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.

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.

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.

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.

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.

Defined and undefined commercial probiotics cultures in the prevention of Salmonella Enteritidis in broilers / Probióticos comerciais de culturas definidas e indefinidas na prevenção de Salmonella Enteritidis em frangos de corte

This study aimed to evaluate the efficacy of probiotics from different formations, defined and undefined cultures, applied in the control of Salmonella Enteritidis in broilers, identifying the compositions and states for which the probiotics are more effective. For that, 390 broilers were inoculated orally with 1.00 ml of Salmonella Enteritidis at a concentration of 1.2x109 CFU (Colony Forming Units). The experimental design used was randomized blocks with 5 treatments and 6 replications, totaling 30 boxes with 13 birds/box (13 birds/m2). The treatments were provided via drinking water 1 hour after inoculation, keeping a daily treatment of 12 hours with probiotics, for 3 consecutive days (birds at 1, 2 and 3 days of age). In general, the five treatments conducted were: T1 - Control without probiotic, T2 - Probiotic A (defined culture - lyophilized form, strain 7), T3 - Probiotic B (defined culture - lyophilized form, strain 11), T4 - Probiotic C (undefined culture liquid form), T5 - Probiotic D (undefined culture - liquid form). After treatments, performance was evaluated through average body weight, feed conversion and mortality counting. Microbiological analysis and Salmonella isolation were performed using MPN (Most Probable Number) and selective enrichment technique methods, respectively. Samples of ileum and liver pool, cecal tonsils, cecum, heart and spleen pool were collected at 5 and 31 days of age. No differences were observed on growth performance and isolation of Salmonella Enteritidis (p≥0.05). All probiotics applied were effective on reducing Salmonella Enteritidis colonization in the ileum, cecal tonsils, and cecum at 5 days of life. Probiotics T2 and T5 has shown effectiveness in reducing colonization at 31 days, being considered the most efficient on Salmonella Enteritidis control, for the intestines segments evaluated. It was not possible to affirm which probiotics formation, defined or undefined, is more efficient for Salmonella Enteritidis control.(AU)

O objetivo deste trabalho foi avaliar a eficácia dos probióticos de diferentes constituições: de culturas definidas e de culturas indefinidas no controle de Salmonella Enteritidis em frangos de corte, identificando qual a constituição e qual ou quais probióticos testados é mais eficaz. Foram inoculados 390 frangos de corte com 1ml de Salmonella Enteritidis, via oral, na concentração de 1,2 x 109 UFC (Unidades Formadoras de Colônia). O delineamento experimental utilizado foi o de blocos casualizados com 5 tratamentos e 6 repetições cada, totalizando 30 boxes com 13 aves/boxe (13 aves/m2). Os tratamentos foram fornecidos via água de bebida 1 hora após a inoculação, com 12 horas de tratamento com probióticos por dia, durante 3 dias consecutivos (1º, 2º e 3º dia de idade das aves). Os cinco tratamentos foram: T1 - Controle sem probiótico, T2 - Probiótico A (cultura definida - forma liofilizada, 7 cepas), T3 - Probiótico B (cultura definida - forma liofilizada, 11 cepas), T4 - Probiótico C (cultura indefinida - forma líquida), T5 - Probiótico D (cultura indefinida - forma liofilizada). O desempenho zootécnico foi avaliado usando o peso médio, a conversão alimentar e a mortalidade. Análises microbiológicas foram realizadas utilizando o método NMP (NMP/g)e isolamento de Salmonella através técnica de enriquecimento seletivo. Amostras de pool de íleo, tonsilas cecais e cecos e pool de fígado, coração e baço foram coletadas aos 5 dias e aos 31 dias de idade. Para desempenho zootécnico e isolamento de Salmonella Enteritidis não foram observadas diferenças (p≥0,05). Todos os probióticos utilizados foram eficazes na redução da colonização de Salmonella Enteritidis no íleo, tonsilas cecais e cecos aos 5 dias de idade e somente os probióticos do T2 (cultura definida) e T5 (cultura indefinida) reduziram a colonização aos 31 dias sendo considerados os mais eficazes no controle de Salmonella Enteritidis nestes segmentos intestinais avaliados. Não se pode afirmar quais das constituições de probióticos, culturas definidas ou indefinidas, são mais eficazes no controle de Salmonella Enteritidis.(AU)

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.

