Comparison of bacteriological culture method and multiplex real-time PCR for detection of mastitis.

This study includes the evaluation of multiplex real-time PCR (rPCR) kit, which was developed to provide rapid diagnosis of mastitis infections, by working with milk samples of 2 different sources of mastitis and comparing the results with the classical bacteriological culture method (BC). A total of 273 bacteria were isolated in 226 samples (47.88%) out of 472 samples by BC. These were 139 (50.91%) Staphylococcus spp., 61 (22.34%) Streptococcus spp., 15 (5.49%) E. coli, 8 (2.93%) Enterococcus spp., 50 (18.31%) other bacteria. When we look at the multiplex rPCR results; 1052 positive were obtained for the gene regions of 14 different bacteria, 1 yeast, and 1 ß-lactamase gene examined in 472 samples. While no searched gene region was found by rPCR in 78 (16.5%) of the 472 samples studied, at least 1 gene was detected in 394 (83.5%) samples. These 1052 positive samples by rPCR were; 263 (28.43%) Staphylococcus spp., 51 (5.51%) S. aureus, 57 (6.16%) Enterococcus spp., 49 (5.29%) C. bovis, 16 (1.73%) S. dysgalactiae, 84 (9.08%) S. agalactiae, 71 (7.67%) S. uberis, 73 (7.89%) E. coli, 14 (1.51%) Prototheca spp., 39 (4.21%) T. pyogenes/P. indolicus, 5 (0.54%) S. marcescens, 15 (1.62%) K. oxytoca/pneumonia, 117 (12.64%) Mycoplasma spp., 31 (3.35%) M. bovis, 40 (4.32%) yeast, and 127 samples (26.90%) were ß-lactamase positive. When the antibiotic resistance of the isolates was evaluated, 78 (31.96%) tetracycline, 72 (29.5%) penicillin, and 60 (24.59%) clindamycin resistance were observed predominantly in Gram-positive isolates, while 6 (23.07%) tigecycline, 6 (23.07%) netilmicin, 6 (23.07%) pipercillin resistance was found in gram-negative isolates. While a bacteria and/or yeast gene was found by rPCR in 187 of 246 (76.01%) samples with no bacterial growth, a bacterium was isolated with BC in only 20 (8.84%) samples whose gene region was not found by rPCR. As a result, the multiplex rPCR system used in the diagnosis of mastitis has been found to be quite reliable as it can detect a large number of bacteria in a very short time compared to classical methods. Therefore, we advise the use of rPCR and/or culture for confirmation of clinical signs in mastitis and at routine mastitis surveillance.

Use of a chromogenic medium with and without selective enrichment to screen for carbapenemase-producing Enterobacterales (CPE) from canine and feline fecal specimens during an outbreak of NDM-5-producing Escherichia coli.

Carbapenemase-producing Enterobacterales (CPE) are one of the most urgent threats to human healthcare globally. Descriptions of CPE outbreaks in veterinary hospitals suggest the need for screening strategies for CPE from companion animals. Our aim was to optimize a chromogenic agar method with and without selective enrichment to isolate CPE from companion animal feces in an ongoing outbreak of New Delhi metallo-ß-lactamse-5 Escherichia coli. A limit of detection (LOD) assay for spiked canine and feline feces was performed for both methods using a carbapenamase-producing E. coli (24213-18); the LOD (1.5 × 103 cfu/g of feces) was equivalent to that reported for human fecal specimens. We screened 1,247 companion animal fecal specimens for carriage of CPE by 1) direct plating to chromogenic agar and 2) plating to chromogenic agar following selective enrichment. Twenty-one specimens were positive for CPE by both direct culture and enrichment culture. No specimens were positive with selective enrichment and negative by direct culture. A selective enrichment step did not result in any increased recovery of CPE from companion animals, which suggests that enrichment broth may not be necessary for outbreak surveillance testing. It is important to continue to validate methods for the detection of CPE in companion animals as outbreaks become more common in veterinary facilities.

Proof of concept for multiplex detection of antibodies against Chlamydia species in chicken serum using a bead-based suspension array with peptides as antigens.

