In Australia, infection of horses with the West Nile virus (WNV) or Murray Valley encephalitis virus (MVEV) occasionally results in severe neurological disease that cannot be clinically differentiated. Confirmatory serological tests to detect antibody specific for MVEV or WNV in horses are often hampered by cross-reactive antibodies induced to conserved epitopes on the envelope (E) protein. This study utilized bacterially expressed recombinant antigens derived from domain III of the E protein (rE-DIII) of MVEV and WNV, respectively, to determine whether these subunit antigens provided specific diagnostic markers of infection with these two viruses. When a panel of 130 serum samples, from horses with known flavivirus infection status, was tested in enzyme-linked immunosorbent assay (ELISA) using rE-DIII antigens, a differential diagnosis of MVEV or WNV was achieved for most samples. Time-point samples from horses exposed to flavivirus infection during the 2011 outbreak of equine encephalitis in south-eastern Australia also indicated that the rE-DIII antigens were capable of detecting and differentiating MVEV and WNV infection in convalescent sera with similar sensitivity and specificity to virus neutralization tests and blocking ELISAs. Overall, these results indicate that the rE-DIII is a suitable antigen for use in rapid immunoassays for confirming MVEV and WNV infections in horses in the Australian context and warrant further assessment on sensitive, high-throughput serological platforms such as multiplex immune assays.
A study to determine the immunoglobulin and cellular responses in the respiratory tract of goats following intranasal exposures to formalin-killed Pasteurella haemolytica A2 was carried out. Forty-two goats were divided into two groups. Goats in Group 1 were subjected to double intranasal exposures to formalin-killed P. haemolytica A2 while goats in Group 2 were the unexposed control. Prior to and at weekly intervals post-exposure, three goats from each group were killed, serum samples were collected while the lungs were flushed with 50 ml normal saline before the right apical lobes were fixed in 10% buffered formalin. Both serum and lung lavage fluid were subjected to enzyme-linked immunosorbent assay (ELISA) to determine the levels of IgA, IgM and IgG while the formalin-fixed tissues were examined histologically. IgA levels in the lung lavage fluid increased rapidly to reach a significantly (p < 0.05) high level as early as Week 2 post-exposure and remained significantly (p < 0.05) high throughout the study period. The IgM levels increased at an intermediate rate to reach a significantly (p < 0.05) high level at Week 3 post-exposure before they decreased to an insignificant (p > 0.05) level the following week and the weeks thereafter. IgG levels increased gradually and only reached a significantly (p < 0.01) high level at Weeks 5 and 6 of the study. The size of the bronchus-associated lymphoid tissue (BALT) and the number of lymphocytes in BALT increased significantly from Week 2 and remained high thereafter. However, differences in the numbers of BALT were insignificant (p > 0.05) initially before becoming significantly (p < 0.05) high at Weeks 5 and 6. The BALT responses were parallel to those of imunoglobulins in the lung lavage fluid.
The outer membrane proteins (OMP) were extracted from the P. haemolytica A2, A7 and A9 to determine their potential as immunogens and their capability for cross-protection. Sixty lambs of approximately 9 months old were divided into four main groups. Animals in Group 1 were vaccinated with 2ml vaccine containing 100microg/ml of the outer membrane proteins of P. haemolytica A2. Animals in Group 2 were similarly vaccinated with the OMPs of P. haemolytica A7 while Group 3 with OMPs of P. haemolytica A9. Animals in Group 4 were unvaccinated control. During the course of the study, serum was collected to evaluate the antibody levels toward each OMP. There appeared to be good immune responses. However, high antibody levels did not necessarily result in good protection of the animals, particularly against cross-infection with P. haemolytica A9 in animals vaccinated with the OMPs of P. haemolytica A2. It seemed that the antibody responses were more specific toward the homologous challenge but generally did not cross-protect against heterologous serotype challenge. However, the OMPs of P. haemolytica A7 produced good in vivo cross-protection and excellent correlations when good antibody responses against all serotypes led to successful reductions of the extent of lung lesions following homologous and heterologous challenge exposures. Thus, the OMPs of P. haemolytica A7 was effective in protecting animals against homologous and heterologous infection by live P. haemolytica A2, A7 and A9.
The relationship between the standard passive mouse protection test or serum antibody titres measured by indirect haemagglutination or enzyme-linked immunosorbent assays and active protection in buffaloes immunized with different types of haemorrhagic septicaemia bacterins was investigated. Groups of 2-3 buffaloes were immunized with the bacterins currently in use in Asia, viz., broth bacterin (BB), alum precipitated vaccine (APV) and oil adjuvant vaccine (OAV) either subcutaneously (BB, APV) or intramuscularly (OAV) and challenged subcutaneously with virulent organisms at different periods post-immunization. Although the passive mouse protection and indirect haemagglutination tests carried out with the pre-challenge sera from vaccinated buffaloes revealed no relationship with active protection in buffaloes, a relationship was observed between the ELISA antibody titres and protection. In contrast, a dose-response relationship was observed between the homologous active and passive mouse protection test.
