Research Accomplishment Reports 2007

Evaluation of Bacterial Endophytes of Grass and Legume Forages as Emerging Causes of Reproductive Loss

T.W. Swerczek
Department of Veterinary Sciences

 

Project Description

An emerging worldwide disease of fetal losses due to equine amnionitis and fetal loss syndrome appears to be affecting other herbivores, including cattle, goats and sheep. A newly recognized, unique micro-organism has now been demonstrated in all affected fetuses with pathognomonic lesions for the syndrome, including equine, bovine, caprine and ovine aborted fetuses. By using filtered fluids to remove bacteria and fungi from affected herbivore fetuses, a filterable agent was inoculated into developing chick embryos. The filterable agent induced the same pathognomonic microscopic lesions in the fetal membranes of the chick embryos that are seen in affected tissues of equine fetuses. Also, the suspect primary pathogen induced early, mid and late embryonic death in chick embryos with lesions consistent with those seen in affected equine fetuses. Since the micro-organism that has been isolated appears to be non-culturable on routinely used culture media, and is difficult to recognize in histologic tissues, alternative methods of tissue preparation and staining techniques on fresh mesothelial tissues were used to demonstrate the micro-organism in affected fetuses. The morphologic characteristics of the micro-organism, and PCR-based method of detection are consistent with those reported for the group of organisms belonging to the class Mollicutes and the genus Mycoplasma.

A micro-organism with the same morphologic characteristics has now been isolated in rabbit tissue culture cells and demonstrated in the mesothelial tissues of the placental membranes, primarily the amnion and umbilical cord, pericardium, eye, gut, lungs and joints of aborted equine fetuses and newborn foals, as well in other herbivores, including bovine, ovine and caprine aborted fetuses. The micro-organism has been demonstrated in the placental membranes, eyes, pericardium and in the mesothelial cells of the gut of aborted fetuses with fetal diarrhea, a syndrome of unknown etiology, but is closely associated with the fetal loss syndrome.

Because the spectrum of microbes present in the tissues of equine fetuses that abort as a result of the equine amnionitis and fetal loss syndrome has not been thoroughly identified, we have commenced a culture-independent, molecular characterization of the total microbial communities (bacteria, fungi, and mycoplasmas) present in such tissues. Internal transcribed spacer (ITS) regions of the ribosomal RNA genes ( rDNA) possess a sequence polymorphism that allows identification of non-culturable pathogens at the species level. The ribosomal ITS region of affected equine fetal tissues was amplified by PCR with pathogen specific primers, cloned into a plasmid vector, and amplified in E. coli to generate rDNA libraries.

Sequencing of individual clones from the rDNA libraries has revealed the existence of multiple species of bacteria, fungi, and mycoplasma in the amnion and internal tissues of the aborted fetuses. Comparative analysis of the microbiota present in tissues from a comprehensive set of unrelated disease cases is underway in an effort to identify one or more micro-organisms common to all of the clinical cases.

Impact

A newly recognized, unique micro-organism has been demonstrated in all cases of fetal losses associated with the disease equine amnionitis and fetal loss, an emerging worldwide fetal loss syndrome that appears to be affecting other herbivores, including cattle, sheep and goats. Since the syndrome was first recognized in 1980 as the cause of spikes in equine fetal losses, researchers have been suspecting an infectious etiologic agent as the primary cause of the fetal loss syndrome, but all attempts have been unsuccessful in recognizing a common infectious agent.

A novel filterable agent from affected aborted fetuses has now been isolated in developing chick embryos where pathognomonic lesions in chick embryos were the same as in aborted equine fetuses as well as other herbivore fetuses including, cattle, sheep and goats. The morphologic characteristics and molecular pattern of the isolated filterable agent are consistent with a group of bacteria in the class Mollicutes and the genus Mycoplasma. The micro-organism appears to be non-culturable on routinely used bacteriologic media, but has been isolated in chick embryos and in rabbit kidney tissue culture cells. By using molecular techniques, attempts will be made to determine if this novel micro-organism seen in all affected fetuses represents a single species, or a variety of similar micro-organisms with the same morphologic characteristics and growth requirements.

Molecular techniques will be utilized to determine the ecology of this micro-organism in the environment. Then procedures, including dietary contributing factors that may be associated with the syndrome, can be implemented to prevent this emerging worldwide fetal loss syndrome, and possibly other disease syndromes closely associated with these fetal losses which may be also associated with this filterable agent. These include fetal diarrhea, funiculitis of the umbilical cord, amnionitis, endo-opthalmitis, pericarditis, pneumonia, arthritis and laminitis, occurring in fetuses, foals and adults, as the micro-organism has now been demonstrated in lesions of organs with these syndromes of unknown etiologies.