Bovine Fecal Samples Research Articles

Technical report: In-gel sample preparation prior to proteomic analysis of bovine faeces increases protein identifications by removal of high molecular weight glycoproteins

Bovine faecal composition is complex and a knowledge gap exists in the understanding of the bovine faecal proteome. In the present study, in-gel sample preparation (IGSP) of faecal samples prior to proteomics showed an increase in the number of proteins identified in faecal samples compared to those processed by filter-aided sample preparation (FASP). The optimised sample preparation method removed high molecular weight glycoproteins as part of the clean-up process of the faecal samples, and in combination with in-gel digestion before liquid chromatography with tandem mass spectrometry (LC-MS/MS). The use of IGSP led to enhanced protein identification with increases in the number of peptides identified and in the percent coverage of proteins in the bovine faecal samples. SignificanceCharacterization of faecal proteins has the potential to increase our understanding of host responses to changes such as diet, disease and drug-treatment. In-gel sample preparation prior to proteomics can be used to remove high molecular weight glycoproteins and reduce protein/peptide loss in FASP. This method of sample preparation will have application not only in the investigation of bovine faecal extracts but also in studies where large molecules such as glycoproteins or oligosaccharides could have detrimental influences on sample preparation involving ultrafiltration.

Whole Genome Sequence-Based Identification of Clostridium estertheticum Complex Strains Supports the Need for Taxonomic Reclassification Within the Species Clostridium estertheticum.

Isolates within the Clostridium estertheticum complex (CEC) have routinely been identified through the 16S rRNA sequence, but the high interspecies sequence similarity reduces the resolution necessary for species level identification and often results in ambiguous taxonomic classification. The current study identified CEC isolates from meat juice (MJS) and bovine fecal samples (BFS) and determined the phylogeny of species within the CEC through whole genome sequence (WGS)-based analyses. About 1,054 MJS were screened for CEC using quantitative real-time PCR (qPCR). Strains were isolated from 33 MJS and 34 BFS qPCR-positive samples, respectively. Pan- and core-genome phylogenomics were used to determine the species identity of the isolates. Average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) were used to validate the species identity. The phylogeny of species within the CEC was determined through a combination of these methods. Twenty-eight clostridia strains were isolated from MJS and BFS samples out of which 13 belonged to CEC. At 95% ANI and 70% dDDH thresholds for speciation, six CEC isolates were identified as genomospecies2 (n=3), Clostridium tagluense (n=2) and genomospecies3 (n=1). Lower thresholds of 94% ANI and 58% dDDH were required for the classification of seven CEC isolates into species C. estertheticum and prevent an overlap between species C. estertheticum and Clostridium frigoriphilum. Combination of the two species and abolishment of current subspecies classification within the species C. estertheticum are proposed. These data demonstrate the suitability of phylogenomics to identify CEC isolates and determine the phylogeny within CEC.

Characterisation of Early Positive mcr-1 Resistance Gene and Plasmidome in Escherichia coli Pathogenic Strains Associated with Variable Phylogroups under Colistin Selection.

An antibiotic susceptibility monitoring programme was conducted from 2004 to 2010, resulting in a collection of 143 Escherichia coli cultured from bovine faecal samples (diarrhoea) and milk-aliquots (mastitis). The isolates were subjected to whole-genome sequencing and were distributed in phylogroups A, B1, B2, C, D, E, and G with no correlation for particular genotypes with pathotypes. In fact, the population structure showed that the strains belonging to the different phylogroups matched broadly to ST complexes; however, the isolates are randomly associated with the diseases, highlighting the necessity to investigate the virulence factors more accurately in order to identify the mechanisms by which they cause disease. The antimicrobial resistance was assessed phenotypically, confirming the genomic prediction on three isolates that were resistant to colistin, although one isolate was positive for the presence of the gene mcr-1 but susceptible to colistin. To further characterise the genomic context, the four strains were sequenced by using a single-molecule long read approach. Genetic analyses indicated that these four isolates harboured complex and diverse plasmids encoding not only antibiotic resistant genes (including mcr-1 and bla) but also virulence genes (siderophore, ColV, T4SS). A detailed description of the plasmids of these four E. coli strains, which are linked to bovine mastitis and diarrhoea, is presented for the first time along with the characterisation of the predicted antibiotic resistance genes. The study highlighted the diversity of incompatibility types encoding complex antibiotic resistance elements such as Tn6330, ISEcp1, Tn6029, and IS5075. The mcr-1 resistance determinant was identified in IncHI2 plasmids pCFS3273-1 and pCFS3292-1, thus providing some of the earliest examples of mcr-1 reported in Europe, and these sequences may be a representative of the early mcr-1 plasmidome characterisation in the EU/EEA.

