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The digestion of protein into peptide fragments reduces the size and complexity of protein molecules. Peptide fragments can be analyzed with higher sensitivity (often > 102 fold) and resolution using MALDI-TOF mass spectrometers, leading to improved pattern recognition by common machine learning algorithms. In turn, enhanced sensitivity and specificity for bacterial sorting and/or disease diagnosis may be obtained. To test this hypothesis, four exemplar case studies have been pursued in which samples are sorted into dichotomous groups by machine learning (ML) software based on MALDI-TOF spectra. Samples were analyzed in ‘intact’ mode in which the proteins present in the sample were not digested with protease prior to MALDI-TOF analysis and separately after the standard overnight tryptic digestion of the same samples. For each case, sensitivity (sens), specificity (spc), and the Youdin index (J) were used to assess the ML model performance. The proteolytic digestion of samples prior to MALDI-TOF analysis substantially enhanced the sensitivity and specificity of dichotomous sorting. Two exceptions were when substantial differences in chemical composition between the samples were present and, in such cases, both ‘intact’ and ‘digested’ protocols performed similarly. The results suggest proteolytic digestion prior to analysis can improve sorting in MALDI/ML-based workflows and may enable improved biomarker discovery. However, when samples are easily distinguishable protein digestion is not necessary to obtain useful diagnostic results.

The Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) showed susceptibility to diverse animal species. We conducted this study to understand the spatial epidemiology, genetic diversity, and statistically significant genetic similarity along with per-gene recombination events of SARS-CoV-2 and related viruses (SC2r-CoVs) in animals globally. We collected a number of different animal species infected with SARS-CoV-2 and its related viruses. Then, we retrieved genome sequences of SARS-CoV-2 and SC2r-CoVs from GISAID and NCBI GenBank for genomic and mutational analysis. Although the evolutionary origin of SARS-CoV-2 remains elusive, the diverse SC2r-CoV have been detected in multiple Rhinolophus bat species and in Malayan pangolin. To date, human-to-animal spillover events have been reported in cat, dog, tiger, lion, gorilla, leopard, ferret, puma, cougar, otter, and mink in 25 countries. Phylogeny and genetic recombination events of SC2r-CoVs showed higher similarity to the bat coronavirus RaTG13 and BANAL-103 for most of the genes and to some Malayan pangolin coronavirus (CoV) strains for the N protein from bats and pangolin showed close resemblance to SARS-CoV-2. The clustering of animal and human strains from the same geographical area has proved human-to-animal transmission of the virus. The Alpha, Delta and Mu-variant of SARS-CoV-2 was detected in dog, gorilla, lion, tiger, otter, and cat in the USA, India, Czech Republic, Belgium, and France with momentous genetic similarity with human SARS-CoV-2 sequences. The mink variant mutation (spike_Y453F) was detected in both humans and domestic cats. Moreover, the dog was affected mostly by clade O (66.7%), whereas cat and American mink were affected by clade GR (31.6 and 49.7%, respectively). The α-variant was detected as 2.6% in cat, 4.8% in dog, 14.3% in tiger, 66.7% in gorilla, and 77.3% in lion. The highest mutations observed in mink where the substitution of D614G in spike (95.2%) and P323L in NSP12 (95.2%) protein. In dog, cat, gorilla, lion, and tiger, Y505H and Y453F were the common mutations followed by Y145del, Y144del, and V70I in S protein. We recommend vaccine provision for pet and zoo animals to reduce the chance of transmission in animals. Besides, continuous epidemiological and genomic surveillance of coronaviruses in animal host is crucial to find out the immediate ancestor of SARS-CoV-2 and to prevent future CoVs threats to humans.

Antibiotics are widely used in commercial fish farms in Bangladesh for therapeutic and prophylactic purpose, raising concerns about antimicrobial resistance (AMR) and environmental contamination. This study used Thin Layer Chromatography to detect antimicrobial residues in four commercially available fish species- Tilapia ( Oreochromis aureus ), Stinging catfish ( Heteropneustes fossilis ), Climbing perch ( Anabas testudineus ), and Pabda ( Ompok pabda )—with 100 samples per species. Ultra High-Performance Liquid Chromatography quantified residues in a subset of 25 samples per species. The prevalence of Ciprofloxacin, Oxytetracycline, and Chlortetracycline residues varied significantly among fish species, with the highest prevalence observed for Ciprofloxacin in Tilapia (42%), Oxytetracycline in Pabda (41%), and Chlortetracycline in Tilapia (49%). Additionally, the prevalence of Levofloxacin and Chlortetracycline differed by sampling location, with the highest levels found in Jhawtala market, 27.5% for Levofloxacin and 53.8% for Chlortetracycline. Furthermore, residue concentrations were highest for Enrofloxacin in Climbing perch (69.32 µg/Kg) and Oxytetracycline in Pabda (88.73 µg/Kg). The highest Hazard Quotient (HQ) was for Enrofloxacin in Climbing perch (0.480), followed by Pabda (0.460), Stinging catfish (0.420), and Tilapia (0.387). While the HQ values were below 1.0, indicating no immediate toxicological risk, residues raise public health concerns due to the chance of potential AMR development. Further research is needed on antimicrobial bioaccumulation, indirect exposure sources, environmental contamination, and antimicrobial resistance in aquaculture and wild fish.

