Development and evaluation of lateral flow assay for sero-diagnosis of Japanese encephalitis in swine.

Japanese encephalitis (JE) is a re-emerging mosquito-borne zoonotic flaviviral disease. Swine sero-convert 2-3 weeks before infection occurs in humans and thus serves as a suitable sentinel for JE surveillance and outbreak prediction in human population. The present study was conducted with the objective of developing a lateral flow assay (LFA) for detecting JEV antibodies in swine sera. Three different formats were tried using recombinant NS1 protein as antigen in order to select the best format. In format I, gold nanoparticles were conjugated with antigen followed by spotting of antigen on NCM as test line and anti-antigen IgG on NCM as control line. In format II, gold nanoparticles were conjugated with antigen followed by spotting of staphylococcal protein A as test line and anti-antigen IgG as control line. Format III used gold nanoparticles conjugated with goat anti-pig IgG followed by spotting of antigen as test line and pig IgG as control line. Amongst the three formats, format II was found to be superior with 100% relative diagnostic sensitivity and 100% relative diagnostic specificity during monsoon and post-monsoon period. A panel of 500 field swine serum samples was tested using format II which revealed sero-positivity of 15.6%, and the format was found suitable to screen swine serum samples during monsoon and post-monsoon period.

Evaluation of carboxyl beads based latex agglutination test for rapid sero-diagnosis of Japanese encephalitis.

Japanese encephalitis (JE) is a major public health problem in the South Asian countries including India. Pigs serve as a relevant sentinel model, the surveillance of which could predict a potential JE outbreak in human population nearby. However, existing serological detection methods like Enzyme-Linked Immuno Sorbent Assay (ELISA), virus neutralization test (VNT) and Haemagglutination Inhibition (HI) require elaborative laboratory facilities which are invariably not available in field conditions. Recognizing the lacunae, attempts were made to develop recombinant antigen (rNS1) based latex agglutination test (LAT) as a rapid on-site test using covalent coupling method. Four different formats were evaluated using different coupling buffers, blocking buffers and reaction conditions. The format in which borate buffer at alkaline pH (8.5) was used for coupling of antigen with carboxylated beads followed by blocking with skimmed milk powder was found to be the best amongst all. Developed latex based test was used for screening of 207 pig serum samples for JE which revealed relative diagnostic sensitivity and specificity of 80.2% and 95.2%, respectively in comparison with indirect IgG ELISA. Hence, the present study demonstrated that covalently coupled recombinant antigen based LAT could be used as a reliable screening test for surveillance of JE in pigs under field conditions.

TaqMan real-time RT-PCR assay for detecting Japanese encephalitis virus in swine blood samples and mosquitoes.

Japanese encephalitis (JE) is an emerging mosquito-borne zoonotic flaviviral disease. The present study was undertaken with the objective to develop TaqMan real-time reverse-transcription polymerase chain reaction (RT-PCR) assay for rapid detection and quantification of Japanese encephalitis virus (JEV) in swine blood and mosquito vectors. The amplification of envelope (E) gene was targeted by designing gene-specific MGB TaqMan fluorescent probe along with the primers. The best performance in terms of sensitivity was achieved by standardized TaqMan real-time RT-PCR with a detection limit of 2.8 copies/reaction and it was found to be 4-log more sensitive than conventional RT-PCR. The applicability of the standardized TaqMan assay was evaluated by screening representative sets of field swine blood samples and mosquito pools for JEV. The viral load ranged between 3.32 × 107-4.2 × 102 copies/ml of swine blood samples, and 5.7 × 109-1.3 × 102 copies/pool of mosquitoes. The standardized assay which is highly sensitive, specific and rapid would aid in screening sentinel swine and mosquitoes under JEV surveillance programs for effective prevention and control of disease in human beings.

Seasonal occurrence of Japanese encephalitis vectors in Bareilly district, Uttar Pradesh, India.