The available differentiating tests for Chlamydia are based on detection of genetic material and only give information about the actual infection status, but reveal nothing of past infections. As the use of serological methods increases the window of detection, the goal of this study was to investigate if it is possible to develop a differentiating serological test for antibodies against Chlamydia species in chicken sera. Focus was on C. psittaci, C. gallinacea, and two closely related species, i.e. C. abortus and C. avium. To enable differentiating serology, a bead-based Luminex suspension array was constructed, using peptides as antigens, derived from known immunoreactive Chlamydia proteins. For the majority of these peptides, species-specific seroreactivity in mammalian sera has been reported in literature. The suspension array correctly identified antibodies against various Chlamydia species in sera from experimentally infected mice, and was also able to differentiate between antibodies against C. psittaci and C. gallinacea in sera from experimentally infected chickens. In field sera, signals were difficult to interpret as insufficient sera from experimentally infected chickens were available for evaluating the seroreactivity of all peptides. Nevertheless, results of the suspension array with field sera are supported by published data on the occurrence of C. gallinacea in Dutch layers, thereby demonstrating the proof of concept of multiplex serology for Chlamydial species in poultry.

Eficácia in sílico do florfenicol no tratamento de pododermatite infecciosa por Fusobacterium necrophorum em ovelhas / In sílico efficacy of florfenicol in the treatment of Fusobacterium necrophorum infectious pododermatitis in sheep

Objetivou-se comparar o efeito in silico do florfenicol nas doses de 20 e 30 mg/Kg em ovinos pelas vias intravenosa (IV) e intramuscular (IM), usando a modelagem PK/PD. Realizou-se uma simulação de Monte Carlo com base nos dados de concentração plasmática de um estudo publicado anteriormente. Calculou-se a área sob a curva (ASC) e as taxas de eficácia do florfenicol para os efeitos bacteriostático, bactericida e de erradicação bacteriológica. A dose de 20 mg/Kg IV demonstrou efeitos de erradicação de 100, 93 e 0% para CIM de 0,5, 1 e acima, respectivamente. O efeito bacteriostático foi de 99 e 90% para CIM de 4 e 2 µg/ml, enquanto o bactericida foi de 14% para CIM de 2 µg/ml. A dose de 30 mg/Kg IV apresentou 100% de erradicação para CIM de 1 µg/mL e 100% de efeito bactericida para CIM de 2 µg/mL. Há 100% de efeito bacteriostático em CIM de 4 µg/ml. As doses de 20 e 30 mg/Kg IM mostraram 100% de erradicação para CIM até 1 µg/mL e 0% para CIM maiores. O efeito bacteriostático foi mantido em 100% para uma CIM de 4 µg/mL em ambas as doses. Este estudo mostra o efeito de erradicação bacteriológica do florfenicol nas doses de 20 e 30 mg/Kg, IV e IM. Recomenda-se que seja feito um estudo de eficácia in vivo com a dose de 30mg/Kg IM em ovinos infectados por F. necrophorum com MIC superior a 2 µg/mL.

We aimed to compare the in silico effect of florfenicol at doses of 20 and 30 mg/Kg in sheep by intravenous (IV) and intramuscular (IM) routes, using PK/PD modeling. We performed a Monte Carlo simulation based on plasma concentration data from a previously published study. We calculated the area under the curve (AUC) and the efficacy rates of florfenicol to bacteriostatic, bactericidal, and bacteriological eradication effects. The dose of 20 mg/Kg IV demonstrated 100, 93, and 0% eradication effects for MICs of 0.5, 1, and above, respectively. The bacteriostatic effect was 99 and 90% for MIC of 4 and 2 µg/ml, while the bactericide was 14% for MIC of 2 µg/ml. The 30 mg/Kg IV dose showed 100% eradication for MIC of 1 µg/mL and 100% bactericidal effect for MIC of 2 µg/mL. There is a 100% of bacteriostatic effect at MIC of 4 µg/ml. Doses of 20 and 30 mg/Kg IM showed 100% eradication for MIC up to 1 µg/mL and 0% for MIC above. The bacteriostatic effect was maintained at 100% for a MIC of 4 µg/mL at both doses. This study shows the bacteriological eradication effect of florfenicol at doses of 20 and 30 mg/Kg, IV, and IM. Therefore, we recommend an in vivo efficacy study with a dose of 30mg/Kg IM in sheep infected with F. necrophorum with MIC greater than two µg/mL.

