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Oval, firm, cutaneous tumours with a rough, hairless, pigmented surface, exhibiting a moderately pronounced papillary structure were detected on the abdominal skin of two young red deer ( Cervus elaphus ). One animal was shot in Lower Austria in 2004, the other at a deer farm in Hungary in 2007. Histological examination of both samples classified the tumours as fibropapillomas, showing marked proliferation of fibroblasts and connective tissue, accompanied by hyperkeratosis, parakeratosis and acanthosis of the overlaying epidermis, and occasional foci of inflammation. The distribution of cytokeratin and vimentin was characterised in the lesion. The presence of papillomavirus (PV) antigen was demonstrated by immunohistochemistry in both cases. Papillomavirus-specific DNA was successfully amplified by PCR from one sample. The obtained partial nucleotide sequence of the L2 ORF exhibited the highest critical identity values with the homologous regions of Delta-papillomaviruses, especially the Roe deer papillomavirus (93%). Phylogenetic analysis of the partial L2 ORF sequence alignment of 10 papillomaviruses by both neighbour-joining and maximum parsimony method confirmed that the Red deer PV is very closely related to the Western roe deer papillomavirus (CcPV1).
Microsporidiosis (nosema disease) of the European honeybee ( Apis mellifera L.) is present in bee colonies worldwide. Until recently, Nosema apis had been regarded as the causative agent of the disease, which may have many negative effects on the colony and cause heavy economic losses in apicultures. Another microsporidium species, Nosema ceranae , was reported to infest the Asian honeybee ( Apis ceranae ), but both honeybee species are susceptible to both microsporidia. In the European honeybee N. ceranae was first detected in Spain in the year 2006. As it is difficult to distinguish N. ceranae and N. apis morphologically, a rapid and accurate assay has been developed to differentiate N. apis and N. ceranae based on polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) of the partial large subunit ribosomal RNA. The assay was tested on 38 Nosema -infested bee samples, which were collected from geographically distant Hungarian bee colonies representing all regions of the country. Only one sample contained N. apis , and in the other 37 samples N. ceranae was detected, which indicates the dominance of N. ceranae in Hungarian apiaries. This is the first report on the presence of N. ceranae in Hungary.
To demonstrate the relationship between tumour development and virus replication, eight specific-pathogen-free pullets of line P2 (Group P; 14 weeks old) and five adult chickens (Group A; 96 weeks old) were inoculated with virulent Marek’s disease virus (vMDV). Five chickens of Group P died or were euthanised due to moribund condition following the development of neoplastic lesions between days 53 and 91. On histopathological examination, these lesions were characterised by the proliferation of lymphoid cells of variable size. On analysis by polymerase chain reaction (PCR), the MDV meq gene was detected in Group P from day 21, and it was continuously identified in five chickens until they died or were euthanised. Abnormal signs and histopathological changes were not observed in chickens of Group A. The MDV meq gene was temporarily detected in some chickens of Group A, but it remained almost undetectable throughout the experimental period. In older chickens inoculated with vMDV, the onset of MD lymphoma development tended to be delayed as compared with the young chicks. The relationship between MD lymphoma development and virus replication in older chickens has been suggested. Our data might indicate the underlying existence of an age-related resistance to vMDV challenge.
Tick-borne haematozoans cause severe diseases in domestic animals, and some of them have zoonotic potential. The results of previous studies in Europe point to the important role of foxes in natural endemic cycles of several tick-borne pathogens, including protozoa. The aim of the present research was to acquire information on the prevalence and distribution of tick-borne protozoan parasites among foxes in Serbia. Legally hunted foxes from 14 localities throughout Serbia were analysed. Spleen samples were collected from 129 animals and tested for the presence of Babesia spp. and Hepatozoon spp. by PCR. In total, 79/129 (61.2%) of the tested foxes were positive for H. canis, while the presence of two Babesia species was confirmed: B. vulpes (37/129, 28.7%) and B. canis (1/129, 0.8%). Coinfection with B. vulpes and H. canis was present in 26/129 (20.2%) foxes and one animal (1/129, 0.8%) was co-infected by B. canis and H. canis. The results of this study indicate the important role of foxes in the epizootiology of B. vulpes and H. canis in the Republic of Serbia and stress the need for further research to clarify all elements of the enzootic cycle of the detected pathogens, including other reservoirs, vectors, and transmission routes.
