Accuracy of two commercial enzyme-linked immunosorbent assays for the detection of antibodies to Salmonella spp. in slaughter pigs from Canada

Large discrepancies are usually found when different ELISAs for the diagnosis of pig salmonellosis are compared. Thus, our main goal was to estimate the diagnostic accuracy through Bayesian approaches of two commercial assays (Svanovir™ “test A” and HerdCheck™ “test B”) for the detection of antibodies to Salmonella spp. in slaughter pigs. Previously, we estimated the agreement between both tests and their relative sensitivity (Se) and specificity (Sp) with respect to bacteriology on caecal content and ileocaecal lymph nodes. Test A, at a cut-off OD% ≥20%, indicated higher prevalence than test B (OD% ≥10%) (14.6% vs. 8.6%). Relative Se with respect to overall bacteriology was low (≈30%) and similar for both tests, but the relative Sp was significantly lower for test A compared to B (88% vs. 95%). Both tests failed to detect some pigs infected with Salmonella serogroups B and C₁, which they were supposed to identify. In general, tests showed only fair-to-moderate agreement when they were compared (kappa: 0.41). In the Bayesian models, Se of test A varied between 63% and 77%, while Se of test B was 73%. Sp of A was always lower than that of test B (89% vs. 95%). The implications derived from the use of these imperfect serological tests will have to be accounted for in large-scale Salmonella-control programs.     

Comparison of bacterial culture and real-time PCR for the detection of Salmonella in grow-finish pigs in Western Canada using a Bayesian approach

The study objective was to evaluate the accuracy of a real-time polymerase chain reaction (RT-PCR) and a culture protocol used to detect Salmonella in the faeces of grow-finish pigs using a Bayesian approach. The RT-PCR was invA-gene-based assay, while the culture protocol included pre-enrichment in buffered peptone water, selective enrichment in tetrathionate and Rappaport-Vassiliadis broths, and isolation on semi-solid (modified semi-solid RV) or solid (XLT4, Rambach) agar plates. Bayesian analysis was performed using a two-test, two-population model with dependence between culture and RT-PCR and compared to a second model with conditional independence between these two tests. Two hundred and ninety three individual faecal and 294 pooled pen samples from grow-finish pig collected from 10 farms were tested and results were divided into two groups according to herd size (five herds <250 sows, five herds with >400 sows). In the dependence model, RT-PCR sensitivity (Se) and specificity (Sp) were estimated to be 90% (95% probability interval 74, 97) and 99% (98, 99), respectively. Culture Se was 92% (75, 99), while culture Sp was considered 100% as all culture-positive samples were confirmed by serotyping. In the conditional independence model, RT-PCR Se and Sp, and culture Se, were 96% (93, 98), 99% (98, 100) and 97% (94, 100), respectively. The dependence model resulted in posterior estimates of Se that were lower and with broader probability intervals than the independence model, indicating that when RT-PCR and culture are evaluated relative to each other, the correlation between these tests is an important source of bias and should be adjusted for during analysis. The RT-PCR evaluated in this study performed almost comparably to culture; given the cost savings associated with using this test and more timely results, the RT-PCR may be a useful alternative to culture for screening large numbers of samples, particularly when Salmonella prevalence is low.     

Comparison of bacterial culture, polymerase chain reaction, and a mix-enzyme-linked immunosorbent assay for the detection of Salmonella status in grow-to-finish pigs in western Canada with a Bayesian approach

Among grow-to-finish pigs from 10 herds in Alberta and Saskatchewan, 23 (16%) of 144 fecal samples were culture-positive and 40 (28%) of 144 pigs were seropositive for Salmonella. With a Bayesian model specifying dependence between the 2 tests, the sensitivity (Se) of culture and real-time polymerase chain reaction (RT-PCR) was 79% to 86%, depending on the cut-off value for the enzyme-linked immunosorbent assay (ELISA). Culture specificity (Sp) was assumed to be 100%; RT-PCR Sp was found to be 94%. The ELISA Se was 76% and 51% at optical density cut-off values ≥ 20% and ≥ 40%, respectively; the Sp was 94% at each cut-off value. The model showed some sensitivity to ELISA prior information, the ELISA Se being approximately 8% lower when informative prior information was specified in the model. When there was no adjustment for dependence between culture and RT-PCR, the posterior estimates for both culture and RT-PCR Se were 11% higher than with the conditional-dependence model and had considerably narrower probability intervals, which suggests that correlation between culture and PCR is important and should be adjusted for in future studies.     

