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Abstract A rat with the mass located on the right lateral aspect of the head in the regio infraorbitalis, maxillaris, lateralis nasi dextra, et regio dorsalis nasi was presented for necropsy. Clinically, a severe form of dyspnea and occasional intermittent stridor were observed. Gross examination and histopathology revealed an abscess on the rat’s head. Hematological examination showed macrocytic regenerative hypochromic anemia. In addition, in blood smears stained with May Grunwald Giemsa, numerous spherocytes and anulocytes as well as reticulocytes indicating anemia were observed. Morphological changes found in the lymphocyte (1,1%) and neutrophil cytoplasm (20%) were most probably the result of adverse, toxic effects of various products of purulent inflammation in the head abscess. Key words: abscess, anemia, reticulocytes, spherocytes, anulocytes

T. Goletić, A. Gagić, V. Savić, E. Rešidbegović, Aida Kavazović, E. Šatrović, T. Harder, S. Prašović et al.

ABSTRACT Background: Towards preparation for a possible influenza pandemic, investigation of the molecular characteristics of the circulating avian H5N1 influenza virus strains is of crucial importance. These H5N1 viruses continue to spread, to infect animals and humans and to evolve and diversify providing so an ever-looming pandemic threat.Aim: To identify genetic structure and molecular biological characteristics of BiH's isolates of H5N1 HPAI as well as to assess the level of pathogenicity, phylogenetic origin and host- specificity of the isolates.Material and Methods: SPF embryonated chicken eggs were used for virus isolation. Viral RNA extracted using QIAamp viral RNA kit and manufacturer’s protocol (QIAGEN®) was used for PCR amplification. cDNA synthesis and PCR amplification of the coding region, using gene specific primer sets (primer sequences available on request), were carried out for all eight viral RNA segments separately. The Prism Big Dye Terminator v1.1 cycle sequencing kit (Applied Biosystems) was used and products were analyzed on an automatic ABI PRISM 3130 genetic analyzer (Applied Biosystems). Nucleotide sequences were analyzed using Bioedit software (v. 7.0.9.0) with an engine based on the ClustalW 1.4 algorithm. MEGA software (v. 4,0), using the neighbor joining tree inference analysis with the Tamura-Nei γ-model, was used to estimate phylogenies and calculate bootstrap values from the nucleotide sequences.Results: Full-length nucleotide sequences of the A/Cygnus olor/BIH/1/2006 (H5N1) strain were deposited in EMBL Nucleotide Sequence Database under accession nos. FN186008 to FN186014 and FM20943. The pathogenicity and host specificity of this strain, as polygenic traits, are determined in silico by the structure of its proteins, especially surface glycoproteins, HA and NA. Multibasic amino acid stretch PQGERRRKKR/GLF, marker of strains highly pathogenic to poultry, was present at the HA cleavage site of BiH strain. The RBS was typical for avian influenza viruses and contained Gln and Gly at positions 238 and 240 (H5 numbering) that is,226 and 228 according to H3 numbering with seven potential glycosylated sites but with increased binding to alpha2-6 sialoglycans thanks to substitutions, as follows, 110N, 171N, 171N, 172A, 205R and 251P. NA structure assigned this strain to the Z genotype, characterized also by the deletion of the five amino acid residues of the NS1 protein (positions 80-84). Amino acid residues, typical for the avian influenza viruses, were revealed in 40 out of 43 positions of M1, M2, NP, PA, PB2 and HA, determining the host range specificity. Phylogenetic analysis of the HA gene revealed that BiH isolates belonged to genetic clade 2.2., and presence of aspartic acid at the position of 403 of HA locate BiH isolates in 2.2.2. sub-clade.Conclusions: The BiH’s isolates were determined as HPAI virus with genes sequences closely related to A/Cygnus olor/Astrakhan/Ast05-2-10/2005 (H5N1). Three residues (M2 - 28V and 78K, NP - 33I), typical of human influenza viruses, were found, indicating a certain degree of intercurrent evolutionary adaptive changes in BiH isolates. Sequence comparison of HA and NA segments with relevant sequences in GenBank revealed that the BiH isolates and the ones from the southern Russia (Astrakhan region) group together phylogenetically, forming a monophyleticcluster in both genes indicating that these isolates have evolved from the same origin. Sequence derived phenotype markers of NA protein (E99, V129, D131, R136, H255 and Y256) as well as of M2 protein (26L, 27V, 30A, S31 and G34) showed that the isolates have an oseltamivir and amantadine sensitive genotype. 