BACKGROUND & OBJECTIVES: Japanese encephalitis (JE) is one of the most common causes of acute encephalitis syndrome in many states of India. Uttar Pradesh state is well known for JE endemicity, contributing 75% of total cases during recent past. Several sporadic cases have been reported from Bareilly region of the state. The disease spread by bite of Culex mosquito. Survey of literature revealed no data on mosquito fauna with reference to JE in this region. Therefore, this study was planned to survey seasonal mosquito population and occurrence of JE vectors in Bareilly region. METHODS: Mosquitoes were sampled on monthly basis from organized pig farm from February 2016 to January 2017 and identified using mosquito identification keys. The meteorological parameters of the area were obtained monthly and standard statistical methods were used to assess the relationship between different weather variables and mosquito population. RESULTS: A total of 4337 mosquitoes belonging to five genera were collected. Mosquitoes of genus Culex were predominant and contributed 84.41% to the total catch. The most dominant species was Cx. tritaeniorhynchus (30.81%), followed by Cx. quinquefasciatus (28.50%), Cx. gelidus (17.24%), Cx. pseudovishnui (11.85%), Cx. vishnui (8.11%), Cx. fuscocephala (2.70%), Cx. infula (0.76%) and Cx. bitaeniorhynchus (0.03%). Pronounced seasonal variation was observed with majority of mosquitoes showing high density in monsoon and post-monsoon period. INTERPRETATION & CONCLUSION: The present study provides knowledge on distribution of JE vector in Bareilly which indicates that the area is at risk of JE outbreak. Abundance of Culex vector clearly demarcates possible threat of JE incidence in the study area. A long-term entomological study is needed to further evaluate the significant role of different weather variables in shaping mosquito densities.

Comparative evaluation of nucleic acid-based assays for detection of Japanese encephalitis virus in swine blood samples.

Japanese encephalitis is an emerging mosquito-borne flaviviral zoonotic disease. The present study was undertaken with the objective of developing rapid and sensitive nucleic-acid-based assays for detection of Japanese encephalitis virus (JEV) in swine blood samples. Three nucleic-acid-based assays, viz., reverse transcription polymerase chain reaction (RT-PCR), reverse transcription loop-mediated isothermal amplification (RT-LAMP), and real-time RT-PCR, were developed and compared in terms of their diagnostic efficacy. All three assays were found to be 100 per cent specific. The minimum detection limit of RT-LAMP and real-time RT-PCR was 12 copies/µl, while RT-PCR could detect 1.2 × 10(5) copies/µl. On comparison, RT-LAMP and real-time RT-PCR were 4-log more sensitive than RT-PCR. The applicability of the assays was evaluated by screening 135 field swine blood samples, of which 24 (17.77 %) were positive by RT-LAMP and real-time RT-PCR and only six (4.44 %) were positive by RT-PCR. The viral load in swine blood samples ranged between 2 × 10(6) and 4.8 × 10(9) copies per ml of blood by real-time RT-PCR. The comparative diagnostic sensitivity and specificity of RT-LAMP vis-à-vis real-time RT-PCR was found to be 100 %, while the sensitivity and specificity of RT-PCR vis-à-vis real-time RT-PCR was found to be 25 % and 100 %, respectively. Thus, the use of RT-PCR may cause the incidence of JEV in the swine population to be underestimated, while the real-time RT-PCR reported here is the test of choice for reference laboratories, and the newly developed one-step RT-LAMP assay will be suitable for field-level testing.

Seroprevalence of Japanese encephalitis virus in pig populations of Tamil Nadu, India: Exploring the tropical endemic link of virus.

