Shiga toxigenic E. coli are important foodborne zoonotic pathogens. The present study was envisaged to standardize loop-mediated isothermal amplification assays targeting stx1 and stx2 genes for rapid and visual detection of STEC and compare its sensitivity with PCR. The study also assessed the effect of short enrichment on the detection limit of LAMP and PCR. The developed LAMP assays were found to be highly specific. Analytical sensitivity of LAMP was 94 fg/µLand 25.8 fg/µL for stx-1 and stx-2 while LOD of 5 CFU/g of carabeef was measured after 6–12 h enrichment. The study highlights the importance of short (6–12 h) enrichment for improving the sensitivity of LAMP. The entire detection protocol could be performed within 9 h yielding results on the same day. The developed LAMP assays proved to be a handy and cost-effective alternative for screening STEC contamination in meat.

Fucosylated chondroitin sulfate (FucCS) is a unique glycosaminoglycan found primarily in sea cucumbers. This marine sulfated glycan is composed of a chondroitin sulfate backbone decorated with fucosyl branches attached to the glucuronic acid. FucCS exhibits potential biological actions including inhibition of blood clotting and severe acute respiratory syndrome coronavirus (SARS-CoV-2) infection. These biological effects have been attributed to certain structural features, including molecular weight (MW), and/or those related to fucosylation, such as degrees of fucosyl branches, sulfation patterns and contents. In a previous work, we were able to generate oligosaccharides of the FucCS from Pentacta pygmaea (PpFucCS) with reduced anticoagulant effect but still retaining significant anti-SARS-CoV-2 activity against the delta strain. In this work, we extended our study to the FucCS extracted from the species Holothuria floridana (HfFucCS). The oligosaccharides were prepared by free-radical depolymerization of the HfFucCS via copper-based Fenton reaction. One-dimensional 1H nuclear magnetic resonance spectra were employed in structural analysis. Activated partial thromboplastin time and assays using protease (factors Xa and IIa) and serine protease inhibitors (antithrombin, and heparin cofactor II) in the presence of the sulfated carbohydrates were used to monitor anticoagulation. Anti-SARS-CoV-2 effects were measured using the concentration-response inhibitory curves of HEK-293T-human angiotensin-converting enzyme-2 cells infected with a baculovirus pseudotyped SARS-CoV-2 wild-type and delta variant spike (S)-proteins. Furthermore, the cytotoxicity of native HfFucCS and its oligosaccharides was also assessed. Like for PpFucCS, we were able to generate a HfFucCS oligosaccharide fraction devoid of high anticoagulant effect but still retaining considerable anti-SARS-CoV-2 actions against both variants. However, compared to the oligosaccharide fraction derived from PpFucCS, the average MW of the shortest active HfFucCS oligosaccharide fraction was significantly lower. This finding suggests that the specific structural feature in HfFucCS, the branching 3,4-di-sulfated fucoses together with the backbone 4,6-di-sulfated N-acetylgalactosamines, is relevant for the anti-SARS-CoV-2 activity of FucCS molecules.

The fusion of haemagglutinin-neuraminidase (HN) protein of peste des petits ruminant (PPR) virus with signaling lymphocyte activation molecules (SLAM) host cell receptor consequences the virus entry and multiplication inside the host cell. The use of synthetic SLAM homologous peptides (i.e., molecular decoy for HN protein of PPR virus) may check PPR infection at the preliminary stage. Hence, the predicted SLAM homologous peptides using bioinformatics tools were synthesized by solid phase chemistry with standard Merrifield's 9-fluorenylmethoxycarbonyl (Fmoc) chemistry and were purified by reverse phase high performance liquid chromatography. The secondary structures of synthesized peptides were elucidated by circular dichroism spectroscopy. The in vitro interactions of these peptides were studied through indirect Enzyme Linked Immuno Sorbent Assay (ELISA) and visual surface plasmon UV-visible spectroscopy. The SLAM homologous peptides were able to interact with the peste des petits ruminant virus (PPRV) with varying binding efficiency. The interaction of SLAM homologous peptide with the PPR virus was ascertained by the change in the plasmon color from red wine to purple during visual detection and also by bathochromic shift in absorbance spectra under UV-visible spectrophotometry. The cytotoxic and anti-PPRV effect of these peptides were also evaluated in B95a cell line using PPR virus (Sungri/96). The cytotoxic concentration 50 (CC50 ) value of each peptide was greater than 1000 μg mL-1 . The anti-PPRV efficiency of SLAM-22 was relatively high among SLAM homologous peptides, SLAM-22 at 25 μg mL-1 concentration showed a reduction of more than log10 3 virus titer by priming of B95a cell line while the use of SLAM-15 and Muco-17 at the same concentration dropped virus titer from log10 4.8 to log10 2.5 and log10 3.1 respectively. The concentration of SLAM homologous peptide (25 μg mL-1 ) to exert its anti-PPRV effect was much less than its CC50 level (>1000 μg mL-1 ). Therefore, the synthetic SLAM homologous peptides may prove to be better agents to target PPRV.