Describing the intestinal microbiota of Holstein Fasciola-positive and -negative cattle from a hyperendemic area of fascioliasis in central Colombia

The ability to identify compositional changes in the intestinal microbiota of parasitized hosts is important for understanding the physiological processes that may affect animal productivity. Within the field of host–parasite interactions, many studies have suggested that helminths can influence the microbial composition of their hosts via their immunomodulatory effects. Bovine fascioliasis is a helminthiasis widely studied by immunologists, but with little information available regarding gut microbial communities. Thus, we aimed to describe the composition of the intestinal microbiota of Holstein Fasciola-positive and -negative cattle using parasitological methods and ELISA (enzyme-linked immunosorbent assay). Bovine fecal samples (n = 65) were obtained from livestock slaughter plants in the Cundi-Boyacense Colombian highlands (a hyperendemic region for bovine fascioliasis) and studied by amplicon-based next-generation 16S-rRNA and 18S-rRNA gene sequencing. From these samples, 35 were Fasciola hepatica-negative and, 30 were F. hepatica-positive in our detection analysis. Our results showed a reduction in the relative abundance of Bacteroidetes and Ascomycota in the Fasciola-positive samples, along with decreased relative abundances of the commensal taxa previously associated with fermentation and digestion processes. However, metabolomic approaches and functional analyzes of the intestinal microbiota are necessary to support these hypothesis. These findings are a small first step in the development of research aimed at understanding how microbial populations in bovines are modulated in liver helminth infections.

Cross-sectional study and genotyping of rotavirus-A infections in ruminants in Kuwait

BackgroundGroup A rotaviruses (RVA) are zoonotic pathogens responsible for acute enteritis in human and neonatal ruminants. This research aimed to determine the prevalence of RVA in ruminants (cattle, sheep, and goats) and investigate the circulating RVA genotypes in these animals in Kuwait. We conducted a cross-sectional study to detect RVA in ruminants, using an immunochromatography test (IC), direct sandwich ELISA test, and real-time RT-PCR (RT-qPCR) assay using fecal samples.ResultsA total of 400 cattle, 334 sheep, and 222 goats were examined. The prevalence of RVA was 5.3, 1.2, and 2.3%, respectively, using IC. The ELISA test detected RVA from 4.3% of cattle, 0.9% of sheep, and 1.8% of goats. There was a significant association between the occurrence of diarrhea and the presence of RVA in bovine fecal samples (p-value = 0.0022), while no statistical association between diarrhea and the presence of RVA in fecal samples of sheep and goats was observed (p-value = 0.7250; p-value = 0.4499, respectively). Twenty-three of the IC-positive samples (17 from cattle, two from sheep, and four from goats) were tested using a RT-qPCR RVA detection assay targeting the NSP3 gene. The results showed that 21 of 23 IC-positive samples tested positive by RT-qPCR. Detection of RVA genotypes revealed that G10P[11] was the predominant strain in cattle (58.8%), followed by G8P[1] (11.7%). One sheep sample was genotyped as G8P[1]. In addition, G6P[1] and G6P[14] were detected in goat samples.ConclusionThe present study revealed that the IC was more sensitive in detecting RVA antigen in fecal samples than the ELISA test. A higher occurrence of RVA infection was observed in cattle than in sheep and goats. This study suggests that RVA might be a risk factor of diarrhea in bovine calves less than 2 weeks old. This research also demonstrates the circulation of RVA in sheep and goat populations in Kuwait. Finally, the G10P[11] RVA genotype was the most prevalent genotype identified from cattle samples.

Genomic Analysis of Antibiotic-Resistant and -Susceptible Escherichia coli Isolated from Bovine Sources in Maputo, Mozambique.