Evaluation of mare endometrial cytology using the novel cytotape technique.

The cytobrush is considered the method of choice to obtain endometrial samples. Rigid brush fibers, however, may induce endometrial irritation and bleeding, or cell fragmentation, decreasing quality and diagnostic value of the samples. It was hypothesized that samples collected using a novel cytotape would provide sample smears of greater quality and less blood contamination than the cytobrush. Endometrial samples were collected with a cytotape and a cytobrush from ten mares without endometritis. Endometritis was then induced with artificial insemination, and samples were again collected 6 h after insemination. A cytology smear and bacterial culture were prepared from each sample. The collection methods and times were compared in terms of number and integrity of endometrial cells; number, integrity, and percentage of neutrophils; number of red blood cells, and number of colony-forming units. Frequency of positive cytology and culture was compared when there was use of each technique. The sensitivity, specificity, positive predictive value, and negative predictive value of cytology and culture for each technique was calculated using endometrial biopsy as the gold standard. While all samples had adequate and comparable cellularity and cell integrity, cytotape samples had less red blood cell contamination compared to cytobrush samples (P < 0.05). The number and percentage of PMNs, frequency of positive cytology diagnosis, number of colony-forming units and frequency of positive cultures did not differ between collection methods. In conclusion, the cytotape is a rapid, easy, and practical technique that can provide endometrial samples with similar diagnostic value to the cytobrush, but with less blood contamination.

A filter-assisted culture method for isolation of Treponema spp. from bovine digital dermatitis and their identification by MALDI-TOF MS.

Digital dermatitis (DD) is a major infectious foot disease of cattle worldwide. Some DD stages are associated with lameness, and the disease has significant economic and animal welfare consequences. The pathogenesis of the disease is not yet fully understood, but Treponema spp. have been associated consistently with clinical cases. Isolation of these fastidious bacteria is difficult and cumbersome. We describe an improved method enabling the culturing of the 3 Treponema spp. (T. pedis, T. phagedenis, and T. medium) from bovine foot specimens derived from DD lesions, using a combination of membrane filtering and subsequent growth on selective agar media. The entire procedure from sampling to verification of individual Treponema spp. takes up to 24 d. In addition, we established a MALDI-TOF MS-based identification method to be applied for confirmation of the different Treponema spp. This scheme provides an unambiguous, simple, and straightforward identification procedure for DD-associated Treponema spp.

Evaluation of a high-throughput nucleic acid extraction method for the detection of Mycobacterium avium subsp. paratuberculosis in bovine fecal samples by PCR.

Johne's disease (paratuberculosis) is an economically important disease of cattle worldwide. The disease is caused by Mycobacterium avium subsp. paratuberculosis (MAP), a fastidious gram-positive bacterium. PCR is increasingly used in diagnostic laboratories for the detection of MAP in fecal samples given the rapid test turnaround time and sensitivity and specificity comparable to fecal culture. However, efficient extraction of DNA for sensitive detection of MAP by PCR is affected by the complex lipid-rich cell wall of MAP and the presence of PCR inhibitors in feces. We evaluated a high-throughput nucleic acid extraction method (MagMAX core nucleic acid purification kit with mechanical lysis module) in conjunction with an hspX gene PCR for the detection of MAP from bovine fecal samples, which resulted in correct identification of all negative (13 of 13) and positive (35 of 35) proficiency test samples obtained from the National Veterinary Services Laboratories. In addition, all 6 negative and 50 of 51 positive diagnostic specimens tested were categorized correctly.

Implementation of a targeted mastitis therapy concept using an on-farm rapid test: antimicrobial consumption, cure rates and compliance.