The novel coronavirus disease of 2019 (COVID-19) pandemic has caused an exceptional drift of production, utilization, and disposal of personal protective equipment (PPE) and different microplastic objects for safety against the virus. Hence, we reviewed related literature on severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA detected from household, biomedical waste, and sewage to identify possible health risks and status of existing laws, regulations, and policies regarding waste disposal in South Asian (SA) countries. The SARS-CoV-2 RNA was detected in sewage and wastewater samples of Nepal, India, Pakistan, and Bangladesh. Besides, this review reiterates the enormous amounts of PPE and other single-use plastic wastes generated from healthcare facilities and households in the SA region with inappropriate disposal, landfilling, and/or incineration techniques wind-up polluting the environment. Consequently, the Delta variant (B.1.617.2) of SARS-CoV-2 has been detected in sewer treatment plant in India. Moreover, the overuse of non-biodegradable plastics during the pandemic is deteriorating plastic pollution condition and causes a substantial health risk to the terrestrial and aquatic ecosystems. We recommend making necessary adjustments, adopting measures and strategies, and enforcement of the existing biomedical waste management and sanitation-related policy in SA countries. We propose to adopt the knowledge gaps to improve COVID-19-associated waste management and legislation to prevent further environmental pollution. Besides, the citizens should follow proper disposal procedures of COVID-19 waste to control the environmental pollution.

This study was carried out to determine the antimicrobial resistance (AMR) genes and mobile genetic elements of 16 Escherichia coli isolates—with reduced susceptibility to ceftazidime and imipenem—that were recovered from the fecal samples of coyotes and wild hogs from West Texas, USA. Whole-genome sequencing data analyses revealed distinct isolates with a unique sequence type and serotype designation. Among 16 isolates, 4 isolates were multidrug resistant, and 5 isolates harbored at least 1 beta-lactamase gene (blaCMY-2, blaCTX-M-55, or blaCTX-M-27) that confers resistance to beta-lactam antimicrobials. Several isolates carried genes conferring resistance to tetracyclines (tet(A), tet(B), and tet(C)), aminoglycosides (aac(3)-IId, ant(3″)-Ia, aph(3′)-Ia, aph(3″)-lb, aadA5, and aph(6)-ld), sulfonamides (sul1, sul2, and sul3), amphenicol (floR), trimethoprim (dfrA1 and dfrA17), and macrolide, lincosamide, and streptogramin B (MLSB) agents (Inu(F), erm(B), and mph(A)). Nine isolates showed chromosomal mutations in the promoter region G of ampC beta-lactamase gene, while three isolates showed mutations in gyrA, parC, and parE quinolone resistance-determining regions, which confer resistance to quinolones. We also detected seven incompatibility plasmid groups, with incF being the most common. Different types of virulence genes were detected, including those that enhance bacterial fitness and pathogenicity. One blaCMY-2 positive isolate (O8:H28) from a wild hog was also a Shiga toxin-producing E. coli and was a carrier of the stx2A virulence toxin subtype. We report the detection of blaCMY-2, blaCTX-M-55, and blaCTX-M-27 beta-lactamase genes in E. coli from coyotes for the first time. This study demonstrates the importance of wildlife as reservoirs of important multi-drug-resistant bacteria and provides information for future comparative genomic analysis with the limited literature on antimicrobial resistance dynamics in wildlife such as coyotes.

The study was aimed to determine the reference values of most commonly used hematological and biochemical parameters of indigenous sheep, reared under semi-intensive backyard farms in Dhaka and Chittagong district, Bangladesh. A total of 41 blood samples were collected from indigenous sheep ( ) from June to December 2016 from Dhaka and Chittagong Districts of Bangladesh. Hematological and serum biochemical parameters such as hemoglobin (Hb), packed cell volume (PCV), erythrocyte sedimentation rate (ESR), total erythrocyte count (TEC), total leukocyte count (TLC), neutrophil, eosinophil, basophil, monocyte, lymphocyte, urea, triglyceride, cholesterol, glucose, albumin, total protein (TP), alanine aminotransferase (ALT), and aspartate transaminase (AST) were determined by biochemical analyzer. 90% reference intervals were calculated for all parameters. The hematologica and serum biochemical profiles of indigenous sheep showed wide range and variation. The results were categorized according to sex and age of the sheep for comparison. Young sheep had significantly higher PCV, eosinophil, triglyceride, and TP level than that of adult (p<0.05), whereas the urea and albumin level was higher in adult than that of juvenile (p<0.05). Most of the values of the parameters are close to each other for both male and female except TEC, urea, cholesterol, triglyceride, glucose, and AST. However, a significant difference was found only for albumin and basophil level between male and female sheep. Hematological and biochemical parameters in Bangladeshi indigenous sheep showed a wide range and variation implicating future study for the prophylaxis of ovine diseases.

