Eight-Year Study of Haemogregarina stepanowi Infection in Poached European Pond Turtles (Emys orbicularis) Held in Belgrade Zoo Quarantine.

The eight-year study (2015-2023) was performed on a large sample of poached European pond turtles infected with Haemogregarina stepanowi and held in a pond that belongs to a quarantine section of Belgrade Zoo. The protected species of European pond turtles have been found in poor health, with general weakness, anorexia, and low motility. Comprehensive cytological, hematological, molecular, and postmortem evaluations have been performed. Initially, Diff Quick staining of the blood smears revealed rounded or elongated erythrocytes, often bearing premeront or U-shaped gamont of the hemogregarines inside. The reduced erythrocyte numbers, hemoglobin, and hematocrit values found in the examined population of infected turtles indicated anemia. Macroscopically, shell necrosis and massive skin hemorrhages were the most prominent findings observed in diseased turtles. Microscopically, the lungs, liver, kidneys, and spleen revealed hyperemia, hemorrhages, and the presence of parasitic stages in tissue samples in 31 of 40 necropsied turtles. Cytological and microscopic examination of the samples proved to be sufficient for establishing the infection, but molecular analyses of the 18S sequence were used for phylogenetic studies. Over the years, the number of diseased and dead turtles has decreased, which could be hypothetically attributed to the elimination of leeches as the definitive host.

African Swine Fever Outbreak in an Enclosed Wild Boar Hunting Ground in Serbia.

African swine fever (ASF) has been detected in many European countries since its introduction in Georgia in 2007. Serbia suffered its first case of ASF in the domestic pig population in 2019. At the beginning of 2020, ASF was detected in wild boars in open hunting grounds in the southeastern region of the country in districts along the country's borders with Romania and Bulgaria. Since then, all ASF outbreaks in wild boar were clustered in the population located in the same bordering areas. Despite the newly implemented biosecurity protocols for hunters in 2019, ASF was detected for the first time in June 2021 in the wild boar population located in an enclosed hunting ground in the northeast region of the country. In this study, we reported the first ASF outbreak in a wild boar population located in an enclosed hunting ground in close proximity to the Serbian-Romanian border. The epizootiological data on the field investigation of the ASF outbreak, with descriptions of the clinical signs and gross pathological lesions detected, including the total number as well as the estimated age, sex, and postmortem interval, were analyzed. Clinical signs were detected only in nine diseased wild boars, while in total, 149 carcasses were found in the open and enclosed part of the hunting ground. In addition, 99 carcasses from which samples (parts of spleen or long bones) were collected for molecular diagnostics (RT-PCR) were confirmed as ASF-positive. The results of the epidemiological investigations indicate the central role of wild boar movements as well as the constant risk of human-related activities in the countries bordering area.

Highly Pathogenic Avian Influenza H5N8 Outbreak in Backyard Chickens in Serbia.

In winter 2016/2017, the highly pathogenic avian influenza virus H5N8 was detected in backyard poultry in Serbia for the first time. The second HPAI outbreak case in backyard poultry was reported in 2022, caused by subtype H5N1. This is the first study that documents the laboratory identification and pathology associated with highly pathogenic avian influenza in poultry in Serbia during the first and second introduction waves. In both cases, the diagnosis was based on real-time reverse transcriptase PCR. The most common observed lesions included subepicardial hemorrhages, congestion and hemorrhages in the lungs, and petechial hemorrhages in coelomic and epicardial adipose tissue. Histologically, the observed lesions were mostly nonpurulent encephalitis accompanied by encephalomalacia, multifocal necrosis in the spleen, pancreas, and kidneys, pulmonary congestion, and myocardial and pulmonary hemorrhages. In H5N8-infected chickens, immunohistochemical examination revealed strong positive IHC staining in the brain and lungs. Following these outbreaks, strict control measures were implemented on farms and backyard holdings to prevent the occurrence and spread of the disease. Extensive surveillance of birds for avian influenza virus did not detect any additional cases in poultry. These outbreaks highlight the importance of a rapid detection and response system in order to quickly suppress outbreaks.

