Comparative susceptibility of eastern cottontails and New Zealand white rabbits to classical rabbit haemorrhagic disease virus (RHDV) and RHDV2.

Rabbit haemorrhagic disease virus (RHDV) is associated with high morbidity and mortality in the European rabbit (Oryctolagus cuniculus). In 2010, a genetically distinct RHDV named RHDV2 emerged in Europe and spread to many other regions, including North America in 2016. Prior to this study it was unknown if eastern cottontails (ECT(s); Sylvilagus floridanus), one of the most common wild lagomorphs in the United States, were susceptible to RHDV2. In this study, 10 wild-caught ECTs and 10 New Zealand white rabbits (NZWR(s); O. cuniculus) were each inoculated orally with either RHDV (RHDVa/GI.1a; n = 5 per species) or RHDV2 (a recombinant GI.1bP-GI.2; n = 5 per species) and monitored for the development of disease. Three of the five ECTs that were infected with RHDV2 developed disease consistent with RHD and died at 4 and 6 days post-inoculation (DPI). The RHDV major capsid protein/antigen (VP60) was detected in the livers of three ECTs infected with RHDV2, but none was detected in the ECTs infected with RHDV. Additionally, RHD viral RNA was detected in the liver, spleen, intestine and blood of ECTs infected with RHDV2, but not in the ECTs infected with RHDV. RHD viral RNA was detected in urine, oral swabs and rectal swabs in at least two of five ECTs infected with RHDV2. One ECT inoculated with RHDV2 seroconverted and developed a high antibody titre by the end of the experimental period (21 DPI). ECTs inoculated with the classic RHDV did not seroconvert. In comparison, NZWRs inoculated with RHDV2 exhibited high mortality (five of five) at 2 DPI and four of five NZWRs inoculated with RHDV either died or were euthanized at 2 DPI indicating both of these viruses were highly pathogenic to this species. This experiment indicates that ECTs are susceptible to RHDV2 and can shed viral RNA, thereby suggesting this species could be involved in the epidemiology of this virus.

Assessment of reproducibility of a VP7 Blocking ELISA diagnostic test for African horse sickness.

The laboratory diagnosis of African horse sickness (AHS) is important for: (a) demonstrating freedom from infection in a population, animals or products for trade (b) assessing the efficiency of eradication policies; (c) laboratory confirmation of clinical diagnosis; (d) estimating the prevalence of AHS infection; and (e) assessing postvaccination immune status of individual animals or populations. Although serological techniques play a secondary role in the confirmation of clinical cases, their use is very important for all the other purposes due to their high throughput, ease of use and good cost-benefit ratio. The main objective of this study was to support the validation of AHS VP7 Blocking ELISA up to the Stage 3 of the World Animal Health Organization (OIE) assay validation pathway. To achieve this, a collaborative ring trial, which included all OIE Reference Laboratories and other AHS-specialist diagnostic centres, was conducted in order to assess the diagnostic performance characteristics of the VP7 Blocking ELISA. In this trial, a panel of sera of different epidemiological origin and infection status was used. Through this comprehensive evaluation we can conclude that the VP7 Blocking ELISA satisfies the OIE requirements of reproducibility. The VP7 Blocking ELISA, in its commercial version is ready to enter Stage 4 of the validation pathway (Programme Implementation). Specifically, this will require testing the diagnostic performance of the assay using contemporary serum samples collected during control campaigns in endemic countries.

Effects of age and macrophage lineage on intracellular survival and cytokine induction after infection with Rhodococcus equi.

Rhodococcus equi, a facultative intracellular pathogen of macrophages, causes life-threatening pneumonia in foals and in people with underlying immune deficiencies. As a basis for this study, we hypothesized that macrophage lineage and age would affect intracellular survival of R. equi and cytokine induction after infection. Monocyte-derived and bronchoalveolar macrophages from 10 adult horses and from 10 foals (sampled at 1-3 days, 2 weeks, 1 month, 3 months, and 5 months of age) were infected ex vivo with virulent R. equi. Intracellular R. equi were quantified and mRNA expression of IL-1ß, IL-4, IL-6, IL-8, IL-10, IL-12 p40, IL-18, IFN-γ, and TNF-α was measured. Intracellular replication of R. equi was significantly (P<0.001) greater in bronchoalveolar than in monocyte-derived macrophages, regardless of age. Regardless of the macrophage lineage, replication of R. equi was significantly (P=0.002) higher in 3-month-old foals than in 3-day old foals, 2-week-old foals, 1-month-old foals, and adult horses. Expression of IL-4 mRNA was significantly higher in monocyte-derived macrophages whereas expression of IL-6, IL-18, and TNF-α was significantly higher in bronchoalveolar macrophages. Induction of IL-1ß, IL-10, IL-12 p40, and IL-8 mRNA in bronchoalveolar macrophages of 1-3-day old foals was significantly higher than in older foals or adult horses. Preferential intracellular survival of R. equi in bronchoalveolar macrophages of juvenile horses may play a role in the pulmonary tropism of the pathogen and in the window of age susceptibility to infection.