BACKGROUND: Aim of the present study was to investigate the implementation of a targeted therapy (tLCT) concept under real-life circumstances, taking both pathogen-related and animal-related factors into account. The reduction of antibiotics without negative effects on cure rates was evaluated as well as the compliance by the farmers. METHODS: After analysing the existing conventional therapy (CT) concepts of five farms, the tLCT concept and a novel on-farm test were introduced. Three treatment groups were compared with respect to bacteriological cure (BC), cytological cure (CYC), full cure (FC), new infection rate (NIR), relapse rate and the treatment approach per mastitis case: the CT group, the tLCT group including all clinical mastitis (CM) cases treated according to the concept, and the modified tLCT group (tLCTmod), including the CM cases in which farmers deviated from the concept. RESULTS: Even so farmers deviated from the treatment concept in 506 out of 909 cases; belonging to one of the three treatment groups had no significant impact on BC, CYC, FC, NIR or relapse rate. The antibiotic usage in the tLCT as well as in the tLCTmod group was significantly lower in comparison to the CT group. CONCLUSION: From this, it can be deduced that farmers will reduce antibiotic doses by implementing a tLCT concept.

Accuracy of PCR, mycobacterial culture and interferon-γ assays for detection of Mycobacterium bovis in blood and milk samples from Egyptian dairy cows using Bayesian modelling.

The number of bovine tuberculosis (bTB) infected dairy herds in Egypt is growing and this calls for accurate and reliable diagnostic methods at cow level for cost-effective bTB eradication as culling of the whole herd is not economically sustainable. The present study aimed to estimate the sensitivity (Se) and specificity (Sp) of PCR, mycobacterial culture and interferon-γ (IFN-γ) assays for Mycobacterium bovis (M. bovis) detection in blood and milk samples from dairy cows in Egyptian dairy herds within a Bayesian framework. As a secondary objective, the distribution of true within-herd prevalence of M. bovis infection was estimated. Blood and milk samples were collected from 245 Holstein dairy cows in 11 Egyptian dairy herds and subjected to PCR, mycobacterial culture and IFN-γ testing. With respect to the detection of M. bovis in blood, IFN-γ recorded higher Se [0.97 (95% Posterior Credible Interval (PCI): 0.87-1.00)] than PCR [0.68 (95% PCI: 0.53-0.95)] and culture [0.22 (95% PCI: 0.13-0.37)]. However, Sp estimates of PCR [0.98 (95% PCI: 0.95-1.00)], culture [0.99 (95% PCI: 0.98-1.00)] and IFN-γ [0.97 (95% PCI: 0.88-1.00)] were comparable. As for milk samples, Se estimate of PCR [0.29 (95% PCI: 0.01-0.60)] was higher than that of culture [0.08 (95% PCI: 0.001-0.23)]. However, the Sp estimates of both tests were statistically similar. The estimated true within-herd prevalences of M. bovis varied across the tested bovine subpopulations and ranged between 0.06 and 0.66. In conclusion, IFN-γ registered a similar overall performance to PCR but was superior to mycobacterial culture. With its good accuracy and wide applicability, IFN-γ lends itself to use in the Egyptian bTB eradication program.

A review on bovine tuberculosis in India.

Bovine tuberculosis (bTB) is a chronic illness in animals, especially in cattle, leading to loss in the productivity and signifies a crucial public health risk. Regardless of the zoonotic threat and significant economic costs associated with the disease, precise estimates of bTB prevalence are deficient in many countries, including India, where national control programs are yet to be instigated. The true burden of the disease remains unknown due to lack of routine surveillance data from most of the developing countries. India is progressing well towards attaining the End TB goal, yet bTB continues to remain largely hidden. Moreover, the paucity of literature on bTB in India might lead to undue complacency and hence has to be scrupulously guarded and prevented from gaining any misconceptions in the minds of the common people. Preventing and controlling bTB at the animal interface is pivotal to evade transmission to human, increase food safety and guard the livelihood of the people. To attain this goal, implementation of strategies based on international norms and a multi-sectoral approach will empower enhanced surveillance and diagnosis of disease in animals and subsequently reduce the risk for humans. As an initiative, we step forward to address this review which briefly summarizes the available data in the literature from early 20th century to date to assess the status of bTB in India. We have discussed in detail, the epidemiology, transmission and diagnosis pertaining to bTB. The review also focuses on the interconnection between the health of people and animal, discuss the preventions and control strategies and recommend the use of vaccination in cattle to reduce the spread of infection among other animals and humans. Implementing One Health approach in India, which recognizes the interdependence of the health of people and animals will help the nation in the fight against TB.