Recent reports indicate Neospora caninum has a possible role in causing abortions in sheep in New Zealand. Knowledge about the epidemiology of neosporosis in sheep is limited. This study aimed to adapt and validate a commercially available ELISA assay as an IgG avidity assay to discriminate between acute (primary and re-inoculated) and chronic N. caninum infections in sheep. In addition, it was used to compare the antibody avidity values between lambs from ewes inoculated with N. caninum either during the pregnancy or in the previous year. The avidity assay was undertaken by using 6M urea for the first wash after incubation with the primary antibody in the commercial ELISA (Chekit* Neospora antibody test kit, IDEXX Laboratories, Australia). Sequential serum samples were obtained from naïve ewes (n=16) experimentally inoculated with live N. caninum tachyzoites. All ewes were seropositive by two weeks post-inoculation and remained seropositive for 20 weeks post-inoculation. There was a linear relationship between time after inoculation and avidity values (p<0.05) over the first 24 weeks. In Week 4, all animals had avidity values <35% and by Week 8, 8/16 animals had avidity values of >35%. These results suggest that an avidity value of <35% indicates a recent primary infection while a value of >35% is indicative of a chronic infection. The assay was then validated using samples from other groups of experimentally inoculated sheep as well as samples from naturally infected ewes. When comparing sample to positive ratio (S/P) and avidity values from lambs born from recently inoculated ewes with those from ewes inoculated the previous year and re-inoculated in the current year, it was possible to differentiate the lambs at 2 weeks of age. Lambs from recently inoculated ewes had low S/P and avidity values at 2 weeks of age which increased by 12 weeks of age. In comparison, lambs from re-inoculated ewes had high S/P and avidity values at 2 weeks of age, due to maternal antibody influence but values were similar to those from lambs that were born from recently inoculated ewes at 12 weeks of age. Avidity values for four naturally infected ewes were all >60% indicating chronic infection. These results suggest that the assay is able to discriminate between recent and chronic infection in sheep as well as able to differentiate lambs with maternal immunity compared to their own de novo immunity. As such it can be utilized to understand the kinetics of N. caninum infection in sheep.
To determine if toltrazuril was effective in eliminating Neospora caninum infection from congenitally infected lambs. Twenty-eight ewes were allocated to 3 groups where animals in Groups A and B were inoculated with 1 × 10(7)N. caninum tachyzoites on Day 120 of gestation and Group C was maintained as a negative control group. Lambs born from ewes in Group A were treated with toltrazuril (20mg/kg) on Days 0, 7, 14 and 21 after birth. Lambs in Groups B and C were untreated. All lambs in Groups A and B were seropositive at 12 weeks of age. At 12 weeks of age, no differences between lambs in Group A and Group B were observed in serological results (ELISA and western blot), presence of N. caninum-related brain histopathological lesions or the number of organisms detected by qPCR. Group C remained negative for serology, detection of N. caninum DNA as well as histopathology throughout the study. Results indicate that N. caninum congenitally-infected lambs had a continuing infection with N. caninum despite being treated with toltrazuril.
Following a series of H5N1 cases in chickens and birds in a few states in Malaysia, there was much interest in the influenza A viruses subtypes that circulate among the local pig populations. Pigs may act as a mixing vessel for avian and mammal influenza viruses, resulting in new reassorted viruses. This study investigated the presence of antibodies against influenza H1N1 and H3N2 viruses in pigs from Peninsular Malaysia using Herdcheck Swine Influenza H1N1 and H3N2 Antibody Test Kits. At the same time, the presence of influenza virus was examined from the nasal swabs of seropositive pigs by virus isolation and real time RT-PCR. The list of pig farms was obtained from the headquarters of the Department of Veterinary Services, Malaysia, and pig herds were selected randomly from six of 11 states in Peninsular Malaysia. A total of 727 serum and nasal swab samples were collected from 4- to 6-month-old pigs between May and August 2005. By ELISA, the seroprevalences of swine influenza H1N1 and H3N2 among pigs were 12.2% and 12.1% respectively. Seropositivity for either of the virus subtypes was detected in less than half of the 41 sampled farms (41.4%). Combination of both subtypes was detected in 4% of all pigs and in 22% of sampled farms. However, no virus or viral nucleic acid was detected from nasal samples. This study identified that the seropositivity of pigs to H1N1 and H3N2 based on ELISA was significantly associated with factors such as size of farm, importation or purchase of pigs, proximity of farm to other pig farms and the presence of mammalian pets within the farm.