The ecology of infectious diseases involves wildlife, yet the wildlife interface is often neglected and understudied. Pathogens related to infectious diseases are often maintained within wildlife populations and can spread to livestock and humans. In this study, we explored the fecal microbiome of coyotes and wild hogs in the Texas panhandle using polymerase chain reactions and 16S sequencing methods. The fecal microbiota of coyotes was dominated by members of the phyla Bacteroidetes, Firmicutes, and Proteobacteria. At the genus taxonomic level, Odoribacter, Allobaculum, Coprobacillus, and Alloprevotella were the dominant genera of the core fecal microbiota of coyotes. While for wild hogs, the fecal microbiota was dominated by bacterial members of the phyla Bacteroidetes, Spirochaetes, Firmicutes, and Proteobacteria. Five genera, Treponema, Prevotella, Alloprevotella, Vampirovibrio, and Sphaerochaeta, constitute the most abundant genera of the core microbiota of wild hogs in this study. Functional profile of the microbiota of coyotes and wild hogs identified 13 and 17 human-related diseases that were statistically associated with the fecal microbiota, respectively (p < 0.05). Our study is a unique investigation of the microbiota using free-living wildlife in the Texas Panhandle and contributes to awareness of the role played by gastrointestinal microbiota of wild canids and hogs in infectious disease reservoir and transmission risk. This report will contribute to the lacking information on coyote and wild hog microbial communities by providing insights into their composition and ecology which may likely be different from those of captive species or domesticated animals. This study will contribute to baseline knowledge for future studies on wildlife gut microbiomes.

Background and Aim: Goat is one of the major livestock species, plays an important role in the economy of Bangladesh. However, the outbreak of different infectious diseases in goats causes high mortality and economic losses due to lack of proper diagnosis and treatment. Conventional culture-based methods for detecting specific pathogens as confirmatory diagnosis are laborious as well as time-consuming in comparison to multiplex polymerase chain reaction (mPCR), by which multiple pathogens can be detected at a time. The present study was aimed to perform faster molecular identification of bacterial pathogens from goats presumed with fever and/or diarrhea and their antimicrobial resistance (AMR) pattern. Materials and Methods: A total of 200 blood samples were collected from goats at S. A. Quaderi Teaching Veterinary Hospital (SAQTVH) in Chattogram Veterinary and Animal Sciences University for the period of July 2017-April 2018. DNA was extracted and subsequently, mPCR assay was performed for the screening of several bacterial pathogens (Salmonella spp., Listeria monocytogenes, Bacillus cereus, Yersinia enterocolitica, Campylobacter jejuni, Campylobacter coli, Clostridium perfringens, Vibrio cholerae, and Staphylococcus aureus). An antimicrobial susceptibility test against ten antimicrobials for positive samples of each organism was conducted using the Kirby–Bauer Disk-Diffusion Method on selective media. Results: S. aureus, C. perfringens, L. monocytogenes, and Salmonella spp. were detected from collected samples and their overall prevalence was 11.5%, 3.5%, 1%, and 20.5%, respectively. The most common clinical signs were mild fever, nasal discharge, dyspnea, and coughing (39.1%) for S. aureus, diarrhea, convulsion, abdominal pain, and incoordination (57.1%) for C. perfringens, fever, protrusion of tongue, and incoordination (100%) for L. monocytogenes, and fever, anorexia, dehydration with mucous feces (36.6%) for Salmonella spp. infection in goats. Antimicrobial diagram of S. aureus showed resistance against Cefotaxime (74%), Cefixime (65%), and Tetracycline (65%); highly sensitive against Amoxicillin (48%), Ciprofloxacin (44%), and Gentamicin (44%). On the other hand, C. perfringens showed highly resistant against Ampicillin (71%), Gentamicin (71%), sensitive against Penicillin (57%), and Cefotaxime (57%). L. monocytogenes were found to be sensitive to Penicillin (100%) and Cefixime (100%) and Salmonella spp. showed resistance to Ampicillin (78%) and Amoxicillin (59%) but sensitive to Ciprofloxacin (54%). Conclusion: This study identified pathogens with their specific clinical signs in goats presumed fever and/or diarrhea through mPCR with their AMR pattern in SAQTVH, Chattogram. Potential risk factors, measuring the strength of association of disease caused by these particular pathogens, were also determined. mPCR may use as an effective tool for rapid detection of pathogens in animal.