Morphological and immunophenotypic characteristics of the liver of swine naturally infected with hepatitis E virus.

Hepatitis E virus (HEV), the zoonotic agent of infectious hepatitis, is present in swine farms in different geographical areas. Little is known about the mechanism of liver damage and type of local immune response by HEV in swine. Therefore, the aim of this study was to determine the morphological and immunophenotypic characteristics of hepatic lesions caused by hepatitis E virus in naturally infected swine. In this study, liver samples of 12 slaughtered 10 weeks old pigs which were RT-PCR positive for HEV RNA in rectal swab samples have been used. Livers were macroscopically examined and samples were taken for histopathological, immunohistochemical (CD3, CD79α and TGF-ß1), semiquantitative, morphometric analysis, RT-nested-PCR, PCR and bacteriological analysis. Microscopically, mild and moderate multifocal lymphoplasmacytic hepatitis was observed. Apoptotic bodies were observed as areas of focal eosinophilic condensation in the cytoplasm of 33.33% liver samples, while in 16.67% liver samples portal fibrosis was detected. Immunohistochemically, portal and lobular lymphocytes in the mononuclear liver infiltrate were predominantly CD3+ T cells (234.80 ± 79.98). An intense TGF-ß1 positive reaction was observed within the mononuclear cell infiltrate as well as polymorphonuclear cells in liver samples with apoptosis of hepatocytes. In all 12 tested liver samples HEV RNA was detected by RT-nested-PCR. HEV is noncytopathic, and this finding provides further evidence for an immune mediated pathogenesis in hepatitis E virus infection in swine. Also, the role of CD3+ cells in hepatocyte damage is clearly demonstrated.

Comparative pathological findings in mute swans (Cygnus olor) naturally infected with highly pathogenic Avian influenza viruses H5N1 and H5N8 in Serbia.

The aim of this study was to compare pathological lesions and viral antigen expression in the organs of mute swans (Cygnus olor) naturally infected with highly pathogenic avian influenza virus subtypes H5N1 and H5N8. The examination was conducted on the carcasses of 22 mute swans which died during the avian influenza outbreaks in Serbia in 2006 and 2016-2017. Avian influenza virus subtype H5N8 isolated from mute swans in 2016-2017 was clustered within the 2.3.4.4 clade group B. After necropsy, lung, liver, spleen, pancreas, kidney and brain tissues were sampled for histopathology and immunohistochemical examination. Avian influenza virus nucleoprotein polyclonal antibodies were used for detecting the viral antigen in the examined tissues. The most significant gross lesions were necrosis and haemorrhages in the pancreas. Major histological lesions were multifocal necroses in the pancreas, spleen and liver, non-purulent encephalitis, lung congestion and oedema. Immunohistochemical demonstration of HPAIV nucleoprotein in pancreas and brain was strongly consistent with histological lesions in both infected groups. Our findings showed that pancreas was the most affected organ in all examined mute swans. In addition to increased mortality rate, similar pathological findings were detected in mute swans naturally infected with highly pathogenic avian influenza viruses H5N1 and H5N8.

Association of the severity of lung lesions with carcass and meat quality in slaughter pigs.

The aim of this study was to determine the association of lung lesions with carcass and meat quality traits in slaughter pigs and to describe the main morphological features associated with lung lesions. Macroscopic lesions on the lungs were detected in 67.09% of a total of 79 pigs examined. Histopathological examination revealed that acute and chronic interstitial pneumonia represented the commonest changes, detected in 26.67% and 33.33% of the cases, respectively. Bronchopneumonia was found in 33.33% of the cases. By immunohistochemical examination, 26.67% of the lungs showed the presence of severe peribronchiolar and perialveolar infiltration composed predominantly of CD3+ T lymphocytes, which finding may be indicative of viral pneumonia. Regarding the production traits, it was confirmed that pigs with severe lung lesions had the lowest liveweight, hot carcass weight and meatiness, the highest pH value 45 min after slaughtering (pH45) and the highest incidence of dark, firm, dry (DFD) and pale, soft, exudative (PSE) meat. The presence of lung lesions significantly downgraded carcass value and caused a significant deterioration in pork quality.

