Twenty years of Hendra virus: laboratory submission trends and risk factors for infection in horses.

Hendra virus (HeV) was first described in 1994 in an outbreak of acute and highly lethal disease in horses and humans in Australia. Equine cases continue to be diagnosed periodically, yet the predisposing factors for infection remain unclear. We undertook an analysis of equine submissions tested for HeV by the Queensland government veterinary reference laboratory over a 20-year period to identify and investigate any patterns. We found a marked increase in testing from July 2008, primarily reflecting a broadening of the HeV clinical case definition. Peaks in submissions for testing, and visitations to the Government HeV website, were associated with reported equine incidents. Significantly differing between-year HeV detection rates in north and south Queensland suggest a fundamental difference in risk exposure between the two regions. The statistical association between HeV detection and stockhorse type may suggest that husbandry is a more important risk determinant than breed per se. The detection of HeV in horses with neither neurological nor respiratory signs poses a risk management challenge for attending veterinarians and laboratory staff, reinforcing animal health authority recommendations that appropriate risk management strategies be employed for all sick horses, and by anyone handling sick horses or associated biological samples.

Responding to the equine influenza outbreak: challenges from a laboratory perspective.

The unique challenges that laboratories in Queensland and New South Wales faced during the response to the 2007 equine influenza outbreak and how these were managed are described.

Validation of an influenza virus A 5'Taq nuclease assay for the detection of equine influenza virus A RNA in nasal swab samples.

OBJECTIVE: Describe the in-house validation of a previously reported influenza virus type A 5'Taq nuclease assay for detecting equine influenza virus A RNA in nasal swab material. METHODS: The validation compares the 5'Taq nuclease assay with a gel-based reverse transcription nested polymerase chain reaction (PCR) previously reported by the Irish Equine Centre for detection of H3N8 and H7N7 equine influenza viruses. This test was chosen because it targets a different region of the viral genome to the real-time test, so it is not merely a repeat of the same test in a different format. Moreover, nested PCRs are commonly considered to have similar sensitivity to real-time PCRs and are therefore ideal for evaluation comparisons. RESULTS: The sensitivity of the nested PCR was comparable to the 5'Taq nuclease test. Known positive samples and known negative samples reacted with both tests with 100% correlation. Parallel testing of 276 nasal swab samples showed 98% agreement. CONCLUSION: The specificity of the nested amplicons was confirmed by nucleotide sequencing and showed >99.5% identity with the same region of previously published equine influenza virus A sequences. The results of this work are appropriate validation for the acceptance of the real-time PCR for equine influenza A virus in equine nasal swabs.

Amelioration of virulent Babesia bovis infection in calves by administration of the nitric oxide synthase inhibitor aminoguanidine.

Calves undergoing initial infection with a virulent strain of the haemoprotozoan parasite Babesia bovis were treated with aminoguanidine (AG), an inhibitor of the inducible form of nitric oxide synthase (iNOS). The mean maximum parasitaemia of the AG treated calves was significantly lower than that of the control cattle. In addition, the febrile response and decrease in packed cell volume (PCV) observed during acute infection were significantly ameliorated in the AG treated cattle relative to the controls. However, AG had no effect on the multiplication of B. bovis in the microaerophilous stationary-phase (MASP) in-vitro culture system. These results provide evidence of a role for nitric oxide (NO) produced in response to acute infection in the pathology of bovine babesiosis.

Vaccination against Babesia bovis: T cells from protected and unprotected animals show different cytokine profiles.

