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.

Higher prevalence of multidrug-resistant extended-spectrum ß-lactamases producing Escherichia coli in unorganized pig farms compared to organized pig farms in Mizoram, India.

AIM: The present study was conducted to record the prevalence of multidrug-resistant (MDR), extended-spectrum ß-lactamases (ESBLs) producing Escherichia coli from pig population of organized and unorganized farms of Mizoram and to record the presence of ESBLs, non-ESBLs, and integrons. MATERIALS AND METHODS: Fecal samples were collected from pigs under organized (n=40) and unorganized (n=58) farms of Mizoram. Samples were processed for isolation and identification of E. coli by conventional techniques, BD Phoenix™ automated bacterial system, and polymerase chain reaction (PCR) based confirmatory test. All the isolates were subjected to antimicrobial sensitivity test by disk diffusion assay and ESBLs production by double-disk synergy test (DDST). The ESBLs producing isolates were subjected to PCR for determination of ESBLs genes and all the isolates were screened for non-ESBLs genes and integrons by PCR. RESULTS: A total of 258 E. coli was isolated and identified from organized (n=120) and unorganized farms (n=138). Majority of the E. coli isolates exhibited high level of resistance against amoxicillin (Ax) (81.78%), cefalexin (85.42%), co-trimoxazole (50.78%), sulfafurazole (69.38%), tetracycline (65.89%), and trimethoprim (TR) (51.94%). Statistically highly significant (p<0.01) variations in resistance among the isolates from organized and unorganized farms were recorded in case of Ax, ampicillin, cephalexin, ciprofloxacin, co-trimoxazole, gentamicin, piperacillin, and TR. By DDST, 65.89% isolates were recorded as ESBLs producer, of which 82/120 (68.33%) and 88/138 (63.77%) were from organized and unorganized farms, respectively. A total of 29/258 (11.24%) isolates were positive for at least one ESBLs gene. blaTEM was most frequently (9.69%) gene, followed by blaCTX -M (5.04%) and blaCMY (0.78%). Altogether, 6 (5.00%), 4 (3.33%), and 2 (1.67%) isolates from the organized farms were positive for blaCTX-M , blaTEM , and blaCMY genes, respectively. Similarly, 21 (15.22%) and 7 (5.07%) isolates from the unorganized farms were positive for blaTEM and blaCTX-M genes, respectively. None of them were positive for blaSHV genes. Altogether 57 (22.09%), 9 (3.49%), 66 (25.58%), 78 (30.23%), 21 (8.14%), and 18 (6.98%) isolates were positive for tetA, tetB, sul1, sul2, aadA, and dfrla genes, respectively. The prevalence of non-ESBLs genes was higher in the E. coli isolates from the unorganized farms than organized farms. CONCLUSION: MDR and ESBLs producing E. coli are circulating among the pigs and their environment in Mizoram. Pigs under unorganized farms exhibited higher level of resistance against majority of the antimicrobials, including third-generation cephalosporins, which might be an indication of overuse or misuse of antibiotics under the unorganized piggery sectors in Mizoram.

Prevalence and molecular characterization of Salmonella species associated with piglet diarrhea in North East India.

Salmonellosis is a public health concern worldwide and also causes huge losses to the piggery industry. A total of 457 fecal samples were collected from organized and unorganized farms including indigenous and crossbreed piglets of North East India. Salmonella isolates were serotyped, screened for their virulence genes, characterized for drug resistance pattern and representative isolates were cloned and sequenced for their partial length enterotoxin (stn) gene. A total of 8.31% Salmonella were identified with higher prevalence observed in unorganized compared to organized farms and higher detection level in cross breed compared to indigenous piglets. Salmonella typhimurium (65.78%) was found to be the predominant serovar and irrespective of serovars high number of isolates (68.4%) harboured enterotoxin gene. The isolates were multidrug resistant showing highest resistance against cefalexin (77.31%). Sequence analysis of stn gene showed two isolates having diverse sequence compared to other isolates. Our study revealed the significance of Salmonella as important pathogen with zoonotic potential between porcine and human populations. This is probably the first systematic study of Salmonella species associated with piglet diarrhea in India.

Extended spectrum beta-lactamase producing Shiga-toxin producing-Escherichia coli in piglets, humans and water sources in North East region of India.

Extended spectrum ß-lactamases (ESBL) producing Shiga toxin producing E. coli (STEC) are posing a constant threat to public health throughout the world leading to serious infections and raising key therapeutic issues. A total of 219 fecal samples were collected from piglets with diarrheoa, pig farmers and water sources in North East India; and were processed for isolation of Escherichia coli. The isolates were screened for antimicrobial resistance and suspected isolates for ESBLs production by double-disk synergy test (DDST). Escherichia coli isolates positive for DDST were subjected for detection of selected ESBL/beta-lactamase genes and virulence associated genes by PCR. By DDST, 337 (67·94%) E. coli isolates were detected as ESBLs producer, of which 211 (66·98%), 117 (70·91%) and 9 (56·25%) isolates were from piglets, humans and water sources respectively. A total of 64 (12·90%) isolates were recorded as STEC, of which 48 (9·68%), 6 (1·21%) and 10 (2·02%) were from human, piglets and water respectively. Majority of the STEC isolates (64·06%) possessed multiple virulence genes, of which 59·38% also harboured ESBL/beta-lactamase genes with 32·81% STEC isolates being positive for multiple ESBL/beta-lactamase genes. SIGNIFICANCE AND IMPACT OF THE STUDY: Multidrug resistant (MDR) enteric bacteria are global concern. Association of MDR traits in STEC isolates are another rising issue in human and animal health perspective. The interaction of such organisms among the human, domestic animals and adjoining water sources require to be analysed systematically. The present study exhibited the possible transmission of MDR-STEC among the human, domestic animals and water sources in the North eastern states of India. To the best of our knowledge, this is the first report of such kind in India.

Prevalence and molecular characterization of porcine Picobirnavirus in piglets of North East Region of India.

Picobirnaviruses (PBVs) have been recognized as one of the important causal viral agents of gastroenteritis in several animal species especially in young immunocompromised hosts. In this study, we report the prevalence and molecular epidemiology of porcine PBVs from North Eastern Hilly region of India. A total of 457 fecal samples from piglets were collected from local (n = 130) and cross (n = 327) breed piglets in different seasons for 2 years. All the samples were subjected to RNA-PAGE and RT-PCR analysis for detection of PBVs. A total of 4.59 and 11.15% samples were recorded as positive for PBVs by RNA-PAGE and RT-PCR, respectively. Rate of detection was higher from diarrhoeic animals (13.56%) compared to non-diarrhoeic (4.23%) animals. Higher prevalence rate was observed from unorganized farms (14.22%) compared to organized farms (8.0%) with slightly higher detection from cross breed (11.62%) compared to local breed (10.0%). Maximum cases of piglet diarrhea associated with PBVs were detected during summer (16.4%) and winter (14.39%) seasons compared to autumn (4.80%) and spring (6.45%). All the samples were positive for PBV genogroup I only. Based upon the sequence analysis, the isolates were unique and placed in separate clad and were not closely associated with any other Indian isolates of PBVs so far. Two isolates were closely related with one Chinese isolate recovered from sewage water. This is the first systematic study of prevalence of PBVs associated with piglet diarrhea.