Validating the Use of Bovine Buccal Sampling as a Proxy for the Rumen Microbiota by Using a Time Course and Random Forest Classification Approach.

Analysis of the cow microbiome, as well as host genetic influences on the establishment and colonization of the rumen microbiota, is critical for development of strategies to manipulate ruminal function toward more efficient and environmentally friendly milk production. To this end, the development and validation of noninvasive methods to sample the rumen microbiota at a large scale are required. In this study, we further optimized the analysis of buccal swab samples as a proxy for direct bacterial samples of the rumen of dairy cows. To identify an optimal time for sampling, we collected buccal swab and rumen samples at six different time points relative to animal feeding. We then evaluated several biases in these samples using a machine learning classifier (random forest) to select taxa that discriminate between buccal swab and rumen samples. Differences in the inverse Simpson's diversity, Shannon's evenness, and Bray-Curtis dissimilarities between methods were significantly less apparent when sampling was performed prior to morning feeding (P < 0.05), suggesting that this time point was optimal for representative sampling. In addition, the random forest classifier was able to accurately identify nonrumen taxa, including 10 oral and putative feed-associated taxa. Two highly prevalent (>60%) taxa in buccal and rumen samples had significant variance in relative abundances between sampling methods but could be qualitatively assessed via regular buccal swab sampling. This work not only provides new insights into the oral community of ruminants but also further validates and refines buccal swabbing as a method to assess the rumen bacterial in large herds.IMPORTANCE The gastrointestinal tracts of ruminants harbor a diverse microbial community that coevolved symbiotically with the host, influencing its nutrition, health, and performance. While the influence of environmental factors on rumen microbes is well documented, the process by which host genetics influences the establishment and colonization of the rumen microbiota still needs to be elucidated. This knowledge gap is due largely to our inability to easily sample the rumen microbiota. There are three common methods for rumen sampling but all of them present at least one disadvantage, including animal welfare, sample quality, labor, and scalability. The development and validation of noninvasive methods, such as buccal swabbing, for large-scale rumen sampling is needed to support studies that require large sample sizes to generate reliable results. The validation of buccal swabbing will also support the development of molecular tools for the early diagnosis of metabolic disorders associated with microbial changes in large herds.

Development of a duplex TaqMan real-time RT-PCR assay for simultaneous detection of newly emerged H5N6 influenza viruses.

BACKGROUND: In 2017-2018, a new highly pathogenic H5N6 avian influenza virus (AIV) variant appeared in poultry and wild birds in Asian and European countries and caused multiple outbreaks. These variant strains are different from the H5N6 virus associated with human infection in previous years, and their genetic taxonomic status and antigenicity have changed. Therefore, revision of the primers and probes of fluorescent RT-PCR is important to detect the new H5N6 subtype AIV in poultry and reduce the risk of an epidemic in birds or humans. METHODS: In this study, the primers and probes including three groups of HA and four groups of NA for H5N6 influenza virus were evaluated. Then a set of ideal primer and probes were selected to further optimize the reaction system and established a method of double rRT-PCR assay. The specificity of this method was determined by using H1~H16 subtype AIV. RESULTS: The results showed that fluorescence signals were obtained for H5 virus in FAM channel and N6 virus in VIC channel, and no fluorescent signal was observed in other subtypes of avian influenza viruses. The detection limit of this assay was 69 copies for H5 and 83 copies for N6 gene. And, the variability tests of intra- and inter-assay showed excellent reproducibility. Moreover, this assay showed 100% agreement with virus isolation method in detecting samples from poultry. CONCLUSION: The duplex rRT-PCR assay presented here has high specificity, sensitivity and reproducibility, and can be used for laboratory surveillance and rapid diagnosis of newly emerged H5N6 subtype avian influenza viruses.

Systematic approach to isolating Batrachochytrium dendrobatidis.

