Diarrhea In Humans Research Articles

Molecular characterization of enterohemorrhagic Escherichia coli isolated from diarrhea samples from human, livestock, and ground beef in North Jordan.

Background and Aim:Enterohemorrhagic Escherichia coli (EHEC) is an important foodborne pathogen with worldwide distribution. Data regarding its presence, distribution, virulence, and antimicrobial susceptibility among various animal species and humans in Jordan are lacking. Therefore, the objectives of this study were to isolate and characterize EHEC from human and animal diarrhea fecal samples and ground beef samples.Materials and Methods:A total of 100 and 270 diarrhea fecal samples from humans and animals, respectively, were collected. In addition, 40 ground beef meat samples were collected from retail markets. EHEC was positively identified by detecting Shiga toxins (stx1 and stx2) genes using multiplex polymerase chain reaction (PCR). Antimicrobial susceptibility patterns were determined using the disk diffusion test. Beta-lactamase production was detected using the double disk diffusion test and the extended-spectrum beta-lactamases (ESBLs) were identified by detection of blaTEM, blaSHV, and OXA-1 genes using multiplex PCR. Pulsed-field gel electrophoresis (PFGE) was used to investigate the relatedness of EHEC isolates from different sources.Results:Out of 410 samples, 194 E. coli isolates were positively identified, of which 57 isolates (29%) were classified as EHEC. Thirty-five (61%) of EHEC isolates were serotyped as O157 (19: O157:H7 and 16: O157:NM). The stx1 gene was detected only among the sheep and goats isolates at a rate of 7.6% and 5.2%, respectively, while the stx2 gene was detected in only one ground beef meat sample. EHEC isolates showed high resistance patterns against amoxicillin, gentamycin, cephalexin, and doxycycline. Twenty-four out of 32 EHEC isolates were determined as ESBL producers, among which 14 isolates expressed the blaSHVgene and 19 isolates expressed the blaTEM while four expressed both genes. PFGE analysis revealed two clusters with high similarity (92%) originated from ground beef meat and cattle fecal samples. No similarities were found between human and animal E. coli isolates.Conclusion:Results of this study indicate widespread ESBL EHEC among humans, animals, and ground beef meat samples. These results represent an important alarm that requires the implementation of appropriate preventative measures by both human and animal health sectors to prevent the transmission of this important foodborne pathogen.

Zoonotic Risk of Encephalitozoon cuniculi in Animal-Assisted Interventions: Laboratory Strategies for the Diagnosis of Infections in Humans and Animals.

The involvement of animals for therapeutic purposes has very ancient roots. To date, it is clear that animal-assisted interventions (AAIs), in addition to ensuring the replacement of missing or deficient affects, improves psychophysiological parameters connected to human health. However, AAI could potentially present risks related to the transmission of infectious agents from animals to humans. Among these microorganisms, E. cuniculi is a microspore which induces pathological effects (fever, headache, nausea, vomiting, diarrhea, breathlessness, respiratory symptoms, and weakness) in both humans and animals. Consequently, an accurate and fast diagnosis of E. cuniculi infection, as well as the identification of new diagnostic approaches, is of fundamental importance. This literature review was carried out to provide an extensive and comprehensive analysis of the most recent diagnostic techniques to prevent and care for E. cuniculi-associated risks in the AAI field.

Evaluation of the Correlation between the mRNA Expression Levels of ystA and ymoA Genes in Y. enterocolitica Strains with Different Enterotoxic Properties.

Yersinia enterocolitica is one of the main causative agents of human diarrhea. Pigs are a reservoir and the most common source of infection for humans. The aim of this study was to analyze the expression of ystA and ymoA genes in Y. enterocolitica strains with different enterotoxic properties, isolated from humans and pigs. The experiment involved two groups of Y. enterocolitica strains producing and not producing enterotoxin YstA, which were isolated from humans and pigs. All strains were ystA- and ymoA-positive. The expression of ystA and ymoA genes was analyzed by quantitative real-time PCR (qPCR). The relative expression level of the ystA gene was significantly higher than the expression level of the ymoA gene in Y. enterocolitica strains isolated from humans with clinical symptoms of yersiniosis. In other strains, a significant decrease in ystA gene transcription was observed, and the relative expression level of the ymoA gene was significantly higher than the expression level of the ystA gene. Statistically significant differences were not observed in either group of strains isolated from pigs. The results of our study revealed a correlation between mRNA expression levels of ystA and ymoA genes in Y. enterocolitica strains isolated from humans.

