Volume 67 (2017): Issue 4 (December 2017)

The main reasons for wildlife forensic research are animal poaching, illegal trade, and falsified game meat products. Small trace amounts, old and degraded materials present the most common samples in revealing criminal activities in this field. This is the reason why it is crucial to use adequate and reliable methods and samples to identify animal species killed outside the hunting season or species protected by law. In this study, different endpoint PCR and real-time PCR protocols were compared in the identification of three Cervidae species (Capreolus capreolus, Cervus elaphus, Dama dama) from old and damaged material found in an enclosed area where the animals were kept. From a total of 129 samples, end point PCR provided results for 119 samples, while real-time PCR was successful in all cases. Also, we created and tested a protocol for simultaneous analyses of different types of samples, which is of great importance as when the amplification is carried out simultaneously it is more cost efficient and speeds up the process.

Experimental animals are still used in today’s medicine to understand better physiological or pathological processes, or to develop, for example better vascular prostheses. For that reason, these animals must show some similarities with humans, from the anatomical to the physiological point of view. When developing vascular prostheses, we have to evaluate if the graft will react in the expected way and if during experimental research there will be some factors that might influence the proper functioning of vascular prostheses in the human body. We observed the consequences of bilateral common carotid artery ligation (BCCAL) or Sham operation in seventeen healthy Merinolandschaf / Württemberg sheep, aged between 2 and 4 years, after testing new types of carbon-coated ARTECOR® and ADIPOGRAFT Ra 1vk 7/350 vascular prostheses. After the follow-up period the prostheses were extirpated, so the blood supply was provided from the vertebral arteries. Sheep in both groups were not sacrificed, but were observed for 18 months. After the observation period all sheep showed no physical or neurological changes and all are still alive. Animal responses to BCCAL are different, depending on the animal species, age, and condition. In sheep, bilateral blocking of the blood fl ow in the carotid bed seems to be conceivable since the brain was sufficiently supplied with blood from the vertebral arteries.

Transitional cell carcinoma (TCC), is the most common form of urinary bladder cancer in dogs and represents 2% of all reported canine cancers. Canine TCC is usually a high-grade invasive cancer and problems associated with TCC include urinary tract obstruction and distant metastases in more than 50% of affected dogs. TCC is most commonly located in the trigone region of the bladder precluding complete surgical resection. Current treatment options for TCC in dogs include medical therapy, surgery or radiation. Mycobacterium Cell Wall Fraction (MCWF) is a biological immunomodulator derived from non-pathogenic Mycobacterium phlei. MCWF possesses a potential in multiple veterinary areas such as anticancer therapy, palliative care and treatment of infectious diseases in both small and large animals. MCWF is considered a bifunctional anti-cancer agent that induces apoptosis of cancer cells and stimulates cytokine and chemokines synthesis by cells of the immune system. Here we report the results from in vitro and in vivo studies that could suggest use of MCWF as an additional treatment option for TCC in dogs. Particularly, we demonstrated that MCWF induces a concentration dependent inhibition of proliferation of K9TCC cells which was associated with the induction of apoptosis as measured by the proteolytic activation of caspase-3 and the degradation of PARP. Furthermore, we demonstrated the safety and potential for in vivo MCWF treatment efficacy in dogs bearing stage T2 TCC by reducing clinical signs, and improving the quality of life in dogs with TCC.

In areas with moderate continental climate, increased average ambient temperature during the summer represents a stressogenic factor that affects the hypothalamo-pituitaryadrenocortical axis in mammals. Therefore, we wanted to examine the effects of 4 days of constant exposure to moderately elevated ambient temperature (35 ± 1oC) on the histomorphometric and immunofl uorescent characteristics, as well as on the hormonal secretion of pituitary corticotropes (ACTH) cells in adult male rats. In comparison with the controls kept at 20 ± 2oC, a signifi cant increase (p<0.05) of the absolute and relative pituitary weight (23.1% and 36.1%, respectively) was registered after exposure to heat. The localization, as well as the shape of the ACTH cells in the heat exposed group was not signifi cantly altered, but their immunopositivity was weaker. After 4 days of heat exposure, a weaker signal confi rmed the relative fl uorescence intensity of the ACTH cells (15.3%, p<0.05). In heat exposed rats, an increase of the cellular and nuclear volumes of immunolabelled ACTH cells and decrease of their volume density (6.9%, 14.3% and 20.0%, respectively; p<0.05) was registered. Observed histomorphometric and immunofl uorescent features of the pituitary ACTH cells were in accordance with the increased (p<0.05) value of plasma adrenocorticotropic hormone (ACTH) by 23.7% compared to the control rats. It can be concluded that the 4-day exposure to moderately elevated ambient temperature intensifi es pituitary ACTH secretion in adult male rats.

