Development and validation of multiplex PCR based molecular serotyping of Salmonella serovars associated with poultry in India.

Salmonella species are Gram-negative bacteria with more than 2600 serovars. Among these serovars, many are associated with various diseases in livestock and humans. White Kauffman Le-Minor (WKL) serotyping scheme applies specific serum to determine the serovars of Salmonella. Recent studies have applied molecular methods for serovar predictions. These methods include PCR, hybridization and sequence data to detect/predict serovar-specific genetic elements. Among these, PCR is a robust method if the unique genetic element is already known. Within this context, also involving novel primers, two multiplex PCR assays were standardized to detect six important Salmonella serovars viz. Typhimurium, Enteritidis, Kentucky, Infantis, Virchow and Gallinarum associated with poultry in India. The developed PCR assays showed targeted serovar specificity. Serial dilution experiments of both kit-based and crude lysate DNA preparations indicated similar applicability of both methods for testing from pure cultures. Further the developed assays were validated with 25 recent field isolates to confirm the applicability in routine diagnosis. The PCR assay could predict all the targeted serovars (17/25) with 100% specificity (CI-95%; 0.63-1). Molecular serotyping can reduce the number of serum used in comparison to the conventional serotyping which involves more random application of serum.

A study on the occurrence of Burkholderia cepacia complex in ultrasound gels used in different veterinary clinical settings in India.

Burkholderia cepacia complex (Bcc) organisms are emerging multidrug-resistant pathogens. They are opportunistic and cause severe diseases in humans that may result in fatal outcomes. They are mainly reported as nosocomial pathogens, and transmission often occurs through contaminated pharmaceutical products. From 1993 to 2019, 14 Bcc outbreaks caused by contaminated ultrasound gels (USGs) have been reported in several countries, including India. We screened a total of 63 samples of USGs from various veterinary and human clinical care centers across 17 states of India and isolated 32 Bcc strains of Burkholderia cenocepacia (46.8%), B. cepacia (31.3%), B. pseudomultivorans (18.8%) and B. contaminans (3.1%) species. Some isolates were co-existent in a single ultrasound gel sample. The isolation from unopened gel bottles revealed the intrinsic contamination from manufacturing sites. The MALDI-TOF analysis to identify the Bcc at the species level was supported by the partial sequencing of the recA gene for accurate species identification. The phylogenetic analysis revealed that isolates shared clades with human clinical isolates, which is an important situation because of the possible infections of Bcc by USGs both in humans and animals. The pulsed field gel electrophoresis (PFGE) typing identified the genetic variation among the Bcc isolates present in the USGs. The findings indicated USGs as the potential source of Bcc species.

Analysis of bluetongue disease epizootics in sheep of Andhra Pradesh, India using spatial and temporal autocorrelation.

Bluetongue (BT) disease poses a constant risk to the livestock population around the world. A better understanding of the risk factors will enable a more accurate prediction of the place and time of high-risk events. Mapping the disease epizootics over a period in a particular geographic area will identify the spatial distribution of disease occurrence. A Geographical Information System (GIS) based methodology to analyze the relationship between bluetongue epizootics and spatial-temporal patterns was used for the years 2000 to 2015 in sheep of Andhra Pradesh, India. Autocorrelation (ACF), partial autocorrelation (PACF), and cross-correlation (CCF) analyses were carried out to find the self-dependency between BT epizootics and their dependencies on environmental factors and livestock population. The association with climatic or remote sensing variables at different months lag, including wind speed, temperature, rainfall, relative humidity, normalized difference vegetation index (NDVI), normalized difference water index (NDWI), land surface temperature (LST), was also examined. The ACF & PACF of BT epizootics with its lag showed a significant positive autocorrelation with a month's lag (r = 0.41). Cross-correlations between the environmental variables and BT epizootics indicated the significant positive correlations at 0, 1, and 2 month's lag of rainfall, relative humidity, normalized difference water index (NDWI), and normalized difference vegetation index (NDVI). Spatial autocorrelation analysis estimated the univariate global Moran's I value of 0.21. Meanwhile, the local Moran's I value for the year 2000 (r = 0.32) showed a high degree of spatial autocorrelation. The spatial autocorrelation analysis revealed that the BT epizootics in sheep are having considerable spatial association among the outbreaks in nearby districts, and have to be taken care of while making any forecasting or disease prediction with other risk factors.

Spatio-temporal and time series analysis of bluetongue outbreaks with environmental factors extracted from Google Earth Engine (GEE) in Andhra Pradesh, India.

This study describes the spatial and temporal patterns of bluetongue (BT) outbreaks with environmental factors in undivided Andhra Pradesh, India. Descriptive analysis of the reported BT outbreaks (n = 2,697) in the study period (2000-2017) revealed a higher frequency of outbreaks during monsoon and post-monsoon months. Correlation analysis of Normalized Difference Vegetation Index (NDVI), Normalized Difference Water Index (NDWI), rainfall and relative humidity (RH) displayed a significant positive correlation with BT outbreaks (p < .05). Retrospective unadjusted space-time, adjusted temporal and spatial analysis detected two, five and two statistically significant (p < .05) clusters, respectively. Time series distribution lag analysis examined the temporal patterns of BT outbreaks with environmental, biophysical factors and estimated that a decrease in 1 unit of rainfall (mm) was associated with 0.2% increase in the outbreak at lag 12 months. Similarly, a 1°C increase in land surface temperature (LST) was associated with 6.54% increase in the outbreaks at lag 12 months. However, an increase in 1 unit of wind speed (m/s) was associated with a 16% decrease in the outbreak at lag 10 months. The predictive model indicated that the peak of BT outbreaks were from October to December, the post-monsoon season in Andhra Pradesh region. The findings suggest that environmental factors influence BT outbreaks, and due to changes in climatic conditions, we may notice higher numbers of BT outbreaks in the coming years. The knowledge of spatial and temporal clustering of BT outbreaks may assist in adopting proper measures to prevent and control the BT spread.