Genotypic analyses and antimicrobial resistance profiles of Campylobacter jejuni from crows (Corvidae) of United States and India reflect their respective local antibiotic burdens.

The study examined the hypothesis that crow-borne Campylobacter can function as environmental reservoirs and indicators of antibiotic resistance (AR) determinants circulating in a human population. Two species of crows from Washington (WA), United States, and Kolkata, India, respectively, were examined for their ability to carry antibiotic resistant Campylobacter. Campylobacter jejuni was the only species isolated by selective agar plating from crow faecal samples. Disk diffusion method used to compare the AR profile of the isolates showed tetracycline (TET) resistance to be the most prevalent (27%) among WA isolates, followed by ciprofloxacin (CIP; 24%). Among Kolkata isolates, nalidixic acid resistance was most common (36%), followed by CIP (27%). The AR profile demonstrated by crow isolates of WA reflects those reported by the US National Antimicrobial Resistance Monitoring System for human isolates (2007-2011), where resistance to TET was most prevalent (≈45%), followed by quinolones (≈24%). The Kolkata crow isolates reflected the AR profile of human clinical isolates from India, where 97% resistance was shown to quinolones, followed by TET (18%). Multilocus sequence typing of 37 isolates, including 11 water isolates from the crow roost area, showed 24 different sequence types (STs). Seventeen of these were previously found in wild birds, 2 in human diarrhoea, 4 in poultry and 8 in environmental water. One isolate was found in both water and faeces, though from different sites within WA. The results indicate that crows most likely acquire the AR from anthropogenic sources. Although they are colonized by specific STs, rarely isolated from humans, they can facilitate the spread of AR. By studying two areas in different continents, this research demonstrates that Campylobacter borne by crows can function as environmental reservoirs and indicators of AR determinants that circulate in a human population. This information will be of importance to scientists from the medical and poultry industries.

Prevalence and antimicrobial susceptibility patterns of Vibrio cholerae in dairy excreta

Vibrio cholerae is a major etiological agent of human diarrhoea and has become epidemic across the world in the recent past. A cross-sectional study was conducted to assess the prevalence of V. cholerae from dairy excreta along with antimicrobial resistant status of the isolates. A total 50 samples were collected from 50 different household manure pit located at Bangladesh Agriculture University (BAU) surrounding area, Mymensingh. Alkaline peptone water was used for enrichment of the samples followed by inoculation onto thiosulfate citrate bile salt sucrose (TCBS) agar media for the isolation of Vibrio spp., which were further confirmed via Vibrio genus specific molecular assay. Biochemical tests were performed to identify V. cholerae from the isolates of Vibrio spp. Out of 50 samples 17 (34%) were confirmed as Vibrio spp. as they produced characteristic yellow colonies on TCBS agar and had found to possess recombinase A gene that confirmed the identity of Vibrio spp. From this 17 Vibrio isolates, 6 (12% in total from 50 samples) were identified as V. cholerae based on different biochemical tests. All the isolates fermented glucose, maltose, sucrose and mannitol with the production of only acid. The isolates were positive in oxidase, gelatinase, methyl-red (MR) and indole test, but negative in case of voges-proskaure (VP) test. In antimicrobial susceptibility testing, V. cholerae isolates showed 100% sensitivity to gentamycin, chloramphenicol and tetracycline with moderate sensitivity to ciprofloxacin and co-trimoxazole. A high level of resistance was observed to ampicillin (100%) followed by moderate resistance to erythromycin and imipenem. In the present study about 33.33% (n = 2) of 6 isolated V. cholerae were found to be multidrug resistant (MDR) as they demonstrated resistant against 3 antimicrobial agents. The findings of this study substantiate the presence of MDR V. cholerae in the dairy excreta, which indicates the role of domestic animals to serve as a reservoir that might pose a health risk to human. Hygienic management of animal waste is needed to reduce the burden of human illness.
 Asian Australas. J. Biosci. Biotechnol. 2021, 6 (1), 40-49