B. melitensis is the most pathogenic zoonotic species of Brucella transmitted to animals through fetal secretions, placenta, and vaginal discharges of infected animals and humans by ingesting unpasteurized milk, dairy products, and raw meat. Early detection of B. melitensis is essential for timely intervention and control of the disease. The gold standard diagnostic methods, such as culture, are time-consuming and may take several weeks aiding to the disease spread. Loop-mediated isothermal amplification assay (LAMP) is widely used to detect infectious pathogens. LAMP can be utilized as a rapid point-of-care test, but has lower specificity which can be enhanced by combining this test with lateral flow immunoassay. No point-of-care test is available for detecting Brucella melitensis in clinical samples. Herein, we developed a LAMP coupled with lateral flow immunoassay (LFIA) for the specific detection of B. melitensis . The sensitivity of LAMP-LFIA was found to be 12.1 fg of genomic DNA isolated from the organism, which is 100-fold more sensitive to conventional PCR and equally sensitive to Real-time (RT-PCR). Moreover, the assay demonstrated high specificity when tested against other Brucella and non- Brucella species. The infective dose of B. melitensis is relatively low for humans, which may remain undetected by conventional PCR, but will be detected using the new technique.

The structure of the sulfated galactan from the red alga Botryocladia occidentalis (BoSG) was originally proposed as a simple repeating disaccharide of alternating 4-linked α-galactopyranose (Galp) and 3-linked β-Galp units with variable sulfation pattern. Abundance was estimated only for the α-Galp units: one-third of 2,3-disulfation and one-third of 2-monosulfation. Here, we isolated again the same BoSG fractions from the anion-exchange chromatography, obtaining the same NMR profile of the first report. More careful NMR analysis led us to revise the structure. A more complex sulfation pattern was noted along with the occurrence of 4-linked α-3,6-anhydro-Galp (AnGalp) units. Interestingly, the more sulfated BoSG fraction showed slightly reduced in vitro anti-SARS-CoV-2 activities against both wild-type and delta variants, and significantly reduced anticoagulant activity. The BoSG fractions showed no cytotoxic effects. The reduction in both bioactivities is attributed to the presence of the AnGalp unit. Docking scores from computational simulations using BoSG disaccharide constructs on wild-type and delta S-proteins, and binding analysis through competitive SPR assays using blood (co)-factors (antithrombin, heparin cofactor II and thrombin) and four S-proteins (wild-type, delta, gamma, and omicron) strongly support the conclusion about the deleterious impact of the AnGalp unit.

Abstract The primary cause of Brucellosis in sheep, goats, human and other animal species is B. melitensis. Despite being widely acknowledged as the gold standard method, the isolation and identification of B. melitensis cannot currently meet the criteria for early diagnostic strategies. Conventional PCR methods and immunological assays can detect B. melitensis, but their use in basic laboratories is constrained by the need for sophisticated instruments. A prompt and accurate diagnosis is essential to prevent the spread of infection to human and animals. The aim of this study was to set up a rapid and specific point of care diagnostic test for the detection of B. melitensis, which could be used routinely. Hence, a Loop mediated isothermal amplification (LAMP) coupled with lateral flow immunoassay (LFIA) was optimized for the specific detection of B. melitensis. The LAMP primers were tagged to generate product labelled with digoxigenin and biotin and the labelled LAMP amplicons were detected using LFIA with streptavidin-gold nanoparticle as a capture reagent. The assembly of gold nanoparticle at the test and control line yielded a characteristic red colour band. The sensitivity of B. melitensis LAMP-LFIA assay was 12.1fg. The assay was 100 fold more sensitive than conventional PCR and was in accordance with RT-PCR. The assay did not exhibit cross reactivity with the non-Brucella pathogens examined in this study or other Brucella species. The assay can be performed in a water bath at 65°C within 60 min and can detect upto12.1fg of genomic DNA isolated from the organism and 102 CFU/ml of B. melitensis in the spiked sample. Thus there is a great deal of potential for this assay to be utilised as a rapid field test for screening different secretions and excretions in suspected animals or their products which can tremendously reduce chances of disease transmission to both animal and human.