Failure of passive transfer (FPT) of immunoglobulins (IgG) is associated with increased morbidity and mortality of calves. In this study we evaluated the digital Brix refractometer usefulness for the assessment of FPT. A number of 16 colostrum samples from the first milking (2-3h post-partum) of Holstein-Friesian dairy heifers and 29 blood sera of 3-6 days old calves were analyzed with a digital Brix refractometer. Total proteins were determined by the biuret reaction. Colostral IgG were determined by radial immunodiffusion (RID), and colostral whey and blood serum γ globulin (composed almost entirely of IgG) were determined by agarose protein gel electrophoresis (APE) and densitometry. Colostral % Brix score was 25.5±3.4%. Concentrations of colostrum IgG and colostral whey γ globulin were 130±33 g/L and 100±24 g/L respectively. The concentration of total proteins in colostral whey was 134±30 g/L. The correlations between Brix values and the concentrations of IgG determined with RID and the concentrations of γ globulin determined with APE were positive and highly significant (P<0.001 and P<0.01). The concentration of serum proteins of new-born calves was 57.75±11.8 g/L, the concentration of γ globulin was 14.4±7.8 g/L, and the Brix score was 8.6±1.0%. FPT (serum γ globulin<10 g/L) was detected in 34.5% (10/29) calves. Brix score correlated with the concentration of blood serum γ globulins in all examined calves. The results have confirmed that digital Brix refractometry allows the producers to use this technique in order to estimate colostral and calf serum IgG, thereby monitoring both colostrum quality and success of passive transfer.

Chytridiomycosis, a disease in amphibians caused by Batrachochytrium dendrobatidis (Bd), has led to a population decline and extinction of frog species since 1996. The objective of this study was to determine the prevalence of and the need for establishing a surveillance system for monitoring chytridiomycosis in five national zoos and five free ranging protected areas across Thailand. A total of 492 skin swab samples were collected from live and dead animals and tested by polymerase chain reaction (PCR) for the presence of Bd. The positive specimens were confi rmed by amplicon sequencing and examined by histopathology and immunohistochemistry. From July 2009 to August 2012, the prevalence of Bd from frog skin samples was low (4.27%), monitored by PCR. All samples from live amphibians were negative. The positive cases were only from dead specimens (21/168, 12.5% dead samples) of two non-native captive species, poison dart frog (Dendrobates tinctorius) and tomato frog (Dyscophus antongilii) in one zoo. Immunohistochemistry and histopathology revealed the typical feature of fl ask-shaped zoosporangia and septate thalli, supporting the PCR-based evidence of chytridiomycosis in captive amphibians in Thailand, but detected Bd in only 7/21 of the PCR-positive samples. Although the introduction of a pathogenic strain of Bd from imported carriers might have a serious impact on the native amphibian populations in Thailand, chytridiomycosis has not currently been detected in native Thai amphibians. An active surveillance system is needed for close monitoring of the fungus crossing into Thai amphibian populations

This study examined the effects of chronic restraint stress (CRS: 2 hours × 14 days) on gene expression of three antioxidant enzymes, copper, zinc superoxide dismutase (SOD 1), manganese superoxide dismutase (SOD 2) and catalase (CAT) in the rat hippocampus. Also, we examined changes in the activities of SOD 1, SOD 2 and CAT in the hippocampus of chronically stressed rats. Investigated parameters were quantifi ed by using real-time RT-PCR, Western blot analysis and assay of enzymatic activity. We found that CRS did not change mRNA and protein levels of SOD 1 and CAT, but increased mRNA and protein levels of SOD 2. However, CRS treatment increased the enzyme activities of SOD 1, SOD 2 and CAT. Our fi ndings indicate that the increased activity of antioxidant enzymes (SOD 1, SOD 2 and CAT) in the hippocampus may be an important adaptive phenomenon of the antioxidant defense system in chronically stressed rats.