First report of Polycystic kidney disease occurrence in Persian cats in Serbia.

Polycystic kidney disease (PKD) is an inherited autosomal disorder in cats, mostly diagnosed in Persian cats. Renal cysts can be diagnosed by ultrasound, but cats must be at least 16 weeks old. The goals of this study were to assess the occurrence of PKD in Serbia using a randomly selected group of Persian cats, to compare the diagnostic efficacy of ultrasound and genetic tests, and to measure haematological and selected biochemical parameters. We examined 70 cats of Persian breed, between 4 months and 8 years of age. Complete blood count and selected biochemical parameters were measured, renal ultrasound was performed. Swabs of the oral cavity were obtained for genetic testing. Percentage of PKD positive cats identified by genetic testing was 48.6%, whilst only 18.6% were detected through ultrasound. Animals that were polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) positive and ultrasound negative ranged from 4 months to 3.5 years. All haematological and biochemical parameters were within the the normal range values in all examined cats. Genetic methods proved to be the most effective for reliable and early diagnosis of PKD in Persian cats. DNA analysis can be used right after birth, and excludes the need for other diagnostic procedures, such as ultrasound.

Skin and skeletal system lesions of european pond turtles (Emys orbicularis) from natural habitats.

Water pollution is known to play an important role in the pathogenesis of plastron, carapace and skin diseases of turtles. In this study, a total of 150 European pond turtles (Emys orbicularis) of different age and both sexes, originating from natural habitats in Serbia, were examined for morphological changes of the skin, plastron, carapace and skeletal system. The turtles were taken out from their natural habitats in Lake Ludas, Lake Palic and Lake Tresetiste. After artificial hibernation, they were subjected to detailed examination, sampled and treated, and finally returned into their natural habitat. Biopsies from the skin and shell were subjected to histopathological examination and microbiological analysis. X-ray scanning was also performed to detect changes in the skeletal system. Macroscopic changes of the skin, most frequently degenerative, inflammatory or neoplastic diseases, were diagnosed in 49.33% of the turtles examined. Dermatitis of different origin and form was the most prominent histopathological finding (28.00%). In the plastron, inflammatory and degenerative processes were frequently found. Osteopathy and mechanical injuries were the dominant findings. Macroscopic changes of the plastron, carapace and skeletal system were diagnosed in 67.33% of the turtles examined. Using X-ray scanning, generalised osteopathy, anomalies and malformations of different aetiology were also diagnosed on the tail and legs. Microbiological examinations showed the presence of a variety of bacterial and fungal agents, either primary pathogens or potential polluters, which invaded the skin and shell, or were present in cloacal swab samples. Bacterial infection was diagnosed in 76.66% of the turtles, first of all in those with skin and shell necrosis. Mycoses were diagnosed in 33.33% of the animals.

The role of hepatic myofibroblasts in liver cirrhosis in fallow deer (Dama dama) naturally infected with giant liver fluke (Fascioloides magna).

BACKGROUND: This paper describes liver cirrhosis in 35 fallow deer infected with the giant liver fluke, as well as the distribution, origin, and role of myofibroblasts in its development. RESULTS: In liver of infected deer, stripes of connective tissue are wound around groups of degenerated and regenerated liver lobuli. In the connective tissue, lymphocytes and macrophages which often contain parasite hematin are also present. The walls of the bile ducts are thickened, the epithelium multiplied with mucous metaplasia, and desquamated cells, parasite eggs and brown pigment are present in their lumen.In the livers with cirrhosis, immunopositivity to α-SMA and desmin was observed in cells in portal and septal spaces, at the edge between fibrotic septa and the surrounding parenchyma and in perisinusoidal spaces. These cells vary in size, they are round, oval, spindle-shaped or irregular in shape, similar to vascular smooth muscle cells. The derangement of epithelial-mesenchymal interactions detected in chronic cholangiopathies is most probably the pro-fibrogenic mechanism in liver cirrhosis of fallow deer (Dama dama) infected with the giant liver fluke (Fascioloides magna). CONCLUSION: Myofibroblasts, especially hepatic stellate cells (HSCs), play an important role in the synthesis of extracellular matrix components in the development of parasitic fibrosis and cirrhosis in the liver of fallow deer.

