi T, Jones B (2005) Prevalence of obesity in dogs examined by Australian veterinary practices and the risk factors involved. Vet Rec 156:695–702 Rijnberk A, Der Kinderen PJ, Thijssen JHH (1968) Spontaneous hyperadrenocorticism in the dog. J Endocrinol 41:397–406 Smith GK, Paster ER, Powers MY, Lawler DF, Biery DN, Shofer FS, McKelvie PJ, Kealy RD (2006) Lifelong diet restriction and radiographic evidence of osteoarthritis of the hip joint in dogs. J Am Vet Med Assoc 229:690–693 Chapter 32 Administration of Essential Oils Cinnamaldehyde, Eugenol, and Capsicum to Beef Cattle: Effects on Health Status and Growth Performance

R. Compiani, C. A. Sgoifo Rossi, A. Pizzi and V. Dell’Orto

Abstract A 53-day field trial was performed to evaluate the effects of the essential oils of cinnamaldehyde, eugenol, and capsicum on the health status and growth performance of 45 Charolais beef cattle (average arrival weight: 422.0 ± 29.3 kg; control: n = 21; treated: n = 24). Each animal was weighed, and blood samples were collected on days 0, 25, and 53. The average daily gain of the treated group was significantly higher (P \ 0.05) than that of the control. The treated group showed a significant increase in antibody for BHV-1 after vacci- nation (P \ 0.05 at day 25 and day 53) and significantly higher serum bactericidal activity (P = 0.01). No differences between groups were observed for serum content of haptoglobin or reactive metabolites of oxygen. The observed improvement in growth performance and health status is due to the capacity of essential oils to optimize rumen fermentations, to increase dry matter intake, and probably to the positive interaction between essential oils and immune system components.

Keywords Beef cattle Á Essential oils Á Growth performance Á Health status

32.1 Introduction

Essential oils are a combination of secondary metabolites obtained from the plant volatile fraction by steam distillation (Benchaar et al. 2006). The many positive health effects of essential oils include antiseptic and anti-inflammatory properties,

R. Compiani (&) Á C. A. Sgoifo Rossi Á A. Pizzi Á V. Dell’Orto Dipartimento di Scienze Veterinarie per la Salute la Produzione Animale e la Sicurezza Alimentare, Università degli Studi di Milano, Milan, Italy e-mail: [email protected]

C. Boiti et al. (eds.), Trends in Veterinary Sciences, 177 DOI: 10.1007/978-3-642-36488-4_32, Ó Springer-Verlag Berlin Heidelberg 2013 178 R. Compiani et al. the ability to inactivate free radicals, and to inhibit membrane lipid peroxidation, chelate metals, and to simulate the activities of antioxidant enzymes (Trouillas et al. 2003; Lee et al. 2003). The effects of dietary administration of several essential oils to ruminants have been widely investigated in vitro (Cardozo et al. 2004, 2005; Busquet et al. 2005, 2006). Essential oils are potentially able to stimulate rumen fermentation by affecting the quantity and quality of volatile fatty acids produced and the nitrogen ruminal metabolism (Cardozo et al. 2004, 2005; Busquet et al. 2005, 2006). This study evaluated the effects of the administration of cinnamaldehyde, eugenol, and capsicum on the growth performance and health status of fattening cattle during the adaptation phase.

32.2 Materials and Methods

A 53-day field trial was performed involving 45 male Charolais cattle imported from France, with an average arrival weight of 422.0 ± 29.3 kg. At arrival (day 0), animals were randomly divided into two groups, control (n = 21) and treated (n = 24), and subjected to vaccination against infectious bovine rhi- notracheitis virus (BHV-1), bovine viral diarrhea Type 1 virus (BVDV), parainfluenza 3 virus (PI3), and bovine respiratory syncytial virus (BRSV). Cattle were also subjected to antiparasitic treatment with ivermectin. A second dose of BHV-1 vaccine was administered at day 25. Both groups were fed with the same unifeed diet administered ad libitum. The diet of the treated group differed only regarding the addition of a mixture of cinnamaldehyde, eugenol, and capsicum (0.8 g/head/day). Each animal was weighed on days 0, 25, and 53 to obtain its average daily gain (ADG). On days 0, 25, and 53, blood samples were collected from 10 animals per group for the evaluation of blood parameters indicating health status, such as titration of antibodies against BHV-1 by serum neutralization test, serum bactericidal activity, haptoglobin, and reactive metabolites of oxygen (ROMs). For statistical analysis, each specific parameter at day 0 was used as a covariate.