The outcome of bacterial infection management relies on prompt diagnosis and effective treatment, but conventional antimicrobial susceptibility testing can be slow and labor-intensive. Therefore, this study aims to predict phenotypic antimicrobial susceptibility of selected beta-lactam antimicrobials in the bacteria of the family Enterobacteriaceae from different beta-lactamase resistance genotypes. Using human datasets extracted from the Antimicrobial Testing Leadership and Surveillance (ATLAS) program conducted by Pfizer and retail meat datasets from the National Antimicrobial Resistance Monitoring System for Enteric Bacteria (NARMS), we used a robust or weighted least square multivariable linear regression modeling framework to explore the relationship between antimicrobial susceptibility data of beta-lactam antimicrobials and different types of beta-lactamase resistance genes. In humans, in the presence of the blaCTX-M-1, blaCTX-M-2, blaCTX-M-8/25, and blaCTX-M-9 groups, MICs of cephalosporins significantly increased by values between 0.34–3.07 μg/mL, however, the MICs of carbapenem significantly decreased by values between 0.81–0.87 μg/mL. In the presence of carbapenemase genes (blaKPC, blaNDM, blaIMP, and blaVIM), the MICs of cephalosporin antimicrobials significantly increased by values between 1.06–5.77 μg/mL, while the MICs of carbapenem antimicrobials significantly increased by values between 5.39–67.38 μg/mL. In retail meat, MIC of ceftriaxone increased significantly in the presence of blaCMY-2, blaCTX-M-1, blaCTX-M-55, blaCTX-M-65, and blaSHV-2 by 55.16 μg/mL, 222.70 μg/mL, 250.81 μg/mL, 204.89 μg/mL, and 31.51 μg/mL respectively. MIC of cefoxitin increased significantly in the presence of blaCTX-M-65 and blaTEM-1 by 1.57 μg/mL and 1.04 μg/mL respectively. In the presence of blaCMY-2, MIC of cefoxitin increased by an average of 8.66 μg/mL over 17 years. Compared to E. coli isolates, MIC of cefoxitin in Salmonella enterica isolates decreased significantly by 0.67 μg/mL. On the other hand, MIC of ceftiofur increased in the presence of blaCTX-M-1, blaCTX-M-65, blaSHV-2, and blaTEM-1 by 8.82 μg/mL, 9.11 μg/mL, 8.18 μg/mL, and 1.04 μg/mL respectively. In the presence of blaCMY-2, MIC of ceftiofur increased by an average of 10.20 μg/mL over 14 years. The ability to predict antimicrobial susceptibility of beta-lactam antimicrobials directly from beta-lactamase resistance genes may help reduce the reliance on routine phenotypic testing with higher turnaround times in diagnostic, therapeutic, and surveillance of antimicrobial-resistant bacteria of the family Enterobacteriaceae.

Hepatitis E virus (HEV) is a waterborne zoonotic disease that can result in a high fatality rate in pregnant women and infants. In 2018, a large HEV outbreak emerged in Chattogram, Bangladesh, resulting in 2800 cases and a significant public health response to mitigate the transmission. While the source of the outbreak remained poorly understood, authorities suggested that possible risk factors for HEV infection included contamination of water supply, exacerbated by concurrent severe flooding events in the community. A cross-sectional study was conducted to investigate the distribution and risk factors for HEV seroprevalence between January and December 2018 in the Chattogram city area. A total of 505 blood samples were collected from symptomatic patients of 10 hospitals who met the case definition for an HEV infection. Standard ELISA tests were performed in all patients to identify anti-HEV antibodies. The size and location of HEV seroprevalence clusters within Chattogram were investigated using SaTScan. We investigated the association between risk of HEV infection and individual and environmentally lagged risk factors using Bernoulli generalised linear regression models. Our results indicate an overall HEV seroprevalence of 35% with significant variation according to sex, source of drinking water, and boiling of drinking water. A positive crosscorrelation was found between HEV exposure and precipitation, modified normalised difference water index (MNDWI), and normalised difference vegetation index (NDVI). Our model indicated that risk of infection was associated with sex, age, source of drinking water, boiling of water, increased precipitation, and increased MNDWI. The results from this study indicate that source and boiling of drinking water and increased precipitation were critical drivers of the 2018 HEV outbreak. The communities at highest risk identified in our analyses should be targeted for investments in safe water infrastructure to reduce the likelihood of future HEV outbreaks in Chattogram.