Veterinary Research Forum

Abstract This study, aimed to investigate Chlamydia abortus, C. pecorum, and C. psittaci in bovine, sheep, and goat abortions sent to the Konya Veterinary Control Institute diagnostic laboratory from various provinces in Central Anatolia and Mediterranean regions of Turkey between 2019 and 2022 via pathological and molecular methods. For this purpose, a total number of 150 abortions (from 58 cattle, 73 sheep, and 19 goats) were analysed via quantitative real-time polymerase chain reaction, histopathology and immunohistochemistry. The chlamydial DNA positivity rate was determined to be 24.00% via quantitative polymerase chain reaction analyses. According to the animal species, the positivity rate was 45.20% in sheep and 15.78% in goats. No cases positive for chlamydial DNA were found in the cattle abortions analyzed in the present study. When chlamydial agents were analysed on a species basis, C. abortus and C. pecorum were detected in 22.66and 1.33% of the cases, respectively. Among the animal species, 42.47% were C. abortus positive, whereas, 2.74% were C. pecorum positive in sheep. In the present study, the percentage of C. abortus-positive goats was 15.78%. and 22.66% of the Chlamydia spp. were positive according to immunohistochemical staining of aborted fetal lung, liver, heart, placenta and umbilical cord samples. This comparative study revealed that C. abortus was the most common chlamydial abortion agent in Türkiye. Chlamydial abortion agents cause abortion mostly in sheep in the region according to the animal species, and immunohistochemical staining contributes to the diagnosis of the agent at the Chlamydia spp. level.

Abstract The aim of this study was to investigate the presence of bovine herpesvirus type 1 and bovine parainfluenza virus type 3 (PI-3) in cattle lungs exhibiting pneumonia symptoms observed during macroscopic examination. Polymerase chain reaction, double immunohistochemistry, and double immunofluorescence methods were used for viral detection. For this purpose, during the summer-autumn period of 2023, a total of 84 lung samples from cattle aged 1 year and older, exhibiting macroscopic signs of pneumonia, were collected in Kayseri province, Türkiye. Pneumonia lesions were recorded according to the anatomical locations. In virus detection, PI-3 was identified at a rate of 8.33% using double immunohistochemistry/immunofluorescence staining methods, while this rate was found to be 6.72% in polymerase chain reaction. Bovine herpesvirus type 1 was not detected by any of the three methods. In the cattle lungs, 44 lesions (52.38%) were detected in the apical lobe, while seven lesions (8.33%) were detected in the middle lobe, eight lesions (9.52%) in the accessory lobe, and 25 lesions (29.76%) in the basal lobe. The PI-3 immunopositivity was observed in alveolar epithelial cells, as well as mononuclear cells in the interstitial and peri-vascular regions. This study is the first to examine bovine herpesvirus type 1 and PI-3 presence in bovine lungs using three distinct validation techniques (polymerase chain reaction, double immunohistochemistry, and double immunofluorescence).

Abstract Porcine reproductive and respiratory syndrome virus (PRRSV) and porcine circovirus 2 (PCV2) are among one of the most prevalent and important infectious agents, which cause a severe economic impact on pig farming worldwide. Circulation of these viruses together in the same pig population may lead to the syndemic infection with altered pathogenicity and thereby pathology and clinical manifestation of the diseases. The present study evaluated syndemic infection between PRRSV and PCV2 in the pig population reared in the backyard farms in Mizoram state of India. The syndemicity between PRRSV and PCV2 was confirmed by clinico-pathological studies followed by simultaneous detection of both the viruses in tissue samples by polymerase chain reaction (PCR) and reverse transcription-PCR (RT-PCR), immunohisto-chemistry and SYBR green-based real-time PCR / RT- PCR. The syndemicity resulted to a more severe respiratory disease in affected pigs. Pathological studies on affected pigs revealed a combined picture of hemorrhagic lesions with lymphadenopathy. The SYBR green-based absolute quantification assay estimated the PCV2 load in more quantities than that of PRRSV in all the tissues. The highest PRRSV load was detected in the lungs, while the highest PCV2 load was detected in mesenteric and inguinal lymph nodes. The co-circulation of different pathogenic viruses and their persistent infections in a population is always a matter of great concern to pig producers across the world. The present findings highlighted the similar situation with syndemic infection of PRRSV with PCV2 for the first time in Mizoram, India, that caused severe respiratory disease in affected pigs.

