Short communication: Evaluation of 5 different ELISA for the detection of bovine leukemia virus antibodies.

Although Canadian dairy herds have been infected with bovine leukemia virus (BLV) for years, recent research has put new emphasis on the potential negative effects of this infection. Consequently, BLV control is becoming more favorable; however, BLV control cannot be successful without identifying infected animals. Bovicheck BLV (Biovet, Saint-Hyacinthe, QC, Canada) is currently the only assay licensed by the Canadian Centre for Veterinary Biologics. The first goal of this study was, therefore, to determine the reproducibility of the Bovicheck BLV assay for serum samples derived from Canadian cattle. The second goal was to evaluate and compare 5 different ELISA and determine their test characteristics using serum samples from Canadian herds. The considered ELISA were Bovicheck BLV, ID Screen BLV Competition (IDvet, Grabels, France), Idexx Leukosis Serum X2 Ab Test (Idexx Europe B.V., Hoofddorp, the Netherlands), Svanovir BLV gp51-Ab (Svanova, Uppsala, Sweden), and the Serelisa BLV Ab Mono Indirect (Synbiotics, Lyon, France). Eighty serum samples from Canadian cattle provided by Prairie Diagnostic Services (PDS; Saskatoon, SK, Canada) and an additional 80 serum samples from Canadian dairy and beef herds were used for the study. The Bovicheck BLV assay yielded the same results for all PDS-derived samples, implying a high level of reproducibility and robustness of this assay. Additionally, the comparison of the assays' results showed high agreement between assays, with Cohen's kappa values between κ = 0.91 and κ = 1. Furthermore, using original test results of the field samples as true status, relative diagnostic sensitivity and specificity were calculated. Relative diagnostic sensitivity of all tests was 100%. False-positive results were probable; therefore, the following relative diagnostic specificities were determined: 100% for Bovicheck BLV, Idexx Leukosis Serum X2, and Svanovir BLV; 95% for ID Screen BLV; and 97% for Serelisa BLV. When considering other test characteristics, ID Screen BLV is exceptional due to considerable practical advantages.

Statistical evaluation of a commercial Neospora caninum competitive ELISA in the absence of a gold standard: application to wild elk (Cervus elaphus) in Alberta.

Neospora caninum is an important abortive agent of domestic ruminants, but few diagnostic tools are available to reliably assess the exposure of wild cervid species such as elk (Cervus elaphus) to this pathogen, which limits our ability to understand their role in the life cycle of this parasite. In the absence of a gold standard test or panels of samples from individuals of known infection status, classical laboratory-based validation methods are not applicable. However, there are a number of statistical methods that can help in selecting an appropriate cut-off value and estimating the resulting diagnostic test performances. In this paper, the performance of a commercial competitive enzyme-linked immunosorbent assay (cELISA) on elk serum samples was evaluated with two statistical approaches: a mixture distribution model fitted to the cELISA results, and a Bayesian latent class analysis combining results from the cELISA and an indirect immuno-fluorescence antibody test. Both methods indicated that the commercial kit could be used on elk serum with the specifications recommended by the manufacturer. In particular, the optimal combination of sensitivity and specificity were obtained for a percentage of inhibition cutoff of 30%. The 95% probability interval of the proportion of elk exposed to N. caninum, adjusting for the sensitivity and specificity of this test in elk, was estimated between 1.3 and 7.4%. There was no association between the serological status of female elk and their pregnancy status. These results point out to the involvement of elk in a sylvatic cycle of N. caninum in this area.

Correction: Novel insights into serodiagnosis and epidemiology of Erysipelothrix rhusiopathiae, a newly recognized pathogen in muskoxen (Ovibos moschatus).

[This corrects the article DOI: 10.1371/journal.pone.0231724.].

Novel insights into serodiagnosis and epidemiology of Erysipelothrix rhusiopathiae, a newly recognized pathogen in muskoxen (Ovibos moschatus).

BACKGROUND: Muskoxen are a key species of Arctic ecosystems and are important for food security and socio-economic well-being of many Indigenous communities in the Arctic and Subarctic. Between 2009 and 2014, the bacterium Erysipelothrix rhusiopathiae was isolated for the first time in this species in association with multiple mortality events in Canada and Alaska, raising questions regarding the spatiotemporal occurrence of the pathogen and its potential impact on muskox populations. MATERIALS AND METHODS: We adapted a commercial porcine E. rhusiopathiae enzyme-linked immunosorbent assay to test 958 blood samples that were collected from muskoxen from seven regions in Alaska and the Canadian Arctic between 1976 and 2017. The cut-off between negative and positive results was established using mixture-distribution analysis, a data-driven approach. Based on 818 samples for which a serological status could be determined and with complete information, we calculated trends in sample seroprevalences in population time-series and compared them with population trends in the investigated regions. RESULTS: Overall, 219/818 (27.8%, 95% Confidence Interval: 24.7-31.0) samples were classified as positive for exposure to E. rhusiopathiae. There were large variations between years and regions. Seropositive animals were found among the earliest serum samples tested; 1976 in Alaska and 1991 in Canada. In Alaskan muskoxen, sample seroprevalence increased after 2000 and, in two regions, peak seroprevalences occurred simultaneously with population declines. In one of these regions, concurrent unusual mortalities were observed and E. rhusiopathiae was isolated from muskox carcasses. In Canada, there was an increase in sample seroprevalence in two muskox populations following known mortality events that had been attributed to E. rhusiopathiae. CONCLUSION: Our results indicate widespread exposure of muskoxen to E. rhusiopathiae in western Canada and Alaska. Although not new to the Arctic, we documented an increased exposure to the pathogen in several regions concurrent with population declines. Understanding causes for the apparent increased occurrence of this pathogen and its association with large scale mortality events for muskoxen is critical to evaluate the implications for wildlife and wildlife-dependent human populations in the Arctic.

