Identification of equine herpesvirus 3 (equine coital exanthema virus), equine gammaherpesviruses 2 and 5, equine adenoviruses 1 and 2, equine arteritis virus and equine rhinitis A virus by polymerase chain reaction

OBJECTIVE: To develop rapid (< 8 hour) tests using polymerase chain reaction (PCR) for the diagnosis of equine herpesvirus 3 (EHV3; equine coital exanthema virus), equine gammaherpesviruses 2 (EHV2) and EHV5, equine adenovirus 1 (EAdV1), EAdV2, equine arteritis virus (EAV), equine rhinitis A virus (ERAV; formerly equine rhinovirus 1) DESIGN: Either single round or second round (seminested) PCRs were developed and validated. METHODS: Oligonucleotide primers were designed that were specific for each virus, PCR conditions were defined and the specificity and sensitivity of the assays were determined. The application of the tests was validated using a number of independent virus isolates for most of the viruses studied. The PCRs were applied directly to clinical samples where samples were available. RESULTS: We developed a single round PCR for the diagnosis of EHV3, a seminested PCR for EHV2 and single round PCRs for EHV5, EAdV1, EAdV2 and RT-PCRs for EAV and ERAV. The PCR primer sets for each virus were designed and shown to be highly specific (did not amplify any recognised non-target template) and sensitive (detection of minimal amounts of virus) and, where multiple virus isolates were available all isolates were detected. CONCLUSION: The development and validation of a comprehensive panel of PCR diagnostic tests, predominantly for viruses causing equine respiratory disease, that can be completed within 8 hours from receipt of clinical samples, provides a major advance in the rapid diagnosis or exclusion diagnosis of these endemic equine virus diseases in Australia.     

High performance liquid chromatographic method using fluorescence detention for quantitative analysis of zearalenone and alpha-zearalenol in blood plasma

A sensitive, high performance liquid chromatographic method is described for quantitative determination of zearalenone and alpha-zearalenol in blood plasma. Blood plasma is extracted with 2-propanol in ether, the extract is evaporated to dryness, and the residue is dissolved in 0.18N NaOH. The aqueous phase is washed with chloroform, dichloromethane, and benzene, neutralized with 0.10M H3PO4, and extracted with benzene. The extract is evaporated, dissolved in methanol, and injected onto a reverse phase column containing LiChrosorb RP-8 under the following conditions: methanol-acetonitrile-water mobile phase, fluorescence detector, excitation wavelength 236 nm, and 418 nm cut-off emission filter. The limit of detectability (twice background) is 0.5 ng standard which is equivalent to 0.6 ng standard/mL blood plasma. Linear standard curves are observed over the range of 0-35 ng of injected zearalenone and alpha-zearalenol. The recoveries from blood plasma are 76-101% in the range of 1.5-6.0 ng standard/mL blood.     

Veterinary attitudes towards pre-pubertal gonadectomy of cats: a comparison of samples from New Zealand,Australia and the United Kingdom

AIM: To compare the attitudes and practices of a sample of veterinarians in New Zealand, Australia and the United Kingdom (UK) towards pre-pubertal gonadectomy of cats.

METHODS: Respondents' demographics were gathered using an electronic questionnaire distributed via professional veterinary associations in the target countries, as were minimum age at gonadectomy and typical age of puberty. Desirability of prepubertal gonadectomy was gauged using three response categories (‘yes’, ‘no’ or ‘sometimes’), respondents were then able to justify the response given. Two-way Analyses of Variance (ANOVA) followed by post hoc Tukey HSD tests were used to test whether there were differences in minimum ages for gonadectomy within and between countries and between providers and non-providers of services to pounds (or animal welfare centres). Views on the desirability of prepubertal gonadectomy relative to demographics were explored using a Likelihood Ratio Test.

RESULTS: The survey received 717 responses. Most respondents believed pre-pubertal gonadectomy was either entirely or ‘sometimes’ desirable (556/621), few thought it was undesirable (65/621). Minimum age at gonadectomy was significantly affected by country surveyed and provision or non-provision of services for pounds. Post hoc Tukey HSD analysis indicated the mean age of both spaying and castration (4.3 months) in the UK was significantly different from both Australia (spaying: 3.4 months, castration: 3.2 months) and New Zealand (spaying: 3.4 months, castration: 3.2 months) (all p<0.001). Mean ages at spaying and castration were also significantly different (p= 0.008; p= 0.019, respectively) for non-providers (spaying: 3.9 months, castration: 3.8 months) of services to pounds when compared to providers (spaying and castration: both 3.6 months).

