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.

Isolation and sero-genomo-epidemiological studies on Brucella infection in dairy cattle in Meghalaya, India.

In this study, we report the serological, bacteriological and whole genome sequencing data of a 6 years study of Brucella abortus in Meghalaya, India. Investigation of 3060 sera samples indicated overall prevalence of 6.4% by Rose Bengal Plate Test and 10.7% by ELISA. Considerably higher prevalence was observed among milk samples (17.5%, n = 362) and in blood samples (37.7%, n = 262) by direct PCR. Clinical samples (n = 94) from late abortion cases yielded 11 B. abortus isolates. Multi-locus sequence typing indicated circulation of single sequence type, ST1. Whole genome sequencing (n = 8) and phylogenomic analysis revealed close clustering of majority of isolates in two clusters alongwith genomes from other countries, indicating global relatedness among B. abortus. Taken together, the results of our study revealed the putative hotspot of infection in the dairy-dominant districts of the state and also calls for concerted One Health based action for prevention and control of this zoonotic disease.

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.].

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.

Harmonia sedecimnotata (F.): Predatory potential, biology, life table, molecular characterization, and field evaluation against Aphis gossypii Glover.

The ladybird beetle, Harmonia sedecimnotata (F.) was studied in biology, life table, consumption rates, molecular characterization, and field evaluation. The net reproductive rate (R0), based on the age-stage and two-sex life table, was 43.2 eggs/individual. The female adults lived longer (68.1 d) than the male adults (62.9 d). The rate of consumption increased with progress in each stage of development. Compared to the other larval stages of the predator, the fourth stadium consumed most quantities of Aphis gossypii Glover nymphs (Hemiptera: Aphididae) (200.4). Both female (2214.6) and male (1792.4) consumed more prey (nymphs) than larvae. The net rate of consumption was 1458.92 nymphs of melon aphids. There was no variation in the sequences of the two nucleotides out of 583 bp, H sedecimnotata China (EU392410) and India (MG720024). Our investigations demonstrated that inoculative release of 30 or 40 or 50 adults per 100 m2 attained high reduction of aphids (>90%). Thus, it may be recommended the release rate of 40 adults per 100 m2 to suppress the eggplant aphid population. H. sedecimnotata is therefore one of the most promising biological control agents for cotton aphids that can be achieved for instant control through an inoculative release of adults.

Identification of a novel cluster of PCV2 isolates from Meghalaya, India indicates possible recombination along with changes in capsid protein.

Documentation of the emergence of Porcine circovirus 2 (PCV2) infection and economic losses incurred due to high mortality has been reported worldwide. The prevalence and genetic diversity of the virus has been reported in Northeast India including the possible chances of Classical swine fever virus (CSFV) vaccine failure in pig population in this region resulting in major disease outbreak. Irrespective of the genetic variability, the emergence of a novel cluster (based on the ORF2 phylogeny) was reported last year. The present study describes a state-wide (Meghalaya, India) molecular epidemiological investigation of PCV2 strains in pig population by amplification, sequencing and undertaking phylogenetic analyses. The results indicate the identification of a novel cluster of PCV2 originating from the inter-genotypic recombination between PCV2c and PCV2d. Multiple sequence alignment of amino acids indicates possible substitution in the A, B and C domains of the capsid protein. Molecular structural modelling of the capsid protein of PCV2 indicated possible motif variations in the secondary structure including presence of a tunnel, encountered at the interface region on each chain facilitating in transportation of molecules and acting as an active site for attachment and penetration. The baseline data strengthens the existing control programme of PCV2 and is possibly helpful in the planning of active surveillance strategy in this region.

Detection of classical swine fever virus E2 gene in cattle serum samples from cattle herds of Meghalaya.

The present study focused on the detection and genetic characterisation of 5' untranslated region (5'UTR) and E2 gene of classical swine fever virus (CSFV, family Flaviviridae, genus Pestivirus) from bovine population of the northeastern region of India. A total of 134 cattle serum samples were collected from organised cattle farms and were screened for CSFV antigen with a commercial antigen capture enzyme linked immunosorbent assay (Ag-ELISA) and reverse transcription-polymerase chain reaction (RT-PCR). A total of 10 samples were positive for CSFV antigen by ELISA, while all of them were positive in PCR for 5'UTR region. Full length E2 region of CSFV were successfully amplified from two positive samples and used for subsequent phylogenetic analysis and determination of protein 3D structure which showed similarity with reported CSFV isolate from Assam of sub-genogroup 2.1, with minor variations in protein structure.

Seroprevalence of peste des petits ruminants and bluetongue in goat population of Meghalaya, India.

