Development of a novel sensitive single-tube nested PCR assay for the detection of African swine fever virus.

African swine fever (ASF) is a highly fatal and contagious viral disease caused by African swine fever virus (ASFV). It has caused significant economic losses to the swine industry and poses a serious threat to food security worldwide. Diagnostic tests with high sensitivity are essential for the effective management of ASF. Here, we describe a single-tube nested PCR (STN-PCR) assay for the detection of ASFV in which two consecutive amplification steps are carried out within a single tube. Two pairs of primers (outer and inner) were designed to target the p72 gene of ASFV. The primer concentrations, annealing temperatures, and number of amplification cycles were optimized to ensure the consecutive utilization of outer and inner primer pairs during amplification while minimizing the likelihood of amplicon contamination. In comparison with two conventional endpoint PCR assays (one of which is recommended by the World Organization for Animal Health), the newly developed STN-PCR assay demonstrated a 100-fold improvement in the limit of detection (LOD), detecting 100 copies of ASFV genomic DNA, whereas the endpoint PCR assays could detect no fewer than 10,000 copies. The clinical performance of the STN-PCR assay was validated using 95 tissue samples suspected of being positive for ASFV, and the assay showed 100% specificity. A Cohen's kappa value of 0.91 indicated perfect agreement between the assays. This new STN-PCR assay is a potentially valuable tool that will facilitate the control of ASF.

Novel sensitive isothermal-based diagnostic technique for the detection of African swine fever virus.

A rapid, simple, and sensitive diagnostic technique for the detection of African swine fever virus (ASFV) nucleic acid was developed for testing clinical samples in the field or resource-constrained settings. In the current study, the saltatory rolling-circle amplification (SRCA) technique was used for the first time to detect ASFV. The technique was developed using World Organization for Animal Health (WOAH)-approved primers targeting the p72 gene of the ASFV genome. The assay can be performed within 90 minutes at an isothermal temperature of 58°C without a requirement for sophisticated instrumentation. The results can be interpreted by examination with the naked eye with the aid of SYBR Green dye. This assay exhibited 100% specificity, producing amplicons only from ASFV-positive samples, and there was no cross-reactivity with other pathogenic viruses and bacteria of pigs that were tested. The lower limits of detection of SRCA, endpoint PCR, and real-time PCR assays were 48.4 copies/µL, 4.84 × 103 copies/µL, and 4.84 × 103 copies/µL, respectively. Thus, the newly developed SRCA assay was found to be 100 times more sensitive than endpoint and real-time PCR assays. Clinical tissue samples obtained from ASFV-infected domestic pigs and other clinical samples collected during 2020-22 from animals with suspected ASFV infection were tested using the SRCA assay, and a 100% accuracy rate, negative predictive value, and positive predictive value were demonstrated. The results indicate that the SRCA assay is a simple yet sensitive method for the detection of ASFV that may improve the diagnostic capacity of field laboratories, especially during outbreaks. This novel diagnostic technique is completely compliant with the World Health Organization's "ASSURED" criteria advocated for disease diagnosis, as it is affordable, specific, sensitive, user-friendly, rapid and robust, equipment-free, and deliverable. Therefore, this SRCA assay may be preferable to other complex molecular techniques for diagnosing African swine fever.

Novel helix loop-mediated isothermal amplification (HAMP) assay for colorimetric detection of Staphylococcus aureus in milk.

Staphylococcus aureus is an important and leading cause of foodborne diseases worldwide. Prompt detection and recall of contaminated foods are crucial to prevent untoward health consequences caused by S. aureus. Helix loop-mediated isothermal amplification (HAMP) is an exciting recent addition to the array of available isothermal-based nucleic acid amplification techniques. This study aimed to develop and evaluate a HAMP assay for detecting S. aureus in milk and milk products. The assay is completed in 75 minutes of isothermal temperature incubation (64 ˚C) and dye-based visual interpretation of results based on colour change. The specificity of the developed assay was ascertained using 27 S. aureus and 17 non S. aureus bacterial strains. The analytical sensitivity of the developed HAMP assay was 9.7 fg/µL of pure S. aureus DNA. The detection limit of the HAMP assay in milk (86 CFU/mL) was 1000x greater than the routinely used endpoint PCR (86 × 103 CFU/mL). The practicality of applying the HAMP assay was also assessed by analysing milk and milk product samples (n = 95) obtained from different dairy farms and retail outlets. The developed test is a more rapid, sensitive, and user-friendly method for the high-throughput screening of S. aureus in food samples and may therefore be suitable for field laboratories. To our knowledge, this is the first study to develop and evaluate the HAMP platform for detecting S. aureus.

