eDNA metabarcoding for diet analyses of green sea turtles (Chelonia mydas)

Understanding sea turtle diets can help conservation planning, but their trophic ecology is complex due to life history characteristics such as ontogenetic shifts and large foraging ranges. Studying sea turtle diet is challenging, particularly where ecological foraging observations are not possible. Here, we test a new minimally invasive method for the identification of diet items in sea turtles. We fingerprinted diet content using DNA from esophageal and cloacal swab samples by metabarcoding the 18S rRNA gene. This approach was tested on samples collected from green turtles (Chelonia mydas) from a juvenile foraging aggregation in the Bijagós archipelago in Guinea-Bissau. Esophagus samples (n = 6) exhibited a higher dietary richness (11 ± 5 amplicon sequence variants (ASVs) per sample; average ± SD) than cloacal ones (n = 5; 8 ± 2 ASVs). Overall, the diet was dominated by red macroalgae (Rhodophyta; 48.2 ± 16.3% of all ASVs), with the main food item in the esophagus and cloaca being a red alga belonging to the Rhodymeniophycidae subclass (35.1 ± 27.2%), followed by diatoms (Bacillariophyceae; 7.5 ± 7.3%), which were presumably consumed incidentally. Seagrass and some invertebrates were also present. Feeding on red algae was corroborated by field observations and barcoding of food items available in the benthic habitat, validating the approach for identifying diet content. We conclude that identification of food items using metabarcoding of esophageal swabs is useful for a better understanding of the relationships between the feeding behavior of sea turtles and their environment.

Detection of invA virulence gene of multidrug-resistant Salmonella species isolated from the cloacal swab of broiler chickens in Blitar district, East Java, Indonesia

Background and Aim: The increasing number of multidrug-resistant (MDR) Salmonella species on poultry farms in Indonesia has caused concern regarding human health. This study was conducted to determine the presence of the virulence gene invA in MDR Salmonella species isolated from the cloacal swab of broiler chickens in Blitar district, East Java Province, Indonesia. Materials and Methods: Cloacal swab samples were collected by purposive sampling from 15 farms in four districts. Isolation and identification of bacteria were performed using standard microbiological techniques. Confirmation of MDR isolates was done using five different classes of antibiotics, including the beta-lactam, aminoglycoside, fluoroquinolone, phenicol, and monobactam groups. An antibiotic susceptibility test was conducted using the Kirby–Bauer disk diffusion method, and a polymerase chain reaction method was used to screen for the presence of invA. Results: It was observed that 32.26% (50/155) of the samples were positive for Salmonella species. Of these 50 Salmonella isolates, 7 (14%) were identified as MDR strains. An important finding was the detection of invA in all the seven MDR Salmonella strains (100%) isolated from the cloacal swab of broiler chickens in Blitar district, East Java Province. Conclusion: Veterinarians have an extremely important role in monitoring the use of antibiotics in farm animals to mitigate the rapid spread of MDR organisms in our environment, which can otherwise cause serious economic losses and also public health issues.

Occurrence of Escherichia coli in cloacal samples of broiler chicken from Kollam and Kottayam districts

Foodborne pathogens like E. coli are considered as the major causes of foodborne illness in humans worldwide. The present study was undertaken to determine the occurrence of E. coli in cloacal samples of broiler chicken from Kollam and Kottayam districts. The occurrence of E. coli in cloacal samples from broiler chicken was 76.5 per cent from Kollam and 79 per cent from Kottayam through culture techniques. Out of the total 400 cloacal swab samples collected from broiler chicken, 77.8 per cent were positive for E. coli. The samples which were subjected to conventional culture techniques were further analysed for PCR confirmation. The study revealed that, 56.5 and 67 per cent samples were positive for E. coli from Kollam and Kottayam, respectively. An overall occurrence of 61.8 per cent out of 400 samples were confirmed for E. coli by PCR. One Health approach can be used as a suitable tool to combat the foodborne zoonotic diseases, since it is an integrated, multidisciplinary, holistic approach. Proper implementation of biosecurity measures in farms is mandatory to control foodborne zoonotic diseases.

Multiple novel filamentous phages detected in the cloacal swab samples of birds using viral metagenomics approach

Members of the family Inoviridae (inoviruses) are characterized by their unique filamentous morphology and infection cycle. The viral genome of inovirus is able to integrate into the host genome and continuously releases virions without lysing the host, establishing chronic infection. A large number of inoviruses have been obtained from microbial genomes and metagenomes recently, but putative novel inoviruses remaining to be identified. Here, using viral metagenomics, we identified four novel inoviruses from cloacal swab samples of wild and breeding birds. The circular genome of those four inoviruses are 6732 to 7709 nt in length with 51.4% to 56.5% GC content and encodes 9 to 13 open reading frames, respectively. The zonula occludens toxin gene implicated in the virulence of pathogenic host bacteria were identified in all four inoviruses and shared the highest amino acid sequences identity (< 37.3%) to other reference strains belonging to different genera of the family Inoviridae and among themselves. Phylogenetic analysis indicated that all the four inoviruses were genetically far away from other strains belonging to the family Inoviridae and formed an independent clade. According to the genetic distance-based criteria, all the four inoviruses identified in the present study respectively belong to four novel putative genera in the family Inoviridae.

