Zoltán Demeter

Genetic diversity of Hungarian canine distemper virus strains

Abstract To achieve proper diagnosis of dogs based on acute clinical symptoms and poorly preserved field samples taken from animals that died due to canine distemper (CD), a new differential diagnostic test has been developed based on polymerase chain reaction (PCR). In this study, more than 150 samples collected from dogs showing respiratory, gastrointestinal and neurological signs suggesting canine distemper virus (CDV) infection were examined. The samples consisted of urine, blood and nasal swabs collected from clinically ill patients, sent to our laboratory by clinicians from various veterinary clinics throughout Hungary. Various organs collected during the necropsy of dogs with pathological changes that suggested CDV infection were also included. Three distinct PCRs were designed. For diagnostic purposes, a primer pair specific to a 409 bases-long segment within the conservative part of the large polymerase region (L) of the CDV genome was designed. Using this test, out of the 150 analyzed samples, 46 (30.66%) proved to be positive for CDV, indicating that CDV still represents a high risk to the canine population in Hungary. For the phylogenetical analysis, a primer pair that completely encompasses the hemagglutinin (H) gene of the CDV genome was designed. The amplicons of this region were sequenced in both directions using the appropriate primers. Our results indicate that several different CDV genotypes are currently present in Hungary. Nine of the analyzed Hungarian strains turned out to belong to the so-called Arctic group of CDVs, and were most closely related to non-European strains from North America, China and Greenland, as well as to the phocine distemper virus 2 (PDV-2) isolated from Baikal seals (Phoca sibirica). One of the Hungarian strains showed high similarity to other European isolates from Denmark, Germany, Italy and Turkey, as well as to other isolates from geographically more distant regions, such as the USA. Three Hungarian strains seem to join a new cluster that is formed by only a couple of strains, one isolated from a mink in Denmark, and another from a dog in North America. Using a third set of primers, a restriction fragment length polymorphism (RFLP) assay has also been designed for the fast and reliable differentiation of the wild-type CDVs from the vaccine strains.

Naturally occurring parvoviral infection in Hungarian broiler flocks

The major enteric disease (ED) complex in broiler chickens is runting–stunting syndrome and in turkey broilers is poult enteritis mortality syndrome. Viruses from numerous families have been identified in the intestinal tracts of poultry with ED, such as Astroviridae, Coronaviridae, Reoviridae, Rotaviridae, and Parvoviridae. The objective of the present study was to directly demonstrate the presence of the scarcely known chicken parvovirus (ChPV) and turkey parvovirus (TuPV) in Hungarian flocks experiencing clinical signs of ED. ChPV and TuPV infection were demonstrated in 15 chicken flocks and two turkey flocks, in intestinal samples collected between 2008 and 2010. The histopathological investigation revealed enteritis in the duodenum and jejunum, and atrophy of the lymphoid organs. Indirect immunohistochemistry (IHC) suggested the intestinal epithelium of chickens and turkeys as a potential replication site of the virus, similarly to other parvoviruses, while in case of the turkey samples IHC positivity was also observed in the bursa of Fabricius, liver and pancreas. However, no direct connection could be established between the presence of the pathogen in the above-mentioned tissues and the histopathological changes observed in the investigated flocks. The phylogenetic analysis performed on the partial nucleic acid sequence of the NS1 gene revealed an evident clustering tendency of the ChPV and TuPV strains, but also highlighted the potential reciprocal role of these two species in the epidemiology of these viruses. The role of the ChPV and TuPV in the ED is far from understood, but the results of the present study emphasize the fact that in certain, still not fully elucidated conditions, ChPV and TuPV may participate in the emergence of ED in chicken flocks, as suggested by previous experimental infections.