This study reports a genomic analysis of Escherichia coli isolates recovered from 25 bovine fecal composite samples collected from four different production units in Maputo city and around Maputo Province, Mozambique. The genomes were analyzed to determine the presence of antibiotic resistance genes (ARGs), genetic relatedness, and virulence factors known to cause diseases in humans. Whole-genome sequencing was conducted on 28 isolates using an Illumina NextSeq 500 sequencing platform. The genomes were analyzed using BLASTN for the presence of resistance genes and virulence factors, as well as to determine their phylogenetic groups, sequence types (ST), and ST complexes (ST Cplxs). The majority of the isolates (85%) were identified as members of phylogenetic groups B1, with fewer isolates identified as members of group A, and a single isolate identified as group "E/Clade I." The ST analysis demonstrated a higher level of diversity than the phylogenetic group analysis. Sixteen different STs, five ST Cplxs, and seven singleton complexes were identified. A strain identified as a novel ST (ST9215) showed a high level of similarity with an isolate recovered from a wild animal in the Gambia. Seven different ARGs were identified, with tet(B) being the most frequently detected, followed by aph(3″)-Ib, aph(6)-Id, sul2, blaTEM-1B, and dfrA1. Three isolates encoded β-lactam-conferring point mutations in the ampC promoter (-42C>T). In total, 51 different virulence factors were identified among the genomes. This study demonstrates that E. coli from bovine sources in Mozambique encoded multiple antibiotic resistance elements, plasmids, and virulence factors. To the best of our knowledge, this is the first genomic description of antibiotic-resistant E. coli isolated from bovine sources in Mozambique.

Preparation of immunomagnetic beads coupled with a rhodamine hydrazine immunosensor for the detection of Mycobacterium avium subspecies paratuberculosis in bovine feces, milk, and colostrum

The aim of this study was to develop and evaluate a method for detecting Mycobacterium avium ssp. paratuberculosis (MAP) bacteria in bovine fecal, milk, and colostrum samples using immunomagnetic beads (IMB) and a rhodamine hydrazone immunosensor. Immunomagnetic beads were prepared by using purified antibodies from hyperimmunized sera that were coupled to Fe nanoparticles with diethylene triamine pentaacetic acid (DTPA) or ethyl (dimethyl aminopropyl) carbodiimide (EDC)-N-hydroxy succinimide (NHS) as linkers. Rhodamine hydrazone particles were synthesized and coupled to IgY anti-MAP antibodies using DTPA or EDC-NHS linkers. Separation efficiency of the IMB was tested on bovine fecal, milk, and colostrum samples experimentally contaminated with MAP. The studied methods were evaluated on their ability to detect MAP and separate bacteria in complex mediums. The ELISA results indicated 95% efficacy in antibody coupling to IMB, with the DTPA-IMB method being more efficient than the EDC-NHS-IMB method. By using the DTPA-IMB method, MAP bacteria were successfully recovered from fecal, milk, and colostrum samples. The DTPA-IMB method used in combination with the rhodamine hydrazone immunosensor had a limit of detection equal to 30 and 30,000 MAP cells/mL using chromogenic and fluorescent properties, respectively. Combining the DTPA-IMB separation method with the rhodamine hydrazone immunosensor provides a fast, sensitive, and cost-beneficial method for detecting MAP in bovine feces, milk, and colostrum.

Prevalence of Schistosoma japonicum in bovines and Oncomelania hupensis quadrasi from ricefields surrounding Lake Mainit, Philippines.

Schistosomiasis is a parasitic disease caused by blood flukes of the genus Schistosoma and involves bovines and snails as a reservoir and intermediate hosts, respectively. This disease is endemic in Lake Mainit, Philippines, as early as 1947. The study was conducted to determine the prevalence of Schistosoma infection in ricefields adjacent to Lake Mainit by collecting Oncomelania hupensis quadrasi snails and bovine fecal samples which are then subjected to laboratory examination and analysis. A total of 345 O. quadrasi individuals and 32 bovine fecal samples were collected from six barangay rice field stations adjacent to Lake Mainit from February and July 2020. Among the six barangays, Alipao had the highest prevalence of Schistosoma in O. quadrasi (39.71%), followed by San Isidro (28.3%), Matin-ao (18%), Poblacion (15%), and Magpayang (2.5%). No Oncomelania snails were collected in the ricefields of Cuyago. Schistosoma japonicum had an overall prevalence of 14.71% in all the bovines sampled, where Cuyago had the highest prevalence (50.0%) followed by Alipao (33.33%). The intensity of infection of Schistosoma eggs per gram (MPEG = 0.70) among bovines is categorized as light. Bovine contamination index calculations revealed that, on average, infected bovines in Lake Mainit excrete ~ 55,000 S. japonicum eggs per day. The study confirms the important role of bovines as a reservoir host for schistosomiasis. The presence of both infected bovines and O. quadrasi in ricefields makes this agricultural venue an important nidus of S. japonicum emergence in Lake Mainit. An integrated approach of mass drug administration (MDA) of praziquantel, implementation of a bovine vaccine-directed control program, and providing farmers with mechanized farm equipment are highly recommended to minimize the risk of exposure and infection to schistosomiasis in rice fields adjacent to the lake.