We developed a protocol for isolating the amphibian chytrid fungus Batrachochytrium dendrobatidis (Bd) from anurans. We sampled skin tissues from 2 common treefrogs, Pseudacris regilla and P. triseriata, collected from populations with high infection prevalence. We sampled tissues from 3 anatomical ventral regions (thigh, abdomen, and foot) where the pathogen is thought to concentrate. To mitigate potential bacterial contamination, we used a unique combination of 4 antibiotics. We quantified infections on frogs as zoospore equivalents (ZE) using a swabbing approach combined with quantitative real-time polymerase chain reaction. We isolated Bd from 68.9% of frogs sampled from both species. Contamination was low (9.7% of all plates), with most contamination presumed to be fungal. We found positive correlations between successful isolation attempts and infection intensity. Our levels of isolation success were 74% for P. triseriata and 100% for P. regilla once Bd detection intensities reached ≥40 ZE. Of the 3 anatomical regions sampled in both species, we had significantly more success isolating Bd from foot tissue. Our results support published recommendations to focus sampling for Bd infection on feet, particularly webbing.

Diagnosis of bovine mastitis: from laboratory to farm.

Accurate diagnosis of disease is the major step between the cause and cure of disease. An economical, reliable, and rapid diagnostic tool is fundamental for the management of udder health. The earlier the disease is identified, the less will be the damage; keeping this in mind, many efforts are being made to develop reliable diagnostic tools for use on farm. However, traditional gold standard methods including somatic cell count and microbial culturing are still in use. They are partially being replaced with polymerase chain reaction and sequencing-based tests. Nanotechnology and protein-based tests have also gained lot of attention and some of them are potential candidate of future diagnostic tests for bovine mastitis. Research laboratories are struggling to develop simple, economical, and user-friendly biosensor-based methods that can be performed on farm for rapid diagnosis. The combination of both genomic and proteomic approaches, together with further involvement of nanotheranostic technologies and other sensors, will assist in the quest of better mastitis diagnostic tools.

The value of pathogen information in treating clinical mastitis.

The objective of this study was to determine the economic value of obtaining timely and more accurate clinical mastitis (CM) test results for optimal treatment of cows. Typically CM is first identified when the farmer observes recognisable outward signs. Further information of whether the pathogen causing CM is Gram-positive, Gram-negative or other (including no growth) can be determined by using on-farm culture methods. The most detailed level of information for mastitis diagnostics is obtainable by sending milk samples for culture to an external laboratory. Knowing the exact pathogen permits the treatment method to be specifically targeted to the causation pathogen, resulting in less discarded milk. The disadvantages are the additional waiting time to receive test results, which delays treating cows, and the cost of the culture test. Net returns per year (NR) for various levels of information were estimated using a dynamic programming model. The Value of Information (VOI) was then calculated as the difference in NR using a specific level of information as compared to more detailed information on the CM causative agent. The highest VOI was observed where the farmer assumed the pathogen causing CM was the one with the highest incidence in the herd and no pathogen specific CM information was obtained. The VOI of pathogen specific information, compared with non-optimal treatment of Staphylococcus aureus where recurrence and spread occurred due to lack of treatment efficacy, was $20.43 when the same incorrect treatment was applied to recurrent cases, and $30.52 when recurrent cases were assumed to be the next highest incidence pathogen and treated accordingly. This indicates that negative consequences associated with choosing the wrong CM treatment can make additional information cost-effective if pathogen identification is assessed at the generic information level and if the pathogen can spread to other cows if not treated appropriately.

Evaluation of various selective media for the detection of Pseudomonas species in pasteurized milk.

Pseudomonas spp. are common gram-negative, post-pasteurization contaminants that contribute to spoilage of pasteurized dairy products. This study evaluated 5 common selective media for detecting Pseudomonas spp. in pasteurized milk. The performance of each selective medium for recovering 12 different Pseudomonas isolates (selected to represent a diversity of pasteurized milk isolates) was compared with that of standard plate count agar pour plates. Pseudomonas isolates showed varying abilities to produce colonies on different selective media. For 2 of 12 isolates, a 48-h incubation time was required for colony formation on any of the media tested. Violet red bile agar and coliform Petrifilm (3M, St. Paul, MN) were less effective than standard plate count agar pour plates at recovering Pseudomonas, regardless of incubation time, and MacConkey agar showed poor detection efficiency compared with SPCP after a 48-h incubation (R(2) = 0.26). Therefore, the use of violet red bile agar, MacConkey agar, or coliform Petrifilm may not be sufficient for detecting common Pseudomonas spp. in milk. The methods showing the highest detection efficiencies were crystal violet tetrazolium agar (CVTA) pour plates (R(2) = 0.95) and CVTA plates inoculated by spiral plating (R(2) = 0.89) incubated at 32 °C for 48 h. Overall, plating milk samples on CVTA followed by a 48-h incubation at 32 °C was the most effective selective method for recovering a diversity of Pseudomonas spp. from milk.