Vaccination of cattle against the haemoprotozoan parasite, Babesia bovis, with the recombinant antigen 11C5 resulted in 9 of 15 cattle being protected against challenge infection. The cellular immune responses of protected and unprotected cattle were compared in order to identify differences in response. No differences were observed in the pattern of change in various blood leukocyte populations throughout challenge infection. FACScan analysis revealed an increase in the proportion of cells bearing the CD2 marker in both protected and unprotected cattle over the course of infection. There were no observable differences in the frequency of various cell-surface markers between the unprotected and protected cattle. During the period of patent parasitaemia, in vitro cultures of peripheral blood mononuclear cells (PBMC) from protected cattle produced significantly more TNF-alpha (P < 0.05) than cultures from unprotected cattle. TNF-alpha concentrations remained at pre-challenge levels until day 10, when levels in the unvaccinated control and vaccinated/unprotected animals dropped. By peak parasitaemia, TNF-alpha production in vitro was significantly greater (P < 0.05) in cultures of PBMCs from protected cattle. Interferon production showed an initial peak at day 5 in all cattle, followed by a decrease and a second peak at days 10-13 in protected cattle only, which coincided with resolution of the infection.

Babesia bovis: biosynthesis and localisation of 12D3 antigen in bovine erythrocytes.

The 12D3 antigen of Babesia bovis was found to be synthesised rapidly in cultured parasites, and localised to both the apical complex of the merozoite and the cytoplasm of the parasitised erythrocyte. Amino-terminal sequencing suggested that the nascent protein had been processed and differences between the predicted and measured molecular weights suggested post-translational modification. The major proportion of 12D3 appeared in the soluble compartment of the parasitised erythrocytes with a molecular weight consistent with no further processing. A significant proportion of the protein required extraction by sodium carbonate, suggesting association with membranous components. The timing of release of soluble 12D3 was coincident with haemoglobin release and this probably reflects a non-specific lysis of the erythrocyte. Synthesis of recombinant BV12D3 was achieved in baculovirus-infected SF9 insect epithelial cells. The product was of the same molecular weight as the native 12D3 and polyclonal antibodies raised against the recombinant protein reacted with both the recombinant and native forms of the antigen.

Low doses of natural human interferon alpha inhibit the development of Babesia microti infection in BALB/c mice.

Studies were undertaken to determine whether treatments with low doses of natural human interferon alpha (HuIFN-alpha) administered by various routes could inhibit the development of the intra-erythrocytic protozoan Babesia microti in BALB/c mice. HuIFN-alpha treatment given intramuscularly significantly inhibited development of the parasitaemia of the parasite compared with infections in control mice.

A study of autoantibodies to phosphatidyl-serine in Babesia bovis and Babesia bigemina infections in cattle.

Sera from cattle infected with Babesia bovis were found to contain antibodies to phosphatidyl-serine (PS), a negatively charged phospholipid normally found on the internal membrane of erythrocytes. In contrast, no autoantibodies were detected following Babesia bigemina infection indicating that the autoimmunity is not genus specific. During infection with Babesia bovis, PS translocates to the external membrane and it is suggested that this may result in PS behaving as an autoantigen owing to a transitional change. These autoantibodies may also play some role in the pathology of infection, especially the disturbed coagulation system associated with acute Babesia bovis infection.

Isolation, initial characterization and serological studies of glycolipid antigens from Babesia bovis-infected erythrocytes.

Serological analysis of Babesia bovis-derived glycolipids by ELISA and the indirect fluorescent antibody technique demonstrated the existence of their antigenic and immunogenic activities not only in B. bovis but also in B. bigemina infections. This indicates that serological cross-reactivity of B. bovis and B. bigemina relates to glycolipids. The negative ELISA reaction obtained with Anaplasma marginale antisera suggested the specificity of the reaction to the genus Babesia. Fractionation of these glycolipids by Florisil Sep-Pak column chromatography with subsequent HPTLC immunostaining and Orcinol staining suggested the presence of carbohydrate antigenic determinants in B. bovis glycolipids.

Cloning and characterisation of cDNA clones encoding two Babesia bovis proteins with homologous amino- and carboxy-terminal domains.

