Volume 63 (2019): Issue 4 (December 2019)

High-throughput sequencing (HTS) identifies random viral fragments in environmental samples metagenomically. High reliability gains it broad application in virus evolution, host-virus interaction, and pathogenicity studies. Deep sequencing of field samples with content of host genetic material and bacteria often produces insufficient data for metagenomics and must be preceded by target enrichment. The main goal of the study was the evaluation of HTS for complete genome sequencing of field-case rabies viruses (RABVs).

The material was 23 RABVs isolated mainly from red foxes and one European bat lyssavirus-1 isolate propagated in neuroblastoma cells. Three methods of RNA isolation were tested for the direct metagenomics and RABV-enriched approaches. Deep sequencing was performed with a MiSeq sequencer (Illumina) and reagent v3 kit. Bioinformatics data were evaluated by Kraken and Centrifuge software and de novo assembly was done with metaSPAdes.

Testing RNA extraction procedures revealed the deep sequencing scope superiority of the combined TRIzol/column method. This HTS methodology made it possible to obtain complete genomes of all the RABV isolates collected in the field. Significantly greater rates of RABV genome coverages (over 5,900) were obtained with RABV enrichment. Direct metagenomic studies sequenced the full length of 6 out of 16 RABV isolates with a medium coverage between 1 and 71.

Direct metagenomics gives the most realistic illustration of the field sample microbiome, but with low coverage. For deep characterisation of viruses, e.g. for spatial and temporal phylogeography during outbreaks, target enrichment is recommended as it covers sequences much more completely.

Porcine circovirus type 3 (PCV3) is a newly discovered porcine circovirus. The molecular characteristics and genetic evolution of PCV3 in Xinjiang province, China still being unclear, the aim of the study was their elucidation.

A total of 393 clinical samples were collected from pigs on commercial farms in nine different regions of Xinjiang and phylogenetic analysis based on full-length Cap genes was performed.

The prevalence at farm level was 100%, while in all the tested samples it was 22.39%. Nine PCV3 strains were detected in Xinjiang province and they shared 98.9–99.3% nucleotide and 97.5–100.0% Cap gene amino acid sequence identities with other epidemic strains from China and abroad. Compared with other epidemic strains of PCV3, there were 26 base mutation sites in the Cap gene in the nine Xinjiang strains, resulting in the mutation of amino acids at positions 20, 24, 75, 77, 108, 111 and 206. Phylogenetic analysis showed that these strains can be divided into two different genetic groups, to the first of which five strains affiliated and divided between subgroups 1.1 and 1.2, and to the second of which the other four strains affiliated and similarly divided between subgroups 2.1 and 2.2.

PCV3 circulates widely among commercial pig farms in Xinjiang province, China, and displays obvious genetic diversity. The results provide epidemiological information useful for the prevention and control of PCV3 infection in the pig industry.

The study evaluated the patterns of local innate immune response in bronchoalveolar lavage fluid (BALF) cells of pigs infected with porcine reproductive and respiratory syndrome virus (PRRSV) alone or co-infected with swine influenza virus (SIV).

The study was performed on 26 seven-week-old pigs in three groups: PRRSV-infected (n = 11), PRRSV and SIV-infected (n = 11), and control (n = 4). BALF was collected post euthanasia at 2 and 4 dpi (three piglets per inoculated group) and at 21 dpi (all remaining pigs). Expression of IFN-α, IFN-γ, IL-1β, IL-6, IL-8, and IL-10 mRNA was quantified in BALF cells. PRRSV RNA was quantified in BALF samples using a commercial real-time RT-PCR kit.

The three cytokines IFN-α, IFN-γ, and IL-1β presented significant expression changes in all experimental pigs. In PRRSV-infected animals IL-8 also did, but in co-infected subjects IL-6 and IL-10 were the additional upregulated cytokines. The highest number of differentially expressed genes was observed at 4 dpi, and significant differences in cytokine gene expression did not occur between the experimental groups at any other time point. The mean PRRSV load in the BALF of PRRSV-infected pigs was higher than that of co-infected pigs at each time point, having statistical significance only at 4 dpi.