An investigation of the environmental niches of blown pack spoilage causing Clostridium estertheticum and Clostridium gasigenes on New Zealand beef and sheep farms

The transfer of blown pack spoilage causing Clostridium spores from the farm to the meat plant is of growing concern to the meat industry. This study investigated the environmental niches of these Clostridium spp., specifically Clostridium estertheticum and Clostridium gasigenes in the beef and sheep farm environments in New Zealand. Faecal, soil, grass, drinking water, puddle water and feed (fodder beet, hay, bailage and silage, where available) samples were collected on five beef and sheep farms during Winter and Spring in 2018, in North and South Island, respectively. Beef and sheep farm samples were tested for C. estertheticum and C. gasigenes using enrichment plus PCR, qPCR and direct plating. C. estertheticum was detected in bovine faecal (4%), soil (2–18%) and grass (0–12%) samples at concentration of up to 2.0 log10 cfu/g. C. gasigenes were found in 18–46% of faecal, 16–82% of soil, 12–44% of grass, 0–44.4% of drinking water and 0–58.3% of puddle water samples tested and the direct counts ranged from 2.4 log10 cfu/ml in puddle water to 3.4 log10 cfu/g in soil. C. estertheticum were detected by qPCR in sheep farms in ovine feces (2.3%), soil (2.3%) and fodder beet (10%). All other sample types (grass, drinking water, puddle water, baleage, hay, silage and fodder beet) were negative using direct and enrichment plus PCR methods. In contrast C. gasigenes was detected in of faecal (22.7–38.6%), soil (22.7–84.1%), grass (17.5–34.1%) drinking water (35.7–78.6%), puddle water (33.3–40%), hay baleage (57%), silage (2%) and fodder beet (10%) at concentrations of up to 3.7 log10 cfu/g/ml. It was concluded that C. estertheticum and C. gasigenes were common on beef and sheep farms with the latter having higher incidence and mean concentration.

A preliminary study of Salmonella spp., Listeria monocytogenes, Escherichia coli O157, Enterococcus faecalis and Clostridium spp. in Irish cattle

Although Salmonella spp., Escherichia coli O157, Listeria monocytogenes, Enterococcus faecalis and Clostridium spp. present a significant food safety and/or spoilage issue for the beef sector, there are limited data on their prevalence in Irish cattle. The objectives of this preliminary study were to investigate the distribution (percentage of farms positive) of Salmonella spp., E. coli O157, L. monocytogenes, E. faecalis and Clostridium spp. and the overall prevalence (%) of these bacteria in cattle on a small cohort of Irish beef farms. A total of 121 fresh bovine faecal samples were obtained on 10 randomly selected beef farms in the Northeast of Ireland and tested for the target pathogens using standard culture-based methods. Presumptive positives were confirmed using previously published polymerase chain reaction (PCR) methods. Salmonella were not detected in any of the samples. E. coli O157, L. monocytogenes, E. faecalis and Clostridium spp. were present on 50%, 40%, 100% and 100% of farms, respectively, with overall (all farms) prevalence rates in cattle of 9%, 8.2%, 61.9% and 87.6%, respectively. This study suggests that E. coli O157 may be more prevalent than previously thought and L. monocytogenes, E. faecalis and Clostridium spp. are widespread in Irish beef animals.

Bovine kobuvirus-A comprehensive review.

Bovine kobuvirus (BKV) is a single-stranded, positive sense, non-enveloped RNA virus in genus Kobuvirus of family Picornavirus. BKV was first identified in the culture media of HeLa cell containing calf serum in 2003. Since then, BKV has been detected in 13 countries of four different continents, suggesting widespread in the world. Herein, we review the detection and genomic characterization of BKV in 13 countries. All studies tested bovine faecal samples for BKV. These studies provide evidence that BKV might be a causative agent for neonatal calf diarrhoea. Therefore, further efforts including animal challenge study are urgently needed to unveil the pathogenicity of BKV.

