Effect of low-pathogenicity influenza virus H3N8 infection on Mycoplasma gallisepticum infection of chickens.

Mycoplasma infection is still very common in chicken and turkey flocks. Several low-pathogenicity avian influenza (LPAI) viruses are circulating in wild birds that can be easily transmitted to poultry flocks. However, the effect of LPAI on mycoplasma infection is not well understood. The aim of the present study was to investigate the infection of LPAI virus H3N8 (A/mallard/Hungary/19616/07) in chickens challenged with Mycoplasma gallisepticum. Two groups of chickens were aerosol challenged with M. gallisepticum. Later one of these groups and one mycoplasma-free group were aerosol challenged with the LPAI H3N8 virus. The birds were observed for clinical signs for 8 days, then euthanized, and examined for the presence of M. gallisepticum in the trachea, lung, air sac, liver, spleen, kidney and heart, and for developing anti-mycoplasma and anti-viral antibodies. The LPAI H3N8 virus did not cause any clinical signs but M. gallisepticum infection caused clinical signs, reduction of body weight gain and colonization of the inner organs. These parameters were more severe in the birds co-infected with M. gallisepticum and LPAI H3N8 virus than in the group challenged with M. gallisepticum alone. In addition, in the birds infected with both M. gallisepticum and LPAI H3N8 virus, the anti-mycoplasma antibody response was reduced significantly when compared with the group challenged with M. gallisepticum alone. Co-infection with LPAI H3N8 virus thus enhanced pathogenesis of M. gallisepticum infection significantly.

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.

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.

Microbiological and pathological examination of fatal calf pneumonia cases induced by bacterial and viral respiratory pathogens.

The infectious origin of fatal cases of calf pneumonia was studied in 48 calves from 27 different herds on postmortem examination. Lung tissue samples were examined by pathological, histological, bacterial culture, virus isolation and immunohistochemical methods for the detection of viral and bacterial infections. Pneumonia was diagnosed in 47/48 cases and infectious agents were found in 40/47 (85%) of those cases. The presence of multiple respiratory pathogens in 23/40 (57.5%) cases indicated the complex origin of fatal calf pneumonia. The most important respiratory pathogens were Mannheimia-Pasteurella in 36/40 (90%) cases, followed by Arcanobacterium pyogenes in 16/40 (40%) cases, Mycoplasma bovis in 12/40 (30%) cases, and bovine respiratory syncytial virus in 4/40 (10%) cases. Histophilus somni was detected in 2/40 (5%) cases, while bovine herpesvirus-1, bovine viral diarrhoea virus and parainfluenza virus-3 were each found in 1/40 (2.5%) case. Mastadenovirus, bovine coronavirus, influenza A virus or Chlamydiaceae were not detected.

Development of a new primer-probe energy transfer method for the differentiation of neuropathogenic and non-neuropathogenic strains of equine herpesvirus-1.

Equine herpesvirus-1 (EHV-1) is a major pathogen of horses with worldwide distribution that can cause various clinical signs ranged from mild respiratory disease to neurological symptoms. Comparison of neuropathogenic and non-neuropathogenic EHV-1 strains revealed that a single non-synonymous nucleotide substitution (A/G2254) in the ORF30 region is associated with the altered functions of the viral DNA polymerase and therefore the neuropathogenicity of EHV-1 virus strains. The aim of the present study was the development of a new differentiation method of this particular single nucleotide polymorphism on the basis of the primer-probe energy transfer (PriProET) technique that has been successfully applied for the detection and classification of various DNA and RNA viruses. The results of melting temperature analysis showed an exact correlation with the sequence variations of the targeted region of ORF30, and the two genotypes (A/G2254) could be easily identified by the different peaks of melting temperatures. The new method is simple, fast, specific and robust as well as more flexible than the previous tests.

Immunogenicity of poxvirus vector avian influenza vaccines in Muscovy and Pekin ducks.

Fowlpox (FP)-vectored avian influenza (FP-AI) vaccines are used in 1-day-old chickens, but they have also recently been shown to be immunogenic in ducks. The objectives of this work were 1) to evaluate safety and to compare the immunogenicity in ducks of three poxvirus vectors (fowlpox, canarypox, and vaccinia) expressing the same hemagglutinin gene from an H5N1 isolate, 2) to study the effect of the dose of the FP-AI and the presence of an adjuvant in 1-day-old Pekin ducks on antibody response after a boost with inactivated vaccine given 3 wk later, and 3) to confirm the immunogenicity of such a heterologous prime-boost vaccination scheme in 1-day-old Muscovy ducks. Immunogenicity induced by the three poxvirus vectors was comparable, and the FP vector was selected for the other studies. As published previously, there was a strong dose effect of the FP-AI priming on the hemagglutination inhibition (HI) titers induced after the boost with an inactivated vaccine. In contrast, the two tested adjuvants did not significantly increase the activity of FP-AI priming. The heterologous prime-boost regimen given to both Muscovy and Pekin ducklings at 1 and 14 or 21 days of age, respectively, was shown to be at least as immunogenic as two administrations of inactivated vaccines given at 2 and 5 wk of age. However, HI antibody titers were of short duration for both vaccine schemes, and their persistence was heterogeneous among individual birds.

A survey of equine abortion and perinatal foal losses in Hungary during a three-year period (1998-2000).

