Epidemiology of Toxoplasma gondii in the pig industry of Yucatan, Mexico

Toxoplasmosis is a worldwide distributed parasitic disease caused by the zoonotic pathogen Toxoplasma gondii. Serological studies have estimated that more than 30% of the human population has been exposed to this protozoan. T. gondii is considered a leading cause of death attributed to foodborne illness. The consumption of infected pork meat is suggested to be an important source for human infection. However, the prevalence of T. gondii in pigs vary greatly between countries, the reasons for this heterogeneity has been addressed to the differences in climate distribution, environments, husbandry systems and fam management. The geographical location of this study is Yucatan, a state located in the south-east of Mexico. Yucatan is considered an endemic area of toxoplasmosis; the last National Seroepidemiological Health Survey (NSHS) revealed more than 70% of prevalence among the human population. Numerous studies suggested that the consumption of pork in this geographical area may be a major source of T. gondii. The aims of this study were to investigate the disease levels in the pig industry of Yucatan, assess an in-home ELISA widely used in this area (ELISA kit Human- GmbH, WB), study the risk factors associated with the disease in theses pig farms and evaluate the contamination with T. gondii in pork meat intended for human consumption. To do that, swine blood samples were collected through a cross-sectional age stratified opportunistic sampling of 12 farms across the state during the year 2014. Farm management and characteristics were obtained by interviewing farmers. In addition, meat and blood samples were collected from a local abattoir from 2013 to 2015. Anti - T. gondii IgG antibody levels were investigated with the well validated MAT (Modified Agglutination Test), with an ELISA (Enzyme-Linked Immunosorbent Assay) for use on pig serum (ID Screen ®, IDVet) and with the gold standard Dye test. The overall seroprevalence was 1.4% (95%CI: 0.6%-2.7%) among 632 pigs sampled. This seroprevalence increased with age (p < 0.05), reaching the 11.5% (95% CI: 2.5%-30%) in pigs older than 20 weeks. The seroprevalence was even higher, 17.8% (95% CI: 6%-37%), in slaughtered animals (n=34). In addition, T. gondii prevalence was investigated using a highly sensitive nested PCR protocol targeting the SAG1 gene. PCR diagnosis revealed the high frequency of 21.2% (CI: 18%-24.6%) of T. gondii DNA circulating in the blood of these pigs (n=632). Furthermore, MLST (Multi-Locus Sequence Typing) of four alleles (SAG1, SAG2, SAG3 and GRA6) allowed, for first time in Yucatan, genetic diversity to be assessed.Data revealed the presence of high genetic diversity among T. gondii strains of this geographical area with shared alleles to strains from both North and South/Central America origin. Moreover, a relatively high number of pigs presented multiple infections with different T. gondii strains suggesting high levels of T. gondii transmission on the intensive pig farms sampled. The frequency of T. gondii DNA was also investigated in pig tongues sampled at the abattoir of which 38.2% (95% CI: 22%-56.4%) were shown to harbour T. gondii DNA in their tissue. The viability of the parasite was also investigated in the tongues intended for human consumption and a total of 8.8% (95% CI: 1.8%-23%) were shown to have viable T. gondii using a mouse bioassay. However, the agreement between serology, PCR and mouse bioassay was low (k=0.12-0.29). Due to the risk of underestimating T. gondii infection by using solely one diagnostic method, a combination of indirect (serology) and direct methods (PCR or/and bioassay) is preferred for a robust diagnosis. This study was pioneering in using a serological test validated in pigs in the state of Yucatan and the data revealed a much lower prevalence than previously reported (95.8%-100%) in market age pigs (Ortega-Pacheco et al., 2013, Hernández- Cortazar et al., 2016a). Although a more optimistic view is obtained; due to the potential of T. gondii to lead to severe illness, measures to control the disease should still be taken. Furthermore, questionnaire results suggested the need for an improvement to the sanitary management of the pig farms of this geographical area not only to prevent T. gondii transmission but also that of other pathogens of zoonotic significance

PRRSV RNA detection in different matrices under typical storage conditions in the UK

In the UK, approximately 40 per cent of the pig breeding herds are outdoors. To monitor their porcine reproductive and respiratory syndrome virus (PRRSV) status, blood is collected commonly from piglets around weaning. Sample collection in British outdoor pigs often occurs during the early morning hours when the piglets tend to accumulate inside sheltered areas. For practical reasons, dry cotton swabs are occasionally used for blood collection and stored at room temperature until arrival in the laboratory. Detection of PRRSV RNA is a function of viral concentration, sample type and storage condition. To evaluate a possible impact of the sampling protocol on PRRSV1 detection, experimentally spiked blood samples using three dilutions of a representative PRRSV1 strain were prepared. In addition, blood samples from pigs naturally infected with PRRSV were obtained from a PRRSV-positive British herd. Spiked blood and blood from infected pigs were used to obtain sera, dry or wet (immersed in saline) polyester or cotton swabs and FTA cards. The different samples were stored for 24 hours, 48 hours or 7 days at 4°C or 20°C and tested by a real-time reverse transcriptase PRRSV PCR assay. Under the study conditions, the best matrix was serum (96.7 per cent), followed by wet swabs (78 per cent), dry swabs (61.3 per cent) and FTA cards (51 per cent). Polyester swabs (76 per cent) showed a better performance than cotton swabs (63.3 per cent). The reduction in sensitivity obtained for swabs and FTA cards was particularly high at low viral concentrations. The results indicate that wet polyester swabs should be used whenever possible