AIM: This study aimed to determine the seroprevalence of peste des petits ruminants (PPR) and bluetongue (BT) in goats' population in the state of Meghalaya of Northeast India. MATERIALS AND METHODS: The serosurveillance study was done from the random sampling (n=598) of blood collected from five districts (Ri-Bhoi, East Khasi Hills, West Khasi Hills, Jaintia Hills and West Garo Hills) of Meghalaya. The presence of antibodies against PPR and BT in the samples was detected by indirect enzyme-linked immunosorbent assay (ELISA) method for PPR and competitive ELISA for BT. RESULTS: The results showed the overall seropositivity of PPR and BT at 7.19% and 60.20%, respectively. West Garo Hills recorded the highest seroprevalence of both PPR (9.81%) and BT (68%) and 3.6% of the samples tested positive for both PPR and BT. CONCLUSION: The random survey results indicating the presence of PPR and BT have specific implication in epidemiological perspectives since it highlights the prevalence under natural situations, where the subclinical, inapparent, or non-lethal or recovery of infection was suspected in unvaccinated animals. It also warrants further studies to suggest appropriate control measures to prevent the spread of infection.

Detection of Peste des petits ruminants virus and goatpox virus from an outbreak in goats with high mortality in Meghalaya state, India.

AIM: We describe a laboratory investigation carried out to confirm the etiology of the heavy mortality (37 animals died out of total 44, i.e. 84%) in goats in Ri-Bhoi district of Meghalaya, Northeast region of India in December 2015. The clinical signs observed were abortion, diarrhea, high fever (up to 104°F), pox lesion in the skin, and respiratory distress. MATERIALS AND METHODS: The samples comprising whole blood, sera, and pox lesion were collected from the animals (n=7) from an outbreak for the screening of peste des petits ruminants (PPR) and poxviruses. The whole blood and sera were used for screening of PPR virus (PPRV) by sandwich enzyme-linked immunosorbent assay (ELISA) and antibody by competitive ELISA as well as detection of PPRV partial N gene by reverse transcription-polymerase chain reaction (PCR). The skin lesions were used for the detection of poxvirus by PCR. RESULTS: The results showed the presence of PPR antigens (58-80%) in the samples by sandwich ELISA and antibody in all the sera samples ranging from 9% to 41% positivity in competitive ELISA. Four samples were positive for PPRV partial N gene. The skin lesion screened for poxvirus was also found to be positive for I3L gene of goatpox virus. CONCLUSION: We confirm the outbreak of disease in goats with high mortality is a case of mixed infection of PPR and goatpox detected for the first time in Northeast India.

Molecular Characterization and Computational Modelling of New Delhi Metallo-ß-Lactamase-5 from an Escherichia coli Isolate (KOEC3) of Bovine Origin.

Emergence of antimicrobial resistance mediated through New Delhi metallo-ß-lactamases (NDMs) is a serious therapeutic challenge. Till date, 16 different NDMs have been described. In this study, we report the molecular and structural characteristics of NDM-5 isolated from an Escherichia coli isolate (KOEC3) of bovine origin. Using PCR amplification, cloning and sequencing of full blaNDM gene, we identified the NDM type as NDM-5. Cloning of full gene in E. coli DH5α and subsequent assessment of antibiotic susceptibility of the transformed cells indicated possible role of native promoter in expression blaNDM-5. Translated amino acid sequence had two substitutions (Val88Leu and Met154Leu) compared to NDM-1. Theoretically deduced isoelectric pH of NDM-5 was 5.88 and instability index was 36.99, indicating a stable protein. From the amino acids sequence, a 3D model of the protein was computed. Analysis of the protein structure elucidated zinc coordination and also revealed a large binding cleft and flexible nature of the protein, which might be the reason for broad substrate range. Docking experiments revealed plausible binding poses for five carbapenem drugs in the vicinity of metal ions. In conclusion, results provided possible explanation for wide range of antibiotics catalyzed by NDM-5 and likely interaction modes with five carbapenem drugs.

Detection of New Delhi metallo-beta-lactamase and extended-spectrum beta-lactamase genes in Escherichia coli isolated from mastitic milk samples.

In this study, eight Escherichia coli isolates were obtained from milk samples of dairy cattle suffering from clinical/subclinical mastitis. Isolates were characterized for antimicrobial resistance traits and virulence genes. Results revealed that one isolate was harbouring New Delhi metallo-beta-lactamase gene (blaNDM ). Cloning and sequencing of the PCR amplicon confirmed the identity of the gene (GenBank accession no. KC769583) having 100% homology with blaNDM-5 (GenBank accession no. JN104597.1), and this isolate was susceptible to colistin, chloramphenicol and tetracycline only. Moreover, another isolate carried extended-spectrum beta-lactamase (ESBL) gene - blaCTX-M , and all isolates possessed blaTEM gene. Of the eight isolates, only one isolate was positive for shiga toxin gene (stx2), and none were harbouring stx1 gene. Occurrence of New Delhi metallo-beta-lactamase (blaNDM ) in one E. coli isolate and ESBL genes in other isolates poses a potential threat to human health following possible entry and spread through food chain.

Studies on the identification and control of pathogen Saprolegnia in selected Indian major carp fingerlings at mid hill altitude.