Japanese encephalitis virus (JEV) is a major cause of encephalitis in Southeast Asia. Tamil Nadu, a state located in the southern part of India, contributes substantially to the national burden of human JE cases every year. However, limited information is available on the epidemiology of JE in pig populations of Tamil Nadu. A cross-sectional study was conducted to assess JEV prevalence in pig populations of Tamil Nadu. A total of 710 pigs reared in 118 farms across 10 districts of Tamil Nadu were sampled using multistage cluster random sampling. Serum samples were analyzed for their JEV status using Immunoglobulin M (IgM) and Immunoglobulin G (IgG) Enzyme-Linked Immunosorbent Assay (ELISA). At the animal-level, the apparent JEV seroprevalence was 60.4% (95% CI: 56.8% - 64.0%) and the true seroprevalence was 50.1% (95% CI: 47.0% - 53.2%). The herd-level apparent seroprevalence was 94.1% (95% CI: 88.1% - 97.5%) and the true seroprevalence was 93.3% (95% CI: 89.5% - 96.2%). The intensity of JEV circulation was high in all the districts, with seroprevalence ranging between 43% and 100%. Pigs across all age categories were seropositive and a high overall seroprevalence of 95.2% (95% CI: 76.2% - 99.9%) was recorded in pigs older than 12 months. JEV seropositivity was recorded in all the seasons but the prevalence peaked in the monsoon (67.9%, 95% CI: 61.1% - 74.2%) followed by winter (65.1%, 95%CI: 57.4% - 72.2%) and summer (53.3%, 95% CI: 47.8% - 58.8%) seasons. The results indicate that JEV is endemic in pigs populations of the state and a one health approach is essential with collaborative actions from animal and public health authorities to control JE in Tamil Nadu, India.

Draft genome sequences of Brucella melitensis from human and animal sources in India.

We present the draft genome sequences of 23 Brucella melitensis isolates derived from human and animal sources across India with genome size predominantly at 3.207 M and uniform GC content (57.24%) across isolates. The accession numbers and detailed sequencing data enhance the utility of this resource for further genomic studies.

Development of immunochromatographic strip assay to detect recent infection of Japanese encephalitis virus in swine population.

Japanese Encephalitis (JE) is a mosquito borne re-emerging viral zoonotic disease. Sero-conversion in swine occurs 2-3 weeks before human infection, thus swine act as a suitable sentinel for predicting JE outbreaks in humans. The present study was undertaken with the objective of developing immunochromatographic strip (ICS) assay to detect recent infection of Japanese Encephalitis virus (JEV) in swine population. The two formats of ICS assay were standardized. In the first format, gold nanoparticles (GNP) were conjugated with goat anti-pig IgM (50 µg/ml) followed by spotting of recombinant NS1 protein (1 mg/ml) of JEV on NCM as test line and protein G (1 mg/ml) as control line. In the format-II, GNP were conjugated with rNS1 protein (50 µg/ml) followed by spotting of Goat anti-pig IgM (1 mg/ml) as test line and IgG against rNS1 (1 mg/ml) as control line. To decrease the non- specific binding, blocking of serum and nitrocellulose membrane (NCM) was done using 5% SMP in PBS-T and 1% BSA, respectively. Best reaction conditions for the assay were observed when 10 µl of GNP conjugate and 50 µl of 1:10 SMP blocked sera was reacted on BSA blocked NCM followed by reaction time of 15 mins. Samples showing both test and control line were considered positive whereas samples showing only control line were considered negative. A total of 318 field swine sera samples were screened using indirect IgM ELISA and developed ICS assay. Relative diagnostic sensitivity and specificity of format-I was 81.25% and 93.0% whereas of format-II was 87.50% and 62.93%, respectively. Out of 318 samples tested, 32 were positive through IgM ELISA with sero-positivity of 10.06% while sero-positivity with format-I of ICS was 8.1%. Owing to optimal sensitivity and higher specificity of format-I, it was validated in three different labs and the kappa agreement ranged from 0.80 to 1, which signifies excellent repeatability of the developed assay to test field swine sera samples for detecting recent JEV infection.

Exploring genetic determinants of antimicrobial resistance in Brucella melitensis strains of human and animal origin from India.

Introduction: Antimicrobial resistance (AMR) in Brucella melitensis, the causative agent of brucellosis, is of growing concern, particularly in low and middle-income countries. This study aimed to explore the genetic basis of AMR in B. melitensis strains from India. Methods: Twenty-four isolates from humans and animals were subjected to antimicrobial susceptibility testing and whole-genome sequencing. Results: Resistance to doxycycline (20.80%), ciprofloxacin (16.67%), cotrimoxazole (4.17%), and rifampicin (16.67%) was observed. Genome analysis revealed efflux-related genes like mprF, bepG, bepF, bepC, bepE, and bepD across all isolates, however, classical AMR genes were not detected. Mutations in key AMR-associated genes such as rpoB, gyrA, and folP were identified, intriguingly present in both resistant and susceptible isolates, suggesting a complex genotype-phenotype relationship in AMR among Brucella spp. Additionally, mutations in efflux genes were noted in resistant and some susceptible isolates, indicating their potential role in resistance mechanisms. However, mutations in AMR-associated genes did not consistently align with phenotypic resistance, suggesting a multifactorial basis for resistance. Discussion: The study underscores the complexity of AMR in B. melitensis and advocates for a holistic multi-omics approach to fully understand resistance mechanisms. These findings offer valuable insights into genetic markers associated with AMR, guiding future research and treatment strategies.