Phenotypic and molecular characterization of ESBLs producing Escherichia coli in bovine faecal and milk samples of North Gujarat

Extended-spectrum β-lactamases (ESBLs) producing E. coli seems to be emerging in veterinary science impacting major threat to public health due to resistance to golden age antibiotics. In this study a total of 109 samples (42 faecal and 67 mastitis milk) of bovines were collected from different regions of North Gujarat. The samples were cultured and identified by standard procedures. The screening for ESBLs production was performed by using Cefotaxime and Cefotaxime+Clavulanate (Combination disc screening method). A total of 71 E. coli isolates were recovered from 109 samples processed, out of which thirty (42.25%) isolates (17 from milk and 13 from faecal) were positive for ESBLs showing multiple resistance to the antibiotics used. The ESBL confirmed isolates were further processed for detection of blaCTX-M, blaTEM, and blaSHV genes. Major gene detected was blaTEM in 17 (23.94%) E. coli isolates. Antibiotic resistance pattern of E. coli isolates was studied against eleven commonly used antimicrobial drugs in the northern region of Gujarat. The results recorded resistance tofollowing antibiotics: tetracycline (100%), ampicillin/sulbactum (83.10%), amoxiclav and gentamicin (83.10%), chloramphenicol (57.74%), ceftriaxone (66.19%), cefoperazone (66.19%), ciprofloxacin (74.65%), amikacin (57.74%), enrofloxacin (74.65%) and, levofloxacin (74.65%).

Distribution of non-aureus staphylococci from quarter milk, teat apices, and rectal feces of dairy cows, and their virulence potential

Non-aureus staphylococci (NAS) are predominantly isolated from bovine milk samples of quarters suffering from subclinical mastitis. They are also abundantly present on dairy cows' teat apices and can be recovered from bovine fecal samples, as recently described. Differences in ecology, epidemiology, effect on udder health, and virulence or protective traits have been reported among the species within this group. The objectives of this study were (1) to describe the species-specific distribution of NAS in 3 bovine-associated habitats, namely quarter milk, teat apices, and rectal feces, and (2) to evaluate the virulence potential of NAS by comparing their distribution in contrasting milk sample strata and the presence of selected virulence genes. A cross-sectional, systematic sampling procedure was followed in 8 dairy herds that participated in the local Dairy Herd Improvement program in Flanders, Belgium. Quarter milk samples (n = 573) were collected from 144 lactating cows in 8 herds. In 5 of the 8 herds, teat apex swabs (n = 192) were taken from 15 lactating cows, before and after milking, and from 18 dry cows. In the same 5 herds, rectal feces were sampled from 80 lactating cows (n = 80), taking into account that a cow could only serve as the source of one type of sample. In addition, milk samples of all clinical mastitis cases were continuously collected during the 1-yr study period from March 2017 to March 2018 in the 8 herds. In total, 1,676 Staphylococcus isolates were phenotypically identified and subjected to MALDI-TOF mass spectrometry. Thirty-three, 98, and 28% of all quarter milk, teat apex, and rectal fecal samples were NAS-positive, respectively, reaffirming the presence of NAS in rectal feces. The overall predominant species in the 3 habitats combined were Staphylococcus haemolyticus, Staphylococcus chromogenes, and Staphylococcus hominis. Four, 16, and 12% of the healthy quarters (quarter milk somatic cell count ≤50,000 cells/mL of milk), quarters with subclinical mastitis (quarter milk somatic cell count >50,000 cells/mL of milk), and quarters with clinical mastitis, respectively, were NAS-positive, suggesting that the potential to cause (mild) clinical mastitis is present among NAS. This was substantiated by comparing the presence of virulence genes of NAS isolates originating from contrasting milk sample strata (healthy quarters and quarters with clinical mastitis).

