Best practices for performance of real-time PCR assays in veterinary diagnostic laboratories.

The exquisite sensitivity of in vitro amplification assays such as real-time polymerase chain reaction (rtPCR) requires the establishment of thorough and robust laboratory practices. To this end, an American Association of Veterinary Laboratory Diagnosticians (AAVLD) committee of subject matter experts was convened to develop a set of best practices for performance of nucleic acid amplification assays. Consensus advice for the performance of preanalytical, analytical, and postanalytical steps is presented here, along with a review of supporting literature.

NeoSeekTM STEC: A Multiplex Molecular Method for Detection and Identification of Select Shiga Toxin-Producing Escherichia coli in Beef.

BACKGROUND: NeoSeekTM STEC is a single-source, service-based method for detection and identification of select Shiga toxin-producing Escherichia coli (STEC), including E. coli O157:H7 and STEC of somatic groups O26, O45, O103, O111, O121, and O145. The method is a multiplex molecular method utilizing more than 80 genetic targets to identify STEC in complex matrices such as food enrichment cultures. OBJECTIVE: A study was conducted to validate the NeoSeek method for detection of select STEC in raw beef trim. METHODS: Performance of the NeoSeek STEC method was compared with that of the U.S. Department of Agriculture, Food Safety and Inspection Service reference methods for E. coli O157:H7 and non-O157 STEC for detection of E. coli O157:H7 and E. coli O26:H11 in raw beef trim. Additionally, inclusivity/exclusivity testing and method robustness testing were performed. RESULTS: Results of raw beef trim testing showed no statistically significant differences in performance between the NeoSeek and reference methods in the ability to detect either E. coli O157:H7 or E.coli O26:H11, as determined by probability of detection analysis. Results of inclusivity and exclusivity testing showed 100% expected results with target and nontarget bacteria, with the exception of a single strain of E. coli O157:H7, which was subsequently verified to be stx-negative by PCR. Conclusions and Highlights: NeoSeek STEC is an accurate, reliable method for rapid detection and identification of select STEC in complex populations such as beef trim enrichment cultures.

Evaluation of Rectoanal Mucosal Swab Sampling for Molecular Detection of Enterohemorrhagic Escherichia coli in Beef Cattle.

Cattle are a primary reservoir of enterohemorrhagic Escherichia coli (EHEC), and contaminated beef products are a source of human infections. The U.S. Department of Agriculture Food Safety and Inspection Service declared seven EHEC serogroups (O26, O45, O103, O111, O121, O145, and O157) as adulterants in raw ground beef. Sampling a large number of animals for EHEC surveillance or evaluations of EHEC-focused preharvest interventions requires a convenient and robust sampling method. We evaluated the diagnostic performance of rectoanal mucosal swab (RAMS) for the detection of the top seven EHEC serogroups. Paired fecal grab (FG) and RAMS samples were collected from 176 beef cattle and tested using the NeoSEEK Shiga toxin-producing E. coli (STEC) confirmation method. The prevalence of virulence-associated genes (stx1, stx2, stx2c, eae, and nleB) was higher in RAMS than in FG samples. The results of the two methods had poor agreement, as indicated by kappa statistics, for the detection of the seven serogroups. When FG and RAMS results were combined for comparison, RAMS was more sensitive than FG for the detection of serogroups O103 (82% versus 39%), O157 (75% versus 67%), and O45 (79% versus 73%) with similar sensitivity for the detection of serogroup O145 (67%). Serogroups O111 and O121 were detected from one and two samples, respectively, by FG and were not detected by RAMS. Serogroup O26 was not detected with either method. RAMS appears to be equivalent or superior to FG sampling for detection of the top seven EHEC serogroups in the feces of beef cattle with the NeoSEEK STEC confirmation test.

Characterization of Enterohemorrhagic Escherichia coli on Veal Hides and Carcasses.