Antimicrobial Activity of Agarwood Oil Against Multiple-Drug-Resistant (MDR) Microbes of Clinical, Food and Environmental Origin.

BACKGROUND AND OBJECTIVES: Multiple-Drug-Resistance (MDR) among bacteria is an imminent problem and alternative therapies are seen as a future abode. Agarwood Oil (AO) is described to possess antimicrobial activity besides many other medicinal utilities. This paper discusses the antimicrobial activity of AO on MDR and non-MDR strains of microbes of 69 genera isolated from clinical and non-clinical samples. METHODS AND RESULTS: In this study sensitivity of microbes was determined for conventional antimicrobials and AO using disc diffusion assay followed by determination of minimum inhibitory concentration (MIC) using agar well dilution assay. A total of 18.5% (522) strains were found sensitive to AO. Carbapenem resistant bacterial strains were more often (p, ≤0.01) resistant to antibiotics with 4.2 times more odds (99% CI, 2.99-5.90) of being MDR than carbapenem sensitive strains but no difference in their AO sensitivity was observed. However, MDR strains were more often (p, <0.001) resistant to AO than non-MDR strains. Bacteria isolated from dogs were more often sensitive to AO than those from buffaloes, human, horse, and cattle. On the other hand, bacteria from pigs were more often (p, ≤0.05) resistant to AO than bacteria from human, cattle, buffaloes, dogs, wild carnivores and birds. Oxidase positive Gram positive bacteria had 4.29 (95% CI, 2.94-6.27) times more odds to be AO sensitive than oxidase negative Gram negative bacteria. Bacillus species strains were the most sensitive bacteria to AO followed by strains of Streptococcus and Staphylococcus. The MIC of AO for different bacteria ranged from 0.01 mg/mL to > 2.56 mg/mL. CONCLUSION: The study concluded that MDR and AO resistance had a similar trend and AO may not be seen as a good antimicrobial agent against MDR strains.

Occurrence of Bordetella infection in pigs in northern India.

Bordetella bronchiseptica infection causing atrophic rhinitis in pigs is reported from almost all countries. In the present study, occurrence of Bordetella infection in apparently healthy pigs was determined in 392 pigs sampled to collect 358 serum samples and 316 nasal swabs from Northern India by conventional bacterioscopy, detection of antigen with multiplex polymerase chain reaction (mPCR), and detection of antibodies with microagglutination test (MAT) and enzyme linked immune-sorbent assay (ELISA). Bordetella bronchiseptica could be isolated from six (1.92%) nasal swabs. Although isolates varied significantly in their antimicrobial sensitivity, they had similar plasmid profile. The genus specific and species specific amplicons were detected from 8.2% and 4.4% nasal swabs using mPCR with alc gene (genus specific) and fla gene and fim2 gene (species specific) primers, respectively. Observations revealed that there may be other bordetellae infecting pigs because about 50% of the samples positive using mPCR for genus specific amplicons failed to confirm presence of B. bronchiseptica. Of the pig sera tested with MAT and ELISA for Bordetella antibodies, 67.6% and 86.3% samples, respectively, were positive. For antigen detection mPCR was more sensitive than conventional bacterioscopy while for detection of antibodies neither of the two tests (MAT and ELISA) had specificity in relation to antigen detection. Study indicated high prevalence of infection in swine herds in Northern India.

Antimicrobial and Herbal Drug Resistance in Enteric Bacteria Isolated from Faecal Droppings of Common House Lizard/Gecko (Hemidactylus frenatus).

From 194 faecal dropping samples of common house geckos collected from offices (60), houses (88), integrated farm units (IFS,18) and hostels, guest houses, and dining rooms of different canteen/mess (HGM, 28), 326 bacterial isolates of enteric bacteria belonging to 17 genera and 34 species were detected. Escherichia coli were the most frequently (39) isolated followed by Citrobacter freundii (33), Klebsiella pneumonia (27), Salmonella indica (12), Enterobacter gergoviae (12), and Ent. agglomerans (11). Other important bacteria isolated from gecko droppings were Listonella damsela (2), Raoultella terrigena (3), S. salamae (2), S. houtenae (3), Edwardsiella tarda (4), Edwardsiella hoshinae (1), and Klebsiella oxytoca (2). Of the 223 isolates tested for antimicrobial drug sensitivity, 27 (12.1%) had multiple drug resistance (MDR). None of the salmonellae or edwardsiellae had MDR however, MDR strains were significantly more common among Escherichia spp. (P = 1.9 × 10(-5)) and isolates from IFS units (P = 3.58 × 10(-23)). The most effective herbal drug, Ageratum conyzoides extract, inhibited growth of only 27.8% of strains tested followed by ethanolic extract of Zanthoxylum rhetsa (13.9%), eucalyptus oil (5.4%), patchouli oil (5.4%), lemongrass oil (3.6%), and sandalwood oil (3.1%), and Artemisia vulgaris essential oil (3.1%).