Zoonotic Transmission and Infection from Bovine Feces in Selected Ricefields of Lake Mainit, Philippines

Ricefields are suitable nidus of active parasite transmission via bovines due to often unprotected exposure by humans and livestock that utilize the habitat for farming and foraging. This study was conducted to assess infection of protozoans, trematodes and nematodes from bovine feces from ricefields of Brgy Magpayang, San Isidro and Matin-ao surrounding Lake Mainit, Philippines. Bovine fecal samples collected from these ricefields were processed using the Formalin Ethyl Acetate Sedimentation (FEA-SD) technique. A total of five species from three major parasite groups (protozoans, trematodes and nematodes) were observed. Bovines from ricefields of Brgy San Isidro harbored the highest number of fecal parasites. The liver fluke Fasciola sp. was the most predominant species observed with the highest prevalence (69%) across study areas. In contrast, multiple parasite burden of Fasciola sp., hookworms, coccidian oocysts and Ascaris sp. eggs were observed from bovines from Brgy San Isidro and Matin-ao. This study is the first report of parasites from bovine feces in selected ricefields of Lake Mainit. The high prevalence of Fasciola eggs in bovines calls for an extensive assessment of possible widespread bovine fascioliasis in the area.

The Isolation and Genome Sequencing of Five Novel Bacteriophages From the Rumen Active Against Butyrivibrio fibrisolvens.

Although the prokaryotic communities of the rumen microbiome are being uncovered through genome sequencing, little is known about the resident viral populations. Whilst temperate phages can be predicted as integrated prophages when analyzing bacterial and archaeal genomes, the genetics underpinning lytic phages remain poorly characterized. To the five genomes of bacteriophages isolated from rumen-associated samples sequenced and analyzed previously, this study adds a further five novel genomes and predictions gleaned from them to further the understanding of the rumen phage population. Lytic bacteriophages isolated from fresh ovine and bovine fecal and rumen fluid samples were active against the predominant fibrolytic ruminal bacterium Butyrivibrio fibrisolvens. The double stranded DNA genomes were sequenced and reconstructed into single circular complete contigs. Based on sequence similarity and genome distances, the five phages represent four species from three separate genera, consisting of: (1) Butyrivibrio phages Arian and Bo-Finn; (2) Butyrivibrio phages Idris and Arawn; and (3) Butyrivibrio phage Ceridwen. They were predicted to all belong to the Siphoviridae family, based on evidence in the genomes such as size, the presence of the tail morphogenesis module, genes that share similarity to those in other siphovirus isolates and phylogenetic analysis using phage proteomes. Yet, phylogenomic analysis and sequence similarity of the entire phage genomes revealed that these five phages are unique and novel. These phages have only been observed undergoing the lytic lifecycle, but there is evidence in the genomes of phages Arawn and Idris for the potential to be temperate. However, there is no evidence in the genome of the bacterial host Butyrivibrio fibrisolvens of prophage genes or genes that share similarity with the phage genomes.

Preliminary evaluation of a novel, fully automated, Telenostic device for rapid field-diagnosis of cattle parasites.

New ideas for diagnostics in clinical parasitology are needed to overcome some of the difficulties experienced in the widespread adoption of detection methods for gastrointestinal parasites in livestock. Here we provide an initial evaluation of the performance of a newly developed automated device (Telenostic) to identify and quantify parasitic elements in fecal samples. This study compared the Telenostic device with the McMaster and Mini-FLOTAC for counting of strongyle eggs in a fecal sample. Three bovine fecal samples were examined, in triplicate, on each of the three fecal egg-counting devices. In addition, both manual (laboratory technician) and automated analysis (image analysis algorithm) were performed on the Telenostic device to calculate fecal egg counts (FEC). Overall, there were consistent egg counts reported across the three devices and calculation methods. The Telenostic device compared very favourably to the Mini-FLOTAC and McMaster. Only in sample C, a significant difference (P < 0.05) was observed between the egg counts obtained by Mini-FLOTAC and by the other methods. From this limited dataset it can be concluded that the Telenostic-automated test is comparable to currently used benchmark FEC methods, while improving the workflow, test turn-around time and not requiring trained laboratory personnel to operate or interpret the results.

Occurrence of Staphylococcus spp. and investigation of fecal and animal viral contaminations in livestock, river water, and sewage from Tunisia and Romania.