32.3 Results

The ADG during the entire study period (d 0–53) was significantly higher (P = 0.0441) in the treated group compared to the control group (Fig. 32.1) and tended to be higher (P = 0.0685) in the early period (days 0–25). Among the blood parameters used as indicators of health status, those that showed significant differences between the two groups were the serum neutralization test (P \ 0.05 at days 25 and 53) and serum bactericidal activity (P = 0.01), with improved values in the treated group (Table 32.1). 32 Administration of Essential Oils Cinnamaldehyde, Eugenol 179

3

2.5 2.43 * 2 2.25 2.24 2.08 2.11 1.99 kg/day 1.5 CONTROL TREATMENT 1

0.5

0 ADG 0-25 ADG 25-53 ADG 0-53

Fig. 32.1 Average daily gain of beef cattle fed a mixture of essential oils cinnamaldehyde, eugenol, and capsicum versus control (*P \ 0.05)

Table 32.1 Blood parameter results Parameters Days Control Treatment P

BHV-1 serum neutralization [log10(dilution)] 0 0 0 – 25 0.57 0.81 0.04 53 0.90 1.02 0.02 Serum bactericidal activity (%) 0 87 91 – 25 87 92 0.01 Haptoglobin (mg/ml) 0 0.36 0.10 – 25 0.13 0.14 0.79

ROMs (mmol H2O2) 0 1.28 1.22 – 25 1.54 1.44 0.64

32.4 Discussion

Growth performance was better among treated cattle as demonstrated by a sig- nificantly greater ADG of 130 g. The improved increase is probably due to the digestive process optimization induced by the treatment. The mixture of essential oils administered has been shown to improve ruminal fermentation activity and reduce methanogenesis, thereby reducing energy waste, reducing ammonia con- centrations, and increasing microbial protein production as demonstrated by in vitro studies (Cardozo et al. 2004, 2005; Busquet et al. 2005, 2006). The effect of essential oils could be particularly interesting in intensive beef cattle rearing, because the antimicrobial effect is directly proportional to the ruminal pH drop from 6.5 to 5.5 (Busquet et al. 2005, 2006; Calsamiglia et al. 2007), a range that characterizes the fattening cattle diets. Concerning the immune response, essential oil administration has helped cattle to quickly recover to physiological conditions after the stressful events typically present in newly received beef calves. Bacte- ricidal serum activity at day 25 was higher than the threshold of 90 %, and an improvement in immune response to vaccination was also observed. These posi- tive effects can be attributed both to the rumen fermentation optimization and to 180 R. Compiani et al. the increase of dry matter intake (Yang et al. 2010a, b), but also to a direct effect of the essential oils with immune system cells. This second mechanism needs to be investigated further. The use of essential oils of cinnamaldehyde, eugenol, and capsicum in newly received beef cattle diet seems to be a successful strategy to improve growth performance and immune response during the adaptation phase.

Acknowledgments Trial conducted in collaboration with New Wave and Phytosolutions. The authors would like to thank Mr. Andrea Zanovello for trial assistance.

