Fecal Culture Research Articles

Implementation of Mini-FLOTAC in Routine Diagnosis of Coccidia and Helminth Infections in Domestic and Exotic Birds.

Mini-FLOTAC (MF) has recently been proposed for the fecal quantification of gastrointestinal (GI) parasites in birds due to its higher sensitivity and precision in comparison with the McMaster method. The current research aimed to test the use of MF in routine diagnosis of coccidia and helminth infections in several domestic and exotic bird collections in Portugal. Between July 2020 and April 2021, a total of 142 fecal samples from organic layers, peacocks and ratites were collected in four Portuguese bird collections and processed using MF and fecal cultures to identify and calculate GI parasite shedding and prevalence. The McMaster method was also used to compare the shedding levels obtained for both quantitative techniques. MF’s relative sensitivity and specificity were also assessed, using McMaster as the reference technique. The implementation of MF resulted in an average Eimeria spp. shedding higher in peacocks from bird collection 2 (502 OPG), followed by peacocks from collection 1 (107 OPG) and organic layers (24 OPG) and peacocks from collection 3 (9 OPG). Peacocks were also positive for Capillaria spp., Trichostrongylus tenuis and Strongyloides pavonis, whereas ostriches and emus were infected by L. douglassii. The MF protocol for exotic animals and the McMaster method did not differ significantly for each parasitic agent and bird species, and MF achieved relative sensitivities and specificities higher than 70% for Galliform Eimeria spp., peacock helminths and ratites’ L. douglassii infections. Higher L. douglassii EPG values were identified using the MF protocol for exotic species (2 g of feces/38 mL of sucrose solution), followed by McMaster 2/28, MF 5/45 and MF 2/18. The use of MF allowed for obtaining different intestinal parasitic populations in several bird species and locations, and MF 2/38 is globally proposed as the most suitable protocol for bird fecal samples as an alternative to the McMaster method in the diagnosis of avian intestinal parasitic infections.

Influence of 2'-Fucosyllactose on the Microbiota Composition and Metabolic Activity of Fecal Cultures from Breastfed and Formula-Fed Infants at Two Months of Age.

Although breast milk is considered the gold standard of nutrition for infant feeding, some circumstances may make breastfeeding difficult. Several commercial milk preparations include synthetic human milk oligosaccharides (HMOs) in their composition. However, the effect of HMOs on the establishment of the intestinal microbiota remains incompletely understood. Independent batch fermentations were performed with feces from six full-term infant donors of two months of age (three breastfed and three formula-fed, exclusively) in the presence of 2′fucosyllactose (2′FL), one of the most abundant HMOs in human milk. Microbiota composition was analyzed by 16S rRNA gene sequencing at baseline and at 24 h of incubation. The 2′FL consumption, gas accumulation, and levels of different metabolites were determined by chromatography. Microbiota profiles at baseline were clearly influenced by the mode of feeding and by the intrinsic ability of microbiotas to degrade 2′FL. The 2′FL degradation rate clustered fecal cultures into slow and fast degraders, regardless of feeding type, this being a determinant factor influencing the evolution of the microbiota during incubation, although the low number of donors precludes drawing sound conclusions. More studies are needed to decipher the extent to which the early intervention with HMOs could influence the microbiota as a function of its ability to utilize 2′FL.

A RETROSPECTIVE REVIEW OF THE CLINICAL UTILITY OF FECAL BACTERIAL ENTERIC PATHOGEN CULTURES IN MAMMALS WITHIN A ZOOLOGICAL COLLECTION AND THE USE OF FECAL CYTOLOGY FOR OPTIMIZATION OF DIAGNOSTIC TESTING.