Cryptosporidium infection in cattle and humans in Ethiopia: A systematic review and meta-analysis

Cryptosporidium is an intracellular coccidian parasite causing gastrointestinal disturbances resulting in diarrhoea in humans and animals. It is more frequently detected in calves and early childhood, and one of the major causes of mortality in low-income countries. National estimates of Cryptosporidium infection rate in cattle and humans are lacking in Ethiopia. Therefore, this systematic review and meta-analysis estimated the prevalence and assess the risk factors of Cryptosporidium infection in cattle and humans over 20 years. Article searches were made using PubMed, HINARI, Research Gates, AJOLs and Google Scholar databases. Studies that met the inclusion criteria under the preferred reporting items for systematic reviews and meta-analyses (PRISMA) checklist were used. Random effects models and Inverse Variance Index were used to calculate the pooled prevalence of cryptosporidiosis and heterogeneity among studies, respectively. A total of 23 eligible studies published between 2000 and 2020 were selected for this study. The estimated pooled prevalence of cryptosporidiosis was found to be 16.2% and 11% in cattle and humans, respectively. Ten Cryptosporidium species were documented with cattle and human-based studies. C. andersoni, C. parvum, C. bovis and C. ryanae were the reported species in cattle. Similarly, in humans, seven types of Cryptosporidium species (such as C. parvum, C. hominis, C. viatorum, C. felis, C. meleagridis, C. canis and C. xiaoi) were recorded. C. parvum and C. hominis were the dominant and responsible species for human illness. Using gp60 gene locus analysis, various zoonotic C. parvum subgenotypes were determined in humans; but it was limited in anthroponotic C. hominis. In conclusion, the overall prevalence of Cryptosporidium infection in cattle and humans was high and linked with several risk factors. Thus, there is a need for further epidemiological and genetic diversity studies, and awareness of creations on the disease to provide strategies that mitigate the disease in cattle and humans.

RETRACTED ARTICLE: Molecular detection of pathogenic Escherichia coli strains and their antibiogram associated with risk factors from diarrheic calves in Jimma Ethiopia

Diarrheagenic Escherichia coli are a number of pathogenic E. coli strains that cause diarrheal infection both in animal and human hosts due to their virulence factors. A cross sectional study was conducted between November, 2016 and April, 2017 to isolate and molecularly detect pathogenic E. coli from diarrheic calves to determine the pathogenic strains, antibiogram and associated risk factors in Jimma town. Purposive sampling technique was used to collect 112 fecal samples from diarrheic calves. Conventional culture and biochemical methods were conducted to isolate E. coli isolates. Molecular method was followed to identify virulence factors of pathogenic E. coli strains. Antimicrobial sensitivity patterns of the isolates were tested using the Kirby–Bauer disk diffusion method. A structured questionnaire was also used to collect information from dairy farms and socio-demographic data. The overall isolation rate of E. coli in calves was 51.8% (58/112) (95% CI 42.0–61.0). The occurrence of the bacterium differed significantly by age, colostrum feeding time, amount of milk given per time and navel treatment (P < 0.05). Multivariable analysis revealed that the odds of being infected was significantly highest in calves which fed 1–1.5 L amount of milk per a time (OR 5.38, 95% CI 1.66–17.45, P = 0.005). The overall virulence genes detection rate was 53.5% (95% CI 40.0–67.0). Eleven (19.6%) of eaeA, 6 (10.7%) of Stx1 and 13 (23.2%) of Stx2 genes were detected from calves isolates. Except ciprofloxacillin, all isolates were resistant to at least one drug. Multi drug resistance was recorded in 68.0% (38/56) of calves isolates. Neomycin, 83.3% (25/30), followed by amoxicillin, 53.3% (16/30) were the highest resisted virulence genes. The study demonstrated considerable isolation rate, multiple antimicrobial resistant isolates and high resistant virulent genes in diarrheic calves. It also indicated that the potential importance of calves as source of pathogenic E. coli strains and resistant genes for human diarrhea infection. Improving the hygienic practice of farms and wise use of antimicrobials could help to reduce the occurrence of pathogenic E. coli in farms. Hence, further studies are needed to describe all virulent factors and serotypes associated with the emergence of drug resistant pathogenic E. coli strains in calves.