References

Benchaar C, Petit HV, Berthiaume R, Whyte TD, Chouinard PY (2006) Effects of addition of essential oils and monensin premix on digestion, ruminal fermentation, milk production, and milk composition in dairy cows. J Dairy Sci 89:4352–4364 Busquet M, Calsamiglia S, Ferret A, Kamel C (2005) Screening for the effects of natural plant extracts and secondary plant metabolites on rumen microbial fermentation in continuous culture. Anim Feed Sci Technol 123(124):597–613 Busquet M, Calsamiglia S, Ferret A, Kamel C (2006) Plant extracts affect in vitro rumen microbial fermentation. J Dairy Sci 89:761–771 Calsamiglia S, Busquet M, Cardozo PW, Castillejos L, Ferret A (2007) Invited review: essential oils as modifiers of rumen microbial fermentation. J Dairy Sci 90:2580–2595 Cardozo PW, Calsamiglia S, Ferret A, Kamel C (2004) Effects of natural plant extracts on protein degradation and fermentation profiles in continuous culture. J Anim Sci 82:3230–3236 Cardozo PW, Calsamiglia S, Ferret A, Kamel C (2005) Screening for the effects of natural plant extracts at different pH on in vitro rumen microbial fermentation of a high-concentrate diet for beef cattle. J Anim Sci 83:2572–2579 Lee SE, Hwang HJ, Ha JS, Jeong HS, Kim JH (2003) Screening of medicinal plant extracts for antioxidant activity. Life Sci 73:167–179 Trouillas P, Calliste CA, Allais DP, Simon A, Marfak A, Delage C, Duroux JL (2003) Antioxidant, anti-inflammatory and antiproliferative properties of sixteen water plant extracts used in the Limousin countryside as herbal teas. Food Chem 80:399–407 Yang WZ, Ametaj BN, Benchaar C, He ML, Beauchemin KA (2010a) Cinnamaldehyde in feedlot cattle diets: intake, growth performance, carcass characteristics, and blood metab- olites. J Anim Sci 88:1082–1092 Yang WZ, Ametaj BN, Benchaar C, Beauchemin KA (2010b) Response to cinnamaldehyde supplementation in growing beef heifers: ruminal and intestinal digestion. J Anim Sci 88:680–688 Chapter 33 Extruded Linseed in the Diet of Grazing Goats: Effects on Milk Conjugated Linoleic Acid

Raffaella Tudisco, S. Calabrò, M. I. Cutrignelli, M. Grossi, N. Musco, V. Piccolo and F. Infascelli

Abstract This research aimed to evaluate the effect of extruded linseed in the diet of grazing goats on milk concentrations of conjugated linoleic acid (CLA). Thirty dairy goats were divided into two groups: C, control and L, which received extruded linseed in the concentrate. Milk yield and fat percentage did not differ between groups, whereas group L showed higher (P \ 0.05) levels of total CLA and c9t11CLA. The results were affected by sampling month; Indeed, the differ- ences were significant in July when linoleic and linolenic acids in the pasture decreased.

Keywords Milk Á Goat Á Linseed Á Fatty acids Á CLA

33.1 Introduction

The increasing interest in the nutritional characteristics of food has led to the study of their lipid components, in particular, to a class of isomers derived from con- jugated linoleic acid (CLA), which are known to have antioxidant and anticarci- nogenic properties in animal models (Parodi 1999). In the rumen, CLA production comes from the bio hydrogenation of linoleic and linolenic acids (both charac- terized by a structure with cis-9 and cis-12 dienes, and a free carboxyl group). Thus, the presence of these precursors in the animal diet affects milk CLA content, with important roles attributed to grazing pasture (Banni et al. 1996; Dhiman et al. 1999; Cabiddu et al. 2004) and/or linseed supplements (Nudda et al. 2006). This

R. Tudisco (&) Á S. Calabrò Á M. I. Cutrignelli Á M. Grossi Á N. Musco Á V. Piccolo Á F. Infascelli Department of Animal Science and Food Control, University of Naples ‘‘Federico II’’, Via F. Delpino 1, 80137 Naples, Italy e-mail: [email protected]

C. Boiti et al. (eds.), Trends in Veterinary Sciences, 181 DOI: 10.1007/978-3-642-36488-4_33, Ó Springer-Verlag Berlin Heidelberg 2013 182 R. Tudisco et al. research aimed to evaluate the effects of adding extruded linseed to the diet of grazing goats on milk concentrations of CLA.