Abstract Contagious caprine pleuropneumonia (CCPP) in goats is defined as a highly contagious and rapidly spreading mycoplasmal disease that is now among the leading causes of major economic losses on many continents (Asia, Africa and the Middle East). In this study, we aimed to evaluate immunohistochemically mast cells (MCs) profile and local interleukin (IL)-17 and IL-1β protein expressions in naturally infected CCPP according to the course of the inflammation (peracute-acute, subacute-chronic). The material of the study consisted of 40 naturally infected CCPP and 6 healthy control goat lung tissues. Appropriate samples were taken from the necropsied goats and subjected to histopathological and immunohistochemical examination. In the histopathological examination of the samples, it was determined that 29 samples had a peracute-acute course and 11 had a subacute-chronic course. In immuno-histochemical examination, MC profile and local IL-17 and IL-1β protein expressions were evaluated in the peracute-acute and subacute-chronic course. Immunohistochemically, significant increases in MC number, local IL-17 and IL-1β scores were detected in the peracute-acute course compared to the control group. There were significant decreases in the relevant scores in the subacute-chronic course compared to the peracute-acute course. Current findings indicated that MC, IL-17, and IL-1β expressions played important roles in the pathogenesis of infection in naturally infected CCPP, especially in the peracute-acute course. Additionally, MC profile was evaluated for the first time in naturally infected CCPP.

Abstract Hydropericardium syndrome (HPS) has caused significant financial losses to the Iranian poultry industry in the past few years. Thirty-two broiler chickens with gross lesions of HPS were inspected histologically and immunohistochemically. Sampling was performed in Sabzevar, Iran. The dead and sick birds from random farms were subjected to necropsy examinations. Only four broiler chickens had no hydropericardium and the other gross findings were similar for birds. Basophilic and eosinophilic intranuclear inclusions, hemorrhages and necrosis in different organs were the primary characteristic histologic lesions. Lymphoid depletion, goblet cell hyperplasia and necrotizing enteritis were some of the findings reported in previous research. Low macrophage infiltration rate and brain lesions were other discoveries in Hematoxylin and Eosin (H&E) examination. Feulgen reaction and Cluster of Differentiation 68 (CD68) immunohistochemical staining were used for a comprehensive investigation and these techniques revealed improved histopathologic details. Feulgen staining confirmed brain lesions and some other changes in different organs. Eventually, the CD68 method revealed low macrophage presence in most organs. This study suggested that HPS might cause brain damage and the susceptibility of the Arian breed to the adenovirus needs further investigation.

Abstract Squamous cell carcinoma (SCC) and more specifically tonsillar SCC are generally regarded as locally invasive with various rates of metastasis. A 13-year-old male Lhasa Apso was referred to the clinic with an unusual mass on his left tonsil and abnormal lumps under the tongue. The dog's food and water intake, breathing and heart rates, body temperature, capillary refill time, and blood tests were normal. A biopsy of abnormal tissue and immunohistochemical analysis were performed. Histopathological and immunohistochemical evaluations confirmed the basaloid SCC. Some treatments like surgery, radiotherapy, and chemotherapy are used to treat a specific tumor or area of the body. However, in this particular case, chemotherapy was not administered. Considering that dogs with tonsillar cancer generally have a poor to guarded prognosis and a short life expectancy, this case was euthanized after one year with the owner's consent due to the significant tumor enlargement.