Single tube allele specific PCR: a low cost technique for molecular screening of sickle cell anaemia in Nigeria.

BACKGROUND: Sickle cell anaemia (SCA) is a hereditary blood disorder caused by a single mutation in the haemoglobin gene. The disease burden of SCA is highest in Nigeria. The allele specific polymerase chain reaction (ASPCR) method is applicable for the direct detection of known single nucleotide polymorphisms (SNPs). OBJECTIVE: To investigate the use of the single tube ASPCR as an accurate and affordable method for SCA screening in Nigeria. METHODS: DNA was extracted from study subjects with normal haemoglobin, HbAA (20), sickle cell anaemia, HbSS (20) and carriers, HbAS (1). Haemoglobin was genotyped by ASPCR using two primer sets that amplifies the wildtype and mutant haemoglobins in each sample. Amplicon sizes were analyzed by gel electrophoresis. RESULTS: Amplicons were visible after electrophoresis at regions 517 base pair (bp) for HbA and 267 bp for HbS. ASPCR correctly and unambiguously detected the presence or absence of haemoglobins A and S from all samples collected, demonstrating its accuracy and precision for the screening of SCA. CONCLUSION: This study validates ASPCR as an effective, low cost approach for the clinical screening of SCA in Nigeria. ASPCR is also applicable for other genetic diseases, paternity testing, and forensics where more expensive fluorescence-based approaches are not obtainable.

Better Alone or in Ill Company? The Effect of Migration and Inter-Species Comingling on Fascioloides magna Infection in Elk.

Migratory movements and alteration of host communities through livestock production are examples of ecological processes that may have consequences on wildlife pathogens. We studied the effect of co-grazing of cattle and wild elk, and of elk migratory behaviour on the occurrence of the giant liver fluke, Fascioloides magna, in elk. Migratory elk and elk herds with a higher proportion of migratory individuals were significantly less likely to be infected with F. magna. This may indicate a decreased risk of infection for migratory individuals, known as the "migratory escape" hypothesis. Elk herds overlapping with higher cattle densities also had a lower prevalence of this parasite, even after adjustment for landscape and climate variables known to influence its life cycle. Serological evidence suggests that even in low-prevalence areas, F. magna is circulating in both elk and cattle. Cattle are "dead-end" hosts for F. magna, and this may, therefore, indicate a dilution effect where cattle and elk are co-grazing. Migratory behaviour and host community composition have significant effects on the dynamics of this wildlife parasite; emphasizing the potential impacts of decisions regarding the management of migratory corridors and livestock-wildlife interface.

Blood collected on filter paper for wildlife serology: detecting antibodies to Neospora caninum, West Nile virus, and five bovine viruses in reindeer.

We compared Nobuto filter paper (FP) whole-blood samples to serum for detecting antibodies to seven pathogens in reindeer (Rangifer tarandus tarandus). Serum and FP samples were collected from captive reindeer in 2008-2009. Sample pairs (serum and FP eluates) were assayed in duplicate at diagnostic laboratories with the use of competitive enzyme-linked immunosorbent assays (cELISAs) for Neospora caninum and West Nile virus (WNV); indirect ELISA (iELISAs) for bovine herpesvirus type 1 (BHV-1), parainfluenza virus type 3 (PI-3), and bovine respiratory syncytial virus (BRSV); and virus neutralization (VN) for bovine viral diarrhea virus (BVDV) types I and II. Assay thresholds were evidence-based values employed by each laboratory. Comparable performance to serum was defined as FP sensitivity and specificity ≥ 80%. Filter-paper specificity estimates ranged from 92% in the cELISAs for N. caninum and WNV to 98% in the iELISAs for PI-3 and BRSV. Sensitivity was >85% for five tests (most ≥ 95%) but was insufficient (71-82%) for the PI-3 and BRSV iELISAs. Lowering the threshold for FP samples in these two ELISAs raised sensitivity to ≥ 87% and reduced specificity slightly (≥ 90% in three of the four test runs). Sample size limited the precision of some performance estimates. Based on the criteria of sensitivity and specificity ≥ 80%, and using adjusted FP thresholds for PI-3 and BRSV, FP sensitivity and specificity were comparable to serum in all seven assays. A potential limitation of FP is reduced sensitivity in tests that require undiluted serum (i.e., N. caninum cELISA and BVDV VNs). Possible toxicity to the assay cell layer in VN requires investigation. Results suggested that cELISA is superior to iELISA for detecting antibodies in FP samples from reindeer and other Rangifer tarandus subspecies. Our findings expand the potential utility of FP sampling from wildlife.