Purpose Staphylococcus aureus is an opportunistic zoonotic organism which secretes around 23 different types of enterotoxins. Classical enterotoxins (SEA, SEB, SEC, SED and SEE) are responsible for >95 % of food poisoning outbreaks of which SEA alone is responsible for >75% of them. The present study was undertaken to develop sandwich ELISA forsensitive, specific and quantitative detection of Staphylococcal enterotoxins-A in food samples. Methods Optimization of sandwich ELISA was attempted in two ways. In first, rabbit polyclonal anti-SEA was used as capture antibody and mouse monoclonal anti-SEA as detector antibody, in second, mouse monoclonal anti-SEA was used as capture Antibody and rabbit polyclonal anti-SEA as detector antibody. Results In the optimization of sandwich ELISA, mouse monoclonal anti-SEA as capture antibody and rabbit polyclonal anti-SEA as detector antibody yieldedhighest sensitivity of 0.5-0.75 ng ml-1. The developed assay was found to be highly specific and having equivalent sensitivity to available commercial kits. Conclusion The developed sandwich ELISA may be utilized as a sensitive, specific and quantitative test for detection of Staphylococcal enterotoxin-A in food samples. The developed ELISA may serve as a cheap alternative to commercial kits which need importation and therefore are relatively costly. The sandwich ELISA developed may be useful for microbiological quality assurance of foods specially in developing countries.

This study explored the transcriptome of lamb testis cells infected with sheeppox virus (SPPV) wild strain (WS) and vaccine strain (VS) at an immediate-early time. Most of the differentially expressed genes (DEGs) and differentially expressed highly connected (DEHC) gene network were found to be involved in SPPV-VS infection compared to SPPV-WS. Further, the signaling pathways were mostly involved in SPPV-VS infection than SPPV-WS. SPPV modulates the expression of several important host proteins such as CD40, FAS, ITGβ1, ITGα1, Pak1, Pak2, CD14, ILK leading to viral attachment and entry; immune-related DEGs such as MAPK, JNK, ERK, NFKB, IKB, PI3K, STAT which provide optimal cellular condition for early viral protein expression; and FOXO3, ATF, CDKNA1, TCF, SRF, BDNF which help in inducing apoptosis and MPTP, BAD and Tp53 inhibits apoptosis or cell death at the immediate-early time. The results captured the specific genes and enabled to understand distinct pathogenic mechanisms employed by VS and WS of SPPV.

Loop-mediated isothermal amplification (LAMP) is a diagnostic method for meat speciation with rapid and minimal equipment requirements. In this study, we developed cattle-specific tube-based LAMP assays targeting mitochondrial Cyt b gene sequence, compared with conventional PCR assay for specificity, sensitivity, and validation of the assay was made. The LAMP reaction was carried at 64 °C for 45 min, and results were confirmed by SYBR Green I dye and agarose gel-electrophoresis. The specificity of the assays was cross-tested with DNA of buffalo, goat, sheep, and pork. The amplification was observed with samples from cattle only without cross-reactivity with other meat species. The analytical sensitivity of LAMP and PCR method for cattle DNA detection was 0.0001 ng and 1 ng, respectively. Repeatability of the assay was achieved on samples from known/blind and admixture meat with other than cattle at the relative percentage of 20%, 10%, 5%, and 1%. The study concluded that the developed assay can be easily employed for the rapid identification of tissue of cattle origin in meat and meat products in low resource areas.