Abstract This study presents the first report of congenital tuberculosis in an 8-month-old male British Shorthair cat. The case was examined using histopathological and immunohistochemical methods. The cat was referred to a private veterinary clinic with general respiratory system problems and subsequent deterioration, leading to death. The cat owner granted permission for the cat necropsy and pathological examinations at Department of Pathology, Faculty of Veterinary Medicine, Selçuk University, Konya, Türkiye. During systemic necropsy, white round foci with diameters ranging from 3.00 to 5.00 mm were observed in the lung and spleen. Tissue samples were collected from the lung, spleen, liver, heart, kidney, mediastinal lymph nodes and brain for histopathological examinations. The tissues were subjected to routine histological tissue processing and sections were stained with Hematoxylin and Eosin and Ziehl-Neelsen. Additionally, Mycobacterium spp. antibodies were used for immunohistochemical staining. Microscopic examination revealed exudative tuberculosis lesions, areas of necrosis without a fibrous capsule and karyorrhectic cells only in the lung and spleen. Acid-resistant bacteria observed by ZN staining in the lesioned areas of the lung and spleen were identified as Mycobacterium spp. using immunohistochemical staining. No positive staining was observed in other organs using ZN and immunohistochemical methods. As a result, congenital tuberculosis was diagnosed in a cat for the first time, especially in relation to lesions in the spleen.

Abstract Tropical or Mediterranean theileriosis in dairy cattle is widely distributed in many tropical regions of the world. The purpose of this study was to evaluate the proliferation status of mononuclear cells infected with Theileria annulata schizonts in different tissues and its relationship with the pathogenesis of the parasite in cattle by histopathology, immuno-histochemistry and polymerase chain reaction (PCR). Blood and tissue samples of eight Holstein cattle that had been lost due to theileriosis and eight healthy slaughtered cattle of the same breed were collected as a control group after necropsy. The piroplasms in the blood smears and the schizonts in the cytoplasm of the lymphocytes and macrophages of the lymph nodes were microscopically detected. Histopathologically, the proliferation of macrophages, lymphocytes, and plasma cells in lymph nodes and the heart, congestion, and bleeding in the red pulp of the spleen, portal tracts of the liver, interstitial tissue of the kidneys, multifocal necrosis and ulceration in the abomasum together with hyperemia and hemorrhages and lymphoblastic infiltration in the submucosa and lamina propria adjacent to these lesions and emphysema with ecchymotic hemorrhage in the lungs were evident. Immunohistochemistry identified the proliferated cells as mostly Cluster of Differentiation 3- Positive T lymphocytes and macrophage marker antibody 387- positive macrophages. Positive results of PCR for the Tams1 30.00 kDa gene were observed in lymph nodes, liver, lung and abomasum. It was concluded that the pathological changes were the result of schizont-infected macrophage proliferation leading to severe uncontrolled proliferation of uninfected T lymphocytes.

Abstract The aim of this study was to determine the prevalence of parainfluenza 3 (PI3) virus antigen through histopathological and immunohistochemical (IHC) methods in sheep lung samples collected from Erzurum province, Türkiye. Between August and November 2017, 1462 sheep were dissected in the slaughterhouse and their lungs were examined macroscopically. In total, 100 of the lung samples with pneumonia were selected. Routine histopathological and IHC analyses of the collected lung tissues with pneumonia were performed. Pneumonia observed through macroscopical and histopathological examinations of the lung samples was classified as purulent-catarrhal bronchopneumonia (14.00%), fibrinous bronchopneumonia (23.00%), interstitial pneumonia (69.00%), granulomatous pneumonia (7.00%), verminous pneumonia (19.00%) and pulmonary adenomatosis (6.00%). Two or three types of pneumonia were observed in many of the same cases. The PI3 virus antigen positivity rate in the IHC analysis of sheep lung samples was 19.00%. In the IHC tracing, positivities were found mostly in the alveolar macrophages and cytoplasm of bronchial, bronchiolar and alveolar epithelial cells. As a result, the prevalence of PI3 virus in sheep in Erzurum province, Türkiye, was determined to be 19.00% using KLN BVB IHC method.

Abstract A 15-year-old male terrier dog with symptoms of lethargy and severe abdominal distension was referred to the polyclinic hospital of the Ferdowsi University of Mashhad, Mashhad, Iran. In addition to numbness and abdominal distension, the dog also had anorexia and severe weakness and some skin masses were observed. Due to the enlarged abdomen, splenomegaly was diagnosed in ultrasonography. Fine needle aspiration was performed on the liver and skin mass and then, neoplastic lesions were reported based on cytology. On the necropsy, two masses were found on the liver and shoulder skin. These masses were well-encapsulated, soft and multi-lobulated. Samples taken from the liver and skin were prepared by Hematoxylin and Eosin staining and then, two different immunohistochemical markers were used to confirm the initial diagnosis. Histopathological examination of these two well-encapsulated, soft and multi-lobulated masses on the liver and skin showed lipid content and liposarcoma was indicated. Immunohistochemical staining using two markers, S100 and MDM2, made a definitive diagnosis and confirmed the diagnosis.

