Study on the epidemiology of equine arteritis virus infection with different diagnostic techniques by investigating 96 cases of equine abortion in Hungary

The occurrence of equine arteritis virus (EAV) induced equine abortions was studied with different laboratory methods during a 3-year period. Tissue samples from 96 aborted equine foetuses or newborn foals were collected from 57 farms located in different parts of Hungary. Virus isolation, polymerase chain reaction (PCR), immunohistochemistry and serology were used for the detection of EAV infection. The overall seroprevalence of EAV infection in mares was 65%. EAV induced abortion was diagnosed in eight (8.3%) cases from six (10.5%) herds. Abortion was sporadic in all herds except for one, where epidemic abortion happened. Fetal serology in six (75%) cases, the virus isolation in one (12.5%) case whereas PCR in all of the four investigated cases were positive. The virus could be observed with immunohistochemistry in seven (87.5%) cases mostly in the spleen followed by other organs and the allantochorion. In conclusion, PCR and immunohistochemistry seem to be the most sensitive and useful tests for the diagnosis of EAV induced equine abortion.     

Advancement in vaccination against Newcastle disease: recombinant HVT NDV provides high clinical protection and reduces challenge virus shedding with the absence of vaccine reactions

Newcastle disease (ND) is a highly contagious disease of chickens causing significant economic losses worldwide. Due to the limitation in their efficacy, current vaccination strategies against ND need improvements. This study aimed to evaluate a new-generation ND vaccine for its efficacy in providing clinical protection and reducing virus shedding after challenge. Broiler chickens were vaccinated in ovo or subcutaneously at hatch with a turkey herpesvirus-based recombinant vaccine (rHVT) expressing a key protective antigen (F glycoprotein) of Newcastle disease virus (NDV). Groups of birds were challenged at 20, 27, and 40 days of age with a genotype V viscerotropic velogenic NDV strain. Protection was 57% and 81%, 100% and 95%, and 100% and 100% after the subsequent challenges in the in ovo and subcutaneously vaccinated chickens, respectively. Humoral immune response to vaccination could be detected from 3-4 wk of age. Challenge virus shedding was lower and gradually decreased over time in the vaccinated birds compared to the unvaccinated control chickens. In spite of the phylogenetic distance between the NDV F gene inserted into the vector vaccine and the challenge virus (genotype I and V, respectively), the rHVT NDV vaccine provided good clinical protection and significantly reduced challenge virus shedding.     

Molecular characterisation of the ORF68 region of equine herpesvirus-1 strains isolated from aborted fetuses in Hungary between 1977 and 2008

Equine herpesvirus-1 (EHV-1) can be classified into distinct groups by single nucleotide polymorphisms (SNPs) in their genomes. Only a few of these can be associated with a special attribute of the virus. Differences in the ORF30 region can determine the neuropathogenic potential, while by substitutions in the ORF68 region several strain groups can be made. In previous studies no connection was found between the neuropathogenic potential and the SNPs in ORF68, but the occurrence of members of distinct groups in different outbreaks can facilitate epidemiological investigations because the geographical distribution of a particular group is very often specific. The present study aimed at the molecular examination and grouping of 35 EHV-1 strains isolated from aborted equine fetuses in Hungary between 1977 and 2008. Genotyping was based on the comparison of nucleotide sequences of a polymorphic segment located in the ORF68 region, which had previously been found to be a useful tool for classification. After sequencing this region, the Hungarian EHV-1 isolates could be classified into seven groups. Only 23 of the 35 isolates belonged to the formerly described groups, while the SNPs of 12 isolates diverged, and four new groups could be set up. In addition, phylogenetic analysis was performed to compare the ORF68 sequences of the Hungarian strains with the sequences of isolates from Europe, America and Australia. The number of newly formed groups suggests that the further analysis of unknown EHV-1 isolates would involve the emergence of extended numbers of new groups, which can impair the usability of this grouping method.     