Simvastatin Reduces Protection and Intestinal T Cell Responses Induced by a Norovirus P Particle Vaccine in Gnotobiotic Pigs.

Noroviruses (NoVs) are a leading cause of acute gastroenteritis worldwide. P particles are a potential vaccine candidate against NoV. Simvastatin is a cholesterol-reducing drug that is known to increase NoV infectivity. In this study, we examined simvastatin’s effects on P particle-induced protective efficacy and T-cell immunogenicity using the gnotobiotic pig model of human NoV infection and diarrhea. Pigs were intranasally inoculated with three doses (100 µg/dose) of GII.4/VA387-derived P particles together with monophosphoryl lipid A and chitosan adjuvants. Simvastatin-fed pigs received 8 mg/day orally for 11 days prior to challenge. A subset of pigs was orally challenged with 10 ID50 of a NoV GII.4/2006b variant at post-inoculation day (PID) 28 and monitored for 7 days post-challenge. Intestinal and systemic T cell responses were determined pre- and postchallenge. Simvastatin abolished the P particle’s protection and significantly increased diarrhea severity after NoV infection. Simvastatin decreased proliferation of virus-specific and non-specific CD8 T cells in duodenum and virus-specific CD4 and CD8 T cells in spleen and significantly reduced numbers of intestinal mononuclear cells in vaccinated pigs. Furthermore, simvastatin significantly decreased numbers of duodenal CD4+IFN-γ+, CD8+IFN-γ+ and regulatory T cells and total duodenal activated CD4+ and CD8+ T cells in vaccinated pigs pre-challenge at PID 28. Following challenge, simvastatin prevented the IFN-γ+ T cell response in spleen of vaccinated pigs. These results indicate that simvastatin abolished P particle vaccine-induced partial protection through, at least in part, impairing T cell immunity. The findings have specific implications for the development of preventive and therapeutic strategies against NoV gastroenteritis, especially for the elderly population who takes statin-type drugs.

Cryptosporidium spp. Infections in Combination with Other Enteric Pathogens in the Global Calf Population.

Simple SummaryThe factors “diagnostic”, “health status of the sampled animals”, and “geographical region” explained the majority of the variance of Cryptosporidium spp. (Crypto) prevalence in the global calf population across the literature. The chance of detecting bovine rotavirus (BRV), bovine coronavirus (BCoV), and enterotoxigenic Escherichia coli F5 (K99) (ETEC) in calves with diarrhoea was lower in the presence of Crypto compared to calves without Crypto. This may indicate an inhibition effect between BRV, BCoV, ETEC, and Crypto. The most common worldwide diarrhoea-causing agents in neonatal calves are Cryptosporidium spp. (Crypto), bovine rotavirus (BRV), bovine coronavirus (BCoV), and enterotoxigenic Escherichia coli F5 (K99) (ETEC). Crypto is a zoonotic pathogen of diarrhoea in humans, particularly for children and immunocompromised adults. Four weighted-stratified random-effect meta-analyses including meta-regression analyses were performed to calculate the worldwide mean prevalence of Crypto and associated concurrent infections with BRV, BCoV and ETEC, as well as their potential influencing factors. The meta-analysis incorporated 28 studies (56 substudies) in 17 countries that determined the presence or absence of concurrent infections with Crypto in the global calf population. Approximately half of all considered studies presented here were conducted in Europe independently of the type of infections with Crypto. Within Europe, the highest estimated mean Crypto-BRV prevalence was identified in Ireland (16.7%), the highest estimated mean Crypto-BCoV prevalence was detected in the United Kingdom (4.3%), and the highest estimated mean Crypto-ETEC prevalence across the literature was determined in Turkey (4.7%). The chance of detecting BRV, BCoV, and ETEC in calves with diarrhoea was 0.8 (confidence interval (CI): 0.6–1.0), 0.7 (CI: 0.5–1.0) and 0.6 (CI: 0.4–0.9) lower in the presence of Crypto compared to calves without Crypto. This may indicate an inhibitory effect between BRV, BCoV, ETEC, and Crypto in calves. The variance in the published prevalence across the literature can mainly be explained by the “diagnostic” factor (R2 min–max: 0.0–40.3%), followed by the “health status of the sampled animals” (R2 min–max: 1.4–27.3%) and “geographical region” (R2 min–max: 5.9–23.6%).