Cloning and sequencing of biofilm-associated protein (bapA) gene and its occurrence in different serotypes of Salmonella.

AIMS: Salmonella spp. has the capability to form biofilm on various surfaces. Biofilm-associated protein (bapA), a large surface protein has been shown to play a leading role in the development of biofilm in Salmonella. Objective of this study was to investigate the presence of bapA gene in different serotypes of Salmonella spp. and to characterize DNA fragment encoding bapA protein of Salmonella Enteritidis. METHODS AND RESULTS: Sixty-seven Salmonella strains belonging to 34 serovars isolated from diverse sources in India were screened for the presence of bapA gene employing a primer designed for the purpose. All the strains yielded a positive amplification indicating that the bapA gene is well conserved in Salmonella spp. The amplified gene fragment of bapA was cloned in Escherichia coli (DH5 α) cells by using pGEM-T easy cloning vector. On partial sequence analysis, the product exhibited 667 base pairs, corresponding to 218 amino acids. CONCLUSIONS: BapA gene was found to be highly conserved in Salmonella. Partial sequence analysis of this gene from a strain of Salm. Enteritidis revealed close association with serotypes of poultry origin and also with some other animal/zoonotic serotypes. SIGNIFICANCE AND IMPACT OF THE STUDY: BapA gene can be targeted for the genus-specific detection of this organism from different sources. Antigenic index of bapA protein indicates its protective and diagnostic potentials.

Development of dipstick enzyme linked immunosorbent assay for on-site sero-diagnosis of Japanese encephalitis in swine.

Japanese encephalitis (JE) is an important viral zoonotic disease in Asia, especially in rural and suburban areas where rice cultivation and pig farming coexist. Pigs serve as a suitable sentinel model, the surveillance of which could predict a potential JE outbreak in human population in the immediate vicinity. However, existing diagnostics like ELISA and VNT require sophisticated laboratory facilities which are more often not available in field conditions. In the present study, we aimed at developing recombinant non-structural (NS1) protein-based dipstick IgG ELISA as an on-site assay for sero-diagnosis of JE in swine. The assay was standardized by optimizing various parameters and the following conditions were found to be ideal including 1 µg of rNS1 protein in carbonate buffer per strip of nitrocellulose membrane comb; bovine serum albumin as blocking agent at 4 °C overnight; serum dilution of 1:10 and conjugate dilution of 1:5000 in skimmed milk powder. Relative diagnostic sensitivity and specificity of dipstick IgG ELISA was 100% and 92.9%, respectively. The dipstick assay was validated in three laboratories as per OIE guidelines. The storage life of dipstick was up to 7 months at 4 °C. The assay is easy to perform and the results can be interpreted with visual observation that precludes the need for absorbance reading equipment. The standardized dipstick assay was found promising for screening swine serum samples in field conditions. Timely detection of JE virus in swine will aid in predicting the outbreak in humans and thus in taking suitable preventive and control measures.

Serological evidence of Japanese encephalitis virus infection in pigs in a low human incidence state, Goa, India.