To determine the diagnostic value of fecal bacterial enteric pathogen cultures (FBEPC) as part of routine preventive medicine protocols in terrestrial mammals housed in a zoological collection, this study investigated the clinical utility of FBEPC results in context of subsequent clinical actions and how its use was rationalized after adjunct use of fecal cytology as a first-line diagnostic tool. Retrospective results (n = 692) that included a routine FBEPC panel of a commercial diagnostic laboratory, including Aeromonas, Salmonella, Campylobacter, Plesiomonas, Shigella, Yersinia, and Edwardsiella, of 417 mammals were organized into preventive (P; n = 485), diagnostic (D; n = 177), or recheck (R; n = 30) samples; for P and D samples, findings were assigned a "clinical significance factor" of 1 to 5 according to culture results and subsequent clinical actions. A score of 3 or higher indicated positive growth of potentially pathogenic bacterial organisms, of which there were 50 FBEPC (P n = 27, D n = 20, R n = 3). The difference in mean clinical significance factor for P versus D samples was significant. Aeromonas spp. were most frequently isolated (n = 32), followed by Salmonella spp. (n = 8) and Plesiomonas shigelloides (n = 8), then Campylobacter spp. (n = 5). There was no growth of Yersinia enterocolitica, Shigella spp., or Edwardsiella spp. In the absence of clinical evidence of gastrointestinal disease, treatment was initiated in only two cases with isolated Campylobacter spp. Implementation of fecal cytology as an initial step in fecal evaluation resulted in a prompt, substantial reduction in number of ordered FBEPC (mean n = 12/month before and n = 5/month after implementation). The findings in this study suggest that FBEPC for these bacterial species has limited value as a screening tool in preventive medicine protocols for the mammalian orders best represented in this study. The use of fecal cytology led to a more targeted and cost-effective use of FBEPC. Fecal cytology as an initial step in preventative and diagnostic testing protocols is recommended.

Microscopic and molecular evaluation of Strongyloides venezuelensis in an experimental life cycle using Wistar rats

Introduction: Strongyloides venezuelensis is a nematode whose natural host is rats. It is used as a model for the investigation of human strongyloidiasis caused by S. stercoralis. The latter is a neglected tropical disease in Ecuador where there are no specific plans to mitigate this parasitic illness.Objective: To evaluate the stages of S. venezuelensis in an experimental life cycle using Wistar rats.Materials and methods: Male Wistar rats were used to replicate the natural biological cycle of S. venezuelensis and describe its morphometric characteristics, as well as its parasitic development. Furthermore, the production of eggs per gram of feces was quantified using two diagnostic techniques and assessment of parasite load: Kato-Katz and qPCR. Results: Viable larval stages (Lr L2, L3) could be obtained up to 96 hours through fecal culture. Parthenogenetic females were established in the duodenum on the fifth day postinfection. Fertile eggs were observed in the intestinal tissue and fresh feces where the production peak occurred on the 8th. day post-infection. Unlike Kato-Katz, qPCR detected parasitic DNA on days not typically reported.Conclusions: The larval migration of S. venezuelensis within the murine host in an experimental environment was equivalent to that described in its natural biological cycle. The Kato-Katz quantitative technique showed to be quick and low-cost, but the qPCR had greater diagnostic precision. This experimental life cycle can be used as a tool for the study of strongyloidiasis or other similar nematodiasis.

Detection of Mycobacterium avium Subspecies Paratuberculosis in Pooled Fecal Samples by Fecal Culture and Real-Time PCR in Relation to Bacterial Density.