Adenovirus vaccination of pregnant ewes and studies on the colostral immunity of their lambs

Pregnant ewes were vaccinated 1 month before parturition with mono or bivalent adenovirus vaccines. Vaccination resulted in increased levels of homologous and heterologous antibody in ewes, with corresponding increases in passive immunity of lambs. Challenge of lambs with homologous or heterologous virus at 21 days of age was associated with significant resistance to development of lesions in lambs challenged with homologous virus, and partial resistance in those challenged with heterologous virus. Bivalent vaccines gave comparable protection to challenge with both virus types.     

Expression of immunoregulatory tumor necrosis factor-like molecule TL1A in chicken chondrocyte differentiation

The proinflammatory cytokine tumor necrosis factor (TNF) alpha is not encoded in the chicken genome. However, 1 member of the TNF family, TNF-like molecule 1A (TL1A), which is an important immunoregulatory protein, has recently been characterized in chickens. In this study, chicken TL1A (chTL1A) and 1 of its receptors, decoy receptor 3 (DcR3) were found to be expressed in developing bone of 14.5-day-old chicken embryos. Chicken chondrocytes were shown to express TL1A by polymerase chain reaction (PCR) amplification of cDNA and by immunohistochemical studies. Tissue expression was localized to the epiphyeal region of tubular bones, particularly cells of the epiphyseal plate, the outer chondrocytes of the cartilage-interfacing synovia, most of the synovial cells, and the stromal fibroblastic cells of the vascular channels of the femoral head. A tissue-specific developmental function of TL1A was supported by the presence of DcR3 in the embryonic connective tissue.     

Retrospective genome analysis of a live vaccine strain of bovine viral diarrhea virus

A live bovine viral diarrhea (BVDV) vaccine, marketed as a derivate of the Oregon C24V strain, was used between the end of the 1960s and the beginning of the 1990s in Central Europe. Since laboratory investigations of mucosal disease cases in vaccinated animals suggested recombinations between the vaccine and wild type variants of BVDV, and recombinational nucleotide sequences seemed distinct from BVDV Oregon C24V, the aim of the present retrospective study was to analyze the genomes of pre-registration (termed here BVDV-Xpre) and of marketed (BVDV-X) batches of the vaccine. The results of the complete genome analysis of BVDV-Xpre confirmed that the original virus strain used at the start of the vaccine production was Oregon C24V. Surprisingly, the analysis of the complete nucleotide sequence of the BVDV-X marketed vaccine revealed that this strain belongs to the BVDV 1b subgroup, with a 93.7% nucleotide sequence homology to BVDV reference strain Osloss. The homology to BVDV Oregon C24V was significantly lower (77.4%), and a thorough sequence scanning showed that the genome of BVDV-X had not derived from Oregon C24V. These data indicate the very likely scenario that a strain different to Oregon C24V was picked up during the in vitro or in vivo passages for vaccine development. Despite of the virus-switch, the BVDV-X vaccine continuously maintained its innocuity and efficacy, as proven by the regular quality testing data, and the presence of the foreign virus remained unnoticed over many years. The results of this work emphasize that the contamination of commercially available live vaccines with exogenous BVDV strains is a real risk factor, and a unequivocal analysis, including molecular methods, is needed to verify their authenticity.     

Prevalence of equine herpesvirus types 2 and 5 in horse populations by using type-specific PCR assays

Equineherpesvirustypes 2 and 5 (EHV-2andEHV-5)have a rather unclearpathogenicity and distribution within the equid population. In order to gain more information on the prevalence of these two viruses, type-specific PCR assays were developed to detect viral DNA in nasal specimens and in peripheral blood leukocytes (PBLs) of adult horses and foals from various regions of Europe, i.e. Sweden, Hungary and the United Kingdom. In adult horses, the prevalence of EHV-2 in PBLs was up to 68% in Sweden and 71% in the United Kingdom. EHV-2 DNA was detected in the PBLs from all the foals tested in all countries and most (93%) of the nasal specimens also yielded positive results. The prevalence of EHV-5 DNA in the PBLs of foals in Hungary was 15 and 24% in adult horses in the United Kingdom. This observation was among the very few reports of the presence of EHV-5 in horses. In summary, the specific PCR assays revealed important data on the occurrence and distribution of EHV-2 and EHV-5 in large horse populations. The findings indicated that infection with EHV-5 occurred later than EHV-2 in foals. This study may contribute to a better understanding of the etiological role of these gammaherpesviruses in equine diseases.     