Bacillus pumilus probiotic feed supplementation mitigates Lawsonia intracellularis shedding and lesions

International audienceAbstractThe causative agent of ileitis, Lawsonia intracellularis, is commonly associated with diarrhea and reduced weight gain in growing pigs. The effect of in-feed probiotics on L. intracellularis infection dynamics was evaluated. In brief, 70 2.5-week-old-pigs were randomly divided into six groups with 10–20 pigs each. All pigs were fed an age appropriate base ration for the duration of the study, which was supplemented with one of three Bacillus strains including B. amyloliquefaciens (T01), B. licheniformis (T02) and B. pumilus (T03). Another group was orally vaccinated with a commercial live L. intracellularis vaccine (VAC) at 3 weeks of age. At 7 weeks of age, T01-LAW, T02-LAW, T03-LAW, VAC-LAW and the POS-CONTROL groups were challenged with L. intracellularis while the NEG-CONTROL pigs were not challenged. All pigs were necropsied 16 days later. By the time of inoculation, all VAC-LAW pigs had seroconverted and at necropsy 10–65% of the pigs in all other challenged groups were also seropositive. The results indicate a successful L. intracellularis challenge with highest bacterial DNA levels in POS-CONTROL pigs, VAC-LAW pigs and T01-LAW pigs. There was a delay in onset of shedding in T02-LAW and T03-LAW groups, which was reflected in less severe macroscopic and microscopic lesions, reduced intralesional L. intracellularis antigen levels and a lower area under the curve for bacterial shedding. Under the study conditions, two of the probiotics tested suppressed L. intracellularis infection. The obtained findings show the potential of probiotics in achieving antibiotic-free control of L. intracellularis

Prevalence of neutralising antibodies against SARS-CoV-2 in acute infection and convalescence: A systematic review and meta-analysis.

BACKGROUND: Individuals infected with SARS-CoV-2 develop neutralising antibodies. We investigated the proportion of individuals with SARS-CoV-2 neutralising antibodies after infection and how this proportion varies with selected covariates. METHODOLOGY/PRINCIPAL FINDINGS: This systematic review and meta-analysis examined the proportion of individuals with SARS-CoV-2 neutralising antibodies after infection and how these proportions vary with selected covariates. Three models using the maximum likelihood method assessed these proportions by study group, covariates and individually extracted data (protocol CRD42020208913). A total of 983 reports were identified and 27 were included. The pooled (95%CI) proportion of individuals with neutralising antibodies was 85.3% (83.5-86.9) using the titre cut off >1:20 and 83.9% (82.2-85.6), 70.2% (68.1-72.5) and 54.2% (52.0-56.5) with titres >1:40, >1:80 and >1:160, respectively. These proportions were higher among patients with severe COVID-19 (e.g., titres >1:80, 84.8% [80.0-89.2], >1:160, 74.4% [67.5-79.7]) than those with mild presentation (56.7% [49.9-62.9] and 44.1% [37.3-50.6], respectively) and lowest among asymptomatic infections (28.6% [17.9-39.2] and 10.0% [3.7-20.1], respectively). IgG and neutralising antibody levels correlated poorly. CONCLUSIONS/SIGNIFICANCE: 85% of individuals with proven SARS-CoV-2 infection had detectable neutralising antibodies. This proportion varied with disease severity, study setting, time since infection and the method used to measure antibodies

A high-resolution melt curve toolkit to identify lineage-defining SARS-CoV-2 mutations.

The emergence of severe acute respiratory syndrome 2 (SARS-CoV-2) variants of concern (VOCs), with mutations linked to increased transmissibility, vaccine escape and virulence, has necessitated the widespread genomic surveillance of SARS-CoV-2. This has placed a strain on global sequencing capacity, especially in areas lacking the resources for large scale sequencing activities. Here we have developed three separate multiplex high-resolution melting assays to enable the identification of Alpha, Beta, Delta and Omicron VOCs. The assays were evaluated against whole genome sequencing on upper-respiratory swab samples collected during the Alpha, Delta and Omicron [BA.1] waves of the UK pandemic. The sensitivities of the eight individual primer sets were all 100%, and specificity ranged from 94.6 to 100%. The multiplex HRM assays have potential as a tool for high throughput surveillance of SARS-CoV-2 VOCs, particularly in areas with limited genomics facilities

A Novel Air-Dried Multiplex High Resolution Melt Assay for the Detection of Extended Spectrum Beta-Lactamase and Carbapenemase Genes.