Simple SummaryParatuberculosis is a worldwide disease causing serious impacts to the dairy industry. Within the context of paratuberculosis control programs, dairy herds have to be classified as either paratuberculosis-positive or paratuberculosis-free with minimum effort but with sufficient reliability. We aimed to estimate the detection rate of positive herds using a combination of random sampling and pooling of five or ten fecal samples. The pooled samples were analyzed with two different laboratory methods (bacterial culture and polymerase chain reaction). Pools of size 10 can be used without significant decrease of detection probability compared with pools of size 5. Analyzing randomly sampled and pooled fecal samples allows the detection of paratuberculosis-positive herds, but the detection probability in herds with only few infected animals (<5.0%) is not high enough to recommend this approach for one-time testing in such herds.Within paratuberculosis control programs Mycobacterium avium subsp. paratuberculosis (MAP)-infected herds have to be detected with minimum effort but with sufficient reliability. We aimed to evaluate a combination of random sampling (RS) and pooling for the detection of MAP-infected herds, simulating repeated RS in imitated dairy herds (within-herd prevalence 1.0%, 2.0%, 4.3%). Each RS consisted of taking 80 out of 300 pretested fecal samples, and five or ten samples were repeatedly and randomly pooled. All pools containing at least one MAP-positive sample were analyzed by culture and real-time quantitative PCR (qPCR). The pool detection probability was 47.0% or 45.9% for pools of size 5 or 10 applying qPCR and slightly lower using culture. Combining these methods increased the pool detection probability. A positive association between bacterial density in pools and pool detection probability was identified by logistic regression. The herd-level detection probability ranged from 67.3% to 84.8% for pools of size 10 analyzed by both qPCR and culture. Pools of size 10 can be used without significant loss of sensitivity compared with pools of size 5. Analyzing randomly sampled and pooled fecal samples allows the detection of MAP-infected herds, but is not recommended for one-time testing in low prevalence herds.

Identification of Streptococcus thermophilus Genes Specifically Expressed under Simulated Human Digestive Conditions Using R-IVET Technology.

Despite promising health effects, the probiotic status of Streptococcus thermophilus, a lactic acid bacterium widely used in dairy industry, requires further documentation of its physiological status during human gastrointestinal passage. This study aimed to apply recombinant-based in vivo technology (R-IVET) to identify genes triggered in a S. thermophilus LMD-9 reference strain under simulated digestive conditions. First, the R-IVET chromosomal cassette and plasmid genomic library were designed to positively select activated genes. Second, recombinant clones were introduced into complementary models mimicking the human gut, the Netherlands Organization for Applied Scientific Research (TNO) gastrointestinal model imitating the human stomach and small intestine, the Caco-2 TC7 cell line as a model of intestinal epithelium, and anaerobic batch cultures of human feces as a colon model. All inserts of activated clones displayed a promoter activity that differed from one digestive condition to another. Our results also showed that S. thermophilus adapted its metabolism to stressful conditions found in the gastric and colonic competitive environment and modified its surface proteins during adhesion to Caco-2 TC7 cells. Activated genes were investigated in a collection of S. thermophilus strains showing various resistance levels to gastrointestinal stresses, a first stage in the identification of gut resistance markers and a key step in probiotic selection.

Early Detection of Mycobacterium avium subsp. paratuberculosis Infected Cattle: Use of Experimental Johnins and Innovative Interferon-Gamma Test Interpretative Criteria.

