Aspects of bovine herpesvirus-1 infection in dairy and beef herds in the Republic of Ireland

Background

Infection with bovine herpesvirus-1 (BHV-1) causes a wide range of disease manifestations, including respiratory disease and abortion, with world-wide distribution. The primary objective of the present study was to describe aspects of BHV-1 infection and control on Irish farms, including herd-level seroprevalence (based on pooled sera) and vaccine usage.

Methods

The characteristics of a diagnostic indirect BHV-1 antibody ELISA test when used on serum pools were evaluated using laboratory replicates for use in the seroprevalence study. The output from this indirect ELISA was expressed as a percentage positivity (PP) value. A proposed cut off (PCO) PP was applied in a cross-sectional study of a stratified random sample of 1,175 Irish dairy and beef cattle herds in 2009, using serum pools, to estimate herd seroprevalence. The study was observational, based primarily on the analysis of existing samples, and only aggregated results were reported. For these reasons, ethical approval was not required. Bulk milk samples from a subset of 111 dairy herds were analysed using the same ELISA. Information regarding vaccine usage was determined in a telephone survey.

Results

A PCO PP of 7.88% was determined to give 97.1% sensitivity and 100% specificity relative to the use of the ELISA on individual sera giving maximization of the prevalence independent Youden''s index, on receiver operating characteristics analysis of replicate results. The herd-level BHV-1 seroprevalence was 74.9% (95% CI - 69.9%-79.8%), with no significant difference between dairy and beef herds. 95.5% agreement in herd classification was found between bulk milk and serum pools. Only 1.8 percent of farmers used BHV-1 marker vaccine, 80% of which was live while 75% of vaccinated herds were dairy.A significant association was found between herd size (quartiles) and seroprevalence (quartiles).

Conclusions

The results from this study indicate BHV-1 infection is endemic, although BHV-1 vaccines are rarely used, in the cattle population in Ireland.     

Malocclusions in the koala (Phascolarctos cinereus)

Malocclusions are a misalignment or incorrect positioning of the teeth when the upper and lower jaws close. These are poorly described in the koala and can result in irregular mastication which can have lifelong effects on body condition and oral health. A total of 370 koalas from two populations in Queensland (295) and one in South Australia (75) were examined for malocclusions. The prevalence of malocclusions in South Australian free‐ranging koalas, captive Queensland koalas and Queensland free‐ranging koalas was 39% (44), 30% (29) and 22% (29) respectively. Four types of malocclusion were identified based on severity of misalignment of the incisor/canine region, types 1, 2, 3 and 4. Maxillary overbite measurements of the molariform teeth were determined and these anisognathic values were then used to describe malocclusions within familial relationships in captive colonies. Captive koalas with a malocclusion had narrower mandibular width that ranged between 0.5 and 1% less than the normal measurements. The specific malocclusions reported in this study affected individuals by leading to tooth rotation, mobility and erosion with inefficient mastication of food and vegetation compaction. These changes increased the oral cavity pathology, by placing animals at risk of periodontal disease. There was evidence of familial links to malocclusion types in captive animals. Therefore captive breeding recommendations should consider known koala malocclusion traits to minimise their effect on future generations.     

Developing high throughput quantitative PCR assays for diagnosing Ikeda and other Theileria orientalis types common to New Zealand in bovine blood samples

AIMS: To develop rapid, quantitative PCR (qPCR) assays using high resolution melt (HRM) analysis and type-specific TaqMan assays for identifying the prevalent types of Theileria orientalis found in New Zealand cattle; and to evaluate their analytical and diagnostic characteristics compared with other assays for T. orientalis.

METHODS: Nucleotide sequences aligned with T. orientalis Buffeli, Chitose and Ikeda types, obtained from DNA extracted from blood samples from infected cattle, were used to design HRM and type-specific probe-based qPCR assays. The three type-specific assays were also incorporated into a single-tube multiplex qPCR assay. These assays were validated using DNA extracted from blood samples from cattle in herds with or without clinical signs of T. orientalis infection, other veterinary laboratory samples, as well as plasmids containing T. orientalis type-specific sequences. Diagnostic specificity (DSp) and sensitivity (DSe) estimates for the qPCR assays were compared to blood smear piroplasm results, and other PCR assays for T. orientalis. Copy number estimates of Ikeda DNA in blood were determined from cattle exhibiting anaemia using the Ikeda-specific qPCR assay.

RESULTS: The T. orientalis type-specific and the HRM qPCR assays displayed 100% analytical specificity. The Ikeda-specific qPCR assay exhibited linearity (R²?=?0.997) with an efficiency of 94.3%. Intra-assay CV were ≤0.08 and inter-assay CV were ≤0.095. For blood samples from cows with signs of infection with T. orientalis, the DSp and DSe of the multiplex probe qPCR assay were 93 and 96%, respectively compared with blood smears, and 97 and 100%, respectively compared with conventional PCR assays. For the Ikeda-specific qPCR assay, the number of positive samples (n=66) was slightly higher than a conventional PCR assay (n=64). The concentration of Ikeda genomes in blood samples from 41 dairy cows with signs of infection with T. orientalis ranged between 5.6?×?10⁴ and 3.3?×?10⁶ genomes per µL of blood.