Japanese encephalitis (JE) is a mosquito-borne zoonosis caused by Japanese encephalitis virus (JEV). It is a leading cause of encephalitis in humans especially children in Asia. Aquatic wading birds are the reservoirs and pigs serve as amplifying hosts for JEV. Humans and horses are dead-end hosts. JE is endemic in several states of India. Goa, a small state on the west coast of India, had witnessed JE outbreaks in the past and as on date human JE cases are reported sporadically. Although human JE cases are well documented in Goa, the status of JEV exposure of pigs has not been well documented. Hence the present study was undertaken with an objective of identifying JEV exposure in the pig population of Goa state in the light of declining human JE cases. To achieve the objective, between January 2017 and May 2019, serum samples from 666 pigs were screened using enzyme-linked immunosorbent assay (ELISA) for the detection of anti-JEV IgG. The apparent prevalence of anti-JEV IgG in pigs was found to be 7.1 % (95 % confidence interval 5.3 %-9.3 %) and true prevalence was 4.6 % (95 % confidence interval 2.7 %-7.1 %). The seroprevalence of JE recorded in pigs of Goa state was low compared to other endemic states in India, which may also be one of the reasons for the lower prevalence of human JE cases in Goa state. Univariate analysis revealed that the age of the pigs and district did not significantly influence the JE seroprevalence in pigs of Goa state. However, in multivariable logistic regression, the North Goa district was found to significantly (p = 0.017) influence the JE seroprevalence in pigs. The study identified that JEV is still circulating in the Goan pig population and hence constant vigil is required to monitor the intensity of JEV circulation in pigs. Besides forewarning possible human outbreaks in the locality, evidence of JEV exposure in pig population provides valuable data on the magnitude and extent of geographical spread.

Detection of recent infection of Japanese encephalitis virus in swine population using IgM ELISA: A suitable sentinel to predict infection in humans.

Japanese encephalitis (JE) is a mosquito-borne flaviviral zoonotic disease and is one of the major causes of encephalitis in children. Swine, being an amplifier host of Japanese encephalitis virus (JEV), play an important role in its epidemiology. Therefore, early detection of either JEV or antibodies against JEV in swine is a feasible alternative for initiating necessary measures to prevent the spread of infection to humans. Since IgM antibodies appear early in swine sera, recombinant NS1 protein based indirect IgM ELISA was developed in the present study with the objective to know the recent infection of swine population with JEV. The relative diagnostic sensitivity and specificity of the developed ELISA was 95.34% and 98.6%, respectively. The developed ELISA was found to have excellent reproducibility on inter-laboratory and inter-institutional validation studies. A total of 3,027 field swine sera samples were screened using the developed ELISA and 488 samples were found positive for IgM against JEV with an overall sero-positivity of 16.12% in swine population of India. The highest sero-positivity was observed in swine population of Eastern zone of India which coincided with the maximum number of human JE cases reported from this zone during the same period. Further, antibody kinetics study revealed that the IgM antibodies against NS1 protein of JEV started appearing in swine sera at day 5 and disappeared completely by day 40. The IgG antibodies started appearing at day 7, and remained for more than 365 days indicating the suitability of IgM ELISA to know the recent infection of JEV. The developed IgM ELISA can be readily incorporated into surveillance programs for detection of JEV activity in swine population so that outbreaks in humans can be prevented by taking suitable preventive measures.

Development of recombinant nonstructural 1 protein based indirect enzyme linked immunosorbent assay for sero-surveillance of Japanese encephalitis in swine.

Japanese encephalitis virus (JEV) causes severe neurological disease in humans, especially among children. The disease is endemic in several South Asian countries including India. Swine play a major role as amplifier host for JEV and act as a source of infection to humans through mosquito bite. Early detection of either virus or antibodies in swine will aid to undertake control measures to prevent virus spread to humans. Swine seldom show symptoms of JEV infection and the viraemic phase lasts for a short period of 3 to 4 days indicating the potential of detection of antibodies, which remain for relatively longer period, as a suitable alternative. Cost effective and sensitive assays for the detection of JEV antibodies in swine are not available indigenously. Hence, we have developed a recombinant nonstructural protein 1 (rNS1) based enzyme linked immunosorbent assay for the detection of IgG antibodies against JEV in swine. The test is robust, highly sensitive (91%), specific (97%), reproducible and affordable. Field validation of the assay was done by screening 3628 swine Serum samples collected from different parts of India. The overall sero-positivity was found to be 32.22%. The developed ELISA can be readily incorporated into surveillance programs for detection of Japanese encephalitis virus activity in swine population thereby aiding in prediction of outbreaks in humans.

Polymerase chain reaction-restriction fragment length polymorphism of mitochondrial 12S rRNA gene: a simple method for identification of poultry meat species.