Paratuberculosis (PTB), also known as Johne's disease, is a chronic proliferative enteritis of ruminants caused by Mycobacterium avium subsp.paratuberculosis (MAP). To date, PTB diagnosis, based on serology, fecal culture, and real-time polymerase chain reaction, has identified animals in advanced stages of infection. To detect MAP infection in animals earlier, the interferon-gamma (IFN-γ) test may be applied. This assay detects cytokines produced by T-lymphocytes of infected subjects after stimulation with purified protein derivatives (PPDs), extracted from Mycobacterium bovis (MB) and from M. avium (MA). The study involved three bovine herds: one PTB-infected herd, one PTB-free herd, and one with an outbreak of bovine tuberculosis. The IFN-γ test was performed on 235 animals, using bovine PPD (PPDB), avian PPD (PPDA), and three experimental PPD Johnins (PPDJs) extracted from a synthetic liquid medium culture of MAP (PPDJ A, B, and C), to assess early MAP detection and avoid false reactions to MB. Furthermore, IFN-γ results were evaluated using 12 interpretative criteria (ICs), based on the differences and ratio between PPD optical density (OD) and IFN-γ basal OD values after lymphocytic stimulation. IC accuracy was expressed as area under the receiver operating characteristic curve. Through a longitudinal study, PPDJs proved to be specific and sensitive in the detection of MAP-infected animals. Among the evaluated ICs, six showed the best performance in terms of accuracy (p < 0.0001), highlighting PTB subclinical infections. In particular, the two best criteria reached sensitivity values of 100% [confidence interval (CI) 95%, 94.1–100%] with a specificity of 91.8% (CI 95%, 81.9–97.3%) and sensitivity levels of 80.6% (CI 95%, 69.1–89.2%) with a specificity of 100% (CI 95%, 94.1–100%). Thus, the IFN-γ assay proved to be a useful diagnostic tool to identify early subclinical MAP-infected animals, in order to manage infected cattle or those exposed to MAP and to monitor younger calves within a herd. Furthermore, the IFN-γ test can be considered an additional test to avoid the introduction of MAP-infected animals, especially in herds where disease has already been eradicated and preservation of the health status is required to maintain the PTB certification level.

Digestion & fermentation characteristics of sulfated polysaccharides from Gracilaria chouae using two extraction methods in vitro and in vivo

The sulfated polysaccharides extracted from Gracilaria chouae using critic acid extraction and water extraction, respectively, and their digestion and fermentation characteristics were compared in vitro and in vivo. The molecular weight of water extracted polysaccharide of G. chouae (WGCP) was 1.73 × 103 kDa while critic acid extracted polysaccharide (CGCP) was 31.5 kDa. During stimulated gastrointestinal digestion in vitro, WGCP and CGCP were lightly degraded. However, the glycemic index (GI) of WGCP and CGCP were 17.7 and 36.12, respectively. After 24 h of fermentation in vitro, the pH values of CGLP in the fecal culture decreased from 6.89 to 4.82, similar to the inulin but significantly (p < 0.05) lower than those of the WGCP and normal control. In addition, CGCP and inulin showed similar microbial fermentation characteristics according to the microbiome compositions and contents of short-chain fatty acids (SCFAs). Nevertheless, CGCP gavage for four weeks could also promote the growth of microbes producing the SCFAs such as Peptococcus, Roseburia and Butyricicoccus in the cecum of KM mice. The present study suggests that polysaccharides prepared by acid-extraction method could potentially be used as a good source of prebiotics.

Representative Bacillus sp. AM1 from Gut Microbiota Harbor Versatile Molecular Pathways for Bisphenol A Biodegradation.

Human gut microbiota harbors numerous microbial species with molecular enzymatic potential that impact on the eubiosis/dysbiosis and health/disease balances. Microbiota species isolation and description of their specific molecular features remain largely unexplored. In the present study, we focused on the cultivation and selection of species able to tolerate or biodegrade the endocrine disruptor bisphenol A (BPA), a xenobiotic extensively found in food plastic containers. Chemical xenobiotic addition methods for the directed isolation, culturing, Whole Genome Sequencing (WGS), phylogenomic identification, and specific gene-encoding searches have been applied to isolate microorganisms, assess their BPA metabolization potential, and describe encoded catabolic pathways. BPA-tolerant strains were isolated from 30% of infant fecal microbial culture libraries analyzed. Most isolated strains were phylogenetically related to the operational taxonomic group Bacillus amyloliquefaciens spp. Importantly, WGS analysis of microbial representative strain, Bacillus sp. AM1 identified the four complete molecular pathways involved on BPA degradation indicating its versatility and high potential to degrade BPA. Pathways for Exopolysaccharide (EPS) and Polyhydroxyalkanates (PHA) biopolymer synthesis were also identified and phenotypically confirmed by transmission electronic microscopy (TEM). These microbial biopolymers could generally contribute to capture and/or deposit xenobiotics.

