A comparison of sampling and testing approaches for the surveillance of SARS-CoV-2 in farmed American mink.

Surveillance for SARS-CoV-2 in American mink (Neovison vison) is a global priority because outbreaks on mink farms have potential consequences for animal and public health. Surveillance programs often focus on screening natural mortalities; however, significant knowledge gaps remain regarding sampling and testing approaches. Using 76 mink from 3 naturally infected farms in British Columbia, Canada, we compared the performance of 2 reverse-transcription real-time PCR (RT-rtPCR) targets (the envelope [E] and RNA-dependent RNA polymerase [RdRp] genes) as well as serology. We also compared RT-rtPCR and sequencing results from nasopharyngeal, oropharyngeal, skin, and rectal swabs, as well as nasopharyngeal samples collected using swabs and interdental brushes. We found that infected mink were generally RT-rtPCR-positive on all samples; however, Ct values differed significantly among sample types (nasopharyngeal < oropharyngeal < skin < rectal). There was no difference in the results of nasopharyngeal samples collected using swabs or interdental brushes. For most mink (89.4%), qualitative (i.e., positive vs. negative) serology and RT-rtPCR results were concordant. However, mink were positive on RT-rtPCR and negative on serology and vice versa, and there was no significant correlation between Ct values on RT-rtPCR and percent inhibition on serology. Both the E and RdRp targets were detectable in all sample types, albeit with a small difference in Ct values. Although SARS-CoV-2 RNA can be detected in multiple sample types, passive surveillance programs in mink should focus on multiple target RT-rtPCR testing of nasopharyngeal samples in combination with serology.

Effect of extrinsic factors on the detection of PRRSV and a porcine-specific internal sample control in serum, oral fluid, and fecal specimens tested by RT-rtPCR.

We characterized the effect of 1) temperature × time, 2) freeze-thaw cycles, and 3) high porcine reproductive and respiratory syndrome virus (PRRSV) RNA concentrations on the detection of PRRSV and a porcine-specific internal sample control (ISC) in serum, oral fluid, and fecal samples using a commercial PRRSV RT-rtPCR assay (Idexx). In study 1, the effect of temperature × time on PRRSV and ISC detection was shown to be specimen dependent. In serum stored at 4, 10, or 20°C, PRRSV detection was consistent for up to 168 h, but storage at 30°C reduced detectable PRRSV RNA. ISC RNA was stable in serum held at 4 and 10°C, but not at 20 and 30°C. In contrast, PRRSV and ISC RNAs in oral fluid and fecal samples continuously decreased at all temperature × time treatments. Based on these data, serum samples should be stored at ≤ 20°C to optimize PRRSV RNA detection. Oral fluid and fecal samples should be frozen in a non-self-defrosting freezer until tested. In study 2, freeze-thaw cycles had little impact on PRRSV and ISC detection, but more so in oral fluids than serum or fecal samples. Thus, freeze-thaw cycles in oral fluids should be minimized before RT-rtPCR testing. In study 3, the ISC was not affected by high concentrations of PRRSV RNA in serum, oral fluid, or fecal samples. It should not be assumed that data from our PRRSV study are applicable to other pathogens; additional pathogen-specific studies are required.

Performance characteristics of the boson rapid SARS-cov-2 antigen test card vs RT-PCR: Cross-reactivity and emerging variants.

Background: SARS-CoV-2 virus has undergone several mutations on its genome, since the onset of the pandemic. Multiple variants of concern (VOC) have emerged including Alpha, Beta, Gamma, and Delta with the more recent one being the Omicron (B.1.1.529). Specific rapid antigen tests (RADs) have been used for the detection of SARS-CoV-2. However, since the emergence of new VOCs, the performance characteristics of these RADs needs to be re-evaluated. Objectives: The main purposes of this clinical study were to determine the diagnostic sensitivity and specificity of the BOSON Rapid Antigen Test compared to the gold standard real time RT-PCR and to determine the ability of the RAD to accurately depict different VOC. Additionally, the cross reactivity to other viruses and pathogen, as well as, the possible interference of non Covid-19 hospitalized patients for various causes, were investigated. Results: A total of 623 individuals (symptomatic) were tested. The sensitivity, specificity and accuracy of the BOSON RAD was 95.27%, 100% and 98.45% (n = 448), meeting the WHO recommended standards. Additionally, the Delta (83.33%, Ct < 34) and Omicron (100%, Ct < 26) VOC were determined with high sensitivity. Also, there was no interference from hospitalized, non-Covid 19 patients, and no cross-reactivity was detected. Conclusions: The study showed that this RAD could rapidly identify individuals with SARS-CoV-2, including those with the new dominant Omicron VOC, with no cross reactivity from other pathogens.

Detection of Tritrichomonas foetus by RT-rtPCR in pooled bovine preputial washings.

Trichomonosis is a venereal disease of cattle caused by the protozoan Tritrichomonas foetus. T. foetus infection in cattle herds can be economically costly for cattle producers; therefore, testing is important for detection of the agent. Given that bulls are considered to be subclinical carriers of T. foetus, it is important to detect T. foetus infection prior to movement and/or breeding season. We have described previously the development of an updated set of PCR primers and probes that offer increased sensitivity of T. foetus detection in preputial washings collected in PBS by utilizing reverse-transcription real-time PCR (RT-rtPCR) that targets the 5.8S ribosomal RNA of the T. foetus organism. Here, we report improvements in the updated RT-rtPCR reagents as well as the evaluation of testing of pooled preputial washings. We found that up to 5 preputial washings can be pooled, similar to routine testing practices (InPouch culture), without reducing the sensitivity of detection of T. foetus.