We evaluated the performances of a newly designed real-time polymerase chain reaction (PCR) assay using TaqMan® probes to detect Salmonella Typhi. TaqMan® real-time PCR assays were performed by designed primers and probe based on the staG gene for detecting S. Typhi. The specificity of the assay was evaluated on 15 Salmonella serovars. The analytical specificity was evaluated on 20 non-Salmonella microorganisms. The analytical sensitivity was assessed using decreasing DNA quantities of S. Typhi ATCC 19430. Finally the detection capability of the TaqMan® real-time PCR assay on isolates recovered from patients with Salmonella infections was compared to the conventional PCR assay. Only S. Typhi strain had positive results when subjected to the assay using Typhi-specific real-time PCR. No amplification products were observed in real-time PCR with any of the non-Salmonella microorganisms tested. The TaqMan® real-time PCR was more sensitive than the conventional PCR. In conclusion, we found that the easy-to-use real-time PCR assays were faster than conventional PCR systems. The staG-based TaqMan® real-time PCR assay showed to be specific and sensitive method for the safe and rapid detection of the S. Typhi.
Kidney samples from chickens diagnosed with acute nephritis and gout were subjected to histological and electron microscopic examination. The investigations revealed cytoplasmic inclusion bodies in the tubular epithelial cells containing round virions of about 30 nm in diameter. Since avian nephritis virus (ANV) is known as a potential causative agent of the so-called baby chick nephropathy, an RT-PCR assay was developed for the molecular detection of ANV-specific nucleic acid in the specimen. The specificity of the assay was confirmed by direct sequencing of the amplicon obtained in the reaction. The nucleotide sequence of the PCR product showed 92% identity with the reference ANV sequence deposited in the GenBank database. After having been validated on some other suspicious cases of avian nephritis, the PCR method described in this study can be a potential tool for routine diagnostic examination of samples submitted from cases of gout and nephropathy in chickens.
The 16 somatic serotype type strains and 60 field isolates of Pasteurella multocida, representing various avian species and geographic regions in Hungary, were characterised by PCR-restriction fragment length polymorphism (PCR-RFLP) analysis of the ompH gene with DraI restriction endonuclease. The type strains yielded eight different (I-VIII) profiles. Strains whose PCR fragment was uncut by DraI (profile IV) could be differentiated with HindIII and PvuII restriction endonucleases. Five of the eight PCR-RFLP profiles (I, III, V, VI and VII) were detected among the field strains. Only a correlation of limited strength was found between the classical somatic serotypes and the PCR-RFLP profiles. However, the results confirmed that molecular methods could confidently distinguish serotype A:1 strains from the other serotypes. Moreover, the specific relationship between somatic serotypes and PCR-RFLP types among isolates from turkey raises the possibility of the existence of host-specific clones within the P. multocida population.
The aim of the present study was to assess the frequency of human herpesvirus 6A (HHV-6A) and human herpesvirus 6B (HHV-6B) infection during pregnancy. 100–100 blood samples were collected from pregnant and non-pregnant women, then nucleic acid was isolated from both plasma and leukocytes fraction. Nested and real-time PCR were used to detect and differentiate HHV-6A and HHV-6B DNA and to determine viral loads. Reverse transcription PCR (RT-PCR) for HHV-6 U79/80 mRNA was performed in order to reveal active HHV-6 replication.HHV-6A and HHV-6B active infections were not detected in blood samples neither from pregnant nor from non-pregnant women. Frequency of HHV-6B and HHV-6A latency did not show difference between the studied groups (15% vs. 16%). HHV-6B latency was dominant in both studied groups (14/15 and 15/16). Beside these results, in leukocyte samples of one pregnant and three non-pregnant women high HHV-6A viral loads (1.28 × 105 − 5.07 × 105 GEq / 1.5 × 106 leukocytes) were detected, and viral DNA was also found in plasma samples. Although RT-PCR did not confirm virus replication, but chromosomal integration was also not proved unequivocally, the number of 0.08–0.33 HHV-6 copy / 1 leukocyte refers more to postnatal infection.
To explore the diversity of some DNA viruses in reptiles, a continuous screening is going on, in our laboratory, by PCR using different consensus primers designed for the detection of the most conserved genome regions of adeno-, herpes- and parvoviruses. The test material consists essentially of dead specimens collected randomly from private pet owners, local pet shops, or at occasional exotic pet fairs. Here we report the partial sequence of a putative novel parvovirus obtained from a dead checkerboard worm lizard (Trogonophis wiegmanni) that had been wild-caught in its native habitat. An in-house-developed PCR with consensus primers targeting the gene of the parvoviral capsid protein was used. Other PCRs, intended to detect certain large DNA viruses, remained negative. The sequence of the PCR product indicated the presence of a hitherto unknown parvovirus in the internal organs of the checkerboard worm lizard. In phylogeny reconstruction, the novel sequence clustered with the members of the Dependovirus genus of the Parvoririnae subfamily, closest to the branch of snake adeno-associated virus. Since we could not demonstrate the presence of a potential helper virus, the putative amphisbaenian parvovirus supposedly can replicate autonomously. This is the first virus infection ever detected in any members of the suborder Amphisbaenia, and only the third parvoviral sequence obtained from any reptilian host.
The lymphatic organs of 50 pigs from a total of eight farms located at different sites in the epizootiological region of North Bačka 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 Bačka 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.