Development and evaluation of a novel polymerase spiral reaction based testing technique for same-day visual detection of Campylobacter coli in pork.

The developed polymerase spiral reaction-based technique specifically amplified the ceuE gene of C. coli and involved a three-step centrifugation method for DNA extraction. PSR, real-time and end-point PCR were able to detect 62 fg, 620 fg and 6.2 pg C. coli DNA/tube, respectively. PSR detection limits for artificially contaminated pork samples without enrichment, with 12 h enrichment and after 24 h enrichment were 1000 CFU/g, 100 CFU/g, and 10 CFU/g samples, respectively which were ten times better than real-time PCR. The detection performance of PSR (with 12 h enrichment) was also compared to culture (ISO10272-1:2017) method using 75 naturally-contaminated samples, which revealed the sensitivity, specificity, PPV, NPV and accuracy of 100% (95%CI, 73.2%-100%), 98.4% (95%CI, 90%-99.9%), 93.3% (95%CI, 66%-99.6%), 100% (95%CI, 92.5%-100%) and 98.7% (95%CI, 92.8%-99.9%), respectively. The advantage and novelty of this assay are its equipment-free nature, dye-based interpretation by the naked eye, and the requirement of one enzyme and one primer pair. This assay could be a better alternative to other molecular methods and may help in reducing the possible troubles (e.g., gastroenteritis, hospitalization, or death) of belated detection of C. coli in food products. This is the primary report applying the PSR for C. coli detection.

Development of a novel visual isothermal amplification assay for rapid detection of Brucella spp.

Brucellosis is an economically important livestock disease worldwide besides having a noteworthy impact on human health. In this study, a rapid, simple, and ultra-sensitive nuclei-acid diagnostic technique was developed for the detection of brucellosis harnessing saltatory rolling circle amplification (SRCA). The diagnostic method was developed using World Organization for Animal Health (WOAH) approved primers targeting the bcsp31 gene of the Brucella genome. The assay can be accomplished within 90 min at a temperature of 65 °C without the requirement of sophisticated instrumentation. The result interpretation can be done with the naked eye with the aid of SYBR green dye. The developed technique displayed 100% specificity by amplifying only 10 reference and field strains of Brucella spp. and there was no cross-reactivity with the other tested pathogens. The lower limit of detections of SRCA and end-point PCR assays were 9.7 fg/µL (2.7 genome copies of Brucella) and 970 fg/µL, respectively. Thus, the developed SRCA assay was found to be 100× more sensitive than the end-point PCR assay. To the best of our knowledge, our study is the first one to develop an SRCA-based assay for the detection of brucellosis and it can be a diagnostic tool for resource-constrained laboratories and veterinary hospitals.

Biofilm-forming antimicrobial-resistant pathogenic Escherichia coli: A one health challenge in Northeast India.

This study aimed to investigate the prevalence of Shiga toxin-producing Escherichia coli (STEC), Enteropathogenic E. coli (EPEC), and Enterotoxigenic E. coli (ETEC) in common food animals (cattle, goats, and pigs) reared by tribal communities and smallholder farmers in Northeast India. The isolates were characterized for the presence of virulence genes, extended-spectrum beta-lactamases (ESBL) production, antimicrobial resistance, and biofilm production, and the results were statistically interpreted. In pathotyping 141 E. coli isolates, 10 (7.09%, 95% CI: 3.45%-12.66%) were identified as STEC, 2 (1.42%, 95% CI: 0.17%-5.03%) as atypical-EPEC, and 1 (0.71%, 95% CI: 0.02%-3.89%) as typical-EPEC. None of the isolates were classified as ETEC. Additionally, using the phenotypic combination disc method (ceftazidime with and without clavulanic acid), six isolates (46.1%, 95% CI: 19.22%-74.87%) were determined to be ESBL producers. Among the STEC/EPEC strains, eleven (84.6%, 95% CI: 54.55%-98.08%) and one (7.7%, 95% CI: 0.19%-36.03%) strains were capable of producing strong or moderate biofilms, respectively. PFGE analysis revealed indistinguishable patterns for certain isolates, suggesting clonal relationships. These findings highlight the potential role of food animals reared by tribal communities and smallholder farmers as reservoirs of virulent biofilm-forming E. coli pathotypes, with implications for food contamination and zoonotic infections. Therefore, monitoring these pathogens in food animals is crucial for optimizing public health through one health strategy.