Enterohemorrhagic Escherichia coli (EHEC) are Shiga toxin-producing E. coli associated with the most severe forms of foodborne illnesses. The U.S. Department of Agriculture, Food Safety and Inspection Service has identified a higher percentage of non-O157 EHEC compared with E. coli O157:H7-positive samples collected from veal trimmings than from products produced from other cattle slaughter classes. Therefore samples were collected from hides and preevisceration carcasses at five veal processors to assess E. coli O157:H7 and non-O157 EHEC contamination during bob veal and formula-fed veal dressing procedures. E. coli O157:H7 prevalence was measured by culture isolation and found to be on 20.3% of hides and 6.7% of carcasses. In contrast, a non-O157 EHEC molecular screening assay identified 90.3% of hides and 68.2% of carcasses as positive. Only carcass samples were taken forward to culture confirmation and 38.7% yielded one or more non-O157 EHEC isolates. The recovery of an EHEC varied by plant and sample collection date; values ranged from 2.1 to 87.8% among plants and from 4.2 to 64.2% within the same plant. Three plants were resampled after changes were made to sanitary dressing procedures. Between the two collection times at the three plants, hide-to-carcass transfer of E. coli O157:H7 and non-O157 EHEC was significantly reduced. All adulterant EHEC serogroups (O26, O45, O103, O111, O121, and O145) were isolated from veal carcasses as well as four other potentially pathogenic serogroups (O5, O84, O118, and O177). Bob veal was found to have a greater culture prevalence of E. coli O157:H7 and greater positive molecular screens for non-O157 EHEC than formula-fed veal (P < 0.05), but the percentage of culture-confirmed non-O157 EHEC was not different (P > 0.05) between the two types of calves. EHEC-O26, -O111, and -O121 were found more often in bob veal (P < 0.05), whereas EHEC-O103 was found more often in formula-fed veal (P < 0.05).

Effects of Moraxella (Branhamella) ovis culture filtrates on bovine erythrocytes, peripheral mononuclear cells, and corneal epithelial cells.

Infectious bovine keratoconjunctivitis (IBK) is a highly contagious ocular disease that affects cattle of all ages and that occurs worldwide. Piliated hemolytic Moraxella bovis is recognized as the etiologic agent of IBK. According to data from the Nebraska Veterinary Diagnostic Laboratory System, however, Moraxella (Branhamella) ovis has been isolated with increasing frequency from cattle affected with IBK. The objective of this study was, therefore, to examine M. ovis field isolates for the presence of the putative virulence factors of M. bovis. Culture filtrates from selected M. ovis field isolates demonstrated hemolytic activity on bovine erythrocytes and cytotoxic activity on bovine peripheral blood mononuclear cells and corneal epithelial cells. The hemolytic activity of the culture filtrates was attenuated after heat treatment. Polyclonal antibodies raised against the M. bovis hemolysin-cytotoxin also recognized a protein of approximately 98 kDa in a Western blot assay. These data indicate that the M. ovis field isolates examined produce one or more heat-labile exotoxins and may suggest that M. ovis plays a role in the pathogenesis of IBK.

Characterization of cytolethal distending toxin of campylobacter species isolated from captive macaque monkeys.

An association between certain Campylobacter species and enterocolitis in humans and nonhuman primates is well established, but the association between cytolethal distending toxin and disease is incompletely understood. The purpose of the present study was to examine Campylobacter species isolated from captive conventionally raised macaque monkeys for the presence of the cdtB gene and for cytolethal distending toxin activity. The identity of each isolate was confirmed on the basis of phenotypic and genotypic analyses. The presence of cytolethal distending toxin was confirmed on the basis of characteristic morphological changes in HeLa cells incubated with filter-sterilized whole-cell lysates of reference and monkey Campylobacter isolates and examinations by light microscopy, confocal microscopy, and flow cytometry. Although cdtB gene sequences were found in both Campylobacter jejuni and Campylobacter coli, the production of cytolethal distending toxin correlated positively (P < 0.0001) only with C. jejuni. We concluded that cytolethal distending toxin activity is a characteristic of C. jejuni. Our C. jejuni cdtB gene-specific PCR assay might be of assistance for differentiating toxigenic C. jejuni from C. coli in clinical laboratories.

Decreased shedding of Escherichia coli O157:H7 by cattle following vaccination with type III secreted proteins.

Cattle are an important reservoir of Escherichia coli O157:H7 leading to contamination of food and water, and subsequent human disease. This pathogen colonizes its hosts by producing several proteins such as Tir and EspA that are secreted by a type III secretion system. These proteins play a role in colonization of the intestine, suggesting that they might be useful targets for the development of a vaccine to reduce levels of this organism in cattle. Vaccination of cattle with proteins secreted by E. coli O157:H7 significantly reduced the numbers of bacteria shed in feces, the numbers of animals that shed, and the duration of shedding in an experimental challenge model. Vaccination of cattle also significantly (P=0.04) reduced the prevalence of E. coli O157:H7 in a clinical trial conducted in a typical feedlot setting. This strategy suggests it is possible to vaccinate cattle to decrease the level of E. coli O157:H7 shedding for the purpose of reducing the risk of human disease.