A. Hodžić, A. Alić, S. Prašović, D. Otranto, G. Baneth, G. Duscher

SUMMARY Based on morphological and genetic characteristics, we describe a new species of Hepatozoon in the European wild cat (Felis silvestris silvestris), herein named Hepatozoon silvestris sp. nov. The study also provides the first data on the occurrence of H. felis in this wild felid. Hepatozoon meronts were observed in multiple cross-sections of different organs of four (44%) cats. Additionally, extracellular forms, resembling mature gamonts of Hepatozoon, were found in the spleen and myocardium of two cats. Furthermore, tissues of six animals (67%) were positive by PCR. Hepatozoon felis was identified infecting one cat (11%), whereas the 18S rRNA sequences of the remaining five cats (56%) were identical, but distinct from the sequences of H. felis. Phylogenetic analyses revealed that those sequences form a highly supported clade distant from other Hepatozoon spp. Future studies should include domestic cats from the areas where the wild cats positive for H. silvestris sp. nov. were found, in order to investigate their potential role to serve as intermediate hosts of this newly described species. Identification of its definitive host(s) and experimental transmission studies are required for elucidating the full life cycle of this parasite and the possible alternative routes of its transmission.

We here describe a case of Mycobacterium terrae infection in a cow in a small dairy household. At necropsy of the cow otherwise positive on routine annual tuberculin skin testing, severe multifocal granulomatous dermatitis and panniculitis, and moderate granulomatous (parasitic) colitis were observed. Histopathology revealed granulomatous dermatitis. Bacteria isolated from regional and thoracic lymph nodes, and skin lesions were identified by colony morphology, biochemical testing and molecular methods as M. terrae. Our findings confirm the difficulties that non-tuberculous mycobacteria can cause in vivo diagnosis of mycobacterial infections, especially in conjunction with parasitic infestations. Key words: Cattle, Mycobacterium terrae, Granulomatous dermatitis, Bosnia and Herzegovina

This paper describes an oral squamous cell carcinoma with metastases into the lung in a 24-years-old female rhesus macaque (Macaca mulatta). At necropsy, 3.5 cm ulcerative, white grey, irregular mass with necrosis and loss of the muscle, and osteolysis of the mandibular bone was observed on the mentum. Histopathologically, the mass consisted of multiple highly cellular lobules of oval to polygonal cells in the abundant connective tissue stroma, and with high mitotic index. There were multifocal pearl-like foci of keratinized cells. Neoplastic cells were PAS negative. Immunohistochemically, positive labeling for high molecular weight cytokeratine (CKhmw) and negative labeling for CA antigen were obtained. Key words: neoplasia, histopathology, immunohistochemistry, monkey

SUMMARY. Streptocara spp. infections are reported to cause gastritis, proventriculitis, esophagitis, and pharyngitis in various waterfowls, especially diving ducks. In the present paper, we describe severe fatal diphtheritic pharyngitis and esophagitis caused by Streptocara incognita in three female mute swans (Cygnus olor) in Bosnia and Herzegovina. Prior to death, the swans were showing signs of lethargy, anorexia, and reluctance to move. At necropsy, in all swans severe diphtheritic pharyngitis and esophagitis with deep, dark red hemorrhagic ulcerations were observed. Numerous thin, white, up to 1-cm-long nematodes, identified as S. incognita, were observed embedded in the pharyngeal and esophageal mucosa under the diphtheritic membranes. Histopathology revealed severe fibrinonecrotic inflammation with numerous cross-sections of the parasites. To the authors' knowledge, this is the first report of severe, fatal streptocariasis in mute swans. RESUMEN. Reporte de Caso—Faringitis y esofagitis verminosas fatales causadas por Streptocara incognita en cisnes vulgares (Cygnus olor). Se ha reportado que las infecciones por Streptocara spp. son causantes de gastritis, proventriculitis, esofagitis y faringitis en aves acuáticas, sobre todo en diversos patos buceadores. En el presente trabajo se describe un caso severo y fatal de faringitis y esofagitis diftéricas causado por Streptocara incognita en tres cisnes vulgares (Cygnus olor) hembras en Bosnia y Herzegovina. Antes de la muerte, los cisnes mostraron signos de letargia, anorexia y renuencia a moverse. Durante la necropsia, se observó en todos los cisnes faringitis y esofagitis diftéricas severas y con profundas ulceraciones de color rojo oscuro y hemorrágicas. Numerosos nemátodos delgados de color blanco y de hasta un centímetro de largo, identificados como S. incognita, se observaron incrustados en la mucosa de la faringe y del esófago por debajo de las membranas diftéricas. La histopatología reveló inflamación fibrinonecrótica severa con numerosas secciones transversales de parásitos. De acuerdo al conocimiento de los autores, este es el primer reporte de streptocariasis grave y mortal en cisnes vulgares.