Comparison of antibody and cell-mediated immune responses of foals and adult horses after vaccination with live Mycobacterium bovis BCG.

BACKGROUND: Equine neonates have reduced humoral and cell-mediated immune responses compared to adult horses after administration of killed vaccines. As a basis for this study, we hypothesized that newborn foals can mount strong immune responses after vaccination with live Mycobacterium bovis BCG. METHODS: Healthy 4-day-old foals (n=7), 4-month-old foals (n=7) and adult horses (n=6) were vaccinated once with live M. bovis BCG. Age-matched animals (n=5 per group) were used as unvaccinated controls. Relative vaccine-specific immunoglobulin concentrations and whole blood mRNA expression of IFN-γ, IL-4, and IL-10 were measured prior to and 2, 4, 6, and 8 weeks after vaccination. Eight weeks after vaccination, delayed type hypersensitivity (DTH) responses were assessed by measuring the increase in double skin thickness after intradermal injection of purified protein derivative. RESULTS: Both groups of foals and adult horses responded with a significant increase in vaccine-specific total IgG, IgGa, IgGc, IgG(T), and IgM concentrations. In contrast, only adult horses mounted significant IgGb responses. Vaccine-specific concentrations of total IgG and IgGa were significantly higher in adult horses than in 4-day-old foals whereas IgGc responses were significantly higher in 4-day-old foals than in the other two age groups. Adult horses had significantly higher basal IFN-γ and IL-4 mRNA expression than both groups of foals but vaccination with M. bovis BCG did not significantly increase expression of these cytokines, regardless of age group. Immunized horses had significantly higher DTH responses than age-matched unvaccinated controls. DTH responses were significantly greater in both groups of vaccinated foals than in vaccinated adult horses. CONCLUSION: Despite a naïve immune system, newborn foals have the ability to mount robust antibody and cell-mediated immune responses to M. bovis BCG.

Macrolide- and rifampin-resistant Rhodococcus equi on a horse breeding farm, Kentucky, USA.

Macrolide and rifampin resistance developed on a horse breeding farm after widespread use was instituted for treatment of subclinical pulmonary lesions in foals. Resistance occurred in 6 (24%) of 25 pretreatment and 8 (62%) of 13 (62%) posttreatment isolates from affected foals. Drug-resistant isolates formed 2 distinct genotypic clusters.

Immunogenicity and clinical protection against equine influenza by DNA vaccination of ponies.

Equine influenza A (H3N8) virus infection is a leading cause of respiratory disease in horses, resulting in widespread morbidity and economic losses. As with influenza in other species, equine influenza strains continuously mutate, often requiring the development of new vaccines. Current inactivated (killed) vaccines, while efficacious, only offer limited protection against diverse subtypes and require frequent boosts. Research into new vaccine technologies, including gene-based vaccines, aims to increase the neutralization potency, breadth, and duration of protective immunity. Here, we demonstrate that a DNA vaccine expressing the hemagglutinin protein of equine H3N8 influenza virus generates homologous and heterologous immune responses, and protects against clinical disease and viral replication by homologous H3N8 virus in horses. Furthermore, we demonstrate that needle-free delivery is as efficient and effective as conventional parenteral injection using a needle and syringe. These findings suggest that DNA vaccines offer a safe, effective, and promising alternative approach for veterinary vaccines against equine influenza.

Effects of inactivated parapoxvirus ovis on the cumulative incidence of pneumonia and cytokine secretion in foals on a farm with endemic infections caused by Rhodococcus equi.