European Surveillance for Pantropic Canine Coronavirus

ABSTRACT Highly virulent pantropic canine coronavirus (CCoV) strains belonging to subtype IIa were recently identified in dogs. To assess the distribution of such strains in Europe, tissue samples were collected from 354 dogs that had died after displaying systemic disease in France (n = 92), Hungary (n = 75), Italy (n = 69), Greece (n = 87), The Netherlands (n = 27), Belgium (n = 4), and Bulgaria (n = 1). A total of 124 animals tested positive for CCoV, with 33 of them displaying the virus in extraintestinal tissues. Twenty-four CCoV strains (19.35% of the CCoV-positive dogs) detected in internal organs were characterized as subtype IIa and consequently assumed to be pantropic CCoVs. Sequence and phylogenetic analyses of the 5′ end of the spike protein gene showed that pantropic CCoV strains are closely related to each other, with the exception of two divergent French viruses that clustered with enteric strains.

High Prevalence of Turkey Parvovirus in Turkey Flocks from Hungary Experiencing Enteric Disease Syndromes

SUMMARY. Samples collected in 2008 and 2009, from 49 turkey flocks of 6 to 43 days in age and presenting clinical signs of enteric disease and high mortality, were tested by polymerase chain reaction and reverse transcription–polymerase chain reaction for the presence of viruses currently associated with enteric disease (ED) syndromes: astrovirus, reovirus, rotavirus, coronavirus, adenovirus, and parvovirus. Turkey astroviruses were found in 83.67% of the cases and turkey astrovirus 2 (TAst-2) in 26.53%. The investigations directly demonstrated the high prevalence of turkey parvovirus (TuPV) in 23 flocks (46.9%) experiencing signs of ED, making this pathogen the second most identified after astroviruses. Phylogenetic analysis on a 527 base pair-long region from the NS1 gene revealed two main clusters, a chicken parvovirus (ChPV) and a TuPV group, but also the presence of a divergent branch of tentatively named “TuPV-like ChPV” strains. The 23 Hungarian TuPV strains were separately positioned in two groups from the American origin sequences in the TuPV cluster. An AvaII-based restriction fragment length polymorphism assay has also been developed for the quick differentiation of TuPV, ChPV, and divergent TuPV-like ChPV strains. As most detected enteric viruses have been directly demonstrated in healthy turkey flocks as well, the epidemiology of this disease complex remains unclear, suggesting that a certain combination of pathogens, environmental factors, or both are necessary for the development of clinical signs.

Controversial results of the genetic analysis of a canine distemper vaccine strain

Canine distemper (CD) is a highly contagious, often fatal, multisystemic viral disease of receptive carnivores. The presence of a PsiI cleavage site on a specific location of the hemagglutinin (H) gene was found to be a hallmark of vaccine strains, thus, a previously published restriction fragment length polymorphism (RFLP) test using PsiI theoretically allows the distinction between all currently used vaccine strains and virulent field strains. The RFLP test was carried out on all brands of CD vaccines available in Hungary. The present work describes the extensive sequencing and phylogenetic study of the strain present in Vanguard (Pfizer Animal Health) vaccines, which following the PsiI based RFLP test reacted as a wild-type strain. Based on the product description provided by the manufacturer, all batches should have contained a virus strain (Snyder Hill) belonging to the group of vaccine strains (America-1). Extensive genetic analysis involving the full nucleic acid sequence of four other genes (N, M, P and F) of the CDV genome revealed that the incriminated virus strain showed a higher level of genetic identity to wild-type strains from the America-2 group than to any of the strains belonging to America-1 group, therefore the vaccine does not contain the virus strain stated by the manufacturer in its product description and has not been containing it since at least 1992.

POXVIRUS INFECTION IN HUNGARIAN GREAT TITS (PARUS MAJOR): CASE REPORT

From a total of 1819 great tits (Parus major) ringed in 2007 in Pilis Mountains, Hungary, 15 birds presented nodular proliferative lesions on different areas of the head and eyelids, suggesting a poxvirus infection. Three birds were submitted for analysis. The presence of avipoxvirus infection was confirmed by histopathology, electron microscopy (EM) and a polymerase chain reaction (PCR) based technique. Nucleotide sequence analysis of a 428 base pairs (bp) fragment of the viral 4b core protein gene revealed 100% identity between two of the Hungarian isolates (PM9 HUN, PM33 HUN) and two great tit poxvirus strains isolated in Norway in 1973 (GTV A256, GTV A311). The third Hungarian isolate (PM34 HUN) was more closely related to a different Norwegian isolate (GTVA310) than to the Hungarian isolates. The nucleotide sequence analysis of a shorter fragment of the viral 4b core protein (227 bp) gene revealed 100% identity between the Hungarian isolates, the same Norwegian isolates and a great tit poxvirus strain detected in Austria in 2007.