The application of innovative diagnostic technologies for the detection of animal pathogens at an early stage is essential in restricting the economic loss incurred due to emerging infectious animal diseases. The desirable characteristics of such diagnostic methods are easy to use, cost-effective, highly sensitive, and specific, coupled with the high-throughput detection capabilities. The enzyme-linked immunosorbent assay (ELISA) and polymerase chain reaction (PCR) are still the most common assays used for the detection of animal pathogens across the globe. However, utilizing the principles of ELISA and PCR, several serological and molecular technologies have been developed to achieve higher sensitivity, rapid, and point-of-care (POC) detection such as lateral flow assays, biosensors, loop-mediated isothermal amplification, recombinase polymerase amplification, and molecular platforms for field-level detection of animal pathogens. Furthermore, animal disease diagnostics need to be updated regularly to capture new, emerging and divergent infectious pathogens, and biotechnological innovations are helpful in fulfilling the rising demand for such diagnostics for the welfare of the society. Therefore, this chapter primarily describes and discusses in detail the serological, molecular, novel high-throughput, and POC assays to detect pathogens affecting farm and companion animals.

Meat fraud and adulteration is a growing concern in the marketplace today. Species identification in food product is among primary duties of food office control. In this study, a one-step, species-specific, sensitive, and rapid loop-mediated isothermal amplification (LAMP) assay was developed for identification of tissues of buffalo origin. A set of six LAMP primers were designed by targeting mitochondrial cyt b gene. The reaction temperature of LAMP and component of reaction mixture were optimized as 64 °C amplification temperature, 6 U/μL concentration of Bst DNA polymerase, 3 mM concentration of MgSO4, 0.4 mM concentration of dNTP, 0.6 M concentration of betaine, and 1:6 ratio of outer and inner primer pair. Successful amplifications were confirmed using SYBR Green I dye and agarose gel electrophoresis. The specificity of assay was tested with DNA of cattle, goat, sheep, and pig where amplification was observed exclusively in buffalo. The analytical sensitivity of the LAMP assay for buffalo DNA detection was 0.1 pg. The applicability check of developed assay carried out on known/coded samples and binary meat admixture substantiated the accuracy of assay. Supernatant of tissue exposed to Phire Animal Tissue Direct PCR master mix treatment was observed rich enough with DNA of target species and successfully amplified under optimized LAMP reaction. The comparison of results on the samples undergone pre-standardized species-specific PCR taken as gold standard shown complete match with developed species-specific LAMP assay. Hence, the developed species-specific LAMP assays are effective in detection of tissue of buffalo origin.

Multi-object tracking is a significant domain in Computer vision applications, which involves giving unique identities to different objects, and maintaining the association between them, for real-time applications. However, most of the trackers fail to achieve decent levels of accuracy as well as speed. In this paper, we propose a tracking technique, which utilizes both high-speed detections from Yolo, as well as deep feature extraction, from a convolutional neural network. The extracted features, along with position vectors and color histograms, are matched between corresponding frames, to develop an association between pedestrians. It can face issues like slight changes in object appearance in continuous frames, such as shape, size or illumination changes, partial occlusions, or re-identification of pedestrians, on re-entering the view, or after being occluded, for a certain length of frames. We have used the Yolo framework for fast object detection, a MobileNet architecture based custom CNN for feature extraction, and a set of algorithms to generate associations between frames. On the publically available Town-centre dataset, our framework can reach a MOTA of 93.2%.

The present study was carried out with the objective of development of species-specific loop-mediated isothermal amplification (LAMP) assay for identification of tissue of cattle origin. The cattle-specific LAMP primer set was designed by targeting mitochondrial D-loop gene. The conditions for LAMP reaction for amplification of template DNA from cattle using designed cattle-specific primer set were optimized for the components of mixture and temperature of reaction. Amplified products were analysed using SYBR Green I dye and by agarose gel electrophoresis. The developed species-specific LAMP assay was evaluated for its specificity, sensitivity and validated in laboratory on samples from known, coded, binary meat admixture with other than cattle at relative percentage of 20%, 10%, 5% and 1%, Phire tissue direct PCR master mix treated tissues of cattle and on species-specific polymerase chain reaction assay positive samples. The developed LAMP assay using self-designed primer set was highly specific, amplifying the DNA template exclusively from cattle tissue under the optimized LAMP reaction conditions. The sensitivity assay using serially diluted DNA templates revealed lowest level of detection as 0.01ng of absolute DNA from target species. Laboratory validation substantiated the accuracy of assay in known/unknown (coded) samples and up to the 1% level of admixture in binary meat sample. DNA present in supernatant of Phire Animal tissue kit treated samples were also amplified successfully eliminating the extra step of extraction of genomic DNA. The developed assays exhibited comparable results with previously established species-specific PCR assay taken as gold standards. Thus, it was concluded that developed species-specific loop-mediated isothermal amplification assay was effective in identification of tissue of cattle origin.