Heat-Labile Toxin from Enterotoxigenic Escherichia coli Causes Systemic Impairment in Zebrafish Model.

Heat-labile toxin I (LT-I), produced by strains of enterotoxigenic Escherichia coli (ETEC), causes profuse watery diarrhea in humans. Different in vitro and in vivo models have already elucidated the mechanism of action of this toxin; however, their use does not always allow for more specific studies on how the LT-I toxin acts in systemic tracts and intestinal cell lines. In the present work, zebrafish (Danio rerio) and human intestinal cells (Caco-2) were used as models to study the toxin LT-I. Caco-2 cells were used, in the 62nd passage, at different cell concentrations. LT-I was conjugated to FITC to visualize its transport in cells, as well as microinjected into the caudal vein of zebrafish larvae, in order to investigate its effects on survival, systemic traffic, and morphological formation. The internalization of LT-I was visualized in 3 × 104 Caco-2 cells, being associated with the cell membrane and nucleus. The systemic traffic of LT-I in zebrafish larvae showed its presence in the cardiac cavity, yolk, and regions of the intestine, as demonstrated by cardiac edema (100%), the absence of a swimming bladder (100%), and yolk edema (80%), in addition to growth limitation in the larvae, compared to the control group. There was a reduction in heart rate during the assessment of larval survival kinetics, demonstrating the cardiotoxic effect of LT-I. Thus, in this study, we provide essential new depictions of the features of LT-I.

A Longitudinal Study on Enteric Virus Contamination in Bivalves along the Coast of Ibaraki Prefecture, Japan

During the 2014 to 2018 seasons, we conducted a longitudinal study involving enteric virus surveillance in bivalves, including natural oysters and clams harvested in Ibaraki Prefecture, Japan. Some norovirus (NoV) contaminations were detected in natural oysters, whereas no enteric virus was found in clams. NoVs detected in oysters were of the genotypes GII.4 and GII.6, both of which are closely related genetically to the NoV strains prevalent in humans. We found low level of enteric virus contamination in bivalves collected along the coast of Ibaraki Prefecture. The possibility of food poisoning caused by these viruses appears low, and few cases of infectious disease have been observed in the surrounding area. The harvest timing was more related to contamination quantity than the harvest area in many enteric viruses. Our results highlight that contamination of bivalves by enteric viruses may depend upon the prevalence of human diarrhea and illness.

