Laura K. Bryan

Panfungal Polymerase Chain Reaction for Identification of Fungal Pathogens in Formalin-Fixed Animal Tissues:

Identification of fungal organisms often poses a problem for pathologists because the histomorphology of some fungal organisms is not specific, fresh tissues may not be available, and isolation and identification in culture may take a long time. The purpose of this study was to validate the use of panfungal polymerase chain reaction (PCR) to identify fungal organisms from formalin-fixed paraffin-embedded (FFPE) tissues. Formalin-fixed paraffin-embedded curls were tested from 128 blocks containing canine, feline, equine, and bovine tissues with cutaneous, nasal, pulmonary, and systemic fungal infections, identified by the presence of fungi in histologic sections. Quantitative scoring of histologic sections identified rare (11.9%), occasional (17.5%), moderate (17.5%), or abundant (53.1%) fungal organisms. DNA was isolated from FFPE tissues and PCR was performed targeting the internal transcribed spacer 2 (ITS-2) region, a segment of noncoding DNA found in all eukaryotes. Polymerase chain reaction products were sequenced and identified at ≥97% identity match using the Basic Local Alignment Search Tool and the NCBI database of ITS sequences. Of the 128 blocks, 117 (91.4%) yielded PCR products and high-quality sequences were derived from 89 (69.5%). Sequence and histologic identifications matched in 79 blocks (61.7%). This assay was capable of providing genus- and species-level identification when histopathology could not and, thus, is a beneficial complementary tool for diagnosis of fungal diseases.

Detection of vapN in Rhodococcus equi isolates cultured from humans

Rhodococcus equi can cause severe infections in people, particularly in immunocompromised individuals. The R. equi virulence plasmids (vap) encoding vapA and vapB are linked to development of infections in domestic animals. Recently, a novel virulence plasmid, vapN, was identified in isolates cultured from cattle, but its prevalence or significance in human R. equi infections has not been extensively studied. To determine the prevalence of vapN in a diverse collection of human-derived isolates from different countries, 65 R. equi isolates collected by various institutions from 1984 to 2002 were screened for the presence of vapN and other virulence plasmids through polymerase chain reaction (PCR) using redesigned primer sets. Of the isolates that carried plasmids, 43% (16/37) were vapN-positive and fewer were vapB or vapA-positive (30 and 16%, respectively). This is the first report of vapN carriage in R. equi isolated from human infections. One isolate (H-30) carried vapN but did not amplify the conjugal plasmid transfer gene traA associated with carriage of vap, which could be explained by sequence variation within the traA gene. Another isolate (H-55) amplified traA, but did not amplify vapA, B, or N (traA+ vapABN-) with previously described primer sets or those developed for this study. The H-55 traA sequence had 98% identity to traA sequences in vapA plasmids, which suggests that it may carry a variant of previously characterized virulence plasmids or a novel virulence plasmid. Carriage of vapN in R. equi isolates derived from people is not uncommon and more research is needed to determine its significance in the epidemiology and pathogenesis of human R. equi infections.

Rhodococcus equi Infections in Dogs

Five cases of Rhodococcus equi infection in dogs were identified from 2003 to 2014. Three of the dogs had severe, internal lesions attributable to R. equi that have not been previously described: endophthalmitis, endocarditis, and suppurative pleuropneumonia. Isolates from 4 of the dogs were analyzed by polymerase chain reaction for Rhodococcus virulence-associated plasmid (vap) genes. One isolate was vapA-positive, 2 lacked a virulence plasmid, and 1 carried the novel vapN-associated plasmid (pVAPN) recently characterized in bovine isolates. The pVAPN plasmid has not been described in isolates cultured from companion animals. Four of the dogs either were receiving immunosuppressive drugs or had endocrinopathies. R. equi has the potential to cause significant infections in dogs, and immunocompromised animals should be considered at risk for infection.

Chagas disease in a Texan horse with neurologic deficits.

A 10-year-old Quarter Horse gelding presented to the Texas A&M University Veterinary Teaching Hospital with a six month-history of ataxia and lameness in the hind limbs. The horse was treated presumptively for equine protozoal myeloencephalitis (EPM) based on clinical signs but was ultimately euthanized after its condition worsened. Gross lesions were limited to a small area of reddening in the gray matter of the thoracic spinal cord. Histologically, trypanosome amastigotes morphologically similar to Trypanosoma cruzi, the agent of Chagas disease in humans and dogs, were sporadically detected within segments of the thoracic spinal cord surrounded by mild lymphoplasmacytic inflammation. Ancillary testing for Sarcocystis neurona, Neospora spp., Toxoplasma gondii and Leishmania spp. was negative. Conventional and real time polymerase chain reaction (PCR) of affected paraffin embedded spinal cord were positive for T. cruzi, and sequencing of the amplified T. cruzi satellite DNA PCR fragment from the horse was homologous with various clones of T. cruzi in GenBank. While canine Chagas disease cases have been widely reported in southern Texas, this is the first report of clinical T. cruzi infection in an equid with demonstrable amastigotes in the spinal cord. In contrast to previous instances of Chagas disease in the central nervous system (CNS) of dogs and humans, no inflammation or T. cruzi amastigotes were detected in the heart of the horse. Based on clinical signs, there is a potential for misdiagnosis of Chagas disease with other infectious diseases that affect the equine CNS. T. cruzi should be considered as a differential diagnosis in horses with neurologic clinical signs and histologic evidence of meningomyelitis that originate in areas where Chagas disease is present. The prevalence of T. cruzi in horses and the role of equids in the parasite life cycle require further study.