Research Note: Tracing pathways of entry and persistence of facultative pathogenic and antibiotic-resistant bacteria in a commercial broiler farm with substantial health problems.

On a commercial broiler farm with substantial health problems, shown by a reported loss rate of approx. 10% and regular antibiotic use, samples were taken at different locations in 2 barns, with the aim of analyzing possible entry routes and persistence of pathogens and antibiotic-resistant bacteria as well as revealing weak points in sanitation. Therefore, swab samples for biofilm and water samples from animal drinking water lines and the spray cooling system were taken twice immediately before restocking. In addition, swab samples from drain holes and air samples were collected. At restocking, hatchlings that died during transportation and chick paper were sampled. All samples were analyzed for the occurrence of Pseudomonas aeruginosa, total coliform count, and antibiotic-resistant bacteria, namely, methicillin-resistant Staphylococcus aureus (MRSA), Escherichia coli, Klebsiella spp., Citrobacter spp., Enterobacter spp., Acinetobacter baumannii, P. aeruginosa, and vancomycin-resistant Enterococci (VRE). No MRSA or VRE were detectable. In all samples from drinking water and sprinkler system pipes, P. aeruginosa was detectable; in most cases, antibiotic-resistant P. aeruginosa was also detected, with varying resistance profiles. Samples from the hatchlings and chick paper were contaminated with antibiotic-resistant Enterobacter spp., with resistance to piperacillin, fosfomycin, and the third-generation cephalosporins cefotaxime and ceftazidime. Therefore, the initial entry of antibiotic-resistant Enterobacteriaceae likely occurred via exposure at the hatchery, resulting in colonization of the chicks. Animals on the fattening farm were treated with colistin, amoxicillin, and lincomycin in the 3 production cycles before sampling. Owing to the frequent administration of several antibiotic classes during the fattening period via piped water in both barns, resistance of isolates from water pipes accumulated, showing additional resistance to chloramphenicol and frequently to ciprofloxacin and levofloxacin. To prevent the development of secondary diseases caused by the facultative pathogen P. aeruginosa in chicks with a weak immune status, the hygiene management for drinking water lines and the spray cooling system was changed. These changes resulted in an improvement in water line sanitation, shown by the absence of antibiotic-resistant bacteria and rare detection of P. aeruginosa.

Feline Sporothrix spp. detection using cell blocks from brushings and fine-needle aspirates: Performance and comparisons with culture and histopathology.

BACKGROUND: Sporotrichosis is an emerging zoonotic mycosis that presents as a cutaneous lymphatic or disseminated disease, caused by fungi from the Sporothrix schenkii (S schenkii) clinical clade. Its importance is growing, primarily due to an outbreak that occurred in Brazil, affecting mainly cats and people. OBJECTIVES: In Brazil, an S schenkii diagnosis is often made using cultures, which allows genus identification and sufficient growth to perform molecular biology testing. Despite its advantages, fungal cultures are slow to develop and can delay public health measures, highlighting the importance of developing additional diagnostics techniques. METHODS: Cell block cytology (CBLC) is an older method that regained importance after liquid-based cytology (LBC) was introduced, and it has been previously and successfully applied to veterinary diagnostics. We aimed to standardize and compare CBLC from cervical brush exfoliation of open wounds and fine-needle aspirates with culture and immunohistochemistry of skin biopsies for sporotrichosis in cats, as a novel method. RESULTS: For this purpose, we selected 40 cats with skin lesions suspected of having sporotrichosis in Guarulhos city, São Paulo state, Brazil. We achieved 97.5% and 95% positivity using CBLC and culture, respectively, and 100% of feline skin biopsies were positive for Sporothrix spp on histopathology/immunohistochemistry. CONCLUSIONS: Cell block cytology is an efficient and rapid tool to diagnose sporotrichosis in cats, particularly during epidemics.

Comparison of culture methodology for the detection of methicillin-resistant Staphylococcus pseudintermedius in clinical specimens collected from dogs.