Abstract Toxoplasma gondii is a protozoon parasite which causes toxoplasmosis both in human and warm-blooded animals. Toxoplasmosis is a worldwide disease and largely threats human and animal health consequently causing economic losses. Also, it affects the visceral organs in different severity degrees according to the strain of parasite and the host. In this study, experimental toxoplasmosis was performed via intra-peritoneal route in 12 gerbils by administrating 5.00 × 10³ tachyzoites of T. gondii RH strain. The gerbils were sacrificed 7 days after inoculation. All systemic organs were obtained via necropsy and examined by immunohistochemical and histopathological methods. Lesions infected with T. gondii mostly observed in the serosa of abdominal cavity organs including stomach, liver, spleen, intestines, and kidneys. The lesions were most severe in liver. The parasite showed an affinity for the hepatic tissue. To our knowledge, this is the first experimental study of acute T. gondii infection in gerbil evaluating macroscopic, microscopic and immunohistochemical findings. It is concluded that Mongolian gerbils can be used as experimental animals to investigate toxoplasmosis. Also, these animals are very suitable hosts to study liver pathology and pathobiology of T. gondii-related hepatitis.

Abstract In addition to the fifty years since the test-and-slaughter program began in Iran and despite a significant reduction in the disease prevalence, positive tuberculosis cases are still being isolated from livestock farms across the country. Tests with 100% sensitivity and specificity are essential features for bovine tuberculosis diagnosis. The relationship between real-time PCR and immunohistochemistry (IHC) as two essential laboratory methods in the diagnosis of bacterial infections were aimed to evaluate single intradermal comparative tuberculin test (SICTT) results. One hundred thirty-eight cows in two groups were examined: Reactors (108 cows) and clean (as a control group; 30 cows). In the reactor group, 58(54.00%) cows were Mycobacterium bovis positive, 46(43.00%) were Mycobacterium avium subsp. paratuberculosis (MAP) positive, and 11(10.00%) were Mycobacterium tuberculosis positive. 32(55.00%) cows were co-infected with M. bovis and MAP and 5(4.55%) cows were co-infected with Mycobacterium tuberculosis and MAP in this group. Of 50 M. bovis negative cows of reactor group were 14(28.00%) MAP positive and 36(72.00%) negative, as well. Concurrent infection with all was observed in one reactor case. Comparing IHC and real-time PCR for the detection of bovine tuberculosis and Johne’s disease showed very good agreement (Kappa values 0.81 - 1.00). The results also provided further confirmation on IHC and real-time PCR as a sensitive and reliable diagnostic screening approach for detection of bovine tuberculosis. The use of one laboratory method to detect bovine tuberculosis is not sufficient alone.

Abstract A 14-year-old female Shih Tzu was presented with conjunctivitis, keratitis, an enlarged mass located on the left lower eyelid and a large subcutaneous cyst in the temporal region. The hematological and biochemical analyses revealed no significant issues. The mass and the cyst were taken from the base with 1.50 cm of the underlying tissue. On histological examination, the mass was made up of adenoid arrangements of sebocytes and basaloid reserve cells with low to moderate mitotic activity of basaloid cells. In addition, a large number of meibomian ducts with marked infiltration of mononuclear inflammatory cells and melanin pigment were observed in the ductal part of the tumor. The cyst was lined by a simple cuboid-like epithelium with no eosinophilic cuticular lining. Immuno-histochemical staining of the mass with CK14 showed positive reaction in the neoplastic basaloid reserve cells and adipocytes; but, not with CK18 and vimentin. Unlike, the luminal cells of the cyst were stained with CK18. The co-existence of mixed meibomian gland and ductal adenoma with an apocrine sweat gland cyst was diagnosed based on the clinical, histological and immunohistochemical findings.