The Indian major carp cultured in ponds in the North Eastern hilly states of India frequently suffer from fungal disease during winter months resulting in mass mortality. This study examined the pathogenic fungi isolated from farmed raised Indian major carp fingerlings and identified as Saprolegnia. For treatment, the diseased fish were exposed to 4g salt per litre of water for 2 min followed by dip treatment with 5ppm KMnO4 for 10 min, thrice every week for a period of 6 weeks. The treatment resulted in recovery from the disease after 6 weeks from the beginning of treatment. Soon after recovery, the pond management practices such as removal of pond bottom soil, application of lime and replenishment with freshwater were followed in the infected ponds. Our study concluded that rapid decrease in pond water temperature from 22 to 8 degrees C that remains low for months together coupled with increased water pH (9) and decreas dissolved oxygen (4ppm) causes saprolegniasis to the fingerlings of Indian major carps.

Clostridial myonecrosis clinically resembling black quarter in an Indian elephant (Elephas maximus).

An Indian elephant (Elephas maximus) which died of acute fatal myonecrosis was examined to determine the aetiology of the infection. The causative organism was identified as Clostridium perfringens type A. Out of five genes encoding for major toxins (cpa, cpb, etx, iA, and cpe genes) the isolate was found to harbour the cpa gene only, as tested by multiplex polymerase chain reaction. It flanks a 324 base pair segment in the cpa gene, indicating the presence of the alpha toxin gene. The organism was sensitive to amikacin, ampicillin, enrofloxacin, gentamicin and norfloxacin but was resistant to bacitracin, oxytetracycline and tetracycline. The acute malignant nature of the myonecrosis and presence of the alpha toxin gene in the isolate suggested that the myonecrosis, although clinically resembling that caused by C. chauvoei in cases of black quarter, was caused by C. perfringens type A.

Detection of multiple virulence-associated genes in Listeria monocytogenes isolated from bovine mastitis cases.

Clinical samples (n=725) were collected from bovines (n=243) which were positive for mastitis using the California mastitis test (CMT) and somatic cell count (SCC). The clinical samples comprising blood (n=239), milk (n=243), and faecal swabs (n=243) were examined for the presence of pathogenic Listeria spp. Isolation of the pathogen was done using selective enrichment in University of Vermont Medium and plating onto Dominguez-Rodriguez isolation agar. Confirmation of the isolates was based on biochemical tests and Christie, Atkins, Munch-Petersen (CAMP) test followed by pathogenicity testing. Pathogenicity of the isolates was tested by phosphatidylinositol-specific phospholipase C (PI-PLC) assay as well as in vivo tests namely, chick embryo and mice inoculation tests. The isolates were subjected to PCR assay for five virulence-associated genes, plcA, prfA, hlyA, actA and iap. Listeria spp. were isolated from 12 (1.66%) samples. Of these 4 (0.55%) and 1 (0.14%) were confirmed as Listeria monocytogenes and Listeria ivanovii, respectively. L. monocytogenes and L. ivanovii were recovered from milk samples (2) and faecal (3) of mastitic cattle (3) and buffaloes (2). L. monocytogenes recovered from the milk of mastitic cattle and L. ivanovii from the faecal swab of buffalo turned out to be pathogenic. However, the remaining three hemolytic isolates exhibiting positive CAMP test turned out to be negative in PI-PLC assay, chick embryo and mice inoculation. L. monocytogenes and L. ivanovii isolates characterized as pathogenic by PI-PLC assay and in vivo pathogenicity tests were found to possess all the five virulence-associated genes and three genes, plcA, prfA and actA respectively. The remaining three hemolytic but non-pathogenic L. monocytogenes isolates were negative for plcA by PCR. It seems that the plcA gene and its expression (in the PI-PLC assay) have an important role as virulence determinants in pathogenic Listeria spp. In conclusion, the PI-PLC assay and virulence genes targeted PCR (plcA, prfA and hlyA genes for L. monocytogenes and plcA, prfA and actA genes for L. ivanovii) hold a good promise as rapid and reliable in vitro alternatives to in vivo pathogenicity tests.

Isolation of Listeria monocytogenes from buffaloes with reproductive disorders and its confirmation by polymerase chain reaction.

Listeria monocytogenes, a gram-positive, facultative intracellular pathogen was isolated from buffaloes with a history of reproductive disorders and polymerase chain reaction (PCR) analyses for the presence of virulence-associated genes were conducted. A total of 530 samples of faecal, nasal, vaginal swabs and blood samples from 135 buffaloes were screened. The prevalence of L. monocytogenes and other Listeria spp. was found to be 4.4 and 7.4%, respectively. All isolates were subjected to PCR for virulence-associated genes (prfA, plcA, hlyA, actA and iap) and to pathogenicity testing by the phosphatidylinositol phospholipase C (PI-PLC) assay and mice and chick-embryo inoculation. All L. monocytogenes isolates were hemolytic and positive for the hlyA gene. One L. monocytogenes isolate possessed all five virulence-associated genes and was also positive in the PI-PLC assay as well as in the in vivo pathogenicity tests. The remaining hemolytic L. monocytogenes isolates lacking the plcA gene and PI-PLC assay activity were, however, non-pathogenic via mice and chick-embryo inoculation tests, in spite of having the hlyA gene. The detection of multiple virulence-associated genes, in combination with in vitro pathogenicity tests, must be performed to identify pathogenic L. monocytogenes.