High-quality DNA isolation protocol for detection of Khapra beetle (Dermestidae: Trogoderma granarium Everts, 1898) in standard wheat germ trap.

BACKGROUND: Khapra beetle (Dermestidae: Trogoderma granarium Everts, 1898) is an internationally significant pest of grain crops and stored grain products. Wheat germ traps, routinely used in surveillance sampling of Khapra beetle provide feed-substrates used by the pest throughout its life cycle. However, Khapra beetle larvae, eggs and other traces of the pest, such as larval frass and exuviae, in wheat germ traps are difficult to sort and taxonomically identify. Additionally, high levels of polysaccharides in wheat germ can inhibit PCR based molecular detection of this pest captured in the traps. METHODS AND RESULTS: We have developed a sensitive and low-cost protocol for extracting trace levels of Khapra beetle DNA from an entire wheat germ trap. Overnight digestion of entire trap contents in 6 mL of ATL buffer, followed by a 40 min lysis step was optimal for DNA extraction. Paired with reported qPCR assays, this protocol allows the detection of a few hairs of T. granarium in a typical 2-gram wheat germ trap. CONCLUSION: This DNA extraction protocol makes it possible to perform a more rapid identification of the pest following wheat germ sample collection. The protocol has potential to improve international efforts for Khapra beetle surveillance.

Efficacy and safety of the direct oral anti-coagulants in patients with cerebral vein thrombosis: A systematic review and meta-analysis.

Vitamin K antagonists (VKAs) are the standard oral anti-coagulant treatment for patients with cerebral venous thrombosis (CVT). However, the direct oral anti-coagulants (DOACs) started replacing VKAs also in this setting. We aimed to evaluate safety and efficacy of the DOACs for CVT treatment. We performed a systematic review and meta-analysis (PROSPERO protocol registration number CRD42020191472). The electronic databases MEDLINE, EMBASE and CENTRAL were searched from inception to January 2022. We included randomised controlled trials (RCTs) and observational studies, enrolling at least 10 adult patients with CVT treated with any DOACs. Twenty-three studies were included, for a total of 618 CVT patients treated with DOACs (treatment duration range 3-12 months). Mortality rate was 1.76% [95% confidence interval (CI) 0.70%-3.24%; I2  = 0%; 5/428 patients, 18 studies]; major bleeding 2.41% (95% CI 1.26%-3.91%; I2  = 1.5%; 12/534 patients, 21 studies); recurrent thrombosis 2.05% (95% CI 1.04%-3.37%; I2  = 0%; 10/577 patients, 21 studies); excellent neurological outcome 85.9% (95% CI 79.0%-91.7%; I2  = 63.7%; 289/340 patients, 13 studies); vessel recanalisation 89.0% (95% CI 82.9%-93.9%; I2  = 62.7%; 316/359 patients, 16 studies). No significant differences emerged by study design (RCTs vs. observational studies) or by treatment (DOACs vs. VKAs). This systematic review showed that the DOACs might represent a reasonable oral anti-coagulant treatment option for CVT patients.

High-throughput direct fecal PCR assay for detection of Mycobacterium avium subsp. paratuberculosis in sheep and cattle.

Johne's disease (JD) is a chronic enteric disease caused by Mycobacterium avium subsp. paratuberculosis that affects ruminants. Transmission occurs by the fecal-oral route. A commonly used antemortem diagnostic test for the detection of M. avium subsp. paratuberculosis in feces is liquid culture; however, a major constraint is the 2- to 3-month incubation period needed for this method. Rapid methods for the detection of M. avium subsp. paratuberculosis based on PCR have been reported, but comprehensive validation data are lacking. We describe here a new test, the high-throughput-Johnes (HT-J), to detect M. avium subsp. paratuberculosis in feces. Its diagnostic accuracy was compared with that of liquid radiometric (Bactec) fecal culture using samples from cattle (1,330 samples from 23 herds) and sheep (596 samples from 16 flocks). The multistage protocol involves the recovery of M. avium subsp. paratuberculosis cells from a fecal suspension, cell rupture by bead beating, extraction of DNA using magnetic beads, and IS900 quantitative PCR. The limit of detection of the assay was 0.0005 pg, and the limit of quantification was 0.005 pg M. avium subsp. paratuberculosis genomic DNA. Only M. avium subsp. paratuberculosis was detected from a panel of 51 mycobacterial isolates, including 10 with IS900-like sequences. Of the 549 culture-negative fecal samples from unexposed herds and flocks, 99% were negative in the HT-J test, while 60% of the bovine- and 84% of the ovine-culture-positive samples were positive in the HT-J test. As similar total numbers of samples from M. avium subsp. paratuberculosis-exposed animals were positive in culture and HT-J tests in both species, and as the results of a McNemar's test were not significant, these methods probably have similar sensitivities, but the true diagnostic sensitivities of these tests are unknown. These validation data meet the consensus-based reporting standards for diagnostic test accuracy studies for paratuberculosis and the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines (S. A. Bustin et al., Clin. Chem. 55:611-622, 2009, doi:10.1373/clinchem.2008.112797). The HT-J assay has been approved for use in JD control programs in Australia and New Zealand.

An epizootic of Chlamydia psittaci equine reproductive loss associated with suspected spillover from native Australian parrots.