Cases of equine abortion and perinatal foal losses were investigated in Hungary during a three-year period (1998-2000). Samples from aborted equine fetuses and newborn foals (total n = 96) were examined using bacteriological, virological, pathological, immunohistochemical (IHC), molecular biological and serological methods. The cause of abortion and perinatal foal loss was identified in 67/96 cases (70%); viral infection was found in 22 (23%), viral and bacterial coinfection in 1 (1%), bacterial infection in 23 (24%), protozoan infection in 1 (1%) and fungal infection in 2 cases (2%). Morphological lesions suggestive of infection were recorded in 2 (2%) and non-infectious causes in 16 cases (17%).

First description of swine Torque teno virus (TTV) and detection of a new genogroup in Hungary: short communication.

Torque teno virus (TTV) belongs to the floating genus of Anellovirus. It was discovered in a human patient, and later it was also found in animals including pigs. The aim of this study was to investigate the presence and estimate the prevalence of swine TTV in Hungarian pig herds for the first time, and to characterise the viruses found. Serum samples of 82 adult swine from 13 piggeries and 44 weaned pigs from one large herd were tested by PCR for the presence of TTV DNA. Viral DNA was found in 30% of the adult swine and 73% of the weaned pigs tested. Liver and intestine of weaned pigs were also tested and found to be infected at a lower rate. The TTV sequences found in sera and intestines were similar and could be clustered as swine genogroup 1. However, the sequences derived from one liver were remarkably different from all other known genogroups and seemed to represent a new genogroup.

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.

A 45-nucleotide insertion in the NS2 gene is responsible for the cytopathogenicity of a bovine viral diarrhoea virus strain.

Cytopathogenicity (cp) markers have recently been investigated in the genomes of field isolates of bovine viral diarrhoea virus (BVDV). Most of the isolates originated from mucosal disease (MD) cases observed after vaccination with a live attenuated vaccine, termed here BVDV-X. The NS2-3 genes of these isolates and of the vaccine proved to be identical, including a 45-nucleotide (nt) viral insertion at nt position 4355. The insertion originated from the NS4B/5A junction region of the BVDV genome. Interestingly, in BVDV strain CP7 a 27-nt insertion originating from the NS2 is located exactly at the same position. Complete genome analysis of BVDV-X did not reveal further potential cp markers. Furthermore, expression studies indicated that the insertion promotes NS2-3 cleavage. In order to examine the possible role of the 45-nt insertion in viral cytopathogenicity in details, a full-length infectious cDNA clone of BVDV-X was generated, and bovine turbinate (BT) cells were transfected with RNA transcribed from the clone. The recovered virus, termed BVDV-XR, showed slight retardation in growth in comparison with the original BVDV-X, and induced cytopathogenic effect (CPE). Since the natural non-cytopathogenic (ncp) counterpart of the vaccine virus was not available, an insertion-negative mutant cDNA clone was generated from BVDV-XR by PCR-directed mutagenesis. The recovered virus, termed BVDV-XR-INS-, showed the same growth characteristics as its cp counterpart BVDV-XR, but caused no CPE. These findings provide a direct proof that the 45-nt insertion at position 4355 has a basic role in the cytopathogenic character of this BVDV strain.

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.

Viral sequence insertions and a novel cellular insertion in the NS2 gene of cytopathic isolates of bovine viral diarrhea virus as potential cytopathogenicity markers.

Cytopathogenicity of bovine viral diarrhea virus (BVDV) has been shown to correlate with the presence of insertions of cellular sequences, duplication of viral sequences with or without insertions, deletions, and point mutations in the genomes of cytopathogenic (cp) strains. In the present study we have investigated cytopathogenicity markers in the genomes of six cp BVDV isolates. The viruses were selected as representatives of various forms of BVDV infection, in some cases presumably induced by vaccination with a live attenuated vaccine. The complete NS2-3 coding region of the six isolates and of the vaccine virus were amplified by reverse transcription-polymerase chain reaction (RT-PCR) and sequenced. In the genomes of four isolates (H6379, H6712, H8427 and H-BVD MD) and of the vaccine virus, a 45-nucleotide viral insertion was found at nucleotide position 4355, encompassing nucleotides 8402-8446, that encode 15 amino acids of the NS4B/NS5A junction region in a normal BVDV genome. Isolate H3887 had a 21-nucleotide insertion of non-viral origin, also located at nucleotide position 4355. This insertion has a high homology with a gene coding for murine interferon-induced guanylate-binding protein 1, and represents the first non-viral insertion identified at this position of the NS2 coding region. Isolate H3142 carries a 42-nucleotide insertion at position 4361, identical to a part of the NS5B gene mapping to position 11078-11119. Additionally, this isolate also has a deletion of three nucleotides (positions 4448-4450). The role of the 45-nucleotide insertion in expression of NS3 was investigated using the vaccine virus. The NS2-3 gene of this virus, and that of a generated insertion-negative variant were cloned in the mammalian expression vector pCI, and expressed in bovine turbinate cells. Western blot analysis revealed that the insertion contributed to a partial cleavage of NS2-3 generating NS3, the marker protein for cytopathogenicity in BVDV. The genome rearrangements found in these isolates occurred preferentially at position 4355, suggesting that this part of the genome could represent a potential hot spot for recombination events in ncp BVDV. The molecular mechanism underlying this phenomenon, however, remains to be elucidated.

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.