33.2 Materials and Methods

The trial was carried out on 30 pregnant Cilentana dairy goats (50 ± 2.5 kg body weight) divided into two groups (C, control, and L, receiving linseed), homoge- neous for parity and milk yield at the previous lactation. All goats were fed ad libitum oat hay, and 200, 300, and 400 g/head/day of concentrate [crude protein (CP) 18 % of dry matter (DM); 1.03 unit feed for lactation (UFL)/kg DM] 45, 30, and 15 days before kidding, respectively. Group L received concentrate containing extruded linseed (30 % as fed). After kidding (first week of February), both groups had free access to pasture (9.00 am–4.00 pm), constituted by 60 % Leguminosae (Trifolium alexandrinum, Vicia spp.) and 40 % grass (Bromus catharticus, Festuca arundinacea, and Lolium perenne). The supply of concentrate was gradually increased to 700 g/head/day. From days 0 to 60, milk was suckled only by the kids; successively, daily milk yield was recorded, and monthly representative milk samples from the two daily milkings were analyzed for protein, fat, and lactose concentrations (Milko Scan 133B, Foss Matic, Hillerod, Denmark) and CLA content (D’Urso et al. 2008). Pasture samples (four samplings) were collected monthly from three areas (2.5 m2 each) at a height of 3 cm above the ground and analyzed for chemical composition (AOAC 2000; Van Soest et al. 1991) and fatty acid profiles (D’Urso et al. 2008). The nutritive value was calculated (MFU/kg DM; INRA 1978). Data were analyzed by the GLM procedure and differences of means compared by the Tukey test (SAS 2000).

33.3 Results

The pasture had 16.6 % CP and 0.76 MFU/kg DM on average. The samples collected in July showed lower values of linoleic and linolenic acids than those in the 2 months before, whereas for both acids, the highest value was registered in September (Table 33.1). Average milk yield and chemical composition (Table 33.2) did not differ between groups. As expected, along the trial, milk yield gradually decreased; the

Table 33.1 Fatty acid profile of pasture (% of total fatty acids) May June July September Linoleic acid 22.6 34.1 16.6 46.3 Linolenic acid 41.2 42.7 36.7 52.4 33 Extruded Linseed in the Diet of Grazing Goats 183

Table 33.2 Milk yield and chemical composition in groups C (control) and L (linseed) Yield Protein Fat Lactose g/day % Group effect C 1,337.2 3.53 4.45 4.59 L 1,432.7 3.58 4.60 4.58 Sampling effect 1 2,043.7A 3.28C 4.23B 4.76A 2 1,703.3B 3.50BC 4.50A 4.74A 3 1,367.8C 3.55B 4.18B 4.60B 4 1,154.6C 3.44BC 3.97C 4.53BC 5 710.8D 4.14A 4.52A 4.43C Significance Group NS NS ** ** Sampling ** ** ** ** Group*sampling NS NS NS * SEM 10.47 9 104 0.14 0.39 0.12 NS not significant; SEM standard error of the mean A, B, C, D P \ 0.01; *P \ 0.05

Table 33.3 CLA content in milk (g/100 g fat) of groups C (control) and L (linseed) C L SEM c9t11CLA 0.779b 0.910a 0.04 t10c12CLA 0.046 0.041 0.9 9 10-3 c9c11CLA 0.021 0.022 0.8 9 10-4 Total CLA 0.845b 0.973a 0.04 SEM standard error of the mean a, b P \ 0.05 percentage of protein decreased slightly at the fourth sampling. Regarding the fat content, the highest values were registered at the second and fifth sampling. Group L showed significantly (P \ 0.05) higher concentrations of total CLA and c9 t11 CLA than group C (Table 33.3).

33.4 Discussion

The influence of diet containing extruded linseed supplementation on CLA content of goat milk has also been reported by others (Nudda et al. 2006). In our trial, the higher value of total CLA in milk of grazing goats fed extruded linseed was attributed to c9 t11CLA content. In addition, the results were affected by the sampling month (Figs. 33.1 and 33.2). Indeed, the differences between groups were significant only in July, when, due to climatic conditions, the pasture is less luxuriant with lower concentrations of both linoleic and linolenic acids. 184 R. Tudisco et al.