The results of the study indicate that infection with PRRSV alone as well as with SIV interferes with innate and adaptive immune response in the infected host. They also showed that co-infection demonstrates additive effects on IL-6 and IL-10 mRNA expression levels.

The study of histopathological changes caused by influenza A (H5N8) viral infection in bird species is essential for the understanding of their role in the spread of this highly infectious virus. However, there are few such studies under natural conditions in minor gallinaceous species. This article describes the pathomorphological findings in Colchis pheasants infected naturally with H5N8 during an epizootic outbreak in Bulgaria.

Samples of internal organs of 10 carcasses were collected for histopathological and immunohistochemical evaluation, virus isolation and identification, and nucleic acid detection.

Consistent macroscopic findings were lesions affecting the intestine, heart, lung, and pancreas. Congestion and mononuclear infiltrate were common findings in the small intestine, as were necrosis and lymphoid clusters in the lamina propria of the caeca. Congestion with small focal necrosis and gliosis with multifocal nonpurulent encephalitis were observed in the brain. Myocardial interstitial oedema and degenerative necrobiotic processes were also detected. Immunohistological analysis confirmed systemic infection and revealed influenza virus nucleoprotein in all analysed organs.

Variable necrosis was observed in the brain, liver, trachea, heart, small intestine, and caeca. Viral antigen was commonly found in the brain, heart, lung and trachea. Contact with migrating waterfowls was suspected as a reason for the outbreak.

Koi herpesvirus (KHV) has infected farmed common carp in Poland clinically and asymptomatically since 2004. The role of non-carp species as vectors of virus transmission is well known except for in the case of KHV. The aim was to better understand this virus’ infection and transmission pathways in common carp, looking at the potential vector role of fishes kept with them.

Eight species were experimentally infected with KHV by immersion in a suspension at 20°C ±1 and transferred to a tank after 45 minutes. Specimens were euthanised at intervals up to 56 days post infection (dpi) and tissue was examined for KHV DNA. Surviving infected fishes were introduced at intervals, each time into a separate tank, to naïve common carp for experimental infection. These were observed daily for symptoms, sacrificed along with controls after three months, and dissected to provide tissue samples. Also fish from 14 species collected from a farm with a history of KHV were sampled from 3 to 22 months after disease was confirmed. Organ sections from single fish were collected in a single tube.

Viral DNA was detected in tench and roach samples up to 49 dpi, but in three-spined stickleback and stone maroko samples only up to 14 dpi. Transmission of KHV to naïve carp occurred after cohabitation. KHV DNA was detected in three fish species three months after the farm outbreak.

We confirmed that grass and Prussian carp, tench, roach, and brown bullhead can transfer the virus to naïve common carp.

Cyprinid herpesvirus 3 (CyHV-3) is a virus infecting carp with disease symptoms of gill necrosis, fish discoloration, sunken eyes, and mortality reaching 90%. Several research groups have examined how to potentially abate the consequences of viral activity. Recently we showed that acyclovir inhibits CyHV-3 replication in vitro and in the present study we examined the anti-CyHV-3 activity of the tricyclic derivative of acyclovir 6-(4-MeOPh)-TACV (T-ACV), a fluorescent molecule known for higher lipophilicity than acyclovir, and therefore potentially better candidate for application in vivo.

CCB and KF1 cell lines were incubated with T-ACV at concentrations of 0, 66.67, and 133.33 μM for three days and toxicity examined with MTT and CV assays. To investigate the antiviral activity of T-ACV, the lines were infected with CyHV-3 or mock infected and incubated for three days with the drug at concentrations of 0 or 66.67 μM. The activity of T-ACV was evaluated by plaque assay and TaqMan qPCR.

T-ACV at a concentration of 66.67 μM displayed low toxicity and inhibited CyHV-3 activity by 13–29%, varying by cell line and method.

The low anti-CyHV-3 activity of T-ACV indicates that it would be reasonable to screen several tricyclic derivatives of acyclovir for such activity.