Evaluation of two herd-level diagnostic tests for Streptococcus agalactiae using a latent class approach

Streptococcus agalactiae mastitis persists as a significant economic problem for the dairy industry in many countries. In Denmark, the annual surveillance programme for this mastitis pathogen initially based only on bacteriological culture of bulk tank milk (BTM) samples, has recently incorporated the use of the real-time PathoProof Mastitis PCR assay with the goal of improving detection of infected herds. The objective of our study was to estimate the herd sensitivity (Se) and specificity (Sp) of both tests of BTM samples using latent class models in a Bayesian analysis while evaluating the effect of herd-level covariates on the Se and Sp of the tests. BTM samples were collected from all 4258 Danish dairy herds in 2009 and screened for the presence of S. agalactiae using both tests. The highest Se of PCR was realized at a cycle threshold (Ct) cut-off value of 40. At this cut-off, the Se of the PCR was significantly higher (95.2; 95% posterior credibility interval [PCI] [88.2; 99.8]) than that of bacteriological culture (68.0; 95% PCI [55.1; 90.0]). However, culture had higher Sp (99.7; 95% PCI [99.3; 100.0]) compared to PCR (98.8; 95% PCI [97.2; 99.9]). The accuracy of the tests was unaffected by the herd-level covariates. We propose that screenings of BTM samples for S. agalactiae be based on the PCR assay with Ct readings of <40 considered as positive. However, for higher Ct values, confirmation of PCR test positive herds by bacteriological culture is advisable especially when the between-herd prevalence of S. agalactiae is low.     

THE ISOLATION OF SALMONELLA FROM JEJUNAL AND CAECAL LYMPH NODES OF SLAUGHTERED ANIMALS

One jejunal and one caecal lymph node were sampled from each of 50 cows, 40 yearling cattle, 25 sheep, 20 lambs and 45 pigs after slaughter. Salmonella, Clostridium perfringens and Staphylococcus aureus , all organisms which cause food poisoning in man, were sought by direct plating methods. The samples were also enriched and cultured for Salmonella. Organisms were cultured from 208 (58%) of the 360 lymph nodes; aerobic plate counts yielded up to 25,000 organisms per gram of tissue, although from most infected samples less than 1000 organisms per gram were cultured. Salmonella was isolated directly from 5% of samples, with counts up to 1,500 per gram. After enrichment Salmonella was isolated from nodes taken from 15 cows, 2 yearling cattle, one sheep and 8 pigs. Cl. perfringens was isolated from the caecal nodes of 2 yearling cattle and 2 pigs; S. aureus was not isolated from any sample. It was concluded that mesenteric lymph nodes may be a significant reservoir of Salmonella for transfer to meat and meat products.     

Improved diagnostic and real-time pcr in rapid screening for Salmonella in the poultry food chain