Methicillin-resistant Staphylococcus pseudintermedius (MRSP) has emerged as a major pathogen in dogs and has been implicated as a hospital-acquired pathogen in veterinary hospitals. We attempted to determine if selective culture methods will detect more MRSP when compared to the traditional culture methods in clinical samples from dogs in Atlantic Canada with a high risk for MRSP infection. Each sample was tested using 4 culture methods: traditional culture; mannitol salt agar with 2 µg/mL of oxacillin (MSAox); enrichment broth (EB) with MSAox; and EB with traditional culture. Detection of penicillin-binding protein 2', via latex agglutination, was used as a confirmatory test for oxacillin resistance. We analyzed 741 samples from 556 dogs between February 2013 and April 2014. The prevalence of MRSP in samples detected by any method was estimated at 13.4% (95% CI: 11.1-16.0%). When the prevalence of MRSP was determined according to culture method, EB with MSAox detected the highest prevalence (11.2% [9.1-13.7%]), followed by EB with traditional (10.8% [8.8-13.2%]), traditional (10.1% [8.1-12.5%]), and MSAox (8.9% [7.1-11.2%]). The prevalence using the traditional culture method did not differ significantly from any of the 3 selective culture methods. Culture with MSAox detected significantly fewer MRSP than either of the EB methods. The addition of EB to current methodology is recommended, particularly for patients considered at high risk for MRSP infection.

Evaluation of MALDI-TOF mass spectrometry for the identification of bacteria growing as biofilms.

We evaluated MALDI-TOF mass spectrometry to identify bacteria from biofilms. We compared three sample preparation procedures on biofilms grown in vitro. The extended direct transfer method was able to identify 13 isolates out of 18 (72%) at the species level and 15 out of 18 (83%) at the genus level.

Conventional identification of Streptococcus uberis isolated from bovine mastitis in Argentinean dairy herds.

The objective of this study was to evaluate a conventional scheme for identifying Streptococcus uberis strains isolated from bovine mastitis. Seventy-five gram-positive, catalase-negative cocci were collected from cows with mastitis from 19 dairy herds located in the east-central region of Argentina. Five American Type Culture Collection strains and bovine isolates were identified by the API 20 Strep system and by restriction fragment length polymorphism analysis of 16S rDNA. A conventional scheme based on 11 biochemical tests was selected for identification of Strep. uberis strains: the Christie-Atkins-Munch-Petersen reaction; hydrolysis of Arg, esculin, and sodium hippurate; growth in inulin, mannitol, raffinose, salicin, and sorbitol; and growth at 45 degrees C and in 6.5% NaCl. Reference strains and 25 bovine isolates were classified accurately to the species level by the conventional scheme in a blind assay. Each reference strain and each bovine isolate were identified as belonging to the same species following these 3 methods. The remaining 50 isolates identified as Strep. uberis by the API 20 Strep system and 16S rDNA RFLP were assayed by the conventional scheme. This scheme correctly identified 47 (94%) of 50 isolates as Strep. uberis by comparing their biochemical profile with that of the reference strain. Three (6%) of the 50 isolates were classified as Strep. uberis by the API 20 Strep system and by 16S rDNA RFLP and were identified as Enterococcus faecalis by the conventional scheme. Thirty percent of the Strep. uberis strains showed biochemical profiles identical to the Strep. uberis American Type Culture Collection 27958 strain. Seventy percent of the Strep. uberis strains demonstrated variability compared with the reference strain, resulting in 19 different biochemical profiles. The conventional scheme proposed in this study resulted in a relatively low number of misidentifications and could biochemically identify not only typical, but also atypical Strep. uberis strains. This conventional scheme can be considered an adequate method for identifying Strep. uberis strains isolated from bovine mastitis because of its affordable cost in developing countries, and it may contribute to determining the frequency of isolation of Strep. uberis strains in Argentinean dairy herds.

Evaluation of bovine abortion cases and tissue suitability for identification of infectious agents in California diagnostic laboratory cases from 2007 to 2012.