Occurrence and multidrug resistance of Campylobacter spp. at duck farms and associated environmental and anthropogenic risk factors in Bangladesh

Background The alarming rise in multi-drug resistant (MDR) zoonotic pathogens, including Campylobacter spp., has been threatening the health sector globally. In Bangladesh, despite rapid growth in poultry sector little is known about the potential risks of zoonotic pathogens in homestead duck flocks. The aim of this study was to understand the occurrence, species diversity, and multi-drug resistance in Campylobacter spp., and identify the associated risk factors in duck farms in Bangladesh. Methods The study involved 20 duck farms at 6 sub-districts of Mymensingh, Bangladesh. Monthly occurrence of Campylobacter spp. in potential sources at the farms during February-September, 2018, was detected by culture and PCR-based methods. Campylobacter isolates were examined for resistance to different antimicrobials. Risk factors, concerning climatic and environmental disposition, farm management, and anthropogenic practices, of Campylobacter infection were estimated by participatory epidemiological tools. Results Occurrence of Campylobacter spp. was detected in overall 36.90% (155/420) samples, more frequently in drinking water (60%, 30/50), followed by cloacal swab (37.50%, 75/200), egg surface swab (35%, 35/100) and soil of the duck resting places (30%, 15/50) but was not detected in feed samples (n = 20). PCR assays distinguished the majority (61.30%, 95/155) of the isolates as C. coli, while the rest (38.70%, 60/155) were C. jejuni. Notably, 41.7% (25/60) and 31.6% (30/95) strains of C. jejuni and C. coli, respectively, were observed to be MDR. The dynamics of Campylobacter spp., distinctly showing higher abundance during summer and late-monsoon, correlated significantly with temperature, humidity, and rainfall, while sunshine hours had a negative influence. Anthropogenic management-related factors, including, inadequate hygiene practices, use of untreated river water, wet duck shed, flock age (1–6 months), and unscrupulous use of antimicrobials were identified to enhance the risk of MDR Campylobacter infection. Conclusion The present study clearly demonstrates that duck farms contribute to the enhanced occurrence and spread of potentially pathogenic and MDR C. coli and C. jejuni strains and the bacterial dynamics are governed by a combined interaction of environmental and anthropogenic factors. A long-term holistic research at the environment-animal-human interface would be integral to divulge health risk reduction approaches tackling the spread of Campylobacter spp. from duck farms.

Isolation and genomic characterization of “unassigned” Salmonella enterica serovars from poultry in Ilorin, north-central Nigeria

Salmonellosis is an important global foodborne disease caused by Salmonella enterica (S. enterica). Strains that are resistant to a variety of antibiotics were known to constitute major hazard to public health. The objectives of this study are to determine the serovar distributions, genomic antimicrobial resistance, prediction of genes conferring resistance to selected antibiotics, the multi-locus sequence typing (MLST), and plasmid replicon typing of “unassigned” S. enterica isolated from poultry. A total of 300 samples comprised of: post-mortem tissues (n = 150), cloacal swabs (n = 30), and poultry environment (n = 120) were aseptically collected and analyzed between January and June, 2017. Presumptive S. enterica isolates were characterized using conventional cultural methods, biochemical tests, and serotyping. The isolates were characterized, using Whole Genome Sequencing (WGS) Method. Five “unassigned” S. enterica serovars were recovered from four matrices (liver, n = 1; water, n = 1; cloacal swab, n = 2; poultry feed, n = 1). Prediction of point mutation in parC (T57S) was reported in two strains which confer resistance to nalidixic acid; in addition to this, prediction of fosA7 that confers resistance to fosfomycin was identified in one of these strains. Three isolates each encoded plasmid mediated quinolone resistance (PMQR) qnrB69 and bla-CMY-98 genes expected to confer decreased susceptibility to ciprofloxacin and resistance to ampicillin, amoxicillin-clavulanic acid, cefoxitin, and ceftriaxone, respectively. Three sequence types, ST-6111, 6114 and 7073 were detected. None of the isolates harbored plasmid replicon. This study highlights the importance of “unassigned” S. enterica serovars in the emergence and spread of S. enterica in poultry. There is a need for the establishment of national collaborative Salmonella program to further investigate the pathogenic and public health risk to humans, of “unassigned” S. enterica serovars in Nigeria.