Evaluation of a real-time RT-PCR panel for detection of SARS-CoV-2 in bat guano.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of coronavirus disease 2019 (COVID-19), which is an ongoing global health concern. The exact source of the virus has not been identified, but it is believed that this novel coronavirus originated in animals; bats in particular have been implicated as the primary reservoir of the virus. SARS-CoV-2 can also be transmitted from humans to other animals, including tigers, cats, and mink. Consequently, infected people who work directly with bats could transfer the virus to a wild North American bat, resulting in a new natural reservoir for the virus, and lead to new outbreaks of human disease. We evaluated a reverse-transcription real-time PCR panel for detection of SARS-CoV-2 in bat guano. We found the panel to be highly specific for SARS-CoV-2, and able to detect the virus in bat guano samples spiked with SARS-CoV-2 viral RNA. Our panel could be utilized by wildlife agencies to test bats in rehabilitation facilities prior to their release to the wild, minimizing the risk of spreading this virus to wild bat populations.

Development and validation of SYBR Green- and probe-based reverse-transcription real-time PCR assays for detection of the S and M segments of Schmallenberg virus.

Schmallenberg virus (SBV), discovered in Germany in 2011, causes congenital malformations in ruminants. Reverse-transcription real-time PCR (RT-rtPCR) assays based on various segments of SBV have been developed for molecular detection. We developed alternative RT-rtPCR assays for SBV detection to avoid earlier reported mutations and hypervariable regions of the S and M segments of the viral genome. For SYBR Green-based detection of the S segment, the R2 value and efficiency of the developed assay were 0.99 and 99%, respectively. For probe-based S segment detection, 2 assays were developed; the first had an R2 value of 0.99 and 102% efficiency, and the second had a R2 value of 0.98 and 86% efficiency. The probe-based M segment assay had an R2 value of 1.00 and 103% efficiency. Detection limits of the RT-rtPCR assays with new primer sets were 102 and 101 copies/µL for the S and M segments, respectively. Field samples from cattle and sheep were also used for primary validation of the developed assays. Our assays should be suitable for SBV detection in ruminants and for in vitro studies of various SBV strains.

Evaluation of six TaqMan RT-rtPCR kits on two thermocyclers for the reliable detection of rabies virus RNA.

Rabies is diagnosed postmortem in animals, based on tests prescribed by the World Organization for Animal Health (OIE), such as the fluorescent antibody test, the direct rapid immunohistochemistry test, or pan-lyssavirus PCR assays. Several reverse-transcription real-time PCR (RT-rtPCR) methods have been developed and validated for rapid and accurate detection of lyssaviruses. We evaluated the performance of 6 TaqMan RT-rtPCR kits using different commercial master mixes and 2 real-time thermocyclers. Changing the master mix overall did not influence the TaqMan RT-rtPCR performance, regardless of the thermocycler used. The limits of detection at the 95% confidence level were 18.1-25.8 copies/µL for the Rotor-Gene Q MDx thermocycler and 16.7-21.5 for the Mx3005P thermocycler. Excellent repeatability was demonstrated for rabies virus (RABV) RNA samples of 100, 50, and 25 copies/µL regardless of the thermocycler used. RABV field samples ( n = 35) isolated worldwide gave positive results using the most efficient of the 6 kits tested, with a copy number of 6.03 × 102 to 6.78 × 107 RNA copies per reaction. The TaqMan RT-rtPCR assay provides sensitive and rapid amplification of RABV RNA.

Effects of sample handling on the detection of porcine reproductive and respiratory syndrome virus in oral fluids by reverse-transcription real-time PCR.

We evaluated effects of handling procedures on detection of porcine reproductive and respiratory syndrome virus (PRRSV) in oral fluids (OFs) by reverse-transcription real-time PCR (RT-rtPCR). The experiments were conducted using a composite sample of PRRSV-positive OF collected from 5-wk-old pigs vaccinated 15 d earlier with a modified-live PRRSV vaccine. Five pre-extraction sample-handling steps and all combinations thereof were evaluated: 1) thaw temperature (4°C or 25°C); 2) sample diluent (1:1 dilution with nuclease-free water or guanidinium thiocyanate-phenol); 3a) sonication of the sample (yes or no); 3b) temperature (4°C or 25°C) at which step 3a was conducted; and 4) temperature at which the sample was maintained after step 3b and until RNA extraction was initiated (4°C or 25°C). All combinations of the 5 sample-handling steps (i.e., 32 unique treatments) were tested in a completely randomized factorial design with 4 replicates and 1 negative control for each treatment. The entire experiment was repeated on 5 separate days to produce a total of 800 PRRSV RT-rtPCR results. Binary (positive or negative) data were analyzed by logistic regression and results (Ct) were analyzed using a generalized linear model. Overall, 1 false-positive result was observed among 160 negative controls (99.4% specificity), and 85 false-negative results were observed among the 640 known-positive samples (86.7% sensitivity). The most significant factor affecting test outcome was thaw temperature (4°C or 25°C); samples thawed at 4°C had higher positivity rate (94% vs. 80%, p < 0.0001) and lower Ct (36.2 vs. 37.5, p < 0.0001).