Establishing a definitive cause of bovine abortion is a challenging problem faced by veterinary practitioners and diagnosticians. Detection of an infectious or noninfectious source for abortion may facilitate interventions that mitigate future fetal loss in the herd. The purposes of this study were to identify the most common causes of bovine abortion in cases submitted to the California Animal Health and Food Safety Laboratory System, Davis (CAHFS) from 2007 to 2013 and to determine if detection of infectious pathogens differed with the fetal tissue evaluated. Records of 665 bovine abortion cases of 709 animals were reviewed for pathologic diagnoses, test methods used to identify causative conditions, and which tissues yielded successful identification of infectious agents associated with abortion. Over 58% of abortions were attributed to an infectious cause and 46.9% had an infectious agent identified. The most common infectious conditions were Epizootic Bovine Abortion (EBA) (16.2% of all fetuses), other fetal bacterial infections (14.7% of all fetuses), and Neospora caninum (9.3% of all fetuses.) The bacterium associated with EBA (currently named Pajaroellobacter abortibovis) was most commonly identified by immunohistochemistry (IHC) in lymphoid organs (thymus and spleen); N. caninum IHC was most frequently positive in brain, kidney, and placenta. In cases of pathogenic and opportunistic bacterial infections, abomasal samples yielded a significantly greater proportion of definitive aerobic culture results than lung or liver tissues. Direct fluorescent antibody test results for Bovine Viral Diarrhea Virus testing were identical between lung and kidney tissues and nearly identical (96.0%) for Bovine Herpesvirus I. Noninfectious abortive conditions included fetal stress (10.5%), dystocia (3.9%), congenital defects (3.3%), toxicological or mineral problems (1.8%), and death of the cow (1.1%). Just over 20% of the aborted fetuses had no gross or histopathological lesions to explain the abortion. This review highlights the need for submission of critical samples including abomasal contents, lymphoid tissues (thymus, spleen, and lymph nodes), and brain to maximize the diagnosticians' ability to identify causes of abortion.

Comparison of immunohistochemistry, acid-fast staining, and cultivation for detection of Mycobacterium paratuberculosis in goats.

Forty-seven paired specimens of ileum and mesenterial lymph nodes from goats originating from 2 herds with paratuberculosis were investigated. Culture of the specimens for Mycobacterium paratuberculosis was compared with Ziehl-Neelsen staining and with immunohistochemical tests on paraffin-embedded tissue sections, using a M. paratuberculosis immune serum and an avidin-biotin-alkaline phosphatase method. Immunohistochemical techniques detected most positive samples and produced more clearly visible reactions than did acid-fast staining. Eighteen of 47 samples were positive by immunohistochemical techniques may represent a valuable adjunct to standard techniques for diagnosis of paratuberculosis in goats.

Identification of field isolates of infectious bronchitis virus by interference with the La Sota of strain of Newcastle disease virus.

The interference phenomenon of infectious bronchitis virus (IBV) with growth of Newcastle disease virus (NDV) in embryonating chicken eggs (ECE) was used as a diagnostic method. Fifteen field isolates obtained from presumptively infectious-bronchitis-affected chickens were analyzed by the IBV-NDV interference test. Eight isolates were capable of interfering with the growth of the La Sota strain of NDV, as measured by hemagglutination (HA) activity when IBV was inoculated 10 hr before NDV into ECE. The interference was considered specific for IBV, because it could be eliminated by adding homologous anti-IBV serum. The sensibility of this method could be demonstrated, because in some cases low-passage levels of IBV isolates showing HA interference ability were not capable of producing lesions in ECE. Furthermore, serologically negative IBV samples did not interfere with NDV growth. From these results, the IBV-NDV interference test appears to be a potential diagnostic alternative for identifying IBV field isolates.

Identifying agent(s) associated with poult enteritis mortality syndrome: importance of the thymus.

Poult enteritis mortality syndrome (PEMS), a highly infectious disease of young turkeys, causes serious financial losses to the turkey industry. Clinically, PEMS is defined by mortality profiles, diarrhea, growth depression, and immunosuppression. Although many viruses, bacteria, and parasites are found in PEMS-infected birds, the inciting agent remains unknown. Experimentally, PEMS can be reproduced by exposing naïve poults to the intestinal contents from infected birds. Previous reports suggest that extraintestinal tissues fail to reproduce the disease. Histopathologic examination of tissues from PEMS-infected poults suggested that the thymus exhibited the earliest signs of pathology. On the basis of these observations, we hypothesized that the thymus harbors an agent(s) involved in PEMS. In these studies, naïve turkey poults were orally inoculated with a bacteria-free filtrate composed of either the intestines and feces or the thymus from PEMS-infected birds and were monitored for clinical signs of PEMS. Poults exposed to a filtrate composed solely of the thymus from PEMS-infected birds exhibited diarrhea, growth depression, mortality, pathology, and, most importantly, immunosuppression similar to poults exposed to the intestinal filtrate. The results of this study suggest that the thymus of infected birds harbors the agent(s) that can reproduce a PEMS-like disease in turkey poults.