Rapid determination of viable but non-culturable Campylobacter jejuni in food products by loop-mediated isothermal amplification coupling propidium monoazide treatment

Campylobacter is the leading cause of foodborne human diarrhea worldwide. This microbe in the viable but non-culturable (VBNC) state can evade detection by routinely used culture-based methods and remain viable for extended periods of time. Bacteria in this dormancy state can resume their metabolic activity and virulence by resuscitation under favorable conditions, and subsequently cause infections. In this study, an assay combining loop-mediated isothermal amplification (LAMP) and propidium monoazide (PMA) treatment was developed for the detection and quantification of VBNC C. jejuni in agri-foods. PMA-qLAMP targeting the hipO gene demonstrated 100% high specificity to C. jejuni. A linear detection of C. jejuni was achieved between 8.77 × 102 and 8.77 × 07 CFU/mL with a coefficient of determination (R2) of 0.9956, indicating a good quantitative capacity. C. jejuni was effectively induced into the VBNC state by osmotic stress (i.e., 7% NaCl, w/v) over 48 h. VBNC C. jejuni cells were spiked into three representative food products and determined by PMA-qLAMP coupled with plating assay. The detection limits of PMA-qLAMP were 1.58 × 102 CFU/mL in milk, 3.78 × 102 CFU/g in chicken breast meat, and 4.33 × 102 CFU/g in romaine lettuce. PMA-qLAMP demonstrated rapid (25–40 min), specific (100% inclusivity and 100% exclusivity) and sensitive (~102 CFU/mL) determination of VBNC C. jejuni. This method can be applied in the agri-food industry to decrease the risks related to the consumption of contaminated agri-foods with pathogenic bacteria in the VBNC state and reduce the burden of C. jejuni infections to public health.

An Escherichia coli Effector Protein EspF May Induce Host DNA Damage via Interaction With SMC1.

Enterohemorrhagic Escherichia coli (EHEC) O157: H7 is an important foodborne pathogen that causes human diarrhea, hemorrhagic colitis, and hemolytic uremic syndrome. EspF is one of the most important effector proteins injected by the Type III Secretion System. It can target mitochondria and nucleoli, stimulate host cells to produce ROS, and promote host cell apoptosis. However, the mechanism of the host-pathogen interaction leading to host oxidative stress and cell cytotoxic effects such as DNA damage remains to be elucidated. Here, we used Cell Counting Kit-8 (CCK-8) assays and 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-OHdG) ELISA to study cell viability and DNA oxidative damage level after exposure to EspF. Western blot and immunofluorescence were also used to determine the level of the DNA damage target protein p-H2AX and cell morphology changes after EspF infection. Moreover, we verified the toxicity in intestinal epithelial cells mediated by EspF infection in vivo. In addition, we screened the host proteins that interact with EspF using CoIP-MS. We found that EspF may more depend on its C-terminus to interact with SMC1, and EspF could activate SMC1 phosphorylation and migrate it to the cytoplasm. In summary, this study revealed that EspF might mediate host cell DNA damage and found a new interaction between EspF and the DNA damage repair protein SMC1. Thus, EspF may mediate DNA damage by regulating the subcellular localization and phosphorylation of SMC1.

First Isolation and Molecular Characterization of bla CTX-M-121 -Producing Escherichia coli O157:H7 From Cattle in Xinjiang, China.

The bovine Escherichia coli O157:H7 is a major foodborne pathogen causing severe bloody diarrhea, hemorrhagic colitis, and hemolytic uremic syndrome in humans. Cattle are recognized major reservoir and source of E. coli O157:H7. We investigated the antibiotic resistance, molecular profiles, and intrinsic relationship between 21 isolates of E. coli O157:H7 from cattle farms and slaughtering houses in Xinjiang. Using pulsed-field gel electrophoresis (PFGE) molecular typing, two types of PFGE were revealed through cluster analysis, including clusters I and II, with 66 and 100% similarity of PFGE spectra between 21 isolates. We also detected that 18 isolates (86%) carried at least one virulence gene, 16 isolates (76%) carried the eae gene, and 7 (33%) carried the stx1 + stx2 + eae + hly + tccp genes. Eighteen isolates were susceptible to antibiotics. Three isolates were resistant to antibiotics, and two were multidrug resistant. One of the two multidrug-resistant isolates detectably carried the blaCTX−M−121 gene. This is the first finding of the blaCTX−M−121 gene detected in E. coli O157:H7 isolated from cattle in Xinjiang. The blaCTX−M−121 gene is transferable between the bacterial strains via plasmid transmission. The results indicated that E. coli O157:H7 may have undergone clonal propagation in cattle population and cross-regional transmission in Xinjiang, China.