OBJECTIVES: This study aimed to develop and evaluate a novel air-dried high-resolution melt (HRM) assay to detect eight major extended spectrum beta-Lactamase (ESBL) (blaSHV and blaCTXM groups 1 and 9) and carbapenemase (blaNDM, blaIMP, blaKPC, blaVIM and blaOXA-48-like) genes that cause antimicrobial resistance to cephalosporins and carbapenems. METHODS: The assay was evaluated using 439 DNA samples extracted from bacterial isolates from Nepal, Malawi and UK and 390 clinical isolates from Nepal with known antimicrobial susceptibility results. Assay reproducibility was evaluated across five different q-PCR instruments (Rotor-Gene Q, QuantStudioTM 5, CFX96, LightCycler® 480 and MIC). Assay stability was also assessed upon the assay storage in the refrigerator (6.2°C±0.9), room temperature (20.4°C±0.7) and oven (29.7°C±1.4) at six time points for eight months. RESULTS: The sensitivity and specificity for detecting the ESBL and carbapenemase genes in comparison to the reference gel-base PCR and sequencing was 94.7% (95%CI: 92.5%-96.5%) and 99.2% (95%CI: 98.8%-99.5%), and 98.5% (95%CI: 97.0%-99.4%) and 98.5% (95%CI: 98.0%-98.9%) when compared to the original HRM wet PCR mix format. The overall agreement was 91.1% (95%CI: 90.0%-92.9%) when predicting phenotypic resistance to cefotaxime and meropenem among Enterobacteriaceae isolates. We observed almost perfect inter-machine reproducibility of the air-dried HRM assay and no loss of sensitivity occurred under all storage conditions and time points. CONCLUSIONS: We present here a ready-to-use air-dried HRM-PCR assay that offers an easy, thermostable, fast and accurate tool for the detection of ESBL and carbapenemase genes in DNA samples to improve AMR

Self-sampling of capillary blood for SARS-CoV-2 serology

Serological testing is emerging as a powerful tool to progress our understanding of COVID-19 exposure, transmission and immune response. Large-scale testing is limited by the need for in-person blood collection by staff trained in venepuncture, and the limited sensitivity of lateral flow tests. Capillary blood self-sampling and postage to laboratories for analysis could provide a reliable alternative. Two-hundred and nine matched venous and capillary blood samples were obtained from thirty nine participants and analysed using a COVID-19 IgG ELISA to detect antibodies against SARS-CoV-2. Thirty eight out of thirty nine participants were able to self-collect an adequate sample of capillary blood (≥ 50 µl). Using plasma from venous blood collected in lithium heparin as the reference standard, matched capillary blood samples, collected in lithium heparin-treated tubes and on filter paper as dried blood spots, achieved a Cohen’s kappa coefficient of > 0.88 (near-perfect agreement, 95% CI 0.738–1.000). Storage of capillary blood at room temperature for up to 7 days post sampling did not affect concordance. Our results indicate that capillary blood self-sampling is a reliable and feasible alternative to venepuncture for serological assessment in COVID-19

Detection of SARS-CoV-2 infection by saliva and nasopharyngeal sampling in frontline healthcare workers: An observational cohort study

Background The SARS-CoV-2 pandemic has caused an unprecedented strain on healthcare systems worldwide, including the United Kingdom National Health Service (NHS). We conducted an observational cohort study of SARS-CoV-2 infection in frontline healthcare workers (HCW) working in an acute NHS Trust during the first wave of the pandemic, to answer emerging questions surrounding SARS-CoV-2 infection, diagnosis, transmission and control. Methods Using self-collected weekly saliva and twice weekly combined oropharyngeal/nasopharyngeal (OP/NP) samples, in addition to self-assessed symptom profiles and isolation behaviours, we retrospectively compared SARS-CoV-2 detection by RT-qPCR of saliva and OP/NP samples. We report the association with contemporaneous symptoms and isolation behaviour. Results Over a 12-week period from 30th March 2020, 40∙0% (n = 34/85, 95% confidence interval 31∙3-51∙8%) HCW had evidence of SARS-CoV-2 infection by surveillance OP/NP swab and/or saliva sample. Symptoms were reported by 47∙1% (n = 40) and self-isolation by 25∙9% (n = 22) participants. Only 44.1% (n = 15/34) participants with SARS-CoV-2 infection reported any symptoms within 14 days of a positive result and only 29∙4% (n = 10/34) reported self-isolation periods. Overall agreement between paired saliva and OP/NP swabs was 93∙4% (n = 211/226 pairs) but rates of positive concordance were low. In paired samples with at least one positive result, 35∙0% (n = 7/20) were positive exclusively by OP/NP swab, 40∙0% (n = 8/20) exclusively by saliva and in only 25∙0% (n = 5/20) were the OP/NP and saliva result both positive. Conclusions HCW are a potential source of SARS-CoV-2 transmission in hospitals and symptom screening will identify the minority of infections. Without routine asymptomatic SARS-CoV-2 screening, it is likely that HCW with SARS-CoV-2 infection would continue to attend work. Saliva, in addition to OP/NP swab testing, facilitated ascertainment of symptomatic and asymptomatic SARS-CoV-2 infections. Combined saliva and OP/NP swab sampling would improve detection of SARS-CoV-2 for surveillance and is recommended for a high sensitivity strategy