The goal of this study was to improve the diagnostic applicability of genus- and serovar- (S. Enteritidis and S. Typhimurium) specific PCR systems in screening faecal and caecal samples of poultry, poultry feed and poultrymeat for Salmonella, by keeping the opportunity to obtain Salmonella cultures from positive samples. Peptone broth pre-enrichment cultures of the samples were tested by PCR. In faecal and caecal samples from broiler chicks a strong inhibitory action was frequently observed. This could be reduced markedly by the addition of bovine serum albumin (BSA) acting as amplification facilitator. The results of testing pre-enrichment cultures from artificially contaminated faecal, poultry feed and poultrymeat samples (using S. Enteritidis, S. Typhimurium and S. Hadar as contaminants) suggest that the sensitivity of the above systems is 10(1)-10(2) CFU g(-1) sample. The testing of 95 caecal samples from slaughtered chicks resulted in 49% culture-positive and 76% PCR-positive samples. The suitability of a generic real-time PCR for testing faecal samples of poultry was also studied. Its detection limit for these samples was found to be lower than that of the diagnostic PCR system. Both methods reduced the time required for Salmonella detection to 24-30 h, and the advantage of the real-time PCR was its increased sensitivity. We have established a diagnostic and a real-time PCR system for rapid and reliable genus- and serovar- (S. Enteritidis and S. Typhimurium) specific detection of Salmonella for monitoring purposes in the poultry food chain. Sensitivity is equal to, or higher than, that of the standard bacterial culture method, and the method still provides the Salmonella culture if needed.     

Prevalence of Salmonella serotypes on pig carcasses from high- and low-risk herds slaughtered in three abattoirs

The aim of this study was to compare the prevalence of Salmonella serotypes at two different sites on pig carcasses from herds classified as high-risk or low-risk and to elucidate the relationship between carcass contamination levels and serological status. Caecal samples and carcass surface swabs were cultured for Salmonella from a total of 210 pigs from low risk herds (< 19% of pigs in herd Salmonella seropositive) and 209 pigs from high risk herds (> 32% of pigs in herd Salmonella seropositive) in three abattoirs. Meat juice samples were collected for analysis by ELISA. The prevalence of Salmonella in the caecal contents of "low-risk" pigs was 10%, which was significantly lower than the 19% prevalence in "high-risk" pigs (p < 0.01). The corresponding figures for skin samples collected immediately post-evisceration were 2% and 12%. The predominant Salmonella serotype in the caecal contents of both the low-risk and high-risk pigs was Salmonella Typhimurium. Salmonella Kentucky and Salmonella Derby were the most frequent isolates from the carcass surface swabs of low- and high-risk pigs respectively. There was a positive association between seropositivity of pigs from high-risk herds and caecal carriage (p < 0.05). Results showed that herd categorisation based on serological results was useful in predicting Salmonella isolation rates from caecal samples and surface swabs of slaughtered pigs.     

Diagnostic performance measures of ELISA and quantitative PCR tests for porcine circovirus type 2 exposure using Bayesian latent class analysis

Porcine circovirus 2 (PCV2) is believed to be a necessary but not sufficient underlying cause of porcine circovirus associated disease (PCVAD) in swine (Opriessnig et al., 2007). Since the potential threat of PCVAD is dependent on the prevalence of PCV2 in swine populations, accurate diagnostic tests are important for epidemiologic surveillance. Therefore, we evaluated the diagnostic sensitivity (Se) and specificity (Sp) of a new indirect ELISA and two quantitative PCR tests for PCV2 in a series of latent class models that used Bayesian estimation procedures. A total of 4140 samples from finisher pigs were tested for evidence of PCV2 by the ELISA and a TaqMan (TM) quantitative PCR, 995 by the ELISA and a SYBR Green (SG) dye-binding PCR, 998 by both PCRs and 993 by all three tests. Overall, the median (95% probability interval) ELISA Se and Sp was 0.85 (0.83-0.87) and 0.74 (0.68-0.79), respectively, when all three tests were analyzed together at an ELISA absorbance (optical density or OD) cutoff of ≥0.3. The TM PCR Se and Sp was 0.86 (0.84-0.88) and 0.94 (0.87-0.97), respectively, and the SG PCR Se and Sp was 0.83 (0.81-0.85) and 0.98 (0.94-1.00), respectively when all three tests were analyzed together at an ELISA OD cutoff of ≥0.3. Sensitivity analysis revealed that Sp estimates in general had less stability than Se estimates, but the SG PCR(Sp) was the most stable. Limited conditional dependence between the two PCR tests was detected. We conclude that the ELISA had the highest diagnostic Se at an absorbance cutoff of ≥0.3, while the SG PCR had the highest diagnostic Sp. The prevalence levels for exposure to PCV2 in finishing swine populations across all analyses ranged from 58 to 100%.     