Rhodococcus equi Infections in Goats: Characterization of Virulence Plasmids:

Rhodococcus equi is an uncommon cause of systemic pyogranulomatous infections in goats with macroscopic similarities to caseous lymphadenitis caused by Corynebacterium pseudotuberculosis. Caprine cases have previously been reported to be caused by avirulent R. equi strains. Six cases of R. equi infection in goats yielding 8 R. equi isolates were identified from 2000 to 2017. Lesions varied from bronchopneumonia, vertebral and humeral osteomyelitis, and subcutaneous abscesses, to disseminated infection involving the lungs, lymph nodes, and multiple visceral organs. Isolates of R. equi from infected goats were analyzed by polymerase chain reaction for R. equi virulence-associated plasmid (vap) genes. Seven of 8 isolates carried the VapN plasmid, originally characterized in bovine isolates, while 1 isolate lacked virulence plasmids and was classified as avirulent. The VapN plasmid has not been described in isolates cultured from goats.

The prevalence of Escherichia coli O157:H7 fecal shedding in feedlot pens is affected by the water-to-cattle ratio: A randomized controlled trial

Escherichia coli O157:H7 fecal shedding in feedlot cattle is common and is a public health concern due to the risk of foodborne transmission that can result in severe, or even fatal, disease in people. Despite a large body of research, few practical and cost-effective farm-level interventions have been identified. In this study, a randomized controlled trial was conducted to assess the effect of reducing the level of water in automatically refilling water-troughs on fecal shedding of E. coli O157:H7 in feedlot cattle. Pens in a feedlot in the Texas Panhandle were randomly allocated as control (total number: 17) or intervention (total number: 18) pens. Fecal samples (2,759 in total) were collected both at baseline and three weeks after the intervention, and tested for the presence of E. coli O157:H7 using immunomagnetic bead separation and selective culture. There was a strong statistical association between sampling date and the likelihood of a fecal sample testing positive for E. coli O157:H7. Pen was also a strong predictor of fecal prevalence. Despite accounting for this high level of clustering, a statistically significant association between reduced water levels in the trough and increased prevalence of E. coli O157:H7 in the feces was observed (Odds Ratio = 1.6; 95% Confidence Interval: 1.2–2.0; Likelihood Ratio Test: p = 0.02). This is the first time that such an association has been reported, and suggests that increasing water-trough levels may be effective in reducing shedding of E. coli O157:H7 in cattle feces, although further work would be needed to test this hypothesis. Controlling E. coli O157:H7 fecal shedding at the pre-harvest level may lead to a reduced burden of human foodborne illness attributed to this pathogen in beef.

Weaned pigs experimentally infected with Salmonella display sexually dimorphic innate immune responses without affecting pathogen colonization patterns123

Abstract Sexually dimorphic innate immune responses have been observed in several species, but have not been studied in response to a live pathogen challenge in pigs. This study aimed to elucidate sexually dimorphic innate immune responses along with Salmonella translocation patterns in newly weaned pigs orally inoculated with Salmonella. Newly weaned pigs (n = 8 gilts and 12 barrows; 6.2 ± 0.2 kg BW) were obtained from a commercial swine facility and were maintained in an environmentally-controlled facility in individual pens equipped with feeders and nipple waterers. Pigs were allowed ad libitum access to a commercial non-medicated starter ration and water throughout the study. On d 12 post-weaning, pigs were anesthetized to allow placement of a temperature measuring device in the abdominal cavity for measurement of intraperitoneal temperature (TEMP). On d 17, pigs were anesthetized and fitted with indwelling jugular vein catheters. On the following day (d 18), pigs were orally inoculated with 4.7x109Salmonella typhimurium. Blood samples were collected at 0.5-h intervals from -2 to 8 h, and at 8-h intervals from 8 to 72 h post-challenge. Whole blood was analyzed for complete blood cell counts. Serum was isolated for measurement of cortisol. Following collection of the 72 h sample, pigs were humanely euthanized and tissues were collected for Salmonella isolation. There was a sex × time interaction (P < 0.001) for TEMP such that gilts had a greater TEMP response to the Salmonella challenge compared to barrows. There was also a sex × time interaction (P = 0.03) for serum cortisol with gilts having decreased cortisol at 16 h yet greater cortisol at 32 h than barrows. Barrows had greater total white blood cells (17.8 vs. 16.2 ± 0.4 103 cells/μL; P < 0.01; respectively) and neutrophils (7.8 vs. 6.1 ± 0.4 103 cells/μL; P < 0.01; respectively) than gilts. However, gilts had greater lymphocytes (9.6 vs. 9.0 ± 0.2 103 cells/μL; P = 0.05; respectively) than barrows. While immune parameters were influenced by sex, there was no effect of sex (P > 0.05) on Salmonella concentrations from fecal shedding 3 d post-inoculation in the cecum, mesenteric and subiliac lymph nodes, liver, spleen, gallbladder, or kidney tissues. These data demonstrate that weaned gilts appear to produce a stronger acute phase response to a Salmonella challenge compared to barrows, without affecting the tissue translocation or shedding of Salmonella.