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.

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.

Somatic cell count and mastitis pathogen detection in composite and single or duplicate quarter milk samples / Contagem de células somáticas e detecção de patógenos causadores de mastite em amostras de leite compostas e individuais simples ou em duplicata

The most acceptable criteria for diagnosing bovine intramammary infections include results of bacteriological culture and measures of inflammation. Therefore, information on the diagnostic characteristics of the procedures used to identify infected quarters is required. Thus, this study was designed to evaluate a set of criteria to classify the infectious status of an udder at the quarter (single and duplicate milk samples) and cow (composite milk sample) levels, and to compare the infectious status with somatic cell counts (SCCs) of the samples. Here, the SCC thresholds determined by receiver operating characteristic curve analysis had a higher Youden index using mammary quarter duplicate milk samples as the gold standard for testing compared with single quarter and composite milk samples, especially for samples for which at least one of the duplicates was microbiologically positive, regardless of the mastitis pathogen isolated. The kappa coefficient for bacteriological results of the single quarter milk samples (single S1 and S2) was 0.85±0.019, indicating that single quarter milk sampling can be useful in mastitis control programs. Therefore, the use of composite milk samples to detect mastitis pathogens may be limited to the detection of major pathogens, given their predictive values. Thus, our findings suggest that the milk SCCs and microbiological examinations, although regarded as the most reliable indicators of ongoing mastitis, should be used in an integrated manner in mastitis control programs. Furthermore, the accuracy of single, duplicate and composite microbiological analyses to diagnosis mastitis should be considered for its implications in mastitis control strategies.(AU)

Os critérios mais aceitáveis para o diagnóstico das infecções intramamárias em bovinos incluem tanto os resultados da cultura bacteriológica e dos indicadores de inflamação. Portanto, a informação sobre os procedimentos mais adequados a serem utilizados para identificação dos quartos infectados é necessária. Assim, o objetivo do presente estudo foi avaliar um conjunto de critérios para identificação da infecção intramamária em bovinos pelo exame microbiológico (amostras individuais de leite simples ou em duplicata, e amostras de leite compostas), e comparar o isolamento do patógeno nas amostras de leite coletadas por distintos critérios com a contagem de células somáticas (CCS). Os valores de corte da CCS determinados pela curva de característica de operação do receptor demonstraram que a coleta de amostras de leite em duplicata apresentou o maior valor do índice de Youden, especialmente quando considerou-se o quarto mamário infectado se pelo menos uma das amostras de leite da duplicata apresentou resultado bacteriológico positivo independentemente do patógeno isolado. O coeficiente kappa dos resultados do exame microbiológico das amostras de leite individuais (amostra simples S1 e S2) foi de 0,85±0,019, indicando que a coleta de amostras de leite individual, ou seja, a coleta de uma amostra de leite por quarto mamário, pode ser utilizada nos programas de controle de mastite. Por outro lado, a coleta de amostras de leite compostas para detectar patógenos causadores de mastite deve ser limitada à detecção dos patógenos principais, considerando os valores preditivos encontrados no presente estudo. Portanto, os resultados do presente estudo indicam que a CCS e o exame microbiológico do leite, embora considerados como os critérios mais aceitos para o diagnóstico da mastite, devem ser utilizados de forma integrada em programas de controle de mastite. Além disto, os critérios de coleta de amostras de leite para o diagnóstico da mastite pelo exame microbiológico e seus valores preditivos devem ser considerados nos programas de controle de mastite.(AU)