Improving Mycoplasma ovipneumoniae culture medium by a comparative transcriptome method.

Mycoplasma ovipneumoniae (Mo) is difficult to culture, resulting in many difficulties in related research and application. Since nucleotide metabolism is a basic metabolism affects growth, this study conducted a "point-to-point" comparison of the corresponding growth phases between the Mo NM151 strain and the Mycoplasma mycoides subsp. capri (Mmc) PG3 strain. The results showed that the largest difference in nucleotide metabolism was found in the stationary phase. Nucleotide synthesis in PG3 was mostly de novo, while nucleotide synthesis in NM151 was primarily based on salvage synthesis. Compared with PG3, the missing reactions of NM151 referred to the synthesis of deoxythymine monophosphate. We proposed and validated a culture medium with added serine to fill this gap and prolong the stationary phase of NM151. This solved the problem of the fast death of Mo, which is significant for related research and application.

Propidium monoazide conventional PCR and DNA sequencing: detection of negative culture bacterial pathogens causing subclinical mastitis.

AIMS: This study was conducted to early detect the negative culture bacterial pathogens causing subclinical mastitis for the fast diagnosis of the disease and the reduction of some milk-transmitted pathogenic bacteria to human consumers. METHODS AND RESULTS: A total of 171 positive California mastitis test (CMT) milk samples collected from asymptomatic dairy cows in Sharkia Governorate, Egypt were examined by conventional bacteriological methods. The obtained results revealed that Streptococcus species (77·2%), followed by Staphylococcus species (48·6%) and Escherichia coli (25·7%) were the most predominant bacterial pathogens isolated from positive culture milk samples, whereas Enterobacter and Pseudomonas species were the lowest ones (1·2%, for each). Herein, 13 (7.6%) negative culture milk samples were subjected to propidium monoazide (PMA) conventional PCR assay, followed by DNA sequencing of purified PCR amplicons. Sequence analysis identified seven different types of negative culture bacterial pathogens comprising as following; 4 Enterococcus hirae, 2 Bacillus cereus, 2 Staphylococcus aureus, 1 Bacillus mycoides, 1 Bacillus subtilis, 1 Enterococcus faecium and 1 Escherichia coli. CONCLUSIONS: All the detected negative culture bacterial pathogens by PMA-PCR assay, followed by DNA sequencing were incriminated in causing subclinical mastitis disease and had serious implications on human public health through consumption of milk contaminated with those recovered bacterial pathogens. SIGNIFICANCE AND IMPACT OF THE STUDY: The used methods could be useful in the routine detection of negative culture bacterial pathogens present in milk and consequently, it will help in the rapid diagnosis of subclinical mastitis disease and the reduction of many milk-transmitted diseases to human.

Comparison of bacterial culture results collected via direct corneal ulcer vs conjunctival fornix sampling in canine eyes with presumed bacterial ulcerative keratitis.

OBJECTIVE: To compare aerobic bacterial culture results between samples obtained from the corneal ulcer versus lower conjunctival fornix in eyes with presumed bacterial ulcerative keratitis. ANIMALS STUDIED: Fifty five client-owned dogs diagnosed with ulcerative keratitis. PROCEDURES: Ophthalmic examinations were performed on each dog including slit-lamp biomicroscopy and indirect ophthalmoscopy. Microbial swabs were collected by direct sampling of the infected corneal ulcer as well as the lower conjunctival fornix, of the same eye, using a sterile rayon-tipped swab. Samples were submitted to an outside reference laboratory for aerobic bacterial culture and sensitivity. RESULTS: One hundred twelve samples were obtained from 56 eyes (55 dogs). Sixty-eight samples yielded bacterial growth. Positive growth from both sites was obtained in 31 eyes (55%). Six eyes yielded bacterial growth from the conjunctival fornix but not from the cornea. No bacterial growth was obtained from either sampling site in 19 eyes. Overall, 31/56 (55%) corneal samples were positive and 37/56 (66%) conjunctival fornix samples were positive. Comparison of organisms isolated from the two collection sites of the same eye revealed an exact correlation in 42/56 (75%) eyes and differed in 14/56 (25%) eyes. Twenty different bacterial isolates were obtained from 68 positive samples. Gram-positive (71%) organisms were more common than Gram-negative (29%). The most commonly isolated organisms were Staphylococcus pseudintermedius (25%), beta-hemolytic Streptococcus spp. (23%), and Pseudomonas aeruginosa (12%). Methicillin-resistant organisms were isolated in 9% of samples. CONCLUSION: Sampling from the conjunctival fornix may be a suitable alternative to direct ulcer sampling in eyes with compromised corneal structural integrity.