Chlamydia psittaci is an avian pathogen capable of spill-over infections to humans. A parrot C. psittaci strain was recently detected in an equine reproductive loss case associated with a subsequent cluster of human C. psittaci infections. In this study, we screened for C. psittaci in cases of equine reproductive loss reported in regional New South Wales, Australia during the 2016 foaling season. C. psittaci specific-PCR screening of foetal and placental tissue samples from cases of equine abortion (n = 161) and foals with compromised health status (n = 38) revealed C. psittaci positivity of 21.1% and 23.7%, respectively. There was a statistically significant geographical clustering of cases ~170 km inland from the mid-coast of NSW (P < 0.001). Genomic analysis and molecular typing of C. psittaci positive samples from this study and the previous Australian equine index case revealed that the equine strains from different studs in regional NSW were clonal, while the phylogenetic analysis revealed that the C. psittaci strains from both Australian equine disease clusters belong to the parrot-associated 6BC clade, again indicative of spill-over of C. psittaci infections from native Australian parrots. The results of this work suggest that C. psittaci may be a more significant agent of equine reproductive loss than thought. A range of studies are now required to evaluate (a) the exact role that C. psittaci plays in equine reproductive loss; (b) the range of potential avian reservoirs and factors influencing infection spill-over; and

The application of the comet assay to assess the genotoxicity of environmental pollutants in the nematode Caenorhabditis elegans.

This study aimed to establish a protocol for cell dissociation from the nematode Caenorhabditis elegans (C. elegans) to assess the genotoxicity of the environmental pollutant benzo[a]pyrene (BaP) using the alkaline version of the single cell electrophoresis assay (comet assay). BaP genotoxicity was assessed in C. elegans (wild-type [WT]; N2, Bristol) after 48h exposure (0-40µM). Induction of comets by BaP was concentration-dependent up to 20µM; comet% tail DNA was ∼30% at 20µM BaP and ∼10% in controls. Similarly, BaP-induced DNA damage was evaluated in C. elegans mutant strains deficient in DNA repair. In xpa-1 and apn-1 mutants BaP-induced comet formation was diminished to WT background levels suggesting that the damage formed by BaP that is detected in the comet assay is not recognised in cells deficient in nucleotide and base excision repair, respectively. In summary, our study provides a protocol to evaluate DNA damage of environmental pollutants in whole nematodes using the comet assay.

Salmonella infection in a remote, isolated wild pig population.

Although wild pig populations are known to sometimes be infected by Salmonella, the situation in Australia has received little attention and few population-based, planned studies have been conducted. Understanding the distribution of Salmonella infections within wild pig populations allows the potential hazard posed to co-grazing livestock to be assessed. We sampled a remote and isolated wild pig population in northwestern Australia. Faecal and mesenteric lymph node samples were collected from 651 wild pigs at 93 locations and cultured for Salmonella. The population sampled was typical of wild pig populations in tropical areas of Australia, and sampling occurred approximately halfway through the population's breeding season (38% of the 229 adult females were pregnant and 35% were lactating). Overall, the prevalence of Salmonella infection based on culture of 546 freshly collected faecal samples was 36.3% (95% CI 32.1-40.7%), and based on culture of mesenteric lymph nodes was 11.9% (95% CI, 9.4-15.0%). A total of 39 serovars (139 isolates) were identified--29 in faecal samples and 24 in lymph node samples--however neither Salmonella enterica serovar Typhimurium nor Salmonella Cholerasuis were isolated. There was a significant (p<0.0001) disagreement between faecal and lymph node samples with respect to Salmonella isolation, with isolation more likely from faecal samples. Prevalence differed between age classes, with piglets being less likely to be faecal-positive but more likely to be lymph node positive than adults. The distribution of faecal-positive pigs was spatially structured, with spatial clusters being identified. Study results suggest that this population of wild pigs is highly endemic for Salmonella, and that Salmonella is transmitted from older to younger pigs, perhaps associated with landscape features such as water features. This might have implications for infection of co-grazing livestock within this environment.

Integrating survey and molecular approaches to better understand wildlife disease ecology.

Infectious wildlife diseases have enormous global impacts, leading to human pandemics, global biodiversity declines and socio-economic hardship. Understanding how infection persists and is transmitted in wildlife is critical for managing diseases, but our understanding is limited. Our study aim was to better understand how infectious disease persists in wildlife populations by integrating genetics, ecology and epidemiology approaches. Specifically, we aimed to determine whether environmental or host factors were stronger drivers of Salmonella persistence or transmission within a remote and isolated wild pig (Sus scrofa) population. We determined the Salmonella infection status of wild pigs. Salmonella isolates were genotyped and a range of data was collected on putative risk factors for Salmonella transmission. We a priori identified several plausible biological hypotheses for Salmonella prevalence (cross sectional study design) versus transmission (molecular case series study design) and fit the data to these models. There were 543 wild pig Salmonella observations, sampled at 93 unique locations. Salmonella prevalence was 41% (95% confidence interval [CI]: 37-45%). The median Salmonella DICE coefficient (or Salmonella genetic similarity) was 52% (interquartile range [IQR]: 42-62%). Using the traditional cross sectional prevalence study design, the only supported model was based on the hypothesis that abundance of available ecological resources determines Salmonella prevalence in wild pigs. In the molecular study design, spatial proximity and herd membership as well as some individual risk factors (sex, condition score and relative density) determined transmission between pigs. Traditional cross sectional surveys and molecular epidemiological approaches are complementary and together can enhance understanding of disease ecology: abundance of ecological resources critical for wildlife influences Salmonella prevalence, whereas Salmonella transmission is driven by local spatial, social, density and individual factors, rather than resources. This enhanced understanding has implications for the control of diseases in wildlife populations. Attempts to manage wildlife disease using simplistic density approaches do not acknowledge the complexity of disease ecology.