Uso de antimicrobiano nanoparticulado para o tratamento da mastite subclínica de ovelhas de corte no período seco / Use of nanoparticulated antimicrobians to treat subclinical mastitis of ewes during the dry period

A inflamação da glândula mamária é uma das principais causas de prejuízo na ovinocultura. Este estudo teve como objetivo investigar as taxas de cura do tratamento da mastite subclínica após infusão intramamária de princípio ativo antimicrobiano no momento da secagem, em formulações convencional e nanoparticulada. Os rebanhos estavam localizados em São Carlos, São Paulo, Brasil. Analisou-se um total de 584 glândulas mamárias de 307 ovelhas de aptidão para produção de carne. Triagem prévia dos casos subclínicos de mastite foi efetuada por meio do California Mastitis Test (CMT) e/ou da contagem de células somáticas (CCS). Análises microbiológicas foram realizadas para confirmação da etiologia infecciosa. As glândulas mamárias com mastite subclínica foram distribuídas em três grupos: G1 (Controle; glândulas mamárias que não receberam tratamento antimicrobiano); G2 (glândulas mamárias em que foi administrado 100 mg de cloxacilina benzatina em estrutura convencional) e G3 (glândulas mamárias em que foi administrado 50 mg de cloxacilina benzatina em estrutura nanoencapsulada). O tratamento aplicado ao G3 mostrou-se mais eficiente (P=0,047) na cura de glândulas mamárias com mastite subclínica. O uso da cloxacilina nanoencapsulada no momento da secagem de ovelhas de corte auxilia no controle da mastite subclínica infecciosa e reduz os prejuízos consequentes.(AU)

Inflammation of the mammary gland is one of the main causes of losses in sheep-rearing. This study aimed to investigate the cure rates from treating subclinical mastitis after intramammary infusion of active antimicrobial agents as conventional formulations or as nanoparticles, at the time when the ewes are being dried off. A total of 584 mammary glands in 307 ewes in meat-producing herds located in São Carlos, São Paulo, Brazil, were analyzed. Prescreening of subclinical mastitis cases was done using the California mastitis test (CMT) and/or the somatic cell count (SCC). Microbiological analyses were performed to confirm the infectious etiology. The mammary glands with subclinical mastitis were distributed into three groups: G1 (control; mammary glands that did not receive any antimicrobial treatment); G2 (mammary glands to which 100mg of benzathine cloxacillin in conventional form were administered); and G3 (mammary glands to which 50mg of benzathine cloxacillin in nanoparticulate form were administered). The treatment applied to G3 was more efficient (P=0.047) in curing mammary glands with subclinical mastitis. Use of cloxacillin nanoparticles at the time when the ewes are being dried off helps to control infectious subclinical mastitis and reduces consequential losses among meat-producing herds.(AU)

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.

O uso da microbiologia como ferramenta para a conservação de aves ameaçadas: dados preliminares para o papagaio-de-cara-roxa, Amazona brasiliensis (aves: Psittacidae) no Paraná / Use of microbiology as a tool for the preservation of threatened bird species: preliminary data for the red-tailed amazon, Amazona brasiliensis (birds: Psittacidae) in Paraná / El uso de la microbiología como herramienta para la conservación de aves amenazadas: datos preliminares para el loro de cara púrpura, Amazona brasiliensis (aves: Psittacidae) en Paraná

Amazona brasiliensis, também conhecido popularmente como papagaio-de-cara-roxa, é uma espécie de Psittacidae endêmica da Mata Atlântica distribuída entre o litoral sul de São Paulo e o litoral Norte de Santa Catarina. Este estudo foi concentrado no estado do Paraná, visando uma prévia caracterização do perfil sanitário natural em filhotes da população por meio de análises microbiológicas. O estudo epidemiológico de uma espécie ameaçada inicia-se com a determinação dos agentes infecciosos comuns na população, que provavelmente co-evoluíram com a espécie e representam baixo risco aos espécimes de vida livre. Do total de colônias isoladas quase 75% foram positivas para a família Enterobacteriaceae. Normalmente, a microbiota entérica de psitacídeos é composta por micro-organismos Gram-positivos, porém a alta porcentagem de Gram-negativas isoladas pode ser explicada pela diferença entre a composição microbiana de adultos e filhotes. Seria interessante um estudo mais detalhado para uma eventual comparação entre possíveis sinais clínicos e micro-organismos presentes em cada indivíduo amostrado.(AU)