The Orobic goat is a hardy breed native to the Orobic Alps (Lombardy, northern Italy). The aim of the study was the assessment of gastrointestinal nematode (GIN) egg excretion in Alpine and Saanen (cosmopolite breeds) and Orobic grazing goats, after a strategic treatment with eprinomectin in late June.

Individual faecal samples from a mixed flock of cosmopolite and Orobic goats were collected and analysed by the FLOTAC double technique every three weeks from June to September.

Strongylida was the primary GIN infection observed in goats that grazed on Alpine pastures; a strategic treatment with eprinomectin led to a prolonged reduction of egg excretion during the whole study period. Egg excretion was also influenced by breed. Pluriparous Orobic does were able to control reinfection better than the pluriparous cosmopolite does. Regarding Nematodirus sp. eggs per gram of faeces (EPG), the autochthonous Orobic breed presented higher values than the cosmopolite breeds. However, cosmopolite goats presented higher EPG values of Strongyloides papillosus than their Orobic counterparts in August.

Further studies on genetic features of local autochthonous goats, such as the Orobic breed, are needed, since they could reveal peculiar characteristics of susceptibility, resistance or resilience to GIN infection, providing genetic resources for selection.

The aim of this study was to investigate Q fever seroprevalence in sheep and goats in the Marmara region. Q fever is a zoonotic disease caused by Coxiella burnetii. In ruminants, the disease causes reproductive disorders, premature births and stillbirths.

Blood samples of sheep and goats were collected from the Marmara region of Turkey and a commercial ELISA was used for detection of specific antibodies to C. burnetii. A total of 832 samples (627 from sheep and 205 from goats) obtained from 126 herds located in 110 villages in 63 municipalities across all 11 provinces were utilised.

Total seroprevalence was found to be 13.22%, while the proportion of seropositive herds was determined to be over threefold higher at 42.85%. The seroprevalence for sheep was found to be 14.19%, and for goats 10.24%. The herd seropositivity rate for sheep of 46.31% and for goats of 32.25% were also over threefold higher than the species-level seroprevalences. The provincial seroprevalence varied between 1.38% and 21.79%.

This study confirms the presence of C. burnetii in sheep and goat herds in the Marmara region and provides original seroprevalence data in hitherto uninvestigated provinces. The data gathered are beneficial for evaluation and elaboration of the seroprevalence of Q fever in sheep and goats in the Marmara region. Surveillance studies should be maintained, particularly in provinces with high seropositivity rates.

Riemerella anatipestifer (RA) infections can lead to high mortality in ducklings. Inactivated vaccines against RA are commercially available, but they fail to provide cross-protection against various serotypes. We have previously demonstrated that a subunit vaccine containing recombinant outer membrane protein A (rOmpA) antigen of serotype 2 formulated with CpG oligodeoxynucleotides (ODN) as the adjuvant was able to stimulate both humoral and cellular immunities.

In the present study, thirty healthy 7-day-old Pekin ducks were randomly assigned to three equal treatment groups: rOmpA-vaccinated, rOmpA + CpG-vaccinated, and control. Vaccine was injected intramuscularly and a booster dose of the same vaccine was given two weeks after primary immunisation. The long-term antibody response and cross-serotype reaction of this vaccine were evaluated in ducks.

Compared to ducks immunised with rOmpA alone, ducks immunised with rOmpA + CpG ODN had significantly (p < 0.05) increased serum antibody titre from two weeks until nine months after primary immunisation. In addition, expression of cytokines including interferon (IFN)-α, IFN-γ, interleukin (IL)-6, and IL-12 was significantly (p < 0.05) enhanced in PBMC of ducks immunised with rOmpA + CpG ODN two weeks after primary immunisation. Antibodies from ducks immunised with the rOmpA + CpG ODN vaccine could also detect RA serotypes 1 and 6 in Western blot analysis.

Combination of rOmpA and CpG ODN could be a feasible strategy for developing a subunit RA vaccine with long term and broader-ranging protection.

Parafasciolopsis fasciolaemorpha is a liver fluke typically parasitising moose in Central and Eastern Europe. The aim of our studies was to describe a case of fatal moose parafasciolopsosis, with special emphasis on the histopathological changes caused in the liver tissue by around 10,000 flukes.