In vitro Efficacy of Albendazole against Strongyle eggs recovered from Trade Goats slaughtered at the Nsukka Abattoir: A Preliminary Survey of Resistance to Albendazole

The efficacy of Albendazole against trichostrongyle nematode parasites in goats presented for slaughter at the Nsukka municipal abattoir was evaluated using the In vitro Egg hatch assay (EHA) model. The abattoir was visited once every week for 4 consecutive months during which a total of 240 goats were sampled. Fecal samples were collected per rectum from a minimum of 15 goats on each day of the visit. Egg Hatch Assay was performed on strongyle eggs recovered from pooled faecal sample on each day of sampling with a 2.5% W/V Albendazole. Faecal culture was also set up from the pooled faecal sample on each sampling day to recover and identify the nematode parasites present in the goats. Among the 240 goats sampled, the prevalence of trichostrongylosis as observed by the presence of strongyle eggs was 94.6% (227/240). Faecal culture and larval identification revealed 69.8% of the strongyles as Haemonchus contortus, while 25.5 and 4.8% were Trichostrongylus colubriformis and Oesophagostomum species respectively. In the EHA, Albendazole had mean LC50 value of 0.16 µg/ml which is slightly in excess of the discriminating dose of 0.1µg/ml as prescribed by the World Association for the Advancement of Veterinary Parasitology (WAAVP) as an indication of anthelmintic resistance. There is therefore an urgent need to screen the nematode parasite population in the Nigeria for the presence Albendazole resistance genes.&#x0D; Key words: GI nematode; egg hatch assay; goat; Albendazole; Resistance; Nigeria

Septicaemia and thrombocytopenia caused by Salmonella gallinarum in a dog fed a raw meat-based diet

In recent years, it has become increasingly popular for pet owners to feed their dogs and cats raw meat as an alternative to processed, manufactured diets. Owners are often unaware of the potential risks this may pose for their animals and for themselves, despite the published literature. This article reports the case of a 5-month-old female French Bulldog referred for investigation of acute vomiting, diarrhoea and pyrexia. She was diagnosed with septicaemia, associated with Salmonella gallinarum (identified on blood culture), as well as a positive faecal culture for Campylobacter coli and severe thrombocytopenia (presumably secondary to infection). She received supportive treatment and marbofloxacin, going on to make a full recovery. Considering the Salmonella serovar identified (which is host-specific to poultry) and the animal's lifestyle, it is possible the septicaemia originated from her raw meat diet. Salmonella spp. is potentially zoonotic and can cause acute enteritis in humans. This is the first report of septicaemia with positive blood culture for Salmonella gallinarum, with secondary severe thrombocytopenia (presumably immune-mediated), in a dog that was fed a raw meat diet.

Washed microbiota transplantation for the treatment of recurrent fungal infection in a patient with ulcerative colitis.

Washed microbiota transplantation for the treatment of recurrent fungal infection in a patient with ulcerative colitis.

Faecal bacterial composition in horses with and without free faecal liquid: a case control study

Free faecal liquid (FFL) is a condition in horses which manifests as differential defecation of solid and liquid phases of faeces. The etiology of FFL is currently unknown, but deviances in the hindgut microbiota has been suggested to be of importance. The present study aimed to compare the faecal bacterial composition of farm-matched horses with (case, n = 50) and without (control, n = 50) FFL. Samples were collected at three different occasions. The V3 and V4 regions of the 16S rRNA gene were amplified and sequenced using Illumina sequencing. Also, samples were cultivated for detection of Clostridioides difficile and Clostridium perfringens. Analysis revealed similar faecal bacterial composition between case and control horses, but an effect of sampling period (p = 0.0001). Within sampling periods, 14 genera were present in higher or lower proportions in case compared to control horses in at least one sampling period. Compared to controls, case horses had higher relative abundance of Alloprevotella (adjusted p < 0.04) and lower relative abundance of Bacillus spp. (adjusted p < 0.03) in at least two sampling periods. All horses tested negative for C. difficile and C. perfringens by culture of faeces. Further studies are required to establish the clinical relevance of specific bacterial taxa in FFL.