Amazona brasiliensis, also popularly known as Red-tailed Amazon, is an endemic species to the Atlantic Forest. This Psittacidae range goes from the south coast of São Paulo state to the northern coast of Santa Catarina state. This study focuses on the population in the state of Paraná, aiming to perform a preliminary characterization of the prevalence of natural pathogens in nestlings through microbiological analyses. The epidemiological study of an endangered species begins with the determination of common infectious agents within the population, which probably co-evolved with the species and represent a low risk to free-living specimens. Almost 75% of the colonies isolated were positive for Enterobacteriaceae. Usually, the enteric microbiota of psittacidae consists of Gram-positive microorganisms, but the high percentage of isolated Gram-negative bacteria can be explained by differences between the microbial composition of adults and nestlings. It would be interesting to further develop this study into a more detailed comparison between possible clinical signs and microorganisms present in each individual sampled.(AU)

Amazona brasiliensis, popularmente conocido como el loro de cara púrpura, es una especie endémica de Psittacidae de la Mata Atlántica distribuido entre la costa sur de São Paulo y la costa norte de Santa Catarina. Este estudio se concentra en el estado de Paraná, buscando caracterizar preliminarmente el perfil sanitario natural en crías de la población, por medio del análisis microbiológico. El estudio epidemiológico de una especie amenazada comienza con la determinación de agentes infecciosos comunes en la población, que probablemente ha evolucionado con la especie y representan un riesgo bajo para especímenes de vida libre. Del total de colonias aisladas casi el 75% fueron positivos para Enterobacteriaceae. Normalmente, la microbiota entérica de loros se compone de microorganismos Gram positivos, pero el alto porcentaje de aislados Gram negativas se puede explicar por la diferencia entre la composición microbiana de adultos y pichones. Sería interesante un estudio más detallado para una posible comparación entre los síntomas clínicos y los microorganismos presentes en cada individuo muestreado.(AU)

Research of Klebsiella pneumoniae in dairy herds / Pesquisa de Klebsiella pneumoniae em rebanhos leiteiros

Klebsiella pneumoniae is a common environmental agent of clinical and subclinical mastitis affecting dairy herds, and may be present in the final product decreasing its quality. Mastitis caused by K. pneumoniae is even more severe due to its poor response to antibiotic therapy, rapid evolution to toxic shock and death of the animal. This paper aimed to study the prevalence of this pathogen among dairy herds in ten farms located in different municipalities of São Paulo State based on size and use of milking technology. All mammary glands of all lactating cows were screened using the California Mastitis Test (CMT) and a strip cup. A single aseptic milk sample (20mL) was collected from all CMT-positive quarters and bulk tanks, whereas swab samples were collected from feces, hind limbs of the animals, bedding and milking parlor. Identification of K. pneumoniae was performed using conventional microbiology culture, biochemical assay and Polimerase Chain Reaction. The primers were designed and tested at the Laboratory of Molecular Biology applied to Zoonoses (FMVZ, Unesp-Botucatu) targeting the 16S rRNA gene. This study included 1067 animals. Six cases of intramammary infection by K. pneumoniae were detected in six different cows in two farms. Moreover, K. pneumoniae was isolated in 77 swabs (34 from bedding in 9 farms, 7 from waiting rooms in 5 farms, 6 from milking parlors in 4 farms, 11 from rectums in six farms, and 19 from hindlimbs in 7 farms. Molecular analysis confirmed the agent was K. pneumoniae. At least one strain of the agent was identified in a certain site in all farms, showing the need of maintaining the hygiene in dairy farms...

