Diagnosis of mycobacteria in bovine milk: an overview

Tuberculosis remains as the world’s biggest threat. In 2014, human tuberculosis ranked as a major infectious disease by the first time, overcoming HIV death rates. Bovine tuberculosis is a chronic disease of global distribution that affects animals and can be transmitted to humans by the consumption of raw milk, representing a serious public health concern. Despite the efforts of different countries to control and eradicate bovine tuberculosis, the high negative economic impact on meat and milk production chains remains, given the decreased production efficiency (approximately 25%), the high number of condemned carcasses, and increased animal culling rates. This scenario has motivated the establishment of official programs based on regulations and diagnostic procedures. Although Mycobacterium tuberculosis and Mycobacterium bovis are the major pathogenic species to humans and bovines, respectively, nontuberculous mycobacteria within the Mycobacterium genus have become increasingly important in recent decades due to human infections, including the ones that occur in immunocompetent people. Diagnosis of mycobacteria can be performed by microbiological culture from tissue samples (lymph nodes, lungs) and secretions (sputum, milk). In general, these pathogens demand special nutrient requirements for isolation/growth, and the use of selective and rich culture media. Indeed, within these genera, mycobacteria are classified as either fast- or slow-growth microorganisms. Regarding the latter ones, incubation times can vary from 45 to 90 days. Although microbiological culture is still considered the gold standard method for diagnosis, molecular approaches have been increasingly used. We describe here an overview of the diagnosis of Mycobacterium species in bovine milk

Nontuberculous mycobacteria in milk from positive cows in the intradermal comparative cervical tuberculin test: implications for human tuberculosis infections

Although the tuberculin test represents the main in vivo diagnostic method used in the control and eradication of bovine tuberculosis, few studies have focused on the identification of mycobacteria in the milk from cows positive to the tuberculin test. The aim of this study was to identify Mycobacterium species in milk samples from cows positive to the comparative intradermal test. Milk samples from 142 cows positive to the comparative intradermal test carried out in 4,766 animals were aseptically collected, cultivated on Lowenstein-Jensen and Stonebrink media and incubated for up to 90 days. Colonies compatible with mycobacteria were stained by Ziehl-Neelsen to detect acid-fast bacilli, while to confirm the Mycobacterium genus, conventional PCR was performed. Fourteen mycobacterial strains were isolated from 12 cows (8.4%). The hsp65 gene sequencing identified M. engbaekii (n=5), M. arupense (n=4), M. nonchromogenicum (n=3), and M. heraklionense (n=2) species belong to the Mycobacterium terrae complex. Despite the absence of M. tuberculosis complex species in the milk samples, identification of these mycobacteria highlights the risk of pathogen transmission from bovines to humans throughout milk or dairy products, since many of mycobacterial species described here have been reported in pulmonary and extrapulmonary diseases both in immunocompetent and immunocompromised people

Patógenos bacterianos isolados de fezes de felinos domésticos, sem sinais entéricos, de ambiente urbano e rural