Latent class analysis of the diagnostic characteristics of PCR and conventional bacteriological culture in diagnosing intramammary infections caused by Staphylococcus aureus in dairy cows at dry off

Background

Staphylococcus aureus is one of the most common causes of intramammary infections in dairy cows at dry off. Reliable identification is important for disease management on herd level and for antimicrobial treatment of infected animals. Our objective was to evaluate the test characteristics of PathoProof ™ Mastitis PCR Assay and bacteriological culture (BC) in diagnosing bovine intramammary infections caused by S. aureus at dry off at different PCR cycle threshold (Ct)-value cut-offs.

Methods

Sterile quarter samples and non-sterile composite samples from 140 animals in seven herds were collected in connection with the dairy herd improvement (DHI) milk recording. All quarter samples were analyzed using BC whereas all composite samples were analyzed with PathoProof ™ Mastitis PCR Assay. Latent class analysis was used to estimate test properties for PCR and BC in the absence of a perfect reference test. The population was divided into two geographically divided subpopulations and the Hui-Walter 2-test 2-populations model applied to estimate Se, Sp for the two tests, and prevalence for the two subpopulations.

Results

The Se for PCR increased with increasing Ct-value cut-off, accompanied by a small decrease in Sp. For BC the Se decreased and Sp increased with increasing Ct-value cut-off. Most optimal test estimates for the real-time PCR assay were at a Ct-value cut-off of 37; 0.93 [95% posterior probability interval (PPI) 0.60-0.99] for Se and 0.95 [95% PPI 0.95-0.99] for Sp. At the same Ct-value cut-off, Se and Sp for BC were 0.83 [95% PPI 0.66-0.99] and 0.97 [95% PPI 0.91-0.99] respectively. Depending on the chosen PCR Ct-value cut-off, the prevalence in the subpopulations varied; the prevalence increased with increasing PCR Ct-value cut-offs.

Conclusion

Neither BC nor real-time PCR is a perfect test in detecting IMI in dairy cows at dry off. The changes in sensitivity and prevalence at different Ct-value cut-offs for both PCR and BC may indicate a change in the underlying disease definition. At low PCR Ct-value cut-offs the underlying disease definition may be a truly/heavily infected cow, whereas at higher PCR Ct-value cut-offs the disease definition may be a S. aureus positive cow.     

Identification of sources of Salmonella organisms in a veterinary teaching hospital and evaluation of the effects of disinfectants on detection of Salmonella organisms on surface materials

OBJECTIVE: To determine sources of Salmonella organisms in a veterinary teaching hospital, compare bacterial culture with polymerase chain reaction (PCR) testing for detection of Salmonella organisms in environmental samples, and evaluate the effects of various disinfectants on detection of Salmonella organisms on surface materials. DESIGN: Prospective study. SAMPLE POPULATION: Fecal samples from 638 hospitalized horses and 783 environmental samples. PROCEDURE: Standard bacterial culture techniques were used; the PCR test amplified a segment of the Salmonella DNA. Five disinfectants were mixed with Salmonella suspensions, and bacterial culture was performed. Swab samples were collected from 7 surface materials after inoculation of the surfaces with Salmonella Typhimurium, with or without addition of a disinfectant, and submitted for bacterial culture and PCR testing. RESULTS: Salmonella organisms were detected in fecal samples from 35 (5.5%) horses. For environmental samples, the proportion of positive bacterial culture results (1/783) was significantly less than the proportion of positive PCR test results (110/783), probably because of detection of nonviable DNA by the PCR test. Detection of Salmonella organisms varied with the surface material tested, the method of detection (bacterial culture vs PCR testing), and the presence and type of disinfectant. CONCLUSIONS AND CLINICAL RELEVANCE: Results of the present study suggested that Salmonella organisms can be isolated from feces of hospitalized horses and a variety of environmental surfaces in a large animal hospital. Although recovery of Salmonella organisms was affected by surface material and disinfectant, bleach was the most effective disinfectant on the largest number of surfaces tested.     