Blood collected on filter paper for wildlife serology: evaluating storage and temperature challenges of field collections.

Filter-paper (FP) blood sampling can facilitate wildlife research and expand disease surveillance. Previous work indicated that Nobuto FP samples from caribou and reindeer (Rangifer tarandus subspecies) had comparable sensitivity and specificity to serum samples (≥ 80% for both) in competitive enzyme-linked immunosorbent assays (cELISAs) for Brucella spp., Neospora caninum, and West Nile virus. The same sensitivity and specificity criteria were met in indirect ELISAs for Brucella spp., bovine herpesvirus type 1 (BHV-1), parainfluenza virus type 3 (PI-3), and bovine respiratory syncytial virus (BRSV), with adjusted FP thresholds used for PI-3 and BRSV. Comparable sensitivity and specificity values to serum were also observed for FP in virus neutralization (VN) assays for bovine viral diarrhea virus types I and II; however, reduced sensitivity is a potential limitation of FP samples in protocols that require undiluted serum (i.e., VN and N. caninum cELISA). We evaluated the performance of FP samples from reindeer and caribou in these nine assays after simulating potential challenges of high-latitude field collections: 1) different durations of storage and 2) different processing/storage regimes involving freezing or drying. Sample pairs (serum and FP) were collected from reindeer and caribou populations in 2007-10 and were tested in duplicate. Comparable performance to serum was defined as sensitivity and specificity ≥ 80%. In the storage experiments, FP performance was determined after 2 mo of storage dry at room temperature, and after two longer periods (variable depending on assay; up to 2 yr). After 1 yr, compared to frozen serum stored for the same period, sensitivity was ≥ 88% for all but two assays (68% BHV-1; 75% PI-3), and specificity remained >90%. A limited trial evaluated the effect of freezing FP samples as opposed to drying them for storage. There were no observed detrimental effects of freezing on FP sample performance, but rigorous investigation is warranted.

Filter-paper blood samples for ELISA detection of Brucella antibodies in caribou.

We evaluated blood collected on Nobuto filter-paper (FP) strips for use in detecting Brucella spp. antibodies in caribou. Whole blood (for serum) and blood-saturated FP strips were obtained from 185 killed arctic caribou (Rangifer tarandus groenlandicus). Sample pairs (serum and FP eluates) were simultaneously tested in duplicate using competitive enzyme-linked immunosorbent assay (c-ELISA) and indirect ELISA (i-ELISA) for Brucella spp. Prior work based on isolation of Brucella spp. revealed sensitivity (SE) and specificity (SP) of 100% and 99%, respectively, for both these serum assays in caribou. Infection status of the animals in the current study was unknown but recent sampling had revealed clinical brucellosis and >40% Brucella antibody prevalence in the herd. To assess the performance of FP relative to serum in these assays, serum was used as the putative gold standard. On both assays, the findings for duplicate runs (A and B) were similar. For c-ELISA run A, the FP Brucella prevalence (47%) was lower than serum prevalence (52%), with SE 89% (95% confidence interval [CI]: 82-95%) and SP 99% (97-100%). For i-ELISA run A, serum and FP Brucella prevalence rates were identical (43%), and the SE and SP of FP testing were 100% and 99% (97-100%), respectively. The findings suggest better FP test performance with i-ELISA than with c-ELISA; however, i-ELISA does not distinguish cross-reacting antibodies induced by Brucella vaccination or exposure to certain other Gram-negative pathogens. Results for duplicate FP eluates (prepared using separate FP strips from each animal) were strongly correlated for both protocols (r=0.996 and 0.999 for c-ELISA and i-ELISA, respectively), indicating minimal variability among FPs from any individual caribou. Dried caribou FP blood samples stored for 2 mo at room temperature are comparable with serum for use in Brucella spp. c-ELISA and i-ELISA. Hunter-based FP sampling can facilitate detection of disease exposure in remote regions and under adverse conditions, and can expand wildlife disease surveillance across temporospatial scales.

Novel multiplex PCR assay for detection of the staphylococcal virulence marker Panton-Valentine leukocidin genes and simultaneous discrimination of methicillin-susceptible from -resistant staphylococci.

We developed a new multiplex PCR assay for detection of Panton-Valentine leukocidin virulence genes and simultaneous discrimination of methicillin-susceptible from -resistant staphylococci. This assay is simple, rapid, and accurate and offers the potential for prompt detection of newly emerging community-associated methicillin-resistant Staphylococcus aureus.