Association of Broiler Litter Microbiome Composition and Campylobacter Isolation.

Infection with Campylobacter species is one of the leading causes of bacterial diarrhea in humans in the US. Chickens, which become colonized on the farm, are important reservoirs of this bacterium. Campylobacter can establish itself in the broiler house via a variety of sources, can survive in the litter of the house, and possibly persist over successive flock cycles. However, the role of the broiler litter microbiome on Campylobacter persistence is not clear. A matched case-control study was conducted to determine whether the broiler litter microbiome composition was associated with Campylobacter isolation within the broiler house. Flocks were classified as cases when either Campylobacter jejuni or Campylobacter coli was isolated in boot sock samples, or as controls otherwise. Case and control flocks were matched at the broiler house level. Composite broiler litter samples were collected and used for DNA extraction and 16S rRNA gene V4 region sequencing. Reads were processed using the DADA2 pipeline to obtain a table of amplicon sequence variants. Alpha diversity and differential bacterial relative abundance were used as predictors of Campylobacter isolation status in conditional logistic regression models adjusting for flock age and sampling season. Beta diversity distances were used as regressors in stratified PERMANOVA with Campylobacter isolation status as predictor, and broiler house as stratum. When Campylobacter was isolated in boot socks, broiler litter microbiome richness and evenness were lower and higher, respectively, without reaching statistical significance. Campylobacter isolation status significantly explained a small proportion of the beta diversity (genus-level Aitchison dissimilarity distance). Clostridium and Anaerostipes were positively associated with Campylobacter isolation status, whereas Bifidobacterium, Anaerosporobacter, and Stenotrophomonas were negatively associated. Our results suggest the presence of bacterial interactions between Campylobacter and the broiler litter microbiome. The negative association of Campylobacter with Bifidobacterium, Anaerosporobacter, and Stenotrophomonas in litter could be potentially exploited as a pre-harvest control strategy.

Preliminary Characterization of Two Small Insulinase-Like Proteases in Cryptosporidium parvum.

Cryptosporidium parvum is a major cause of moderate-to-severe diarrhea in humans and animals. Its compact genome contains 22 genes encoding divergent insulinase-like proteases (INS), which are poorly characterized. In this study, two small members of this family, INS-21 encoded by cgd7_2080 and INS-23 encoded by cgd5_3400, were cloned, expressed, and characterized to understand their functions. Recombinant INS-21 and INS-23 were expressed in Escherichia coli and polyclonal antibodies against these two proteins were prepared. The cgd7_2080 gene had a high transcription level during 0–2 h of in vitro C. parvum culture, while cgd5_3400 was highly transcribed at 0–6 h. INS-21 was mostly located in the apical region of sporozoites and merozoites whereas INS-23 was found as spots in sporozoites and merozoites. The immunoelectron microscopy confirmed the expression of INS-21 in the apical region of sporozoites while INS-23 appeared to be expressed in the dense granules of sporozoites. The neutralization efficiency was approximately 35%, when the cultures were treated with anti-INS23 antibodies. These results suggest that INS-21 and INS-23 are expressed in different organelles and might have different functions in the development of C. parvum.

Metagenome-Assembled Genomes Isolated from a Human Fecal Diarrhea Sample.

Diarrheal infection is the second leading infectious disease that is killing children under the age of 5 years. This study investigates the microbial community within a fecal sample from a diarrhea-affected child through shotgun metagenomic sequencing.

Thermostable, Dissolvable Buccal Film Rotavirus Vaccine Is Highly Effective in Neonatal Gnotobiotic Pig Challenge Model.