Investigation of an outbreak of mycobacteriosis in pigs.

BACKGROUND: A high proportion of pigs imported to Serbia from a Lithuanian breeding herd reacted positively against avian and/or bovine tuberculin. The pigs were euthanized and lesions characteristic for mycobacterial infection were detected. An investigation of potential mycobacteriosis in the pigs imported to Serbia and the possible source of infection in the Lithuanian herd were therefore initialised. RESULTS: Formalin fixed, paraffin embedded lymph nodes from tuberculin positive animals were examined by real-time PCR for IS1245 and IS6110. IS1245 was detected in 55% and IS6110 in 11% of the samples. Seven of the ten IS6110 positive samples were positive for IS1245. Eleven lymph nodes from 10 pigs and 15 environmental samples were collected from the Lithuanian breeding herd and cultured for mycobacteria. M. avium subsp. hominissuis was detected in all lymph nodes and from eight samples of peat and sawdust. Isolates with identical and related IS1245- and IS1311 RFLP profiles were detected from swine and peat. CONCLUSIONS: This study demonstrated cross reactions between avian and bovine tuberculin in pigs. Real-time PCR indicated infection with M. avium in the Serbian pigs. However, as a small proportion of the lymph nodes were positive for IS6110, infection with bacteria in the M. tuberculosis complex could not be ruled out. Analyses confirmed the presence of M. avium subsp. hominissuis in porcine and environmental samples from the Lithuanian breeding herd. The results indicate peat as a source of M. avium subsp. hominissuis infection in these pigs, and that the pigs imported to Serbia were infected with M. avium subsp. hominissuis.

Distribution of porcine circovirus 2 cap antigen in the lymphoid tissue of pigs affected by postweaning multisystemic wasting syndrome.

The lymphatic organs of 50 pigs from a total of eight farms located at different sites in the epizootiological region of North Backa County were studied to obtain data on the prevalence of circoviral infections in Serbia. All of the pigs examined had clinical signs suggestive of postweaning multisystemic wasting syndrome (PMWS). All pigs underwent necropsy and tissue samples were taken for histopathological, immunohistochemical (IHC) and PCR analysis. The presence of porcine circovirus 2 (PCV2) was established by PCR analysis in the organs of the pigs tested. The most frequent histopathological lesions of lymphoid tissue linked with the presence of positive immunostaining for PCV2 Cap antigen confirmed the existence of PMWS in all farms tested in North Backa County. Using PCR, histopathological and IHC techniques, the presence of PMWS was proved in the Republic of Serbia. During necropsy, generalised enlargement of the lymph nodes was evident. The most common histopathological finding was lymphocyte depletion in the follicular and perifollicular areas of lymph nodes. Infiltration by macrophages was also recorded. By IHC analysis, the cytoplasm of macrophages was shown to contain a large amount of the ORF2-coded Cap antigen of PCV2. Lymphocyte depletion and large numbers of macrophages were recorded in the tonsils, spleen, intestinal lymphatic tissue, Peyer's patches and ileocaecal valve. The presence of typical granulomatous lesions with multinuclear giant cells (MGCs) was also recorded in the lymphatic tissue. Cap antigen was shown to be present in macrophages and less often in lymphocytes.

Biological properties of a naturally attenuated infectious bursal disease virus isolated from a backyard chicken flock.

