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Trichuris Ovis Trakia Journal of Sciences, No 2, pp 174-178, 2017 Copyright © 2017 Trakia University Available online at: http://www.uni-sz.bg ISSN 1313-7050 (print) ISSN 1313-3551 (online) doi:10.15547/tjs.2017.02.012 Case Report SOME PARASITOLOGICAL, PATHOLOGICAL AND IMMUNOHISTOCHEMICAL EXAMINATIONS IN SHEEP NATURALLY INFECTED BY TRICHURIS OVIS P. T. Iliev1*, A. Ivanov1, Z. Kirkova1, K. Hristov2, K. Dinkova3, J. Ananiev3 1Department of Veterinary Microbiology, Infectious and Parasitic Diseases, Faculty of Veterinary Medicine, Trakia University, Stara Zagora, Bulgaria 2Department of Obstetrics, Gynecology, Biotechnology of Reproduction, Pathological Anatomy and Biochemistry, Faculty of Veterinary Medicine, University of Forestry, Sofia, Bulgaria 3Department of General and Clinical Pathology, Medical Faculty, Trakia University, Stara Zagora, Bulgaria ABSTRACT A case of Trichuris ovis infection in a lamb is presented. In June 2012 a herd of 12 infected with T. ovis lambs in Brestnik (Southern Bulgaria) were discovered. A diarrhea and loss in body weight in 2 lambs have been observed. Despite antibiotic treatment one of the animals died. Parasitological examination was done. During necropsy of the gastro-intestinal tract, adult worms in the caecum were found. Standart staining and immunohistochemistry were performed to detect the cell population in the affected areas of the caecum. Key words: Trichuris ovis, immunohistochemistry, sheep INTRODUCTION embedded in the intestinal mucosa by their Trichuris ovis (Adenophorea: Trichuridae) is a anterior end (3, 4, 5). Тhe penetration of T. caecal parasite most prevalent in ruminants ovis and T. scrjabini into the mucosa causes irrespective of age, gender and breed of the not only mechanical damage but also hosts (1). It is usually detected incidentally histological changes corresponding to the during routine diagnostic or necropsy. The degree of infection. The main changes consist heavy infections are rare but may be seen in of а local lymphocyte infiltration, mucosal very young lambs (2). Generally, natural desquamation, erosions in the place of infections are seldom severe enough to cause attachment and hypersecretion of the intestinal clinical disease (3). Due to its thin, thread-like glands (6). anterior end of the body, T. ovis is more commonly known as ovine whipworm. This The information about the histological parasite is soil-transmitted helminth with direct disorders during T. ovis infection in the life cycle (4). Eggs contain an unsegmented caecum is limited. In contrast, many studies of embrio when they laid. Larvae are formed and the inflammatory cell infiltration in T. vulpis, become infective within the shell of the eggs T. suis and T. muris infection have been done. after disposing them into the environment. The Here, we describe and compare the hosts become infected orally by ingesting food, immunohistology and histopathology of the contaminated with eggs. Having reached in caecum infested by sheep whipworm T. ovis, small intestine by gastric passage, the eggs using canine, porcine and murine whipworms hatch and the released larvae pass to the as comparative models. caecum. There, they burrow into the mucosal CASE HISTORY layer and turn into adult worms. After reaching In June 2012 the owner of herd of 12 lambs sexual maturity, the worms remain deeply observed that in 2 of the animals diarrhea and ___________________________ faintness were manifested with short periods of *Correspondence to: Petar T. Iliev, Department of Veterinary Microbiology, Infectious and improvement of the general condition. Parasitic diseases, Faculty of Veterinary Medicine, Satisfactory outcome after treatment with Trakia University, 6000 Stara Zagora, e-mail: Tylovet® has not been observed and one of the [email protected] 174 Trakia Journal of Sciences, Vol. 15, № 2, 2017 ILIEV P.T., et al. animals died. The carcass was deposited at phosphate buffered saline (PBS), pH 7.4, Trakia University for necropsy. incubated in 1.2 % hydrogen peroxide in methanol for 30 min, and rinsed in 0.1 M PBS, The partial necropsy and parasitological pH 7.4, for 15 min. Immunohistochemical observations were made in Veterinary Faculty, reaction were carried out using Monoclonal Department of Parasitology, and Mouse Anti-Human CD4, Clone 4B12 immunohistochemistry - in Medical Faculty, (DAKO), Monoclonal Mouse Anti-Human Department of General and Clinical Pathology, CD68, Clone KP1 (DAKO) and Monoclonal Trakia University. Mouse Anti-Human Granzyme B, Clone GrB- 7 (DAKO). After washing three times in PBS, MATERIALS AND METHODS the slides were incubated with DAKO- Parasitological examinations REALTM En-VisionTM detection system Flotation technique (DAKO) for 30 min, then visualized with 3,3’- Eleven individual