A dextran sulphate protein (DSP) fraction derived from Babesia bovis has previously been shown to induce a protective immune response in cattle. A B. bovis cDNA library was screened with both the complete anti-DSP serum and a subfraction of the anti-DSP serum affinity purified on a native B. bovis protein of approx. 80 kDa. cDNA clones encoding two different B. bovis proteins were identified. The product of one gene, Bv80, has a single divergent copy of a sequence of 149 amino acids (approx. 30% amino acid identity) in both the amino- and carboxy-terminal domains. These domains are separated by an array of short variant repeat sequences rich in proline and glutamic acid. The product of the other gene, BvVAl (homologous to the previously described 225-kDa B. bovis protein)[19], is predicted to have a single divergent copy of a sequence of 170-171 amino acids (approx. 35% amino acid identity) in both the amino- and carboxy-terminal domains. These domains are also separated by an array of repeats. The 73-amino acid repeat unit of this array is composed of a number of variant derivatives of shorter repeat units. Detailed analysis of genomic clones flanking two alleles of the gene encoding BvVAl/225 kDa identified further members of a multi-gene family. This region of the genome of B. bovis has been subject to a large number of amplification processes.

The development of a recombinant Babesia vaccine.

Crude extracts of Babesia bovis parasites were shown to induce levels of protection in susceptible cattle equivalent to that resulting from natural infection. The crude material was systematically fractionated and tested in numerous sequential vaccination/challenge experiments in adult cattle. Antigens in protective fractions were then purified by affinity chromatography with monoclonal antibodies. Three highly protective (more than 95% reduction in parasitaemias) antigens were thus identified. None of these antigens was immunodominant; a number of immunodominant antigens were identified and all were immunosuppressive and/or non-protective. The three protective antigens were cloned and expressed as either beta-galactosidase or glutathione-S-transferase (GST) fusion proteins. Two of these, GST-12D3 and GST-11C5, when used in combination were almost as protective as has been previously shown for the commercially available live attenuated vaccine. A short fragment of a third antigen (21B4) has also been shown to be protective. In two of the antigens, repetitive segments have been shown to be non-protective while the third antigen (12D3) does not contain repetitive domains. Homologues of these antigens exist in other Babesia species and it is anticipated that these may be candidate antigens for protective vaccines against those species.

Vaccination of cattle with dextran sulphate-binding Babesia bigemina antigens.

Dextran sulphate-bound Babesia bigemina antigens were used in a preliminary vaccination study and were shown to elicit a protective immune response in cattle. A dextran sulphate-binding fraction of B. bigemina was further subfractionated on a Phenyl Sepharose column to give two fractions--one that strongly bound to the column (bound fraction) and one that did not (unbound fraction). Two groups of cattle were each vaccinated with either the bound or the unbound fraction. These two groups of animals along with a control group were then challenged with B. bigemina-infected erythrocytes. Both groups of vaccinated animals showed considerably lower mean daily parasitaemias as compared to the control group.

Serological and immunological studies with a hexane extract of Babesia bovis-infected erythrocytes.

Antigenic and immunogenic activities of a hexane extract from Babesia bovis-infected erythrocytes were investigated. Positive ELISA and IFAT reactions were obtained with bovine antisera to B. bovis and B. bigemina produced by natural infection and rabbit antisera to the hexane extract, respectively. In contrast, negative ELISA reactions were obtained with Anaplasma marginale antisera indicating that the antigen(s) is specific for the genus Babesia. The IFAT clearly demonstrated that the antigen was associated with the parasite and the infected erythrocyte and not present in uninfected erythrocytes. Furthermore, cross-reactions with Babesia bigemina antisera suggested that serological cross-reactivity in bovine Babesia species is at least due in part to lipid or lipid-associated antigens.

Babesia bovis: dextran sulphate as an adjuvant for and precipitant of protective immunogens.

Dextran sulphate, a chemical with some specificity for lipoproteins, was used to precipitate a fraction from a soluble extract of Babesia bovis-infected erythrocytes. The precipitate, in combination with dextran sulphate as an adjuvant, was used to vaccinate naive calves. The vaccinates and a group of control calves were challenged with virulent homologous B. bovis. The vaccinates showed delayed and decreased parasitaemias comparative to the controls. The antibody response to vaccination was primarily against the infected erythrocyte being of both IgG1 and IgG2 classes. We believe this is the first report of B. bovis antibody being detected in the IgG2 class. Lipase inhibition and chemical analysis suggested babesial lipid or lipoprotein was sufficiently immunogenic to produce serologically detectable antibody and presumably to elicit immunity.