Wild birds as reservoirs of multidrug-resistant enterobacteria in Mulungu, Brazil

Caatinga is a biome unique to Brazil that is degraded by anthropogenic actions, which lead to the loss of biodiversity putting many species at risk of extinction. The Ceará State is located in the Caatinga and has a rich avifauna comprised of 433 species including 13 species that are threatened with extinction, which are found in the Baturité Massif. The aim of this study was to investigate the frequency and diversity of enterobacteria in wild birds and to determine their susceptibility to antimicrobials. Cloacal swab samples were collected from 50 individuals of 28 different species, including the Ceara Gnatheter ( Conopophaga cearae ) and Red-necked Tanager ( Tangara cyanocephala cearensis ), which are classified as vulnerable (VU) by the Brazilian Ministry of the Environment. A total of 55 isolates belonging to 14 different species of Enterobacteriaceae were identified. Among these, Pantoea agglomerans and Escherichia coli were the most prevalent species with isolation rates of 36% and 26%, respectively. The highest rate of antimicrobial resistance found was to ampicillin (41.8%), followed by nalidixic Acid (36.3%) and amoxicillin associated with clavulanic acid (32.7%). The drugs with the best efficacy were tobramycin (96.4%), ciprofloxacin (92.6%) and tetracycline (90.9%). Multidrug resistance was observed in 23.5% of the analyzed strains. This research provides important information about the composition of the cloacal microbiota of wild birds in Mulungu, Brazil, as well as their health status. In addition, these results demonstrate that they harbor multidrug-resistant strains of Enterobacteriaceae.

Molecular Characterization of Hemagglutinin-Neuraminidase Protein Virus Newcastle disease (ND) in Surabaya during 2003 and 2016

This study aimed to determine the mutation of amino acid, nucleotide homology, phylogenetic tree, epitope prediction of Hemagglutinin-Neuraminidase protein Newcastle Disease (ND) Virus isolated from traditional market around Surabaya. Samples were from 37 chicken with cloacal swab and one positive samples for control (LaSota). Samples were inoculated on embyonate chicken eggs and identified with HA test confirmed with HI test. Positive samples processed by PCR using forward and reverse primer with 503 bp RNA target. The PCR result then analyzed with sequencing. Result of sequencing analysis showed that theres similiarity between samples amino acid and vaccine isolate its effect the percentage of nucleotide homology and phylogenetic relation between isolate. Epitope NGAANNSGWGAPIHDPDYIGG have high immunogenic value at all of isolate which good as vaccine candidate.

Helicobacter cyclurae sp. Nov., Isolated From Endangered Blue Iguanas (Cyclura lewisi)

Blue iguanas (Cyclura lewisi) are endangered reptiles found only on Grand Cayman. Previously, DNA for a novel Helicobacter species GCBI1 was detected in sick and dead iguanas. In the current study, fecal and cloacal swab samples were obtained from 25 iguanas. Through molecular and microbiological techniques, a novel Helicobacter species was cultured from feces and characterized, for whom we propose the name Helicobacter cyclurae. This novel helicobacter had a prevalence of 56% by PCR and 20% by culture in samples analyzed. The type strain MIT 16-1353 was catalase, oxidase, and gamma-glutamyl transpeptidase positive. By electron microscopy, H. cyclurae has a curved rod morphology and a single sheathed polar flagellum. Phylogenetic analysis using 16S rRNA, gyrB, and hsp60 indicated that these strains were most closely related to Helicobacter sp. 12502256-12 previously isolated from lizards. H. cyclurae has a 1.91-Mb genome with a GC content of 33.37%. There were 1,969 genes with four notable virulence genes: high temperature requirement-A protein-secreted serine protease, gamma-glutamyl transpeptidase, fibronectin/fibrinogen binding protein, and neutrophil-activating protein. Whole-genome phylogeny, average nucleotide identity, and digital DNA–DNA hybridization analysis confirmed that H. cyclurae is a novel species, and the first helicobacter cultured and characterized from blue iguanas.

Antibiotic Resistant Pattern and Resistant Gene Identification of Staphylococcus aureus from Chicken Farm in Bogor

Chicken is one of the important protein source in Indonesia. Moreover, the largest population of chicken layer and poultry in Indonesia is known situated at West Java province with Bogor manicipality as the main producer. The aims of this study were to determine the antibiotic resistance pattern of Staphylococcus aureus isolated from poultry and layer farm in Bogor. The study also identified gene encoded the resistance. Cloacal swab samples were collected from chicken broiler and layer farm in Bogor manicipality. The samples were then cultured in Mannitol Salt Agar (MSA) medium to obtain S. aureus. Suspected colony was then confirmed by biochemical test. Positive strains were tested against several antibiotics and the diameter of clear zone arround of colony was interpreted based on Clinical and Laboratory Standard Institute. Furthermore, the DNA from resistant strains were then extracted, followed by detection of the resistance gene by using polymerase chain reaction (PCR) method. A total of 14 isolates of S. aureus were positive from poultry farm, and 15 isolates from layer farm. Most of all were resistant to tetracycline, ampicillin, oxytetracycline, erythromycin and nalidixic acid. On the other hands, several strains were sensitive to gentamycin and chloramphenicol. The study showed 28 isolates out of them were multi-drug resistant. Resistant gene such as blaTEM, gyrA and tetA were also identified in some isolates except for ErmB gene which was found in isolates originated from poultry farm. In conclussion, S. aureus in both farm showed mostly multi-drug resistant to several antibiotics which were supported by identification of resistant gene among isolates.