fecal samples were diaminobenzidine and counterstained with examined by standard saline flotation hematoxylin. We used a known tissue to technique. exhibit high levels of marker for positive Species identification control and for negative control, the primary For this task we used some morphological and antibody was replaced with PBS. morphometrical characteristics of adult whipworms such as total body length, length of RESULTS the anterior esophageal section (LES), length Parasitological findings of the posterior section (LPS), ratio between Coprological examination showed the presence the LES and LPS, collocation and shape of the of Trichuris spp. eggs in all examinated vulva, length and shape of the spicules, all animals. Species identification of 13 female described by Soulsby (3), Charakchiev (7) and and 7 male adult parasites confirmed the Kuchai et al. (1). monoinfection by T. ovis. Pathological examination Pathological findings The necropsy was made by routine protocol Macroscopically the mucosa of the caecum and only the gastro-intestinal tract was was oedematous and some petechial examined. hemorrhages were presented. We found plenty of mucus and worms deeply embedded in the Staining studies caecal mucosa. No necroses were discovered. Standard staining Histologically, hematoxylin and eosin stained For standard staining we used hematoxylin sections revealed the parasite body surrounded and eosin stain (H&E stain) on the paraffin by numerous macrophages, lymphocytes, some sections with 5 μm thick. neutrophils, and single eosinophilic leukocytes Immunohistochemisry (Figures 1 and 2 a, b). Mucosa of the caecum For immunohistochemical staining, the underwent intense inflammatory infiltration paraffin blocks were prepared using caecal accompanied by rising in the number of tissues. Paraffin sections 5 μm thick were lymphocytes and neutrophils. dewaxed in two xyllenes and were rehydrated in ethanol. Later, they were washed in 0.1 M Figure 1. Caecum. Anterior thin end of T. ovis surrounded by dystrophic mucosal layer. H/E, 200X Trakia Journal of Sciences, Vol. 15, № 2, 2017 175 ILIEV P.T., et al. Figure 2a. Caecum. Inflammatory cell infiltration consisting of macrophages, lymphocytes and a number of neutrophilic leukocytes. H/E, 400X Figure 2b. Caecum. Mucosal fibroblast cells and eosinophil infiltration. H/E, Bar = 25 µm. Immunohistochemistry investigation occasionally observed in this region (Figure Immunohistochemical studies confirmed 4). There are no NK positive cells (Granzime strong staining for CD68+cells in caecal B negative) in investigated tissue. mucosa (Figure 3). CD4+ cells were also Figure 3. Caecum. Presence of intraepithelial CD68+ cells in the mucosa. Immunohistochemical staining, 200X 176 Trakia Journal of Sciences, Vol. 15, № 2, 2017 ILIEV P.T., et al. Figure 4. Caecum. Presence of CD4+ cells in the mucosa. Immunohistochemical staining, 200X DISCUSSION changes were observed in T. suis infection in It is known that the adult trichurids parasitize pigs (9). In our case a moderate infection in the deeply embedded in bowel. According to dead lamb was discovered. A diarrhea was Kirkova et al. (8) both mature parasites and found only in 2 of 12 infected animals. Given larvae are able to cause mechanical changes in the weak pathological changes in the lamb we the intestinal mucosa. In contrast, Efremov and assume that the cause of death was not due to Shihobalova (see 6) report that T. muris whipworm infection. Likely, a secondary infection does not induce any macroscopic microflora has been implicated. According to changes in the cecum. Our results were similar Smith et al. (10) Trichuris spp. do not play a to those of Bratanov et al. (6). The authors significant pathogenic role, especially in found that the degree of infection with ovine ruminants which is in agreement with our whipworms is able to cause from slight findings. changes as petechial hemorrhages to better expressed hemorrhages of the bowel. Similar Figure 5. Caecum. Dystrophic changes in the mucosal epithelium layer. H/E, 10X Based on the results we assume that T. ovis Kirkova et al. (8) on histopathology in T. infection is accompanied by weak dystrophic vulpis infection in dogs have shown that changes in the mucosal epithelium (Figure 5) massive colonization of the intestinal mucosa and inflammatory infiltrate consisting of leads to extensive local inflammation which macrophages, lymphocytes, neutrophils and supports our results. We found that the cellular single eosinophils. The investigations of infiltration of macrophages and lymphocytes Trakia Journal of Sciences, Vol. 15, № 2, 2017 177 ILIEV P.T., et al. are mainly focused on areas around the physiological solutions at constant parasite bodies. Surrounding mucosa was temperature,
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