The viruses under Orthoreovirus genus of Reoviridae family are non-enveloped, segmented double-stranded RNA virus, possess icosahedral symmetry, and replicate in the cytoplasm. The avian reovirus (ARV) is ubiquitously distributed worldwide in poultry and in other wild birds and causes severe arthritis and tenosynovitis in the affected birds. The clinical manifestation in the affected birds is lameness, malabsorption-related enteric dysfunction, runting-stunting syndrome (RSS), respiratory infections, and immunosuppression. Infection with ARV can incur production losses that are estimated to be $23,000 per affected flock (28,000 birds/flock). Several methods for diagnosis of ARVs are reported, viz., virus isolation, immunofluorescent staining, and immunoperoxidase histochemistry offer straight detection of viral antigens in tendon tissues. Our laboratory has applied a real-time loop-mediated isothermal amplification technique to develop a rapid, sensitive, and specific method for virus detection and quantification. Additionally, we have standardized sigma B protein-based dot-ELISA which can be used as a simple, reliable, and inexpensive alternative to commercial ELISA kits for serodiagnosis of ARV. A number of high-throughput -sequencing studies deciphers the host pathogen interaction in ARV infection. Our laboratory has established the role of sigma B protein in ARV pathogenesis. Various live and inactivated vaccines are available for prevention of the disease. Currently, there has been a dramatic increase in the number of clinical cases of reoviruses in poultry and commercial vaccines are unable to provide adequate levels of protection against disease. Research focused on new-generation diagnostics, and vaccine may provide easy and effective substitute as vaccines and diagnostics candidates for these highly divergent viruses.

Background and Aim: Campylobacteriosis finds its place among the four important global foodborne illnesses. The disease, though self-limiting, needs antibacterial therapy in extraintestinal complications. Therefore, the present study was designed to estimate the prevalence of thermophilic Campylobacters in poultry, animals, and humans of the Kumaon region of Uttarakhand. Materials and Methods: A total of 609 samples comprising of poultry ceca (n=116), poultry droppings (n=203), and feces of pigs (n=71), cattle (n=61), sheep (n=19), goat (n=17), human beings (n=88), and laboratory animals (n=34) (rats, rabbits, and guinea pigs) were collected. The thermophilic Campylobacters, Campylobacter jejuni and Campylobacter coli were confirmed using multiplex polymerase chain reaction. The isolates were also screened for the presence of virulence genes, and their antibiotic susceptibility testing was done against eight antibiotics. Results: An overall prevalence of 6.24% was revealed with highest from poultry ceca (15.52%), followed by poultry droppings (5.91%), cattle feces (4.92%), human stools (3.40%), and pig feces (2.82%). The virulence genes, namely cadF, flaA, virB11, and pldA, were present in 38 (100%), 37 (97.37%), 7 (18.42%), and 14 (36.84%) isolates, respectively. All the isolates were resistant to nalidixic acid, while all were sensitive to erythromycin and co-trimoxazole. Conclusion: It was concluded that the animals and humans in the region harbored the thermophilic Campylobacters which may contribute to the human illness. Resistance shown among the isolates may complicate the antimicrobial therapy.