Chicken (Gallus gallus), duck (Anas platyrhynchos), turkey (Meleagris gallopavo), guinea fowl (Numida meleagris) and quail (Coturnix japonica) are the common poultry species consumed as meat throughout the world. In this work, a molecular technique has been developed for identification and differentiation of meat originating from these species. This tool helps in detection of misrepresentation of different poultry meats. The technique involves the extraction of DNA from the given sample, polymerase chain reaction (PCR) amplification of mitochondrial 12S rRNA gene using universal primers, restriction analysis with selected restriction enzymes, followed by identification of meat species based on restriction fragment length polymorphism (RFLP) pattern. In this study, we used HinfI, Mph1 103I, MvaI, and Eco47I to identify and differentiate to poultry species referred to above. This species identification technique has also been applied successfully to processed meat products including those cooked at 120 degrees C for 30 min. Simplicity of interpretation of results combined with versatility makes this a convenient and appropriate technique in the hands of meat analysts for identifying poultry meat species.

Comparative study of cytotoxicity of Aeromonas spp. on four different cell lines.

In vitro cytotoxicity is an important virulence property of motile mesophilic Aeromonas species. Cell-free supernatant prepared from 55 Aeromonas isolates including one A. hydrophila type strain (MTCC 646) were examined for their cytotoxic potential on four different cell lines (Vero, BHK-21, MDBK, B 95a). Results of the study revealed cytotoxic potential in 92.72% of the isolates. Analysis of data exposed significant variation among isolates in respect of their cytotoxicity. Vero cells proved to be most sensitive to aeromonal toxins and B 95a cells showed significantly (P<0.01) lower response compared to other cell lines. Sensitivities of BHK-21 and MDBK cell lines were in between Vero and B 95a.

Occurrence of Campylobacter jejuni in vegetables.

In order to understand the importance of vegetables in the transmission of thermophilic Campylobacter, 56 samples of different vegetables were screened. Out of these, 2 samples (1 spinach and 1 fenugreek) revealed the presence of Campylobacter jejuni biotype I. Both the isolates were enteropathogenic in rat ileal loop test.

Preservative effect of combinations of acetic acid with lactic or propionic acid on buffalo meat stored at refrigeration temperature.

A silverside of buffalo was cut in 15 equal-sized steaks and divided into five groups, each group containing three steaks. The steaks from groups 1,2,3 and 4 were treated with 1, 2, 3 and 4% acetic:lactic acid combinations, respectively, and the fifth group was kept as a control. Similar treatments were also given with acetic: propionic acid mixtures. The microbial analysis and changes in colour and odour were noted at 0, 24, 72 and 168 h. The bacteriostatic and bacteriocidal action of the acid mixtures increased with increasing concentration but the effect was reduced as the time advanced. Both acid mixtures had pronounced antibacterial effect on gram negative organisms than gram positive ones. The 3% acetic: lactic acid combination showed reduction in bacterial numbers without affecting the colour and odour of buffalo meat and is recommended for decontamination and preservation of meat for up to seven days at refrigeration temperature (7 ± 1°C).

Effect of nisin and its combination with sodium chloride on the survival of Listeria monocytogenes added to raw buffalo meat mince.

Antilisterial activity of nisin (Nisaplin), alone at concentrations of 400 and 800 IU/g and in combination with 2% sodium chloride was incorporated in raw buffalo meat mince. Samples of the raw meat mince were inoculated with 10(3) colony forming units (cfu)/g of L. monocytogenes and stored at 4°C for 16 days and at 37°C for 36 h. Initial estimates of pH, extract release volume, mesophilic and psychrophilic counts were found to be 5.74, 48 ml, 3.5×10(5) and 1.0×10(5) cfu/g of meat, respectively. The growth of L. monocytogenes in the treated groups was significantly (P<0.05) inhibited compared to the control group. The degree of inhibition increased with increasing concentration of nisin and decreasing storage temperature. Addition of 2% sodium chloride in combination with nisin increased the efficacy of nisin at both storage temperatures. The pH in the treated groups remained significantly lower (P<0.01) than in the control groups at both 4 and 37°C.