CONCLUSIONS: All qPCR assays had improved specificity and sensitivity over existing conventional PCR assays for diagnosis of T. orientalis Ikeda. The burden of Ikeda DNA in blood was demonstrated using an Ikeda-specific qPCR assay with titrated synthetic gene target.

CLINICAL RELEVANCE: Adoption of high-throughput DNA extraction and qPCR reduced T. orientalis and Ikeda diagnosis times. The Ikeda-specific qPCR assay provides a specific diagnosis for Ikeda in animals with signs of infection with T. orientalis and can be used to monitor the parasite load of Ikeda in blood.     

Clinicopathological features of 11 suspected outbreaks of bovine adenovirus infection and development of a real-time quantitative PCR to detect bovine adenovirus type 10

CASE HISTORY: A retrospective study was conducted to investigate 11 outbreaks of presumptive fatal adenovirus infection diagnosed through two New Zealand diagnostic laboratories during 2014 and 2015. Outbreaks occurred in 6–12-month-old Friesian or Friesian cross cattle during autumn, winter and spring. Individual outbreaks were short in duration, with mortality rates ranging from 3/250 to 20/600 (1.2 to 3.3%).

CLINICAL AND PATHOLOGICAL FINDINGS: Clinical signs included severe diarrhoea, depression, recumbency, and death. Post-mortem examination revealed congestion and oedema of the alimentary tract and fluid to haemorrhagic intestinal contents. Histopathological lesions were characterised by congestion and haemorrhage of the alimentary tract mucosa, oedema of the submucosa, and mild interstitial inflammation in the kidneys. Large basophilic intranuclear inclusion bodies were identified in vascular endothelial cells of the alimentary tract in 11/11 cases and of the kidney in 8/9 cases.

MOLECULAR TESTING: A real-time quantitative PCR (qPCR) assay was designed to detect bovine adenovirus type 10 (BAdV-10) using hexon gene sequences available in GenBank. DNA extracted from a field case and confirmed by sequencing was used as a positive control. The qPCR had a reaction efficiency of 101% (R²=0.99) and the limit of detection was <10 DNA copies/reaction. The qPCR detected BAdV-10 in formalin-fixed paraffin-embedded (FFPE) tissue from 10/11 cases. DNA sequencing of PCR products from nine of these cases showed them to be identical to BAdV-10 sequences in GenBank. For the PCR-negative case, the PCR product had a hexon sequence 99% similar to bovine adenovirus Wic isolate Ma20-1, a close relative of BadV-10.

DIAGNOSIS: Bovine adenovirus type 10 was identified in FFPE tissues from cattle with histopathological evidence of adenovirus infection.

CLINICAL RELEVANCE: Bovine adenoviruses, and especially BAdV-10, should be considered in the differential diagnosis for acute enteric disease and death in young cattle. The qPCR detected BAdV-10 from FFPE tissue of cattle with suspected adenoviral infection diagnosed by histopathology. However results should be interpreted in light of clinical and pathological findings due to the possibility of adenovirus shedding by healthy cattle and the presence of pathogenic adenoviruses other than BAdV-10.     

Freezing equine semen: the effect of combinations of semen extenders and glycerol on post-thaw motility

Objective   We evaluated combinations of two commercial semen extenders and three concentrations of glycerol to determine the combination that yielded the highest post-thaw sperm motility.
Design   A randomised 2 × 3 block design was used.
Procedure   Semen was collected from four stallions (6 collections per stallion). The sample was diluted with either a dried skim-milk glucose extender (EZ Mixin Original Formula) or a chemically defined, milk-free diluent (INRA 96), and each was used in combination with 2%, 3% or 4% glycerol in standard commercial freezing medium. Sperm motility was assessed by microscopy in fresh and post-thaw semen.
Results   There was a significant difference between the two extenders in the motility of spermatozoa after cryopreservation (48.9% for INRA 96; 38.6% for EZ Mixin OF; P < 0.0001). Glycerol at 4% in freezing medium yielded the highest post-thaw motility, significantly better than 2% ( P < 0.05). Three of four stallions had significantly higher post-thaw motility using INRA 96 relative to EZ Mixin OF ( P < 0.01), and two of four stallions had significantly higher post-thaw motility using 4% glycerol ( P < 0.05). The combination of INRA 96 and 4% glycerol in freezing medium gave the highest average post-thaw motility of 51.5%.
Conclusion   In this study, INRA 96 combined with 4% glycerol yielded an average recovery of progressively motile sperm consistently above the 35% target.