1.2

1

0.8 C 0.6 L 0.4

0.2

0 April May June July September

Fig. 33.1 Milk c9t11CLA (g/100 g fat) as a function of sampling month

1.2

1

0.8

0.6 C L 0.4

0.2

0 April May June July September

Fig. 33.2 Milk total CLA (g/100 g fat) as a function of sampling month

References

AOAC 2000. Official methods of analysis, 17th edn. Association of Official Analytical Chemists, Arlington Banni S, Carta G, Contini SM, Angioni E, Deiana M, Dessı‘ MA, Melis P, Corongiu FP (1996) Characterization of conjugated diene fatty acids in milk, dairy products, and lamb tissues. J Nut Biochem 7:150–155 Cabiddu A, Addis M, Spada S, Sitzia M, Molle G, Piredda G (2004) The effect of different legumes-based pastures on the fatty acid composition of sheep milk with focus on CLA. In: Luscher A, Jeangross B, Kessler W, Huguenin O, Lobsiger M, Millar N, Suter D (eds) Proceedings of the 20th meeting of European Grassland Federation, Switzerland. Blackwell Publishing Ltd, Oxford, pp 1133–1135 D’Urso S, Cutrignelli MI, Calabrò S, Bovera F, Tudisco R, Piccolo V, Infascelli F (2008) Influence of pasture on fatty acid profile of goat milk. J Anim Physiol Anim Nutrition 92:405–410 Dhiman TR, Anand GR, Satter LD, Pariza MW (1999) Conjugated linoleic acid content of milk from cows fed different diets. J Dairy Sci 82:2146–2156 INRA (1978) Alimentation des Ruminants. INRA, Paris Nudda A, Battacone G, Usai MG, Fancellu S, Pulina G (2006) Supplementation with extruded linseed cake affects concentrations of conjugated linoleic acid and vaccenic acid in goat milk. J Dairy Sci 89:277–282 33 Extruded Linseed in the Diet of Grazing Goats 185

Parodi PW (1999) Conjugated linoleic acid and other anticarcinogenic agents of bovine milk fat. J Dairy Sci 82:1339–1349 SAS (2000) SAS/STAT Software: changes and enhancements through release 8.1. SAS Institute Inc., Cary, NC Van Soest PJ, Robertson JB, Lewis BA (1991) Methods for dietary fiber, neutral detergent fiber, and nonstarch polysaccharides in relation to animal nutrition. J Dairy Sci 74:3583–3598 Index

A Capsicum, 178 Abdominal lymph nodes, 119–123 Cardiac troponins, 130 Acanthocheilonema reconditum, 76 Casein, 21–25 Actin, 44, 153–155 Casein dephosphorylation, 24 ADaM, 98 Casein isoforms, 22, 25 Adult stem cells, 33 Cat, 142, 143 Aflatoxin M1, 110 Certification, 160, 162 Algal biotoxins, 107 Chordoid Meningioma, 58 Algal toxicity tests, 99 Chronic arthritis, 173, 174 Antibacterial, 98 Cinnamaldehyde, 178 Antibacterial compounds, 97, 99, 101 Ciprofloxacin, 98 Antifolic compounds (sulphonamides), 101 Conjugated linoleic acid (CLA), 181–183 Aonchotheca putorii, 86–88 Cortisol, 4, 7, 52–54 Aquaculture, 97, 101 Crustaceans, 98 Aquaporin 1, 16 Aquatic organism, 98 Aquatic toxicology, 97 D Aquatic toxicity tests, 98 Dairy cows, 125 Arachidonate, 37 D6-desaturase, 38 Arachidonic acid, 38 Daphnia magna, 97–100 Autonomic neurons, 28, 29 Daphnids, 98, 99 Dicentrarchus labrax, 4 Diplodus sargus, 16–18 B Dirofilaria immitis, 73 Beef cattle, 179 Dirofilaria repens, 75 Behavioral changes, 97 Docohexaenoate (DHA), 37 Behavioral endpoints, 97, 98 Dog, 58–60, 85–88, 116, 117, 137–139, 141, Beta lactam antibiotics, 109, 111, 112 171–174 Biological networks, 42 Dog heartworms, 73 Boar, 28, 29 Donkey milk, 22, 24, 159–162 Brachydanio rerio, 99 Drugs, 99 Dry-cured ham, 153, 154

C Calabrian capocollo, 165–169 E Calcium, 44 EC50, 98–100 Capacitation, 42 Eco toxicology, 97 Capillaria spp., 85–87 Eicosapentanoate (EPA), 37