Influence of faecal culture media and incubation time on the yield of infective larvae of Haemonchus contortus (Rudolphi 1803)

The aim of this experiment was to determine the yield of Haemonchus contortus third-stage larvae (L3) in faecal cultures in different conditions, including incubation time (7 or 14 days), the addition of inert additives (polystyrene pellets, vermiculite or no additive) and physical condition of the incubated faeces (ground or whole pellets). Twelve groups of 10 cultures each were arranged and incubated at 24 °C to evaluate the interaction of the above-mentioned conditions. Significantly, more L3 (p=0.0019 to p=0.0200) were recovered from cultures incubated for 7 days than for 14 days, except for the groups containing whole pellets with no additives (p=0.53) or with vermiculite (p=0.41). Larval yields from 7-day incubated cultures did not differ between groups (p=0.47), but for the whole pellets with vermiculite group, which yielded significantly less L3 (p<0.0001) than the rest of the cultures. Incubation for 14 days showed that cultures containing whole pellets with no additives yielded significantly more L3 (p<0.05) than the rest. Culturing faeces with H. contortus seems not to require inert additives or extra manipulation to obtain good L3 yields.

In vitro Selection of Probiotics for Microbiota Modulation in Normal-Weight and Severely Obese Individuals: Focus on Gas Production and Interaction With Intestinal Epithelial Cells.

The intestinal microbiota plays important roles in the maintenance of health. Strategies aiming at its modulation, such as probiotics, have received a deal of attention. Several strains have been studied in different in vitro models; however, the correlation of results obtained with the in vivo data has been limited. This questions the usefulness of such in vitro selection models, traditionally relying on over-simplified tests, not considering the influence of the accompanying microbiota or focusing on microbiota composition without considering functional traits. Here we assess the potential of six Bifidobacterium, Lactobacillus and Lacticaseibacillus strains in an in vitro model to determine their impact on the microbiota not just in terms of composition but also of functionality. Moreover, we compared the responses obtained in two different population groups: normal-weight and severely obese subjects. Fecal cultures were conducted to evaluate the impact of the strains on specific intestinal microbial groups, on the production of short-chain fatty acids, and on two functional responses: the production of gas and the interaction with human intestinal epithelial cells. The response to the different probiotics differed between both human groups. The addition of the probiotic strains did not induce major changes on the microbiota composition, with significant increases detected almost exclusively for the species added. Higher levels of gas production were observed in cultures from normal-weight subjects than in the obese population, with some strains being able to significantly reduce gas production in the latter group. Moreover, in obese subjects all the Bifidobacterium strains tested and Lacticaseibacillus rhamnosus GG were able to modify the response of the intestinal cells, restoring values similar to those obtained with the microbiotas of normal-weight subjects. Our results underline the need for the screening and selection of probiotics in a target-population specific manner by using appropriate in vitro models before enrolling in clinical intervention trials.

Detection of Mycobacterium avium ssp. paratuberculosis in Cultures From Fecal and Tissue Samples Using VOC Analysis and Machine Learning Tools.