Air sampling of pigs infected with foot-and-mouth disease virus: comparison of Litton and cyclone samplers.

The air in looseboxes containing groups of pigs in the acute stage of foot-and-mouth disease was sampled simultaneously with two air-sampling devices: a large volume sampler (Litton) and a cyclone sampler. Although the cyclone sampler was slightly less efficient at trapping airborne virus it was easier to operate. When pigs were sampled individually within a 610 litre cabinet using the cyclone sampler, the mean recovery of virus over a 24 hour period was log10 8 X 6 TCID50 per animal. This figure is four times higher than the maximum amount estimated in previous studies in which groups of pigs held in looseboxes were sampled with a Litton apparatus.

Rapid diagnosis of Aujeszky's disease in pigs by immunofluorescence.

Direct immunofluorescence on impression smears of brain and pharynx was compared with virus isolation in cell culture for the diagnosis of Aujeszky's disease in experimentally and naturally infected pigs. Pharyngeal impression smears were more sensitive than virus isolation in two pigs killed 10 and 12 days after experimental infection. Both methods were of similar sensitivity in the detection of virus from field cases of disease. Smears of brain and pharynx were more sensitive than virus isolation for tissue which had been stored at room temperature (approximately 20 degrees C) for up to 48 hours. Some reduction in the amounts of virus recovered from tissues and the intensity of fluorescent staining occurred in these samples.

Erysipelothrix tonsillarum isolated from dogs with endocarditis in Belgium.

Five strains of Erysipelothrix tonsillarum were isolated from dogs with endocarditis in Belgium. The identity and validity of the species was proved by serotyping, and biochemical and pathogenicity tests. All the isolates belonged to serovar 7 (E tonsillarum serovars); they produced acid from saccharose but did not induce any clinical sign of erysipelas in swine. These results suggest that some strains of E tonsillarum are a canine pathogen.

Survival and development of ivermectin-resistant or susceptible strains of Haemonchus contortus under field and laboratory conditions.

The free-living development of three strains of Haemonchus contortus was studied in two experiments. Day 21 faecal samples were collected from lambs infected with either a susceptible strain, a laboratory-selected ivermectin (IVM) resistant strain or a South African field strain showing multiple anthelmintic resistance, which included IVM. No eggs hatched in samples cultured at 4 or 10 degrees C. At 22 degrees C the laboratory-selected strain showed the highest rate of development while at 27 degrees C the susceptible strain produced the highest yield of third stage larvae (L3): at both temperatures the field strain showed the lowest percentage development to L3. The second experiment was a field study carried out in southern Brazil. Faeces containing either an IVM-susceptible or an IVM-resistant strain of H contortus were placed in two series of grass plots during each of three summer months. Soil subsequently yielded more larvae than did grass suggesting migration or mechanical transport into the soil. For plots contaminated during the first two months there was no significant difference in recovery rate between the two strains (P > 0.05). When contamination occurred during the third month, the IVM-resistant strain produced significantly higher recovery rates (P < 0.05) from both pasture and soil.

Generation times of Epidinium caudatum and Entodinium caudatum, determined in vitro by transferring at various time intervals.

Generation times were determined in vitro with a pure culture of Epidinium caudatum and a mixed culture of Epidinium caudatum and Entodinium caudatum. Measurement of logarithmic growth from a small inoculum for Epidinium caudatum alone, or in coculture, resulted in generation times of 30.8 and 19.5 h, respectively. Epidinium concentrations, either alone or in coculture, were maintained when cultures were transferred every 12 h; however, concentrations decreased rapidly with transfers at 4, 6, or 8 h. For Entodinium caudatum, a generation time of 16.3 h was obtained from measurement of logarithmic growth. Based on sequential transfer data at varying time intervals, Epidinium caudatum and Entodinium caudatum seem to be capable of doubling in approximately 12 to 13 h. These values are markedly less than those previously reported and help explain the ability of these protozoa to maintain themselves in the rumen.