Likelihood Ratio Tests indicated significant effects amongst countries and between genders relative to the desirability of pre- pubertal gonadectomy. Respondents from the UK were more likely to answer ‘no’ (p = 0.004) or ‘sometimes’ (p = 0.050) as compared to those from New Zealand or Australia. Females were more likely to respond with ‘sometimes’ as opposed to ‘yes’ than males. Reasons for considering pre-pubertal gonadectomy desirable or sometimes desirable focussed on reducing unwanted pregnancies and improving population control, as well as improving rates of adoption, owner compliance and cat behaviour and health.

CONCLUSIONS AND CLINICAL RELEVANCE: In general, pre-pubertal gonadectomy is considered a desirable procedure by those practitioners that responded to the survey. However age at which any such procedure occurs differs depending upon a number of factors. Differences among countries may relate to the specific veterinary association's guidelines or possibly differences in social discourse which affect perception of cats. There is substantial overlap between the reported minimum age of gonadectomy and the age at which cats can enter early puberty, allowing a window for unintentional pregnancy when pre-pubertal gonadectomy does not occur.     

Evidence for absence of equine arteritis virus in the horse population of New Zealand

AIM: To summarise investigation and laboratory data collected between 2001 and 2011 to provide evidence that equine arteritis virus is not present in the horse population of New Zealand.

METHODS: Analysis was carried out on results from laboratory tests carried out at the Ministry for Primary Industries Animal Health Laboratory (AHL) for equine arteritis virus from horses tested prior to being imported or exported, testing of stallions as part of the New Zealand equine viral arteritis (EVA) control scheme and testing as part of transboundary animal disease (TAD) investigations for exclusion of EVA. Horse breeds were categorised as Thoroughbred, Standardbred or other.

RESULTS: A total of 7,157 EVA serological test records (from import and export testing, EVA control scheme testing and TAD investigations) were available for analysis between 2005 and 2011. For the three breed categories a seroprevalence of ≤1.6% at the 95% confidence level was determined for each category. Between 2001 and 2011, as part of the EVA control scheme, the EVA status of 465 stallions was determined to be negative. During 2005–2011 EVA was excluded from 84 TAD investigations.

CONCLUSIONS: There was no evidence of equine arteritis virus being present in the general horse population outside of carrier stallions managed under the EVA control scheme.

CLINICAL RELEVANCE: Equine arteritis virus is absent from the general horse population of New Zealand.     

Effects of Gonadotropins on In Vitro Maturation and of Electrical Stimulation on Parthenogenesis of Canine Oocytes

The objective of this study was to determine the effects of gonadotropins on in vitro maturation (IVM) and electrical stimulation on the parthenogenesis of canine oocytes. In experiment I, cumulus oocyte complexes were collected from ovaries at a random phase of the oestrus cycle and cultured on maturation medium treated with hCG or eCG for 48 or 72 h. There were no significant differences in the effects on the metaphase II (MII) rate between the hCG and eCG treatment groups over 48 h (5.4% vs 5.5%). The MII rate in the co-treatment group of hCG and eCG for 48 h was higher than in each hormone treated group (15.5%, p < 0.05). In experiment 2, the parthenogenetic effect on oocyte development, at various electrical field strengths (1.0, 1.5, 2.0 kV/cm DC) for 60 or 80 μs with a single DC pulse after IVM on the co-treatment of hCG and eCG, was examined. The rate of pronuclear formation (37.1%) in electrical activation at 1.5 kV/60 μs without cytochalasin B (CB) was higher than that of oocytes activated in the other groups (p < 0.05). However, we did not observe the cleavage stages. Also, CB did not influence parthenogenesis of canine oocytes. The results showed that the pronucleus formation rate, indicative of the parthenogenesis start point, could be increased by electrical stimulation. Therefore, these results can provide important data for the parthenogenesis of canine oocytes and suggest the probability of parthenogenesis in canines.