Klebsiella pneumoniae é um agente ambiental comum de mastite clínica e subclínica que afetam vacas leiteiras e pode estar presente no produto final, reduzindo a sua qualidade. Mastite causada por K. pneumoniae é ainda mais grave devido à sua má resposta à antibioticoterapia, rápida evolução para choque tóxico e morte do animal. Este trabalho teve como objetivo estudar a prevalência deste patógeno entre os rebanhos leiteiros em dez fazendas localizadas em diferentes municípios do Estado de São Paulo com base no tamanho do rebanho e uso de tecnologia de ordenha. Todas as glândulas mamárias das vacas em lactação foram examinadas usando o California Mastitis Test (CMT) e caneca de fundo telado. Foram colhidas amostras de leite (20mL) de todos os quartos CMT- positivos e dos tanques de expansão, também foram colhidos swab de fezes, membros posteriores dos animais, cama dos animais e sala de ordenha. O isolamento e identificação de K. pneumoniae foi realizada através de cultura microbiológica convencional, ensaio bioquímico e Reação em Cadeia da Polimerase, utilizando primers desenhados e testados no Laboratório de Biologia Molecular aplicada à Zoonoses (FMVZ, Unesp-Botucatu) com base na região do gene de 16S rRNA. Este estudo incluiu 1067 animais. Foram detectados seis casos de infecção intramamária por K. pneumoniae em seis diferentes animais em duas fazendas. Ainda, K. pneumoniae foi isolada em 77 swabs (34 de camas em 9 propriedades, 7 de salas de pré-ordenha em 5 propriedades, 6 de salas de ordenha em 4 propriedades, 11 do reto de animais em 6 propriedades e 19 de membros posteriores em 7 propriedades. A análise molecular confirmou o agente K. pneumoniae. K. pneumoniae foi isolada pelo menos em uma localização em todas as propriedades leiteiras., salientando a necessidade de manter a higiene nas fazendas leiteiras a fim de controlar a mastite por esse patógeno...

The Rivalta's test as a diagnostic variable in feline effusions--evaluation of optimum reaction and storage conditions.

OBJECTIVE: The Rivalta's test is used to diagnose feline infectious peritonitis (FIP) in cats with effusion. Only little information on the influence of sample storage and reaction conditions on test results is available, and diagnostic sensitivity and specificity to diagnose FIP vary considerably between few available studies. This study determined the influence of storage of effusion, modifications on reaction conditions, and inter-observer variation. MATERIAL AND METHODS: The Rivalta's test was repeated up to 21 days after storage at room temperature, in the refrigerator, or freezer. The test was performed by two independent, blinded investigators. It was also performed using different volumes of acetic acid, different acids, and different kinds of water. RESULTS: Even after storage for 21 days, test results were comparable. While inter-observer variation revealed substantial disagreement, different modifications in performance showed no major influence on test outcome. CONCLUSION: The Rivalta's test seems to be a very robust test concerning storage conditions. Modifications in reaction condition also do not substantially influence outcome. However, the test is subjective and depends on the evaluating person.

Trends in diagnosis and control of bovine mastitis: a review.