Abstract Canine lymphoma is the most common neoplasm of the hematopoietic system with the most frequently diagnosed malignancy. Also, dogs affected by T-cell lymphoma displayed a lower rate of complete chemotherapy response and have a high risk of death in the early stage of the disease. A 5-year-old male mixed dog was presented to the Small Animal Hospital, School of Veterinary Medicine, Shiraz University, Shiraz, Iran with a history of left testicle swelling for four months. The testicle had approximately 13.00×10.00 cm size with a greyish-white cut surface. Histopathologically, the testicular tissue was infiltrated by neoplastic cells as only a few degenerated seminiferous tubules have remained. The neoplastic cells were round to oval with pleomorphic nuclei and single or multiple prominent nucleoli. Immunohistochemical analysis revealed positive and negative immunoreactivity for CD3 and CD20, respectively. According to histopathological and immunohistochemical features, the tumor was concluded as a testicular T-cell lymphoma. It seems that recognition of lymphoma type could be helpful for clinicians therapeutic protocols.

Abstract We report the occurrence of lung cancer in a six months old lamb of Ouled Djellal breed from Algeria. The main clinical sign was a considerable amount of whitish foamy fluid discharge from the nostrils when the animal head was lowered and the rear end was lifted. The post-mortem examination revealed the presence of enlarged, heavy and edematous lungs with diffuse or foci areas, reddish or white-gray in color. The gross and histological lesions of the lungs were compatible with pulmonary adenocarcinoma. Lung adenocarcinoma in sheep is caused by jaagsiekte sheep retrovirus (JSRV) and originated from differentiated alveolar type II cells and non-ciliated bronchiolar epithelial Clara cells. We evidenced the expression of the oncogenic JSRV by immunostaining of lung slides with specific antibodies against the JSRV envelope. The viral proteins were expressed only in the tumor cells from the affected areas. As already described in other countries, JSRV-induced lung adenocarcinoma is present in the sheep population in Algeria.

Abstract The aim of this study was to find a proper method for improvement of ischemic condition in the rat hind limb and also to observe the efficacy of cell engraftment with alginate/gelatin three-dimensional scaffolds. Eighteen male Wistar rats weighing 200 to 250 g were randomly divided into three groups (n = 6) including a) ischemia group; in which femoral artery was removed after ligation at the distance of 5 mm, b) scaffold group; in which hydrogel scaffold was added to the site of transected femoral artery and c) test group; in which in addition to hydrogel scaffold, mast cells (MCs) were also added (1 × 10⁶ cells). Analysis of capillary density, artery diameter, histomorphometric parameters and immunohistochemistry in transected location were done on day 14 after femoral artery transection. The average number of blood capillary was significantly higher in the test group than other groups. Also, the average number of medium and large blood vessels was significantly higher in the test group compared to ischemia and scaffold groups. Application of MCs through the use of hydrogel scaffolds (alginate/gelatin) can be considered as a new approach in the application of stem cells for therapeutic angiogenesis under ischemic conditions which can improve the angiogenesis process in patients with peripheral artery diseases.

Abstract A four-year-old male Persian cat was referred with three weeks history of progressive lameness due to a rigid osseous mass with 3.50×2.50×2.00 cm in dimensions in his left arm. In the histopathological evaluation of bone biopsy, two distinct populations of cells including multinucleated giant cells and oval cells which embedded in a fibro-osseous stroma and surrounded by lamellar bone trabeculae were observed. At necropsy, multiple metastatic nodules with different sizes unveiled in the liver and spleen. Microscopically, those tumor cells which already described in the bone lesion were also infiltrated to the liver and spleen. The neoplastic cells had no immunoreaction to CD68, desmin, alpha smooth muscle actin, S100, CD20, CD3 and pancytokeratin, but only a few cells had reaction to vimentin were similar to fibroblasts.

Abstract Astrocytoma as one of the most common central nervous system (CNS) tumors is rarely reported in veterinary literature. A 7-year-old Persian Lori-Bakhtiari ewe was presented to the clinic with a two months history of progressive blindness, nystagmus to the right, bilaterally decreased pupillary reflexes, head pressing and paddling. At necropsy, a whitish well-circumscribed mass with dimensions of 3.50×2.50×1.50 cm was observed in the dorsal parietal lobe of the left cerebral hemisphere. Microscopically, the mass was well-circumscribed and highly cellular, consisted of round to elongated cells with scant and vacuolated cytoplasm with few, flaccid processes. The nuclei were round to oval with densely stippled chromatin and indistinct nucleoli. Immunohistochemical analyses showed positive staining for vimentin, S100 and glial fibrillary acidic protein. Definitive diagnosis of cerebral protoplasmic astrocytoma was made on the basis of the histopathological and immunohistochemical findings. This type of neoplasm should be included in the differential diagnosis of CNS lesions in the sheep.