The biological properties of an infectious bursal disease (IBD) virus isolated from bursas collected during an outbreak in a village chicken flock in Macedonia are described. The mortality rate was 50%. Two viruses coexisted in the bursas of infected chickens (IBDVwt and IBDVtc). The virus termed IBDVtc grows on chicken embryo fibroblast (CEF) cells from the first passage. Specific pathogen free chickens inoculated with IBDVtc at passage level 4 did not develop any clinical signs of disease. Some discrete bleeding on the leg muscles was seen and the bursa of Fabricius revealed pathological lesions similar to those caused by classical strains. However, the bursa recovered quickly (bursa lesion score 2) by 14 days post infection (PI). We also found evidence of bursal repopulation by means of perinuclear antigen staining. Strong CD3 influx was evident at 4 days PI, and at 33 days PI the CD3+ cell finding was comparable to the control. The mean antibody titre was 9.2 log 2 at 14 days PI. The amino acid composition of VP2 in IBDVwt (222 Ala, 242 Ile, 253 Gln, 256 Ile, 279 Asp, 284 Ala, 294 Ile and 299 Ser) is described. The same sequence was found in IBDVtc, except for two point mutations, at Gln253→His and Ala284→Thr. Such amino acid substitution is responsible for partial attenuation and the ability of the strain to replicate in cell culture. None of the commercial vaccine viruses has a similar arrangement of amino acids in the variable domain of IBDV. This strongly suggests that IBDVtc originates from a very virulent strain. To the best of our knowledge, this is the first report of a concomitant infection of chickens with highly pathogenic IBDV and its mutant counterpart.

Cell proliferation in pathogenesis of esophagogastric lesions in pigs.

Esophagogastric ulcer is an independent disease in swine that is characterized by ulcerous autodigestion of the cutaneous mucosa, which does not exhibit a tendency to recover, but, on the contrary, a tendency toward severe hemorrhaging, with a predominantly lethal outcome. Since it develops in the part of the stomach that is morphologically and functionally different from other glandular mucosa, it was questioned earlier whether it could be a peptic ulcer based on its nature. Spontaneous ulcers, usually of the stomach, commonly occur in many domestic animals. Some of these lesions are chronic and they may occur in either the glandular or squamous-lined regions of the stomach. As with the human disease, the pathogenesis in domestic animals is multifactorial, poorly understood, and variable between and within species. Environmental stress and dietary factors are very important in the ulcer disease in swine. It has been shown that the Helicobacter spp. is strongly associated with naturally occurring ulcer and preulcer lesions of the pars esophagea in swine, which raises the possibility that Helicobacter spp. is an important factor in the pathogenesis of these lesions. The dynamics of the development of esophagogastric ulcers imply hyperplastic lesions (parakeratosis and hyperkeratosis), keratolysis, erosions, peptic necrosis, and the development of ulcers with all the characteristics of peptic ulcerations in other localities. In addition, K6 is expressed in association with the mucosal changes. The pattern of the intermediate filaments of keratin suggests that epithelial proliferation, which leads to visible hyperkeratosis, constitutes the essence of gastric ulcers in swine.

Cellular basis of chronic obstructive pulmonary disease in horses.

Chronic obstructive pulmonary disease (COPD) is an inflammatory obstructive disease of the airways characterized with hypersensitivity of the airway tissues to various allergens, most commonly the fungi contained in the poor-quality hay and straw bedding-Saccharopolyspora rectivirgula, Aspergillus fumigatus, and Thermoactinomyces vulgaris. It is manifested clinically in middle-aged horses with recurrent episodes of dyspnea, chronic cough, and their reduced athletic and working capacity. Pulmonary emphysema and lack of pulmonary collapse are the most common gross lesion. Pathohistological findings in horses with COPD are chronic bronchitis/bronchiolitis, with characteristic changes in lumen, mucosa, submucosa, and smooth muscle layer and alveolar emphysema, both distensive and destructive form. Increased immunoreactivity in lungs and tracheobronchial lymph nodes is also noted. Most common lesions seen on cytology imprint smears from tracheal bifurcation is thick, viscous, PAS-positive mucus that forms Curschmann's spirals. Dominant cell population consists of desquamated airway epithelial cells, as well as eosinophils, neutrophils, mast cells, erythrocytes, and alveolar macrophages. Primary pulmonary pathogens as well as potential contaminants and secondary infection agents were isolated bacteriologically from lung samples. All of the aforementioned findings correlate pointing to the fact that chronic bronchitis/bronchiolitis represents a basic substrate of COPD, which have combined inflammatory and immunological etiology, and emphysema is secondary to airway obstruction.