The goal of this study was to compare the results of serological and conventional bacteriological methods with the results obtained using multiplex PCR Bruce-ladder assay. Based on the obtained results, the usability of the assay was assessed in regard to rapid diagnosis of canine brucellosis directly from the samples of reproductive organs of infected dogs. Out of 225 blood samples, 33 (14.67%) had a positive agglutination reaction. In this study, out of the 225 assayed reproductive organs of dogs, B. canis was isolated from 3 samples (1.33%), while the PCR Bruce-ladder assay detected two positive samples (0.88%). Two dogs from which B. canis was isolated, an antibody titer of 1/200 was established in blood serums, and third dog from which B. canis was isolated was negative using the tube agglutination test. From a total of 225 assayed organ samples, a positive PCR reaction was obtained from two samples. The obtained results show that the tube agglutination method remains the first choice for the detection of dogs infected with B. canis. In addition, whenever possible, it is necessary to try isolation. It is desirable to attempt the detection of B. canis in tissues using PCR, but the results may not be treated as definitive and reliable.

This study describes a concurrent occurrence of lower respiratory aspergillosis and pneumoconiosis in a turkey flock. From one flock of 1000 turkeys, 4 clinically affected turkeys were examined pathologically and microbiologically. Clinically affected turkeys showed anorexia, lethargy, drooping of the wing, vomiting, dyspnea and open-beak breathing, or sudden dead. Gross lesions were observed in the air sacs and lungs which presented white to yellowish granulomas of varying size. Microscopic there was multifocal granulomatous airsacculitis and pneumonia. At the center of the granulomas, fungal hyphae were clearly highlighted in the lungs and air sacs by Gomori methenamie silver (GMS) staining. In the microbiological cultivation of the affected lungs and air sacs, Aspergillus fumigatus was isolated on Sabouraud dextrose agar. Furthermore, dark black dust accumulations were present in the cytoplasm of macrophages or interstitial tissue in the lungs and air sacs. There were also fibrosis and lymphohistiocytic cell infiltrations around the particles. Overall, this study could implicated that pneumoconiosis might be a predisposing factor in the development of respiratory aspergillosis in turkeys.

Equine glanders is a severe bacterial disease known since ancient times. Although eradicated in the most part of the world it is now considered re-emerging. Considering very scarce literature data, we used from formalin fixed collection material: nasal septum, lung and skin specimens from naturally infected horses. Tissues were grossly examined and photographed. Tissue samples, after standard processing, were stained with HE, Congo red and Groccot and microscopically examined. Gross changes include nodules and ulcers in the nasal mucosa with granulation and scarring, pyogranulomas in the lung tissue and nodules and ulcers of the skin. Microscopically marked inflammation of affected tissues with neutrophilic and mononuclear infiltration and fibrous tissue proliferation were seen. As a potent zoonotic agent it has been already used as a biological weapon in the past.

Feline injection-site sarcomas were initially described by Hendrick and Goldschmidt (1991). The link between vaccination site and sarcoma occurrence suggested the term of vaccine-associated sarcomas. Our paper describes an unusual feline injection-site fibrosarcoma with cerebellar metastasis. A 7-year-old female domestic short-hair cat was submitted to the Pathology Department (Faculty of Veterinary Medicine Cluj- Napoca, Romania) for necropsy. A subcutaneous tumor (relapse) in the interscapular region and multiple metastatic masses in the lungs, kidneys, subcutaneous tissue (scapular and thigh regions) and cerebellum were observed. Cytological, histological and immunohistochemical (for vimentin, desmin, multi-cytokeratin, α-smooth muscle actin, S100 and CD45) analyses from all tumors were performed. Cytological examination identifi ed highly pleomorphic spindle-shaped cells admixed with neoplastic multinucleated giant cells. Histologically, all neoplastic masses were composed of numerous spindle cells arranged into interlacing bundles. Extensive intratumoral areas of necrosis along with a neutrophilic infiltrate were also detected. A fibrillary material was present among neoplastic cells (green stained by Masson’s trichrome method), suggesting a collagenous structure. In all tumors assessed, immunohistochemistry showed an intense reaction only for vimentin in numerous neoplastic cells. Based on the history, gross, cytological, histological and immunohistochemical data, the final diagnosis was recurrent feline injection-site fibrosarcoma, with multiple metastases (including in the cerebellum). Overall, an uncommon case of feline injection-site fibrosarcoma has been reported. This is the first consistent record of an injection site sarcoma in a cat with cerebellar metastasis.

A 12 year old male Fox Terrier (case 1) and a one year old female Poodle (case 2) were presented with a history of dysorexia and vomiting, and case 2 also with distress associated with eating. Both dogs were diagnosed with acute pancreatitis based on clinical signs, blood analysis and ultrasonography which revealed the presence of a vegetable foreign body in the proximity of the pancreatic right lobe. Laparatomy enabled the gramineae awns to be removed which led to full resolution of clinical signs. We believe that these are the first reported cases of acute pancreatitis due to grass awns.