Abstract In the present study, we described cyto-histopathological features and immunophenotyping of the large B-cell lymphoma in an 8-year-old mixed breed dog with applying the World Health Organization (WHO) system of classification of canine lymphomas. In fine-needle aspiration (FNA), lymph nodes were involved by neoplastic cells of intermediate to large size with deep blue cytoplasm; consist of centroblasts, immunoblast and medium-sized cells. Histopathologically, the follicles and sinuses of lymph nodes were replaced by sheets of numerous immunoblasts (less than 90.0% of total cells) and centroblasts. Numerous mitotic figures were also observed. Immunohistochemical analysis presented that the neoplastic cells express B-cell phenotype CD20 and CD79a, but do not stain for T phenotype CD3. On the basis of cytology, histopathology and immunohistochemical findings, the present tumor was diagnosed as diffuse large B-cell lymphoma, high-grade centroblastic type (DLBCL-CB) according to WHO histological classification. Applying this classification system for diagnosis of canine lymphomas is very useful and has a high accuracy and consistency. However, further co-operative studies between clinicians and pathologists should be performed, in order to improve the effectiveness of this classification.

Abstract In the present study, we describe a subcutaneous mass between the left flank and hip in a 2-year-old male Great Dane dog. Histopathologically, cells appeared to be spindle shaped around a central capillary together with a fingerprint pattern. Immunohistochemical analysis presented that the neoplastic cells expressed vimentin, but did not stain for S-100 protein. On the basis of histopathology and immunohistochemical findings, the present tumor was diagnosed as canine hemangiopericytoma. Hemangiopericytoma could be considered in differential diagnosis list of any mass in the skin (even in young dogs) and must be identified histopathologically.

Abstract An 11 year – old mixed female Labrador was presented with two masses in trunk and neck. The tumoral masses were excised and sent for histopathological and immunohistochemical analyses. Histopathological examination of masses revealed diffuse infiltration of small sized lymphoid cells in subcutaneous tissue which were intense around the blood vessels. More than 10% lymphoid cells were CD3 positive in the immunohistochemical staining and most of them were accumulated around vessels. Protein 53 (p53) expression was detected by brown nuclei in immunohistochemical staining. Subcutaneous lymphoma was diagnosed according to histopathological results. After 6 months the case was referred with multicentric lymphoma and based on the owner request euthanasia was performed. These findings emphasize on poor prognosis for tumors with p53 mutation.

Abstract Tendon never restores the complete biological and mechanical properties after healing. Bone marrow and recently adipose tissue have been used as the sources of mesenchymal stem cells, which have been proven to enhance tendon healing. Stromal vascular fraction (SVF), derived from adipose tissue by an enzymatic digestion, represents an alternative source of multipotent cells, which undergo differentiation into multiple lineages to be used in regenerative medicine. In the present study, we investigated potentials of this source on tendon healing. Twenty rabbits were divided into control and treatment groups. Five rabbits were used as donors of adipose tissue. The injury model was unilateral complete transection through the middle one third of deep digital flexor tendon. Immediately after suture repair, either fresh stromal vascular fraction from enzymatic digestion of adipose tissue or placebo was intratendinously injected into the suture site in treatments and controls, respectively. Cast immobilization was continued for two weeks after surgery. Animals were sacrificed at the third week and tendons underwent histological, immunohistochemical, and mechanical evaluations. By histology, improved fibrillar organization and remodeling of neotendon were observed in treatment group. Immunohistochemistry revealed an insignificant increase in collagen type III and I expression in treatments over controls. Mechanical testing showed significant increase in maximum load and energy absorption in SVF treated tendons. The present study showed that intratendinous injection of uncultured adipose derived stromal vascular fraction improved structural and mechanical properties of repaired tendon and it could be an effective modality for treating tendon laceration.