Analysis of volatile organic compounds (VOCs) is a novel approach to accelerate bacterial culture diagnostics of Mycobacterium avium subsp. paratuberculosis (MAP). In the present study, cultures of fecal and tissue samples from MAP-infected and non-suspect dairy cattle and goats were explored to elucidate the effects of sample matrix and of animal species on VOC emissions during bacterial cultivation and to identify early markers for bacterial growth. The samples were processed following standard laboratory procedures, culture tubes were incubated for different time periods. Headspace volume of the tubes was sampled by needle trap-micro-extraction, and analyzed by gas chromatography-mass spectrometry. Analysis of MAP-specific VOC emissions considered potential characteristic VOC patterns. To address variation of the patterns, a flexible and robust machine learning workflow was set up, based on random forest classifiers, and comprising three steps: variable selection, parameter optimization, and classification. Only a few substances originated either from a certain matrix or could be assigned to one animal species. These additional emissions were not considered informative by the variable selection procedure. Classification accuracy of MAP-positive and negative cultures of bovine feces was 0.98 and of caprine feces 0.88, respectively. Six compounds indicating MAP presence were selected in all four settings (cattle vs. goat, feces vs. tissue): 2-Methyl-1-propanol, 2-methyl-1-butanol, 3-methyl-1-butanol, heptanal, isoprene, and 2-heptanone. Classification accuracies for MAP growth-scores ranged from 0.82 for goat tissue to 0.89 for cattle feces. Misclassification occurred predominantly between related scores. Seventeen compounds indicating MAP growth were selected in all four settings, including the 6 compounds indicating MAP presence. The concentration levels of 2,3,5-trimethylfuran, 2-pentylfuran, 1-propanol, and 1-hexanol were indicative for MAP cultures before visible growth was apparent. Thus, very accurate classification of the VOC samples was achieved and the potential of VOC analysis to detect bacterial growth before colonies become visible was confirmed. These results indicate that diagnosis of paratuberculosis can be optimized by monitoring VOC emissions of bacterial cultures. Further validation studies are needed to increase the robustness of indicative VOC patterns for early MAP growth as a pre-requisite for the development of VOC-based diagnostic analysis systems.

Isolation of Mycobacterium avium and other nontuberculous mycobacteria in chickens and captive birds in peninsular Malaysia

BackgroundMycobacterium avium complex (MAC) causes a chronic infectious in the birds known as avian mycobacteriosis. Almost all species of the birds are susceptible to MAC which consists of two closely related species of mycobacteria, that is, M. avium and M. intracellulare. This study aimed to determine the occurrence of Mycobacterium avium subsp. avium (MAA) in chickens and captive birds in selected states of Peninsular Malaysia.ResultsA 300 fecal samples were collected from village chickens (n = 100), layer chickens (n = 100) and captive birds (n = 100). Fecal samples were split into two aliquots for microbiological and molecular detection of MAA. Microbiology detection consisted of microscopy (Ziehl-Neelsen staining) and culture of samples decontaminated with 1% Cetylperidinium chloride and vancomycin, nalidixic acid and amphotericin B (VNA) antibiotic cocktail [vancomycin (VAN) 100 μg/ml, nalidixic acid (NAL) 100 μg/ml and amphotericin B (AMB) 50 μg/ml] onto Löwenstein-Jensen (L-J). Molecular detection (PCR-IS901) was performed to detect MAA DNA from the feces and PCR-16S rRNA and IS901 for identification of genus Mycobacterium and Mycobacterium avium sub species avium isolated onto L-J. All samples (296) were AFB negative smear. M. avium was isolated in 0.3% (1/296) samples by culture and detected in 2.5% (6/242) samples by PCR (IS901). Other mycobacteria were found in 1.7% (5/296) chickens. Of five isolates, two were identified as Mycobacterium terrae and M. engbaekii and remaining isolates were not sequenced. Birds positive for M. avium included White Pelican (n = 1) Black Hornbill (n = 1), Macaw (n = 2), Cockatoo (n = 2) and village chicken (n = 1).ConclusionIt is concluded that chickens and birds were infected with M. avium in selected areas of Peninsular Malaysia. Although, PCR is rapid, reliable and cost effective method for detection of M. avium in a subclinical stage, the culture of the avian feces should still be used as a reference test for the diagnosis of avian tuberculosis.