H. Beširović, A. Alić, S. Špičić, Ž. Cvetnić, S. Prašović

Non-tuberculous Mycobacteria (NTM) are worldwide opportunist pathogens which can infect both humans and animals. Infections with these mycobacteria can have various clinical manifestations and may cause allergic sensitisation of the infected animals. Besides the potential zoonotic risk these infections in cattle can interfere with intradermal tuberculin test and hamper the in vivo diagnosis of bovine tuberculosis resulting in significant economic losses due to unnecessary restrictions and culling of reactor animals. Here we describe a case of Mycobacterium terrae infection in a cow on a small dairy household in Central Bosnia and Herzegovina. For the identification of the disease and causative agent comparative tuberculin skin test (TST), pathomorphology, microbiology and molecular methods were applied. At necropsy of a cow that was positive on annual routine tuberculin skin testing, severe multifocal granulomatous dermatitis and paniculitis were observed. Furthermore, moderate granulomatous (parasitic) colitis was also noted. Histopathology revealed granulomatous dermatitis. Ziehl-Neelsen staining was negative for acid fast bacteria. Regional and thoracic lymph nodes, and skin lesions were submitted for microbiology examinations. Isolated bacteria were identified by molecular methods as Mycobacterium terrae. Our findings confirms the difficulties that non- tuberculous mycobacteria can cause in the in vivo diagnosis of mycobacterial infections, especially in conjunction with parasitic infestations.

N. Obradovic, S. Kovačević, S. Prašović, D. Lalošević, A. Stancu

Bovine tuberculosis is a chronic disease of a zoonotic character that occurs in cattle and a wide range of domestic and wild animals. In this paper, bovine tuberculosis caused by M. caprae in six small household herds in Bosnia and Herzegovina was described. For the identifi cation of the disease and determination of the causative agent comparative tuberculin skin test (TST), pathomorphology, microbiology and molecular methods were applied. Twenty-eight cows from fi ve households from the Livno region (25/28 animals) and one household from the Vitez region (3/28 animals) were euthanized after being found positive by the comparative TST test. Eleven animals from fi ve households from the Livno region (9/11 animals) and one household from the Vitez region (2/11 animals) were subject to fi eld necropsy, and lesions consistent with tuberculosis were observed on the lymph nodes of the thoracic cavity and lungs in all carcasses. Histopathologic examination by hematoxylin and eosin staining confi rmed the presence of specifi c granulomatous lesions, while Ziehl- Neelsen staining demonstrated the presence of very few acid fast bacteria. Mediastinal lymph nodes from seven necropsied animals (two animals from the Vitez region and fi ve animals from fi ve fl ocks from the Livno region) were submitted for bacteriology. Acid fast bacteria from fi ve out of the seven submitted samples from one animal from the Vitez region and four animals from four households from the Livno region were isolated. All isolates were identifi ed as M. caprae. By MIRU typing we found two different M. caprae genotypes, unique to Bosnia and Herzegovina. Our fi ndings represent the fi rst evidence of bovine tuberculosis caused by M. caprae in Bosnia and Herzegovina.

D. Lalošević, S. Prašović, V. Lalosevic, Verica Simin, I. Capo, N. Obradovic, M. Božić, S. Putić et al.

Summary An increase of the red fox (Vulpes vulpes) population has been documented in Europe after the success of the oral vaccination against rabies, and this trend is now becoming evident in Serbia as well. The role of the red fox as reservoir for zoonotic parasites becomes very important in suburban areas, especially for Echinococcus multilocularis and other parasitic diseases. Foxes (N = 90) were collected during the hunt from every part of Vojvodina, northern province of Serbia, over a period from winter 2008 to January 2012. At the necropsy, carcasses were opened and the trachea from the larynx to the bifurcation was collected and preserved in 30% ethanol. Tracheas were opened on the anterior side by scissors and parasites were collected under a stereomicroscope and wet-mounted in glicerine-ethanol or lactophenol. Nasal cavity was opened through the palate, and the nasal mucosa was scrapped off and conserved in 30% ethanol. Pulmonary parenchyma are compressed and examined as wet-mount slides. Sections of lungs and trachea were also histologically examined. Faecal samples and urine were collected and then tested using the glycerin flotation method and wet-mount slides, respectively. Collected parasites were counted, and measured in the ―Image J‖ free programme. The estimated prevalence of the infection with Capillaria aerophila, Capillaria boehmi and Crenosoma vulpis was 84.3%, 90%, and 13.15%, respectively. Zoonotic potential of pulmonary capillariasis for humans and pet animals and crenosomosis for domestic and stray dogs are discussed. Identification of pulmonary capillariasis in one human patient increased interest for this zoonosis in Serbia.

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