Acceleration of the identification of sepsis-inducing bacteria in cultures of dog and cat blood.

OBJECTIVES: To evaluate matrix-assisted laser desorption ionisation time of flight mass spectrometry (MALDI-TOF MS) combined with the Sepsityper kit (Bruker Daltoniks GmbH, Bremen) for the direct detection of bacterial species from inoculated blood cultures from dogs and cats. MATERIALS AND METHODS: Canine and feline blood samples were inoculated with typical sepsis-causing bacteria such as Staphylococcus intermedius, Staphylococcus aureus, Streptococcus canis, Enterococcus faecalis, Escherichia coli and Pseudomonas aeruginosa at two distinct concentrations (each in triplicate), resulting in 72 blood culture bottles incubated at 37°C. Samples were comparatively analysed with MALDI-TOF MS after preparation with the Sepsityper kit and also by standard bacteriology (culturing and biochemical characterisation). RESULTS: Bacterial species identified from agar plates and by MALDI-TOF MS from blood culture bottles were identical for all samples. The MALDI Biotyper software (Bruker Daltoniks) correctly identified all bacterial strains from inoculated canine and feline blood with analysis indicating very good precision. CLINICAL SIGNIFICANCE: MALDI-TOF MS analysis combined with the Sepsityper kit is a reliable tool for a quick detection of veterinary-relevant bacterial species directly from blood culture bottles. This approach could reduce the time for identification of critical species to only 24 hours.

Growth of adenocarcinoma from canine pleural fluid on aerobic bacterial culture.

We report a case of canine adenocarcinoma with multi-organ metastasis in which colonies of adenocarcinoma cells grew upon aerobic bacterial culture of pleural effusion. Stained agar colonies were highly similar to rare suspicious cells seen on cytologic examination of the pleural effusion, as well as rare cells seen on cytologic examination of pancreatic and gastric wall fine-needle aspirates. Cells from colonies growing on agar media were mildly immunoreactive for cytokeratin. Histologic examination of tissues obtained at autopsy revealed pancreatic adenocarcinoma with vascular invasion and nodal, gastric, pulmonary, and pleural metastasis.

Test Characteristics of Assays to Detect Mycobacterium bovis Infection in High-Prevalence African Buffalo (Syncerus caffer) Herds.

A herd of African buffaloes (Syncerus caffer) was tested for Mycobacterium bovis infection using three cytokine release assays. All animals were subsequently euthanized and mycobacterial culture determined the infection prevalence (52%) and diagnostic characteristics. Sensitivities were lower than previously reported and results provide new insight into the practical utility of these assays.

Development of novel aptamer-based enzyme-linked apta-sorbent assay (ELASA) for rapid detection of mariculture pathogen Vibrio alginolyticus.

As the major opportunistic pathogen to both marine animals and humans, Vibrio alginolyticus (V. alginolyticus) has caused heavy economic losses to mariculture. ssDNA aptamer VA2 targeting live V. alginolyticus was generated by systematic evolution of ligands by exponential enrichment (SELEX) technology in our previous study. In this study, we first developed aptamer (VA2)-based enzyme-linked apta-sorbent assay (VA2-ELASA) for rapid detection of mariculture pathogen V. alginolyticus. The VA2-ELASA could achieve the rapid detection for V. alginolyticus infection with high specificity and sensitivity. The VA2-ELASA could specifically identify V. alginolyticus, but not other non-target bacterial strains. VA2-ELASA could detect V. alginolyticus at the concentration of 5 × 104 /ml, the incubation time short to 1 min and the incubation temperature as high as 45°C, which proved sensitivity and stability of the novel VA2-ELASA in this study. It took less than one hour to accomplish the detection process by VA2-ELASA. The characteristics of specificity, sensitivity and easy operation make VA2-ELASA a novel useful technology for the rapid diagnosis of pathogen V. alginolyticus in mariculture.