Accuracy of the composite somatic cell count to detect intra-mammary infection in dairy cows using latent class analysis

The somatic cell count (SCC) is considered an important indicator of intra-mammary infection (IMI). The purpose of this study was to determine the accuracy of both SCC and culture to detect IMI and their conditional dependence by means of latent class methods. This study involved 175 dairy cows from 2 herds with different udder infection prevalences. Quarter and composite milk samples were collected for SCC and bacteriological culture. Latent-class models using Bayesian methods were used to estimate test sensitivity (Se) and specificity (Sp) and population prevalence. The models ran involved only major mastitis pathogens and composite SCC (CSCC). Five thresholds between 100,000 and 300,000 cells/mL were evaluated and the receiver operating characteristics (ROC) curve analysis was performed. Fifty-five percent of the cows had CSCC ≥200,000 cells/mL and 95.4% of the cows had at least one infected quarter either with minor or major pathogens. Considering a threshold of 150,000 cells/mL, the estimated Se and Sp for the CSCC were, 0.80 (95% CrI 0.71–0.88) and 0.57 (95% CrI 0.44–0.71), respectively. The estimated culture Se and Sp were 0.83 (95% CrI 0.73–0.93) and 0.89 (95% CrI 0.74–0.98), respectively. There was no evidence of dependence between CSCC and culture. The area under curve for CSCC was 0.72. To the best of our knowledge, this is the first report of the CSCC accuracy to detect IMI for major pathogens considering the effect of culture misclassification. The estimates provided here could help to examine the performance of sampling schemes based on CSCC to manage udder health.     

Validation of a method for the detection of virulent Yersinia enterocolitica and their distribution in slaughter pigs from conventional and alternative housing systems

Various methods have been described in the literature for the detection of virulent Yersinia enterocolitica in pigs. The risk factors for pig herd contamination have yet to be determined. The objective of this study was to validate a sensitive method for the detection of Y. enterocolitica and to describe the distribution of the bacteria in pigs at slaughter from conventional and alternative ("organic") housing systems. First, samples were collected from tonsils, caecum with caecal contents, and the caecal lymph nodes of 60 slaughter pigs. These samples were used to compare the sensitivity of six different laboratory culture methods either in common use or described in the literature with that of a polymerase chain reaction with two primer pairs (multiplex PCR). Then, only PCR was used to examine tonsils, caecum and caecal lymph nodes from two groups of slaughter pigs: 210 from six conventional fattening farms and 200 from three with alternative housing. The results of the multiplex PCR were positive in 28 cases. All culture methods proved inferior to PCR in sensitivity. In the second part of the study, PCR detected 36 (18%) positive pigs from alternative housing and 60 (29%) from conventional housing (p<0.05). The highest rate of Y. enterocolitica contamination was found in tonsils (11% alternative, 22% conventional; p<0.05), followed by caecum (5%, 11%) and lymph nodes (2%, 7%). The housing system appears to be one important factor in the prevalence of this common pathogen in pig herds, as we found important differences between the two systems studied here. In the conventional system, the main risk factors appeared to be sourcing pigs from different pig suppliers, use of commercial feed and transportation to slaughter.     

Examining heterogeneity in the diagnostic accuracy of culture and PCR for Salmonella spp. in swine: a systematic review/meta-regression approach