Difficulties related to storage and transport of currently available live oral rotavirus vaccines can have detrimental consequences on the efficacy of the vaccines. Thus, there is a great need for thermostable vaccines that can eliminate the necessity for cold chain storage or reconstitution before administration. In this study, we developed a dissolvable oral polymeric film comprised of a live attenuated thermostable tetravalent rhesus-human reassortant rotavirus vaccine (RRV-TV) powder and antacid (CaCO3). Immunogenicity and protective efficacy of the vaccine after buccal delivery was evaluated in the gnotobiotic pig model of human rotavirus (HRV) infection and diarrhea. Two doses of the vaccine were highly immunogenic and conferred strong protection against virus shedding and diarrhea upon challenge with a high dose of a virulent G1 HRV in gnotobiotic pigs. Those pigs vaccinated with the preserved film vaccine had significantly delayed onset of diarrhea; reduced duration and area under the curve of diarrhea; delayed onset of fecal virus shedding; and reduced duration and peak of fecal virus shedding titers compared to pigs in both the placebo and the reconstituted liquid oral RRV-TV vaccine groups. Associated with the strong protection, high titers of serum virus neutralization antibodies against each of the four RRV-TV mono-reassortants and G1 HRV-specific serum IgA and IgG antibodies, as well as intestinal IgA antibodies, were induced by the preserved film vaccine. These results demonstrated the effectiveness of our thermostable buccal film rotavirus vaccine and warrant further investigation into the promise of the novel technology in addressing drawbacks of the current live oral HRV vaccines.

Comparation Between Mac conkey and Coconut Water Medium as a Growth Medium for Escherichia coli

Escherichia coli is the bacteria that can cause diarrhea in humans and often used as a parameter of stool environmental pollution. Culture of E. coli from the sample often requires Mac Conkey as commercial media which is able to distinguish it from other bacteria in the Enterobacteriaceae group. Commercial media such as Mac Conkey certainly has a price that is quite expensive because of its ability as a growth medium for Enterobacteriaceae. Therefore, in the study tested natural ingredients that can be used for growth media, such as coconut water. The purpose of this study was to compare the ability of Mac Conkey media and coconut water to support the growth of E. coli. This research is an experimental study with a completely randomized design. The concentration of coconut water tested was 0%, 20%, 40%, 60%, 80%, and 100%. The results showed that at the concentration of coconut water 20% to 60% the number of E. coli colonies on coconut water media was slightly below the Mac Conkey Agar media, while in coconut water a concentration of 80% showed a greater number of colonies than Mac Conkey. The Mann Whitney test showed a significant difference between the number of colonies on 80% coconut water media and Mac Conkey Agar, which was equal to 0.004 (p &lt; 0.05). Based on these results, coconut water has the potential to be used as a growth medium for E. coli.

Genome-wide screening of vaccine targets prioritization and reverse vaccinology aided design of peptides vaccine to enforce humoral immune response against Campylobacter jejuni

Campylobacter jejuni, gram-negative bacteria, is an infectious agent of foodborne disease-causing bloody diarrhea, abdominal pain, fever, Guillain-Barré syndrome (GBS) and Miller Fisher syndrome in humans. Campylobacter spp. with multidrug resistance to fluoroquinolones, tetracycline, and erythromycin are reported. Hence, an effective vaccine candidate would provide long-term immunity against C. jejuni infections. Thus, we used a subtractive proteomics pipeline to prioritize essential proteins, which impart a critical role in virulence, replication and survival. Five proteins, i.e. Single-stranded DNA-binding protein, UPF0324 membrane protein Cj0999c, DNA translocase FtsK, 50S ribosomal protein L22, and 50S ribosomal protein L1 were identified as virulent proteins and selected for vaccine designing. We reported that the multi-epitopes subunit vaccine based on CTL, HTL and B-cell epitopes combination possess strong antigenic properties and associates no allergenic reaction. Further investigation revealed that the vaccine interacts with the immune receptor (TLR-4) and triggered the release of primary and secondary immune factors. Moreover, the CAI and GC contents obtained through codon optimization were reported to be 0.93 and 53% that confirmed a high expression in the selected vector. The vaccine designed in this study needs further scientific consensus and will aid in managing C. jejuni infections.