Methods and basic concepts for microbiota assessment.

There has been a marked increase in interest regarding complex microbial populations in recent years. The methodology used for microbial assessment has drastically changed over the last two decades and continues to advance at a rapid pace. Culture-based studies have been superseded by those based upon molecular methods, which have been largely used to discover new species and to better characterize complex communities, mainly driven by the advances in DNA sequencing, termed 'next generation sequencing'. These methodologies have allowed for a better understanding of the relationship between hosts and their microbiotas, which have important roles in health maintenance and in the pathophysiology of wide ranging conditions such as obesity, diabetes, allergic diseases and even behavioural changes. While most widely used in humans, these approaches are now commonly used in veterinary research, with increasing interest in direct clinical applications. As these methods provide novel insights that will constitute the basis for the development of new therapeutic and prevention strategies, and as commercial efforts to offer microbiota assessment as a clinical tool expand, it is essential for researchers and clinical veterinarians to understand and have the tools to be able to interpret research performed in this new fascinating field. The objective of this review is to describe some of the most common methods for characterization of microbial communities and to provide an overview of the basic concepts necessary for good interpretation of the research performed in this field.

Optimizing identification of Mycoplasma bovis by MALDI-TOF MS.

Fast and accurate identification of Mycoplasma bovis in cattle samples is of great importance for rational treatment and control of pneumonia, arthritis and mastitis. However, which growth conditions will allow the fastest identification of M. bovis with MALDI-TOF MS remains unclear. Therefore, growth conditions and incubation time were investigated to optimize identification of M. bovis with MALDI-TOF MS and an in-house library was constructed. Nine different M. bovis strains were inoculated in triplicate in three liquid media (B1-3). Basic broth (B1) consisted of pleuropneumonia-like organism broth, enriched with 25% horse serum and 0.7% yeast extract. B2 and B3 were additionally supplemented with 0.5% pyruvate or 520 µg/mL ampicillin, respectively. Protein extraction was performed after 24, 48, 72, 96 and 120 h of incubation (37 °C, 5% CO2) and processed with Autoflex III smartbeam. Identification scores ≥1.7 were interpreted as reliable. The present study showed reliable identification of M. bovis with MALDI-TOF MS as early as 24 h after inoculation, and in broth supplemented with pyruvate, up to 120 h after inoculation. Serial dilutions showed improved survival of M. bovis in broth with pyruvate. The addition of ampicillin to prevent contamination, did not impair identification of M. bovis and state-of-the-art in-house libraries contributed to higher identification scores for M. bovis with MALDI-TOF MS.

Effect of Specimen Type and Processing on the Detection of Clostridioides [Clostridium] difficile in Piglet Fecal Samples.

Subclinical Clostridioides difficile colonization in piglets could be a potential source of this bacterium for community-acquired C. difficile infection. The purposes of this study were to assess the effect of specimen type and processing on C. difficile isolation, culture, and detection by polymerase chain reaction (PCR), and to determine the occurrence of C. difficile in piglets of different ages. We compared different culture procedures-direct plating, ethanol shock, and an enrichment step-to isolate C. difficile from swine feces and rectal swabs. DNA was isolated directly from feces, processed feces, and bacterial isolates to detect the triose phosphate isomerase (tpi) gene and identify the toxins A and B genes. The results show that ethanol shock increased the C. difficile isolation from feces, while it decreased it for rectal swabs, in comparison with direct plating. The use of the enrichment broth gave the highest C. difficile recovery from both types of specimen. Our findings show low sensitivity for tpi gene detection after the DNA extraction directly from feces and an increase in PCR-positive samples when feces were processed before the DNA extraction. The overall prevalence of C. difficile was 16.9% (22/130), of which 100% were found to be toxigenic as assessed by the enrichment culture of fecal samples. The rate of isolation of positive samples decreased with the animal age, regardless of the presence or absence of diarrhea. Our results demonstrate the persistent reservoir of toxigenic C. difficile in fecal samples of piglets and support the impact of specimen processing on its isolation.