A male moose, found dead in Polesie National Park, eastern Poland, was subjected to parasitological necropsy. Macroscopic and histopathological examination of the liver was performed.

Over 10,000 flukes identified as P. fasciolaemorpha were isolated from the liver parenchyma. Histopathological examination of the liver revealed the presence of multiple cavities, which were filled with flukes and cellular detritus and encysted with a layered capsule of connective tissue. Extensive liver fibrosis with signs of incomplete septal cirrhosis was also observed.

Parafasciolopsosis with accompanying diarrhoea was the most probable reason for the moose’s death. However, it is possible that most moose are able to survive extremely intensive P. fasciolaemorpha infection by formation of extensive fibrosis, which isolates flukes from the liver parenchyma and therefore retards the failure of the organ. To the best of our knowledge, this is the first histopathological description of changes in the liver of a moose infected with P. fasciolaemorpha.

This study aimed to characterise the effects of ketosis on milk yield and composition and digestive capacity in transition dairy cows.

Seven ketotic and seven healthy cows were housed in individual stalls for six days. Samples of plasma, milk, refused total mixed ration, and faeces were collected, and the blood biochemical parameters, milk yield and composition, dry matter intake, and faecal dry matter (FDM) production were determined.

Compared with healthy cows, the ketotic cows had significantly higher concentrations of milk fat and citrate, but lower levels of milk protein and lactose. The cows exhibited a need for acid detergent fibre in forage and better digestion of neutral detergent fibre, starch, crude protein, and phosphorus than healthy cows, but more fat and gross energy were excreted in their faeces. Ketotic cows had higher energy-corrected milk yields and lower FDM than healthy cows.

Lower feed intake coinciding with the requirement to maintain high milk production is considered to be the cause of ketosis in dairy cows. Ketotic cows exhibited lower dry matter fat digestion.

The use of growth promoters in animal husbandry to increase weight gain and efficiency of feed conversion into muscle has been banned in the European Union since 1988, and under Directive 96/23/EC, surveillance for anabolic steroid hormones is obligatory. The hormones present in animal tissues may be of endogenous origin or may result from illegal administration. Steps have been taken to determine selected steroids in the form of esters in the alternative matrix of animal hair. Their detection in biological material is direct proof of the illegal use of anabolics.

The procedure for the determination of steroid esters in animal hair, based on digestion, extraction, purification, and liquid chromatography with tandem mass spectrometry was validated under the current regulations. In total, 348 samples of animal hair were examined using this method.

Good recoveries and precision values (RSD) were obtained during validation. Decision limits (CCα) and detection capabilities (CCβ) were in the ranges of 2.57–4.18 μg kg⁻¹ and 4.38–7.12 μg kg⁻¹, respectively. The method met the criteria for confirmation techniques with respect to Commission Decision 2002/657/EC.

Testing for steroid esters in animal hair was introduced into the National Residue Control Programme in 2017. Steroid esters were not found in any hair samples above the CCα, which indicates that illegal use of anabolics was not confirmed.

Viral infections are the greatest threat to waterfowl and cause significant economic losses. Diagnosis and differentiation of three goose viruses is difficult in the field and often requires laboratory confirmation. Therefore, the aim of the study was to develop a triplex PCR and optimise its parameters for simultaneous detection of DNA of goose parvovirus (GPV), goose polyomavirus (GHPV), and goose circovirus (GoCV).

The DNA of viruses isolated from field cases from the National Veterinary Research Institute’s own collection was used for the study. The primer attachment temperature, the number of reaction cycles, and the Taq DNA polymerase and Mg²⁺ concentrations were optimised. The sensitivity and specificity of this triplex PCR was also determined.

Based on the obtained results, triplex PCR parameters were optimised for simultaneous detection of DNA of GPV, GHPV, and GoCV in one sample. The following PCR products of the expected size were obtained: GPV DNA of 806 bp, GoCV DNA of 571 bp, and GHPV DNA of 180 bp.

The developed triplex PCR method proved to be useful for simultaneous detection of infections with three waterfowl viruses and will be used in relevant laboratory diagnostics.