Prevalence of antibodies against Toxoplasma gondii and Neospora caninum in Hungarian red foxes (Vulpes vulpes)

In the present study we have investigated the seroprevalence to the protozoan parasites Toxoplasma gondii and Neospora caninum in 337 red foxes (Vulpes vulpes) from 16 out of 19 counties in Hungary. The foxes were originally collected within a National vaccination program against rabies. Antibodies to T. gondii were detected in as many as 228 (68%) of the foxes using a commercial direct agglutination test (DAT). In an indirect iscom ELISA, five foxes (1.5%) were positive for antibodies against N. caninum. The high prevalence of foxes positive for T. gondii might be explained by the widespread occurrence of the parasite in the diet of foxes. As a contrast, latent infections of N. caninum among red foxes in Hungary are much less common.     

Effect of dietary electrolyte balance on production, immune response and mineral concentrations of the femur in broilers

The aim of this study was to assess the age-related effects of dietary electrolyte balance (DEB) on the performance, immune response (from day 0 to 42) and macromineral content of femur ash of broilers. The DEB values of the purchased commercial broiler diets were modified with the addition of NH4Cl or NaHCO3 to formulate the diets (DEB 325, 250, 175, 100, 25 and -50 mmol/kg) for this investigation. A total of 396 chickens were divided into 6 treatment groups and fed with the experimental diets for 6 weeks. During the first two weeks of life, DEB did not influence feed intake and body weight gain; however, by the 21st day of age DEB 175 and between 22 and 42 days of age DEB 250 mmol/kg gave significantly better results than the control. DEB did not affect the macromineral concentrations of bone ash. The immune response of broilers on low DEB (< 175 mmol/kg) was faster and more intensive than that of chickens on diets with medium or high DEB (> 175 mmol/kg). It can be concluded that the optimal DEB value required for the best body weight gain is significantly influenced by the age of broilers. Our results call attention to the discrepancy between the decreasing DEB level of commercial broiler diets and the age-related increase of 'electrolyte requirements' of broilers. It is also interesting that DEB may influence not only the performance but also the immune response of broilers.     

Molecular identification of Anaplasma marginale and rickettsial endosymbionts in blood-sucking flies (Diptera: Tabanidae, Muscidae) and hard ticks (Acari: Ixodidae)

In an attempt to identify the main vector and possible transmission routes of Anaplasma spp. in a region of Hungary with high prevalence of ovine and bovine anaplasmosis, DNA was extracted from 316 haematophagous arthropods (individually or in pools), including 4 species of ixodid ticks, 6 species of tabanid flies and hornflies. Midichloria-like organisms were identified with PCR (amplifying a portion of the 16S rRNA gene) and sequencing from Dermacentor marginatus and Ixodes ricinus. Significantly higher 16S positive D. marginatus individuals were collected in March than in April, suggesting earlier questing of ticks that contain rickettsial agents (thus endosymbionts). Midichloria- and Wolbachia-like organisms were also found in randomly caught horse flies (Tabanus bovinus and T. tergestinus) as well as hornflies (Haematobia irritans), respectively, with 97-99% similarity to sequences deposited in the GenBank. Although all ticks were negative in the Anaplasma spp.-specific msp4 PCR, four individuals of T. bovinus collected near to grazing cattle were positive for Anaplasma marginale. The results of the present study provide the first molecular evidence for the potential mechanical vector role of T. bovinus in the transmission of A. marginale, and broaden the range of haematophagous arthropods harbouring Midichloria-like bacteria, for the first time in any Dermacentor or Tabanus species.