Misleading results of the MboII-based identification of type 2a canine parvovirus strains from Hungary reacting as type 2c strains

Type 2 canine parvovirus (CPV2) infection is one of the most frequent causes of death in the young, susceptible canine populations worldwide. Since its emergence in the 1970s, several variants have been described. In the present study the authors describe the genetic analysis of 24 Hungarian CPV2 strains collected from 2004 to 2008. Surprisingly, the genetic and phylogenetic investigations of all these strains revealed that all of them were type 2a CPVs. On the other hand, the genetic analysis provided substantial evidence to demonstrate that due to a seemingly constant point mutation present in most of the Hungarian CPV2a strains, a previously described MboII-based rapid identification of CPV2c strains unfortunately cannot be reliably used any more.

Feline parvovirus infection in an Asian palm civet (Paradoxurus hermaphroditus).

FELINE panleukopenia virus (FPV) is a single-stranded DNA virus belonging to the family Parvoviridae ([Truyen and others 1995][1]). FPV is closely related to other parvoviruses, such as mink enteritis virus, raccoon parvovirus (RPV), canine parvovirus (CPV) and blue fox parvovirus (BFPV) ([Parrish

Novel mastadenovirus infection and clinical disease in a pygmy marmoset (Callithrix [Cebuella] pygmaea).

We describe the detection and successful isolation of a novel mastadenovirus from a pygmy marmoset (Callithrix [Cebuella] pygmaea) that died following an episode of severe respiratory signs. Pathologic/histopathologic examination revealed hydrothorax and catarrhal bronchopneumonia with pronounced desquamation of the bronchiolar epithelial cells, while in other airways a marked hyperplasia of the epithelial lining and numerous giant cells could be observed. We obtained partial sequence data from the adenoviral DNA-dependent DNA-polymerase gene of the isolated strain and analyses of this region showed the highest level of identity to the recently described bat adenoviruses (strains PPV1 and TJM) and the type 2 canine adenovirus. Similar results were gained by phylogenetic calculations indicating that this novel marmoset adenovirus is only distantly related to reference Old and New World primate adenoviruses and formed a monophyletic group with bat and canine adenoviruses and the equine adenovirus 1. Even though the source of the infection remained unknown, our results could imply the possibility of a cross-species transmission of the virus from an anonymous host to the pygmy marmoset.

Postmortem small babesia-like morphology of Babesia canis — Short communication

Here we report a case of canine babesiosis with unusual morphology of the causative agent. A male, seven-week-old Labrador retriever puppy, exhibiting severe anaemia and haemoglobinuria, was presented at the Clinic of Internal Medicine in February 2011. The puppy was euthanised. The most relevant pathological changes were icterus, severe splenomegaly, generalised lymphadenopathy and haemoglobin nephrosis. Samples were collected from various organs for histology within one hour post mortem. Impression smears were also prepared from the spleen after overnight storage at 4 °C. Tissue sections and smears showed the presence of multiple, coccoid intraerythrocytic bodies that measured 1-2 μm and resembled small babesiae. No large piroplasms were seen. DNA was extracted from the spleen, and a conventional PCR was performed for the amplification of a 450-bp region of the 18S rRNA gene of piroplasms. The causative agent was identified as Babesia canis canis, with 99% sequence identity to other European isolates. Sequence identity to B. gibsoni was only 91%. This is the first account to verify that the morphology of the large canine piroplasm, B. canis, can be uniformly small babesia-like post mortem or following the storage of tissue samples.