Prevalence of bovine tuberculosis and analysis of risk factors among the dairy farms in and around Guwahati metropolitan city, India.

Bovine tuberculosis (bTB), a neglected zoonotic disease caused by Mycobacterium bovis is being reported worldwide. The present work was carried out from December 2020 to November 2021 to assess the prevalence and risk factors of bTB in peri-urban and urban dairy farms of Guwahati, Assam, India. A questionnaire was used to collect data on knowledge about bTB on 36 farms, and ten animals per farm were screened by single intradermal comparative cervical tuberculin test (SICCT) to determine the prevalence of bTB, giving a total of 360 animals. The demographic data of the farmers revealed that 61.1% respondents were illiterate, 66.7% had no awareness about bovine tuberculosis and 41.7% consumed unpasteurised milk and milk products. SICCT showed that 38 cattle from 18 of the farms were positive reactors for bTB, yielding an overall animal level prevalence of 10.55% (95% confidence interval (CI = 7.58-14.2%) and a 50% herd prevalence (95% CI 32.9-67.1%). Animals 5 years and above were found to be more likely to be positive for bTB (17.18%). The study highlighted the widespread prevalence of bovine tuberculosis in peri-urban and urban dairy farms of Guwahati which gives a picture also about other major cities of India. Hence, it is of utmost importance to undertake a comprehensive epidemiological study in such cities for effective control and prevention of bTB in a one health approach.

Development of a novel polymerase spiral reaction (PSR) assay for rapid and visual detection of Clostridium perfringens in meat.

C. perfringens is a widespread foodborne pathogen and one of the major concerns in the meat industry. There is a need for a simple, rapid and equipment free detection system for C. perfringens as conventional anaerobic culture method is labour and resource intensive. Here, we applied a novel polymerase spiral reaction phenomenon to develop and evaluate an assay for effortless and visual detection of C. perfringens in meat foods employing pork as a representative model. Specificity of the assay was determined using 51 C perfringens and 20 non- C. perfringens strains. Analytical sensitivity of the developed test was 80 fg DNA per tube indicating 100 times more sensitivity than end-point PCR assay. The detection limits were 980 CFU/g and 9.8 × 104 CFU/g of pork for PSR and PCR assays, respectively. The operation time of the PSR assay including DNA extraction was 120 min. The developed PSR assay was accurate and effective in comparison to culture method, in detecting C. perfringens in 38 of 74 pork samples. Therefore the specificity, sensitivity, negative predictive value, positive predictive value and accuracy rate of the developed PSR assay were 100%. The developed PSR assay is easy to perform, rapid, affordable, permitting sophisticated-equipment free amplification and naked eye interpretation. This is the initial report in which the PSR assay was optimized for the detection of C. perfringens.

Erratum to "Development of a novel polymerase spiral reaction (PSR) assay for rapid and visual detection of Clostridium perfringens in meat" [Heliyon 7 (1) (January 2021) Article e05941].

[This corrects the article DOI: 10.1016/j.heliyon.2021.e05941.].

Genome wide host gene expression analysis in mice experimentally infected with Pasteurella multocida.

Pasteurella multocida causes acute septicemic and respiratory diseases, including haemorrhagic septicaemia, in cattle and buffalo with case fatality of 100%. In the present study, mice were infected with P. multocida (1.6 × 103 cfu, intraperitoneal) to evaluate host gene expression profile at early and late stages of infection using high throughput microarray transcriptome analyses. Several differentially expressed genes (DEGs) at both the time points were identified in P.multocida infected spleen, liver and lungs. Functional annotation of these DEGs showed enrichment of key pathways such as TLR, NF-κB, MAPK, TNF, JAK-STAT and NOD like receptor signaling pathways. Several DEGs overlapped across different KEGG pathways indicating a crosstalk between them. The predicted protein-protein interaction among these DEGs suggested, that the recognition of P. multocida LPS or outer membrane components by TLR4 and CD14, results in intracellular signaling via MyD88, IRAKs and/or TRAF6 leading to activation of NFκB and MAPK pathways and associated cytokines.