Examination of hydrogen cross-feeders using a colonic microbiota model

BackgroundHydrogen cross-feeding microbes form a functionally important subset of the human colonic microbiota. The three major hydrogenotrophic functional groups of the colon: sulphate-reducing bacteria (SRB), methanogens and reductive acetogens, have been linked to wide ranging impacts on host physiology, health and wellbeing.ResultsAn existing mathematical model for microbial community growth and metabolism was combined with models for each of the three hydrogenotrophic functional groups. The model was further developed for application to the colonic environment via inclusion of responsive pH, host metabolite absorption and the inclusion of host mucins. Predictions of the model, using two existing metabolic parameter sets, were compared to experimental faecal culture datasets. Model accuracy varied between experiments and measured variables and was most successful in predicting the growth of high relative abundance functional groups, such as the Bacteroides, and short chain fatty acid (SCFA) production. Two versions of the colonic model were developed: one representing the colon with sequential compartments and one utilising a continuous spatial representation. When applied to the colonic environment, the model predicted pH dynamics within the ranges measured in vivo and SCFA ratios comparable to those in the literature. The continuous version of the model simulated relative abundances of microbial functional groups comparable to measured values, but predictions were sensitive to the metabolic parameter values used for each functional group. Sulphate availability was found to strongly influence hydrogenotroph activity in the continuous version of the model, correlating positively with SRB and sulphide concentration and negatively with methanogen concentration, but had no effect in the compartmentalised model version.ConclusionsAlthough the model predictions compared well to only some experimental measurements, the important features of the colon environment included make it a novel and useful contribution to modelling the colonic microbiota.

Infection of Immunocompetent Conventional Mice with Shiga Toxin-Producing E. coli: The DSS+STEC Model.

Previous methods of infecting mice with Shiga toxin-producing E. coli (STEC) required suppression of host immune function or ablation of the gut microbiota to induce susceptibility to gastrointestinal colonization. Consequently, many pathogen-host interactions occurring in immunocompetent hosts during STEC infection and Shiga toxicosis have remained unclear. The following protocol describes the use of dextran sulfate sodium (DSS) to induce a mild colitis in immunocompetent conventional C57BL/6 mice to facilitate susceptibility to STEC infection by oral gavage. STEC colonization in infected mice was confirmed by recovery of live STEC via fecal cultures and quantified via quantitative polymerase chain reaction of fecal DNA for the STEC-specific gene eae. DSS colitis is well established, broadly reproducible, and does not require specialized equipment or high levels of technical proficiency to be a useful method of inducing susceptibility to gastrointestinal STEC colonization. The DSS+STEC mouse model provides a novel and useful tool for the exploration of local STEC-host interactions in the gut environment and the pathogenesis of Shiga toxicosis.

Anthelmintic resistance of Haemonchus contortus from sheep flocks in Brazil: concordance of in vivo and in vitro (RESISTA-Test©) methods.

This study evaluated the resistance status of Haemonchus contortus from sheep flocks in the state of São Paulo, Brazil, through comparison between the fecal egg count reduction test (FECRT) and the larval development test (LDT). For the FECRT, 35 sheep were selected in each of five flocks and divided into groups treated with: benzimidazole, levamisole, ivermectin, monepantel and control. Feces were collected for EPG and fecal cultures. The LDT was performed using thiabendazole (TBZ), levamisole (LEV), ivermectin aglycone (IVM-A) and Zolvix (ZLV). Resistance to all drugs was detected using FECRT in 100% of the flocks, except in relation to ZLV (40% resistant and 20% suspected of resistance). LDT indicated resistance to TBZ and IVM-A in all flocks, to LEV in 80% of flocks and to ZLV in 10%. Total agreement was obtained between the two tests for TBZ and IVM (k = 1.0), while for LEV (k = 0.8) and ZLV (k = 0.9), substantial and almost perfect agreement were obtained, respectively. The concordance between the tests was significant, thus showing that it is possible to use the outcome of the LDT to predict the FECRT, and hence validating the former as a fast diagnostic test for use by sheep farmers in Brazil.