A nested-PCR strategy for molecular diagnosis of mollicutes in uncultured biological samples from cows with vulvovaginitis.

Bacteria classified in Mycoplasma (M. bovis and M. bovigenitalium) and Ureaplasma (U. diversum) genera are associated with granular vulvovaginitis that affect heifers and cows at reproductive age. The traditional means for detection and speciation of mollicutes from clinical samples have been culture and serology. However, challenges experienced with these laboratory methods have hampered assessment of their impact in pathogenesis and epidemiology in cattle worldwide. The aim of this study was to develop a PCR strategy to detect and primarily discriminate between the main species of mollicutes associated with reproductive disorders of cattle in uncultured clinical samples. In order to amplify the 16S-23S rRNA internal transcribed spacer region of the genome, a consensual and species-specific nested-PCR assay was developed to identify and discriminate between main species of mollicutes. In addition, 31 vaginal swab samples from dairy and beef affected cows were investigated. This nested-PCR strategy was successfully employed in the diagnosis of single and mixed mollicute infections of diseased cows from cattle herds from Brazil. The developed system enabled the rapid and unambiguous identification of the main mollicute species known to be associated with this cattle reproductive disorder through differential amplification of partial fragments of the ITS region of mollicute genomes. The development of rapid and sensitive tools for mollicute detection and discrimination without the need for previous cultures or sequencing of PCR products is a high priority for accurate diagnosis in animal health. Therefore, the PCR strategy described herein may be helpful for diagnosis of this class of bacteria in genital swabs submitted to veterinary diagnostic laboratories, not demanding expertise in mycoplasma culture and identification.

Association of Histophilus somni with spontaneous abortions in dairy cattle herds from Brazil.

This study investigated the participation of infectious agents in spontaneous abortions and reproductive problems at eight dairy cattle herds from three geographical regions of Brazil. Fourteen aborted fetuses and the organ sections of one cow with history of repeated abortions were received for pathological evaluations and molecular diagnostics. PCR/RT-PCR assays targeted specific genes of abortifacient agents of cattle: bovine viral diarrhea virus (BVDV), bovine herpesvirus 1 (BoHV-1), Listeria monocytogenes, Neospora caninum, Leptospira spp., Brucella abortus, and Histophilus somni. Six fetuses were adequate for pathological investigations; one of these did not demonstrate remarkable pathological alterations. Significant histopathological findings included vasculitis, hemorrhage, and fibrinous thrombosis of the cerebrum (n = 4); necrotizing myocarditis (n = 3); and hemorrhagic enteritis (n = 3). The placenta and uterus of the cow as well as the kidney, pancreas, and liver of her aborted fetus contained H. somni DNA and demonstrated histopathological evidence of histophilosis. All fetuses contained H. somni DNA in multiple organs. Coinfections of H. somni with B. abortus (n = 2), N. caninum (n = 2), BVDV (n = 1), and BoHV-1 (n = 1) were identified; two fetuses demonstrated three pathogens. These findings suggest that H. somni was associated with the spontaneous abortions and reproductive problems of these herds. However, the exact cause of fetal death might not be attributed only to H. somni in all aborted fetuses, since some of these were infected with other abortifacient agents.