The accuracy of bacterial culture and PCR for Salmonella in swine was examined through systematic review of existing primary research in this field. A replicable search was conducted in 10 electronic databases. All steps of the review were conducted by two reviewers: to identify relevant publications, to assess their methodological soundness and reporting, and to extract raw data or reported test accuracy estimates. Meta-analyses and meta-regression were performed: to evaluate pooled estimates of test sensitivity (Se) and specificity (Sp), to identify variables explaining the variation in reported test estimates, and to evaluate the association between these variables and reported test Se and Sp. Twenty-nine studies were included in the review. Unique test evaluations reported in these 29 studies were categorized according to the type of test comparison: culture versus culture (n = 134 test evaluations) and PCR versus culture (n = 21). We identified significant heterogeneity among evaluations for each test category. For culture, more heterogeneity was caused by differences in individual test protocols (52%) than overall differences between studies (16%). Enrichment temperature, study population, agar and enrichment type were significantly associated with variation in culture Se. Furthermore, interaction between enrichment temperature and enrichment type was detected. For PCR, most of the heterogeneity was caused by overall differences between studies (65-70%); sample type and study size were associated with variation in reported PCR Se and Sp. The overall methodological soundness and/or reporting of primary studies included in this review were poor, with variable use of reference standards, and consistent lack of the use or reporting of blinding, randomization and subject (sample) selection criteria. Consequently, the food safety and veterinary public health research community should formally consider ways for standardizing the conduct and reporting of this type of research.     

Effects of feed particle size at dietary presence of added organic acids on caecal parameters and the prevalence of Salmonella in fattening pigs on farm and at slaughter

In a field study with fattening pigs, effects of feed particle size at the dietary presence of organic acids on Salmonella prevalence were measured. On two farms (f1/f2), each holding ～800 pigs, diets based on finely ground (control) or coarsely ground ingredients (experiment) were fed as crumbs. On f1 both control and experimental grower and finisher diets contained identical concentrations of formic and propionic acid (0.4% and 0.2% respectively). On f2 only finisher diet of the experimental group contained 1.2% potassium diformate. At the start of the fattening period no statistical differences were measured between Salmonella prevalence in animals fed control and experimental diets on both farms. At slaughter Salmonella prevalence in caecal contents was lower (p < 0.05) on f1 in animals fed the experimental diet. Furthermore, the number of seronegative meat juice samples taken from these animals [optical density (OD) <10] was higher (p < 0.001); seropositive as well as distinct seropositive samples (OD ≥20 and ≥40 respectively) were less frequent (p < 0.01) compared to samples from animals fed the control diet. Feeding the experimental diet on f2 resulted in a lower Salmonella detection rate in faeces before slaughter (p < 0.01). Salmonella prevalence was lower in caecal content at slaughter for pigs fed the experimental diet compared to those fed control diet (p < 0.0001). The number of distinct seropositive meat juice samples (OD ≥40) was lower (p < 0.01) for pigs fed the experimental than for those fed the control diet. In comparison to pigs in the control group, starch concentrations in the caecal content from pigs in the experimental groups on both farms were higher (p < 0.05) and the pH values lower (p < 0.05). Propionate (p < 0.0001) and butyrate (p < 0.01) concentrations were higher in the caecal content taken from pigs in the experimental group on f2.     

Evaluation of a PCR to detect Salmonella in fecal samples of horses admitted to a veterinary teaching hospital.

The diagnostic accuracy of a PCR used to identify horses shedding Salmonella spp. in their feces during hospitalization was estimated, relative to bacterial culture of serially collected fecal samples, using longitudinal data. Five or more fecal samples were collected from each of 116 horses admitted as inpatients, for reasons other than gastrointestinal disease, between July 26, 2001 and October 25, 2002. All 873 fecal samples collected were tested with a PCR based on oligonucleotide primers defining a highly conserved segment of the histidine transport operon gene of Salmonella typhimurium, and each sample was cultured for Salmonella spp. One or more samples from 87 (75%) horses were PCR positive, and Salmonella was cultured from 1 or more samples from 11 (9.5%) horses. All culture-positive horses had at least 1 PCR-positive result, whereas only 29 (28%) culture-negative horses were PCR negative on all fecal samples tested. The PCR was most specific, relative to bacterial culture of serially collected fecal samples, when used to test samples from Quarterhorse or breeds other than Thoroughbred or Standardbred, or from clinical (vs. healthy, accompanying horses) cases. Overall, the PCR had the greatest agreement (70%), compared with bacterial culture of serially collected fecal samples, using a cutoff of 2 or more positive PCR test results to define a Salmonella-positive horse. The reasons why some fecal samples, from which Salmonella organisms cannot be isolated, are PCR positive need to be determined before the PCR can be incorporated into Salmonella surveillance programs for hospitalized equine populations.     

