ABSTRACT
Neosporosis is one of the major causes of abortion in cattle, and it is responsible for significant economic losses in those animals. Thus, this study aimed to evaluate indirect ELISA using subcellular fractions of Neospora caninum obtained via sucrose gradient separation. Eighty-five sera from dairy cattle previously tested using indirect immunofluorescence assay (IFA) were used. Three distinct bands were separated at 1.0 M, 1.4 M, 1.6 M, and the pellet at 1.8 M, which were identified as fractions one (F1), two (F2), three (F3), and four (F4), respectively. These fractions showed parasite membranes in the F1, rhoptry and conoids in the F2, mitochondria in the F3, and tachyzoite ghosts remain in F4. Indirect ELISAs for IgM, and IgG were performed. Additionally, sensitivity, specificity, and kappa values were defined considering the IFA as the gold standard. The highest and lowest specificities were observed for F1 (76 %) and F3 (16 %), respectively. F2 and F4 showed the highest sensitivity (93.3 %), kappa agreement (0.46), and Negative Preventive Value (NPV) (73 %) respectively. It was possible to standardize indirect ELISAs using whole soluble antigen and subcellular fractions of N. caninum, and F2 and F4 showed higher sensitivity (93.3 %), kappa (0.41), and NPV values (75 %) than F1, and F3, which could be used for epidemiology studies such as screening.
ABSTRACT
The Macavirus genus, Gammaherpesvirinae subfamily, Herpesviridae family, contains ovine gammaherpesvirus 2 (OvGHV2), the cause of sheep-associated malignant catarrhal fever (SA-MCF). Members of the Macavirus genus associated with the development of malignant catarrhal fever (MCF) in their respective hosts share the 15A antigenic epitope, are conserved within the DNA polymerase gene and are collectively referred to as the malignant catarrhal fever virus (MCFV) complex. The ability of MCFV and/or OvGHV2 to produce abortions in ruminants is currently unknown, with little documentation of infections by these agents in bovine fetuses. This report presents the findings observed due to the detection of OvGHV2 DNA and MCFV tissue antigens in aborted bovine fetuses from southern Brazil. Four aborted bovine fetuses from three farms, located in a geographical region of Paraná State with elevated immunohistochemical (IHC) prevalence of MCFV tissue antigens, with gestational ages varying between 78 to 208 days were investigated. Significant gross and histopathological alterations were not observed in any of these fetuses. An IHC assay using the 15A-monoclonal antibody (15A-MAb), which is based on the 15A antigenic epitope of Macavirus, identified MCFV tissue antigens in multiple organs from two fetuses (#1 and #4); however, positive immunoreactivity to the 15A-MAb IHC assay was not detected in Fetus #2 and #3. Molecular testing amplified OvGHV2 DNA only from the myocardium and lungs of Fetus #1 that had positive intracytoplasmic immunoreactivity to the 15A-MAb IHC assay in these tissues. Furthermore, infections by Leptospira spp. were confirmed by molecular assays in fetuses #1, #3, and #4, while PCR detected Neospora caninum in the myocardium of Fetus #2. Additionally, molecular assays to identify well-known fetopathy agents of cattle, including bovine viral diarrhea virus, bovine alphaherpesvirus 1, Histophilus somni, and Listeria monocytogenes, did not amplify the nucleic acids of these pathogens. PCR assays to identify bovine gammaherpesvirus 6 (BoGHV6), another Macavirus known to infect cattle in Brazil, were unsuccessful. These findings confirmed that the 15A-MAb IHC assay can be efficiently used to detect MCFV antigens in organs of aborted bovine fetuses. The identification of MCFV antigens with the simultaneous detection of OvGHV2 DNA confirmed that Fetus #1 was infected by OvGHV2 and added to the few descriptions of this infection in aborted fetuses of ruminants worldwide. Moreover, the IHC detection of MCFV in multiple organs of Fetus #4, without the molecular detection of OvGHV2 or BoGHV6, may suggest that this fetus was infected by a Macavirus that was not previously diagnosed in cattle herds from Brazil. These findings strongly suggest that OvGHV2 and MCFV can produce transplacental infections in cattle.