C. Boiti et al. (eds.), Trends in Veterinary Sciences, 187 DOI: 10.1007/978-3-642-36488-4, Ó Springer-Verlag Berlin Heidelberg 2013 188 Index

E (cont.) L Enrofloxacin, 98 Legislation, 144 Environment, 101 Lethality, 98, 99 Equine tenocytes, 47, 48 Liguria, 87, 88 Erythrocyte, 136–139 Linseed, 181–183 Essential oils, 177 Lipid, 36 Eucoleus aerophilus, 86–88 Lowest observable effect concentration Eucoleus boehmi, 86, 87 (LOEC), 97 Eugenol, 178 Lysozyme activity, 4, 8 Ewes, 52, 53 Exercise test, 131, 132 Fatty acids, 182 M Meat juice, 80–82 Metabolic disturbance, 100 F Metabolic profile, 125–128 Fertilization, 42, 149 Metaphylactic treatments, 101 Fish, 3, 7, 8, 98, 99 Methanogenesis, 179 Fish skin, 149, 150 Microbial protein, 179 Folate, 100 Milk, 68–71, 181–183 Food safety, 160, 161 Morphology, 136–138 Freezing, 100 MS-222, 98 Mycobacterium avium subsp., 68

G Gills, 150–152 N Gilthead seabream, 152 Nervous system, 100 Glutathione, 4 Neutering, 172, 174 Goat, 182, 183 Neutralizing antibodies (NA), 64–66 Grading, 59, 60 No observable effect concentration (NOEC), 97, 99

H Half-life, 101 O Ham, 153 Obesity, 171–174 Heart failure, 131 Organisms, 100 Heavy metals, 115 Ostreopsis ovata, 103, 104, 107 Helminths, 93, 94 Ovine, 68–70 Hematocrit, 4, 7 Ovine amniotic epithelial stem cells, 46–48 Hip dysplasia, 36–38 Ovine tenocytes, 48 Hormone disorders, 172, 173 Oxidative stress, 3, 7, 8 Horse, 10–12

P I Pain, 142–44 Immobilization, 100 Papillary, 58, 59 Immobilization test, 100 Paratuberculosis, 68, 69 Immune response, 179, 180 Pearsonema plica, 86 Immunohistochemistry, 28 Photoperiod, 3, 7, 8 In vitro co-culture, 46, 47 Pigs, 64 Innate immunity, 3 Plasma serotonin, 11 Italy, 86–88 Platelet-rich plasma, 32 Index 189

Poecilia reticulata, 97–99 Spermatozoa, 41 Poikilocytosis, 136 Sublethal effects, 98 Poly-unsaturated fatty acids, 36, 38 Sublethal endpoin, 100 Porcine circovirus type 2 (PCV2), 63–66 Sublethal, 100 Predation avoidance, 97, 100 Sulphaguanidine (SGD), 98, 99 Pregnancy, 51–53 Sulphamethazine (SMZ), 98, 99 Protein kinase A, 44 Sulphaquinoxaline, 98 Protein kinase C, 44 Surface waters, 101 Proteolysis, 153, 154 Swimming activity, 97, 98, 100 Public health, 68 Puppy, 120, 123 T Technological losses, 167–169 Q Temperature, 7, 8 Quality, 160–162 Tendon regeneration, 33 Quality index method, 150 Tenogenic differentiation, 46, 48 Quatrefoil erythrocyte, 136 Therapy, 142–144 Total volatile basic nitrogen (TVBN), 150–152 Toxicity, 104, 105 R Toxicity test, 97, 98 Reproduction, 100 Toxoplasma gondii, 79–82 Retrograde neuronal tracer, 28 Trichuris vulpis, 85–88 Trimethoprim (TMP), 98–101 Trimethylamine nitrogen (TMA-N), 149–152 S Two-dimensional electrophoresis, 22 Sardinia Island, 74 Scale-free topology, 43 Sea bass, 4, 7 V Sea urchins, 42 Video automation, 98 Serum bactericidal activity, 178 Video tracking, 98 Serum neutralization test, 178 Virus neutralization test (VNT), 64–66 Sheep, 92 Shelf life, 149, 152 Signaling system, 42 W Sparus aurata, 150 Wild boar, 79–82 Specific spoilage organisms (SSO), 149, 150