Avian reovirus (ARV) causes significant economic losses to the poultry industry worldwide. The ARV proteins fall into three different classes based on their sizes:lambda. (large); mu (medium) and sigma (small). sigma B, an outer capsid protein of the ARV contains group specific neutralizing epitopes and induces strong immune response in naturally infected chickens. This study describes the development of a rapid dot-enzyme linked immunosorbent assay (dot-ELISA) using recombinant sigma B protein antigen of 54 kDa (approx). The assay is rapid (4-5 h) and results can be read by the naked eye. Sixteen ARV positive serum samples (group A) produced strong reaction in the dot-ELISA while twenty of the ARV negative serum samples (group B) collected from SPF chickens showed no reaction. Seventy six randomly collected serum samples were tested with a commercial indirect ELISA kit and the in-house developed dot-ELISA. A total of sixty eight serum samples were found to be positive by indirect ELISA and sixty five serum samples were found to be positive by dot-ELISA. Therefore, using the commercial ELISA as the reference test, the dot-ELISA had a diagnostic sensitivity of 83.8% and specificity of 88.6%. This dot-ELISA can be used as a simple, reliable and inexpensive alternative to commercial ELISA kits for serodiagnosis of ARV where the facilities for standard ELISA are not available.

Human papillomavirus (HPV) is a viral infection with skin-to-skin based transmission mode. HPV annually caused over 500,000 cancer cases including cervical, anogenital and oropharyngeal cancer among others. HPV vaccination has become a public-health concern, worldwide, to prevent the cases of HPV infections including precancerous lesions, cervical cancers, and genital warts especially in adolescent female and male population by launching national programs with international alliances. Currently, available prophylactic and therapeutic vaccines are expensive to be used in developing countries for vaccination programs. The recent progress in immunotherapy, biotechnology, recombinant DNA technology and molecular biology along with alternative and complementary medicinal systems have paved novel ways and valuable opportunities to design and develop effective prophylactic and therapeutic vaccines, drugs and treatment approach to counter HPV effectively. Exploration and more researches on such advances could result in the gradual reduction in the incidences of HPV cases across the world. The present review presents a current global scenario and futuristic prospects of the advanced prophylactic and therapeutic approaches against HPV along with recent patents coverage of the progress and advances in drugs, vaccines and therapeutic regimens to effectively combat HPV infections and its cancerous conditions.

Endoparasitic diseases are commonly encountered in free-ranging birds. Although not all endoparasites cause disease, persistent infection with large numbers of parasites almost always affects normal physiological functions, leading to deleterious effects on the host. This paper describes the anatomopathological alterations caused by the renal trematode Paratanaisia bragai in Indian peafowl (n = 3) and examines the phylogeny of these and related parasites. Peafowl from forests in and around the Bareilly region, Uttar Pradesh, India, were necropsied, and microscopic and molecular investigations were performed. The peafowl were confirmed to be infected with P. bragai. Significant gross pathological lesions suggested nephrosis, and microscopic findings indicated a mild-to-moderate degree of nephrosis caused by the parasites in the tissue. The parasites were identified as P. bragai by histomorphological analysis of adult and eggs in the ureters, and the identification was confirmed by PCR and phylogenetic analysis. Nucleotide sequencing of the PCR products from the renal trematodes recovered from Indian peafowl revealed a close association with P. bragai from Columbiformes in the United Kingdom and Spain. The pathology and molecular epidemiology of parasitic diseases affecting peafowl is not well understood in India. This is the first report from India and the second report worldwide to document P. bragai infection in peafowl.

Marek's disease (MD) and lymphoid leucosis (LL) are the major diseases causing lymphoid tumors in chickens accounting for high economical losses. Gross examination could not yield definite diagnosis owing to their similar presentation of lesions. Thus present work was aimed for diagnosis and differentiation of MD and LL by utilizing simple cytology and novel immunocytology techniques. Cytological examination was carried out on slides with tumor touch imprints stained by simple Giemsa staining. The diagnosis was mainly achieved based on morphology of cell population. In the present study, out of a total of 595 cases examined, 502 cases had pleomorphic lymphocytic cell population suggestive of MD and 53 cases had uniform lymphocytic/lymphoblast cell population suggestive of LL, while the rest 40 cases remained inconclusive. A definitive diagnosis was achieved after performing immunocytology using specific antibodies that revealed 518 cases had reactivity for Meq oncoprotein specific for MD and 77 cases showed immunoreactivity for IgM in transformed B-cells confirming LL. The technique of immunocytology which has been useful for detecting human viral pathogens and MD in poultry has been applied for the first time as a novel, simple, rapid and inexpensive technique that could be used as an alternate test to effectively detect and differentiate MD and LL in poultry.