Babesia bovis: analysis of and preliminary vaccination studies with a defined infected erythrocyte membrane binding antigen.

Murine monoclonal antibodies (MAB) were produced against the 'beta' fraction of Babesia bovis. A MAB, W11C5, selected on the criterion of its staining of erythrocytes infected with B. bovis, was purified. The antigen identified by MAB W11C5 was extracted from B. bovis infected erythrocytes by affinity chromatography and used in a vaccination trial to test its vaccine efficacy against homologous B. bovis infection in splenectomized calves. The vaccinated group showed significantly different parasitaemias from the control group and it was concluded that the B. bovis antigen 11C5 induced a protective immune response when used as a vaccine. This antigen should be synthesized using recombinant DNA techniques to determine its efficacy and suitability as a commercial vaccine against B. bovis infection.

An ELISA for the diagnosis of Babesia ovis infection utilizing a synthetic, Babesia bovis-derived antigen.

The development of an enzyme-linked immunosorbent assay (ELISA) for the detection of Babesia ovis antibodies is described. In an initial study, a crude Babesia bovis antigen and a synthetic B. bovis-derived antigen (designated 11C5) were used to screen 46 B. ovis-positive and 55 negative sheep sera. A 95% correlation between the two antigenic preparations was found with the positive sera; no negative sera gave positive reactions. The synthetic antigen was then used in the screening of 1466 sera collected from sheep from 18 regions of Turkey. A high incidence of B. ovis-positive reactions was found from all regions (60-80%) in sheep over 1 year old, while from two smaller samples the incidence in young sheep was much less (28 and 52%). This test is superior to existing ones because the synthetic antigen can be produced in a highly reproducible state, is specific and is stable over extended periods of time.

Babesia bovis: immunity induced by vaccination with a lipid enriched fraction.

A chloroform extract from Babesia bovis-infected erythrocytes was used to vaccinate a group of five naive cattle. Following vaccination, the vaccinates, along with a group of control cattle, were challenged with a virulent heterologous strain of B. bovis. The vaccinates, comparative to the controls, showed delayed as well as decreased parasitaemias. The serological and initial biochemical studies suggested that the immune response was elicited by lipid of babesial origin.

Babesia bovis--effects of phospholipid translocation and adenosine tri-phosphate consumption.

The adenosine tri-phosphate concentration of Babesia bovis-infected erythrocytes was significantly (P less than 0.001) less than that of pre-infection erythrocytes. In addition, phosphatidyl serine was detected on the plasmatic surface of the infected erythrocyte. These two related findings could play important roles in the microvascular stasis characteristic of acute B. bovis infection.

An improved ELISA for the detection of antibodies against Babesia bovis using either a native or a recombinant B. bovis antigen.

Two new enzyme-linked immunosorbent assays (ELISA) for the diagnosis of Babesia bovis in cattle are described. The ELISA using a native antigen is more sensitive and less laborious than the assays described previously, because it does not require adsorption of sera with bovine erythrocytes. The second ELISA, using a recombinant B. bovis antigen expressed in Escherichia coli, was both sensitive and specific. It is suitable to replace the native antigen, thus avoiding large batch-to-batch variations in antigen preparations and the need to sacrifice experimental cattle.

Immunopathophysiology of babesial infections.

Humans infected with Plasmodium falciparum and bovines infected with Babesia bovis display severe haemolysis, alterations in red cell deformability and rigidity, and endothelial cell damage leading to pulmonary oedema and cerebral dysfunction. Much of the pathology associated with these infections is not easily attributable to the relatively small numbers of parasites present. This paper considers the possible roles of soluble mediators released from macrophages, and chemical and physical changes in the infected red cell membrane, in the pathogenesis of babesial infections, and also discusses the effects of prior immunization with various immunogens.