Estimation of sensitivity and specificity of five serological tests for the diagnosis of porcine brucellosis

While serological tests are essential in surveillance and control programs of animal diseases, to date none of the common serological tests approved in the EU (complement fixation test or Rose-Bengal test) has been shown to be reliable in routine individual diagnosis of porcine brucellosis, and some more recent tests like ELISA have not been fully evaluated yet. In the absence of a gold standard, this study allowed the estimation of sensitivities and specificities of these tests with a Bayesian approach using Markov Chain Monte Carlo algorithms. The pig population that was tested included 6422 animals from Metropolitan France. Serum samples were collected from a large population of pigs, representative of European swine population and tested with five brucellosis serological tests: Rose-Bengal test (RBT), fluorescence polarization assay (FPA), indirect ELISA (I-ELISA) and two competitive ELISAs (C-ELISA). The sensitivity and the specificity of each test were estimated. When doubtful results were excluded, the most sensitive and specific test was C-ELISA(2) (Se C-ELISA(2)=0.964, [0.907; 0.994], 95% credibility interval (CrI); Sp C-ELISA(2)=0.996, [0.982; 1.0], 95% CrI). When doubtful results were considered as negative, C-ELISA(2) was still the most sensitive and specific test (Se C-ELISA(2)=0.960, [0.896; 0.994], 95% CrI and Sp C-ELISA(2)=0.994, [0.977; 0.999], 95% CrI). The same conclusions were reached when doubtful results were considered as positive (Se C-ELISA(2)=0.963, [0.904, 0.994], 95% CrI and Sp C-ELISA(2)=0.996, [0.986; 1.0], 95% CrI).     

Estimation of the diagnostic accuracy of the glyco-C and US9 gene-based polymerase chain reaction technique for the detection of bovine Herpesvirus type 5 DNA in decomposed brain suspension from a slaughter house using Bayesian analysis, Brazil

Brazil represents the greatest beef producer among tropical countries, and the major obstacle for meat international trade is sanitary problems especially closely related to viral encephalitis. The goal of this study was to estimate the accuracy of the glycol and US9 gene-based polymerase chain reactions (PCRs) for the detection of bovine Herpesvirus type 5 (BoHV-5) from decomposed brain samples (n = 95). For this purpose, a latent-class (Bayesian) approach was used. Sensitivity (Se) was estimated to be 70% (95% probability interval, 40–80) and specificity (Sp) 100% in the statistical analysis for both PCRs used. Accordingly, a minimum of ≥40% of the calves was estimated to harbor BoHV-5 DNA even after 72 h of decomposition at room temperature. It was concluded that US9 gene-based PCR could also be considered a cost-effective alternative in sanitary programmers. However, given the importance of veterinary diagnoses, PCR-positive samples should be further confirmed through in vitro isolation and/or sequencing.     

Evaluation of three serological tests for diagnosis of Maedi-Visna virus infection using latent class analysis