Mastitis (inflammation of mammary gland) is a most devastating disease condition in terms of economic losses occurring throughout the world. The etiological agents may vary from place to place depending on climate; animal species and animal husbandry and include wide variety of gram positive and gram negative bacteria; and fungi. They may be either contagious viz. Staphylococcus aureus; Streptococcus agalactiae or environmental viz. S. dysgalactiae, S. uberis, Corynebacterium bovis and Coagulase negative Staphylococcus. Conventional diagnostic tests viz. California Mastitis Test (CMT); R-mastitest and Mast-O-test methods are applied under field conditions; whereas somatic cell count and Bulk Tank Somatic Cell Count (BTSCC) are useful for early mastitis detection and detection of sub clinical or chronic mastitis respectively. In vitro culture based diagnosis require further study as they can detect only viable cells. The advent of Polymerase Chain Reaction (PCR) technology along with its various versions like multiplex and real time PCR has improved the rapidity and sensitivity of diagnosis. Circulating micro RNA (miRNA) based diagnosis; immune assay and proteomics based detection along with biochips and biosensors prove to be asset to diagnosticians for advanced diagnosis of this economically important condition. Improvement of milking hygiene; implementation of post-milking teat disinfection; regular control of the milking equipments; implementation of milking order; Improvement of bedding material are the general measures to prevent new cases of mastitis. The use of antibiotics (intramammary infusions; bacteriocins) and herbs (Terminalia spp.) are important for prophylaxis and therapeutics. Vaccines viz. cell based; Recombinant (staphylococcal enterotoxin type C mutant) or chimeric (pauA); live (S. uberis 0140J stain based) and bacterial surface extract based; DNA-based and DNA-protein based have greatly aided in management of bovine mastitis. Quorum sensing and disease resistant breeding using novel biomarkers viz. toll like receptors (TLR) 2 and 4, interleukin (IL) 8; breast cancer type 1 susceptibility protein (BRCA1) and calcium channel voltage-dependent alpha 2/delta sub unit 1 (CACNA2D1) are also indispensable. This mini review gives an overview of all these different aspects that act as trend setters as far as the diagnosis and control of bovine mastitis is concerned to help the diagnosticians; epidemiologists and researchers not to remain ignorant about this grave condition.

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.

Microbiological identification and analysis of swine tonsils collected from carcasses at slaughter.

The primary objective of this 7-month study was to determine the prevalence of porcine pathogens of the tonsil of the soft palate of swine at slaughter. Additional objectives were to determine if sampling the carcasses of normal or abnormal hogs provided different microbiological profiles and if the slaughter plant provides a feasible sampling frame and environment for detecting and monitoring important pathogens in tonsils that have health implications for both swine and humans. A total of 395 samples were collected from 264 farms. Of these, 180 tonsils were collected from normal carcasses and 215 tonsils were collected from carcasses that were diverted to the hold rail. Laboratory testing included bacteriological culture and identification as well as real time-polymerase chain reaction (PCR) testing for porcine reproductive and respiratory syndrome virus (PPRSV) and immunohistochemistry (IHC) for porcine circovirus-2 (PCV-2). The most commonly isolated bacteria included: Streptococcus suis (53.7%), Arcanobacterium pyogenes (29.9%), Pasteurella multocida (27.3%), and Streptococcus porcinus (19.5%). Virus screening revealed evidence of PRRSV and PCV-2 in 22.0% and 11.9% of the samples, respectively. Salmonella Typhimurium and Yersinia enterocolitica were isolated in 0.5% and 1.8% of the samples, respectively. Tonsils collected from the hold rail were more likely to be positive for Staphylococcus hyicus [odds ratio (OR) = 7.51, confidence interval (CI) = 2.89 to 19.54], Streptococcus porcinus (OR = 9.93, CI = 4.27 to 23.10), and Streptococcus suis (OR = 2.16, CI = 1.45 to 3.24). Tonsils collected from abnormal carcasses were less likely to be positive for Staphylococcus aureus (OR = 0.05, CI = 0.005 to 0.482).

Infectious and zoonotic disease testing in pet birds.

Pet bird ownership and the veterinary diagnostic market for avian and exotic species testing have grown markedly during the past 20 years. Birds present with both unique infectious diseases and other diseases that are known to the human medical community, including aspergillosis, mycobacteriosis, chlamydophilosis, and bornavirus infection, some of which have clear zoonotic implications. Although diagnostic testing for these avian infectious diseases has grown considerably and includes the newer technology of polymerase chain reaction as well as traditional serologic testing, guidelines for the use and interpretation of these tests and standardization of tests among veterinary laboratories remains an unmet challenge.