The objectives of the present study were to determine if administration of inactivated parapoxvirus ovis (IPPVO) can decrease the cumulative incidence of pneumonia and increase the number of IFN-γ- and IL-4-secreting cells among foals. Fifty-nine foals were randomly assigned to 2 treatment groups (IPPVO or placebo) prior to birth. At 24-48 h of age, foals received 2 ml of either IPPVO or a placebo by intramuscular injection. Injections were repeated 24h and 8 days later. The number of IFN-γ- and IL-4-secreting cells was measured using a validated ELISPOT assay on blood mononuclear cells collected when the foals were 1-14 days old. Foals were monitored daily for clinical signs of pneumonia and biweekly for lung lesions by ultrasonography. The proportion of foals that developed clinical or ultrasonographic evidence of pneumonia was not significantly different between IPPVO (16 of 28) and placebo (14 of 31). IFN-γ- and IL-4-secreting cells were detected in only 22 and 15 foals, respectively. There was a significant effect of treatment with IPPVO on the number of IFN-γ secreting cells in foals 7- to 14-days-old but not in younger foals. There was no significant effect of treatment with IPPVO on the number of IL-4-secreting cells. The odds of detecting IFN-γ (5.1; 95% CI: 1.5-15) and IL-4 (3.5; 95% CI: 1.1-12) were significantly higher in foals 7-14 days than in younger foals regardless of treatment group. There was no significant association between IFN-γ or IL-4 secretion early in life and subsequent development of pneumonia.

Humoral and cell-mediated immune responses of old horses following recombinant canarypox virus vaccination and subsequent challenge infection.

Equine influenza virus is a leading cause of respiratory disease in the horse population; however, the susceptibility of old horses to EIV infection remains unknown. While advanced age in horses (>20 years) is associated with age-related changes in immune function, there are no specific recommendations regarding the vaccination of older horses even though a well-characterized effect of aging is a reduced antibody response to standard vaccination. Therefore, we evaluated the immunological and physiological response of aged horses to a live non-replicating canarypox-vectored EIV vaccine and subsequent challenge infection. Vaccination of the aged horses induced EIV-specific IgGb and HI antibodies. No specific increase in cell-mediated immune (CMI) response was induced by the vaccine as determined by EIV-specific lymphoproliferation and the detection of EIV-specific IFNγ(+) CD5(+)T cells, IFNγ, IL-2, IL-4 and IL-13 mRNA expression. Non-vaccinated aged horses exhibited clinical signs of the disease (coughing, nasal discharge, dyspnea, depression, anorexia) as well as increased rectal temperature and viral shedding following challenge. Concomitant with the febrile episodes, we also observed increased production of pro-inflammatory cytokine mRNA production in vivo using RT-PCR. Naïve horses were included in this study for vaccine and challenge controls only. As expected, the canarypox-vectored EIV vaccine stimulated significant CMI and humoral immune responses and provided significant protection against clinical signs of disease and reduced virus shedding in naive horses. Here, we show that aged horses remain susceptible to infection with equine influenza virus despite the presence of circulating antibodies and CMI responses to EIV and vaccination with a canarypox-vectored EIV vaccine provides protection from clinical disease.

Development and evaluation of one-step TaqMan real-time reverse transcription-PCR assays targeting nucleoprotein, matrix, and hemagglutinin genes of equine influenza virus.

The objective of this study was to develop and evaluate new TaqMan real-time reverse transcription-PCR (rRT-PCR) assays by the use of the minor groove binding probe to detect a wide range of equine influenza virus (EIV) strains comprising both subtypes of the virus (H3N8 and H7N7). A total of eight rRT-PCR assays were developed, targeting the nucleoprotein (NP), matrix (M), and hemagglutinin (HA) genes of the two EIV subtypes. None of the eight assays cross-reacted with any of the other known equine respiratory viruses. Three rRT-PCR assays (EqFlu NP, M, and HA3) which can detect strains of the H3N8 subtype were evaluated using nasal swabs received for routine diagnosis and swabs collected from experimentally inoculated horses. All three rRT-PCR assays have greater specificity and sensitivity than virus isolation by egg inoculation (93%, 89%, and 87% sensitivity for EqFlu NP, EqFlu M, and EqFlu HA3 assays, respectively). These assays had analytical sensitivities of >or=10 EIV RNA molecules. Comparison of the sensitivities of rRT-PCR assays targeting the NP and M genes of both subtypes with egg inoculation and the Directigen Flu A test clearly shows that molecular assays provide the highest sensitivity. The EqFlu HA7 assay targeting the H7 HA gene is highly specific for the H7N7 subtype of EIV. It should enable highly reliable surveillance for the H7N7 subtype, which is thought to be extinct or possibly still circulating at a very low level in nature. The assays that we developed provide a fast and reliable means of EIV diagnosis and subtype identification of EIV subtypes.