Maedi-Visna virus (MVV) infection in sheep is present in several European countries, including Norway. The current Norwegian surveillance and control programme for MVV infection uses three serological tests: an agar gel immunodiffusion test (AGID) and two commercially available indirect ELISAs (Institut Pourquier, P-ELISA and HYPHEN BioMed, H-ELISA). From 18 flocks with suspected or confirmed MVV infection, sera from naturally infected sheep were obtained, and sensitivity (Se) and specificity (Sp) of the three tests were estimated in absence of a perfect reference test using latent class models in a Bayesian analysis. The AGID had higher Sp (95% posterior credibility interval (PCI) [98.4; 99.9]) than either ELISA (95% PCI: P-ELISA, [95.1; 99.0]; H-ELISA, [91.4; 96.6]), but much lower Se (95% PCI: AGID, [41.4; 59.8]; P-ELISA, [92.7; 100.0]; H-ELISA, [90.9; 99.4]). Currently the P-ELISA is used for screening and positive samples are subsequently confirmed by a setup using all three tests in a serial reading. The Se and Sp of the serial interpretations with and without the H-ELISA were estimated. The results suggested that the H-ELISA could be dropped as a confirmatory test as the Se of the three test serial reading was reduced significantly without adding a significant improvement of the Sp compared to the serial reading of the P-ELISA and AGID alone. However, the perceived cost of false positives versus false negatives will influence this decision. Estimates of the predictive values for the tests and combinations suggested that the P-ELISA is a good choice of screening, but confirmatory tests are needed to achieve acceptable levels of positive predictive values.     

Characteristics and comparative performance of direct culture, direct PCR and enumeration methods for detection and quantification of Campylobacter spp. in broiler caeca

Detection and enumeration of Campylobacter spp. in broiler chicken flocks are key components of research and surveillance studies aimed at reducing Campylobacter infections in people. Direct culture of caecal contents onto selective agar is the typical method used to confirm flock colonisation. Modified charcoal cefoperazone deoxycholate agar (mCCDA) is commonly used for this method, although alternative selective media have been used. Additionally, PCR methods to detect Campylobacter DNA from caecal contents may provide a rapid alternative. However comparative performance data for these methods is limited and therefore required to ensure optimal detection methods for this sample type. In this study, 306 broiler caeca were tested for Campylobacter using direct culture on mCCDA, Skirrows and Preston agars and two real-time PCR methods, one specific for mapA/ceuE regions and another for the flaA gene region. Additionally, the suitability of spread plating and spiral plating methods for enumeration of Campylobacter and the impact of sample storage were assessed. This study confirmed modified CCDA as an optimal media for detection of Campylobacter in broiler caeca. It was significantly more sensitive than Skirrows or Preston agars. This study also demonstrated that the mapA/ceuE PCR had excellent agreement with culture on mCCDA and is a genuine alternative method. Spread plating and spiral plating methods were suitable for enumeration although spiral plating appeared more sensitive for stored samples (72h). A 1 log reduction in viable Campylobacters was observed in stored samples, therefore storage effects should be considered for quantitative studies with broiler caeca.     

Impact of the Salmonella status of market-age pigs and the pre-slaughter process on Salmonella caecal contamination at slaughter

The aim of this study was to evaluate the impact of the pre-slaughter process on Salmonella caecal contamination of pigs at slaughter. An observational study was carried out in 2001 on 101 conventional farrow-to-finish pig farms. On each farm, one batch of contemporary pigs was followed from the end of the fattening period until slaughter. The Salmonella bacteriological status of the batches was assessed by environmental samples of faecal material. The serological Salmonella status was obtained on 30 individually identified market-age pigs using an indirect ELISA test. At the slaughterhouse, 25 g of caecal contents were taken from 10 of the identified pigs. Faecal and caecal material were analysed according to a classical bacteriological method. A questionnaire was designed to obtain information about the type of feeding during the fattening period (dry versus wet), the duration of fasting on the farm before leaving for the slaughterhouse, the duration of transport between the farm and the slaughterhouse, the holding time in lairage at the slaughterhouse and loading and unloading conditions on the farm and at the slaughterhouse. To assess the relationships between these factors and the Salmonella caecal status of the pigs and the batches, two logistic models were fitted at the individual and at the batch level, respectively. The first analysis was performed using a random effects logistic regression model. The second analysis was based on a cumulative logit model with a positive caecal rate classified into three classes as the outcome variable. The results showed that the Salmonella status of market-age pigs assessed on the farm either by serological or bacteriological examinations and the time spent in lairage before slaughtering played a crucial role on caecal contamination. In the light of these results, actions should be considered both on the farm and at the slaughterhouse to decrease the risk of Salmonella contamination of the caecal contents.