The objective of this study was to determine the occurrence of Staphylococcus spp., Escherichia coli, somatic coliphages, F-specific RNA bacteriophages, hepatitis E virus (HEV), and bovine enterovirus (BEV) in fecal and water samples. The occurrence of Staphylococcus spp. was investigated in treated wastewater samples collected from slaughterhouse of Tunisia. Results showed that Staphylococcus spp. were detected in the totality of collected samples with an average mean of 5.44Log10 (CFU/100ml). Regarding fecal indicator bacteria, E. coli was detected in the totality of water samples and was more abundant in Tunisian samples than in samples collected from Romania (P < 0.05). Concerning somatic coliphages and F-specific RNA bacteriophages used as viral indicators, they were detected in all raw and treated wastewaters. Bovine enterovirus (BEV) was detected in 20.1% and 28% of bovine stool samples collected from Tunisia and Romania, respectively. BEV was also detected in 60% of porcine stool samples from Romania. BEV was absent in all treated sewage samples. HEV was detected in raw sewage and bovine fecal sample from Romania with low occurrence and none sample from Tunisia was positive. This study may give us an insight into the monitoring of water quality in Tunisia and Romania.

Regression of viral pathogen indicators due to improvement of hygiene protocols on boots in a bovine farm.

Rotavirus A (RVA), bovine torovirus (BToV), bovine enterovirus (BEV) and bovine coronavirus (BCV) at a bovine farm in Ibaraki prefecture were monitored by one-step multiplex reverse transcription polymerase chain reaction (RT-PCR), with the aim of confirming the reduction of “viral pathogen indicators”. A total of 960 bovine fecal samples were collected from calves less than 2 month-old within the period from October 2016 to October 2018 every 2 months at the bovine farm. In each sampling, 40 samples were taken from calves 3 week-old or less, and 40 samples from calves over 3 week-old, in principle. At the end of September 2017, the farm introduced improvement of hygiene protocols on boots by exchanging boots and appropriate usage of a footbath at the entrance of calf sheds. In the comparison of the virus detection by RT-PCR, prevalence of all 4 viruses was significantly reduced (P<0.01) in calves 3 week-old or less after the improvement. The mortality of calves less than 2 month-old was also significantly reduced after the improvement of hygiene protocols. These data suggest that the proper control of boots at calf sheds is important, perhaps even vital, for rearing hygiene measures at bovine farms so as to attain substantial decrease in the prevalence of pathogens.

Longitudinal and cross-sectional detection of four bovine enteric viruses by multiplex- reverse transcription polymerase chain reaction: Identification of possible indicator viruses to assess biosecurity level at bovine farms.

It can be judged that if the detection frequency of prevalent pathogenic viruses decreases, biosecurity has been enhanced. To monitor bovine farm biosecurity levels, one-step multiplex reverse transcription polymerase chain reaction (RT-PCR) for the simultaneous detection of group A rotavirus (RVA), bovine torovirus (BToV), bovine enterovirus (BEV), and bovine coronavirus (BCV) was designed, with the aim of configuring candidates for “viral pathogen indicators”. A total of 322 bovine fecal samples were collected from calves aged less than three months at 48 bovine farms in Ibaraki and Chiba prefectures. At farm A, 20 calves were selected and sampled weekly for 12 weeks (184 samples); at farm B, 10 calves were selected and sampled for five weeks (50 samples); and at the rest of the 46 farms, 88 calves were sampled once. The screening on the 358 field samples proved positive for 27 RVA, 4 BToV, 55 BEV, and 52 BCV. In the successive sampling, RVA was detected once but not continuously, whereas BEV and BCV were detected in succession for up to five weeks. The results revealed that RVA was the primary agent among the positive samples obtained from calves aged three weeks or less, while BEV was the primary among those from the older than three weeks old. They can be employed as useful viral pathogen indicators for soundly evaluating biosecurity at bovine farms.

Application value of a dynamic automatic identification system in Schistosoma japonicum miracidium hatching test

To evaluate the value of a dynamic automatic identification system in routine miracidium hatching test with nylon gauzes. Different quantities of fresh Schistosoma japonicum eggs were added to bovine fecal samples and divided into the low-infection group, medium-infection group and high-infection group, while the bovine feces without S. japonicum eggs served as negative controls. The detection efficiency and accuracy were compared between the identification system and manual detection in different groups. The identification system can automatically identify S. japonicum miracidium. The detection rate and efficiency of S. japonicum miracidium in bovine fecal samples were both higher by using the identification system than by manual detection. Notably in the low-infection group, the identification system had a significantly higher rate of detection of S. japonicum miracidium than manual detection (χ2 = 10.769, P = 0.002). The identification system completed the detection of bovine fecal samples in the field within 1 min. The dynamic automatic identification system may effectively improve the detection efficiency and accuracy of routine miracidium hatching test with nylon gauzes, and it may replace manual detection to be used in the field schisotsomiasis examinations and related researches.