Os patógenos entéricos são um grupo complexo de micro-organismos que causam doenças em animais e humanos. Os gatos domésticos podem ser considerados carreadores de patógenos entéricos com potencial zoonótico devido ao hábito de auto-limpeza, estabelecer amplo território, pelo instinto de caça e eliminar subclinicamente patógenos. O presente estudo investigou a ocorrência de Escherichia coli, Salmonella sp., Clostridium spp. e Rhodococcus equi nas fezes de 200 gatos domésticos sem sinais entéricos, dos quais 100 pertenciam ao ambiente urbano e 100 à áreas rurais. Marcadores de virulência de E. coli, Clostridium spp. e R. equi também foram investigados. Do total de 200 amostras fecais avaliadas foram identificadas 175 (88%) linhagens de E. coli das quais 93 (93%) de gatos de ambiente urbano e 82 (82%) de gatos de área rural. Houve diferença significante (p=0,03) entre os grupos para frequência mais alta de isolamento de E. coli em gatos de ambiente urbano. Dentre os marcadores de virulência de E. coli foram identificados os genes eae (13%), escN (13%), stx1 (1%), stx2 (0,6%), aatA (0,6%) e ipaH (0,6%). Foram isoladas 86 (43%) linhagens de C. perfringens A, das quais em 21 (24%) foi detectado o gene cpb2, que codifica a toxina beta 2 e em uma (1%) linhagem foi identificado o gene cpe, responsável pela codificação da enterotoxina. Também foram identificados cinco (2%) isolados de C. difficile, dos quais um (20%) apresentou os genes tcdA e tcdB, codificadores das toxinas A e B, respectivamente. Foram isoladas ainda sete (4%) linhagens de R. equi, com diferença significante (p=0,01) para o isolamento do patógeno em animais de ambiente rural. Os isolados de R. equi foram classificados como avirulentos pois não foram detectados os genes vapA, vapB ou vapN, associados à virulência. No entanto, dentre a literatura consultada o presente estudo identificou pela primeira vez R. equi nas fezes de gatos, cujas linhagens foram submetidas a caracterização do perfil de virulência associado aos plasmídios. As principais coinfecções nos gatos de ambiente urbano foram observadas entre E. coli e C. perfringens A (26%), E. coli e C. perfringens A cpb2 + (8%) e E. coli eae+ /escN+ e C. perfringens A (5%), enquanto nos animais de ambiente rural foram E. coli e C. perfringens A (21%), E coli e C. perfringens A cpb2 +(8%), E. coli e R. equi (4%) e entre E. coli e C. difficile tcdA-/tcdB-/cdtB- (3%). No entanto, não houve diferença estatística (p>0,05) entre a identificação de coinfecções e os ambientes amostrados. A presença de E. coli e Clostridium spp., contendo marcadores de virulência, e R. equi nas fezes de gatos domésticos, sem sinais entéricos, indica o potencial dessa espécie animal como reservatório de patógenos de veiculação fecal, para humanos, principalmente pelo crescente estreitamento da relação entre humanos e animais de companhia, particularmente gatos.Enteric pathogens are a complex group of microorganisms associated to diseases in animals and humans. Domestic cats may be considered carriers of enteric pathogens with zoonotic potential, due to habit of self-cleaning, to establish wide territory of life, by hunting behavior, and subclinical elimination of pathogens through feces. The aim of present study was investigate the occurrence of Escherichia coli, Salmonella sp., Clostridium spp., and Rhodococcus equi in 200 fecal samples of cats without enteric signs, of which 100 cats belonging to urban area and 100 to farm environment. Virulence markers from E. coli, Clostridium spp. and R. equi were also tested. Among 200 fecal samples were identified 175 (88%) E. coli strains, from these, 93 (93%) were from feces of cats breeding in urban areas, whereas 82 (82%) were from animals from farms. Statistical difference was observed (p=0.03) to higher identification of E. coli from cats breeding in urban area. Genes eae (13%), escN (13%), stx1 (1%), stx2 (0,6%), aatA (0,6%) e ipaH (0,6%) were indentified from virulent markers in E. coli strains. Eighty-six (43%) C. perfringens A were isolated. From these, cpb2 gene (which encodes beta 2 toxin) was detected in 21 (24%) isolates, and cpe gene (which encodes enterotoxin) was identified in another (1%) isolate. Five (2%) strains of C. difficile were also identified. From these, only one (20%) presented tcdA e tcdB genes, which encodes A and B toxins, respectively. Were isolated also uncommon presence of seven (4%) R. equi strains exclusively from cats of rural environment (p=0,01). All R. equi were characterized as avirulent, because none isolate were detected with vapA, vapB, and vapN genes, associated to virulence. However, to the best of our knowledge, the present study report by first time isolation of R. equi from feces of cats, where virulence plasmid profile was assessed. The main co-infection observed between microorganism identified in cats from urban area were represented by: E. coli and C. perfringens A (26%); E coli and C. perfringens A cpb2+ (8%); E. coli eae+ /escN+ and C. perfringens A (5%). Whereas in animals from farms co-infections were comprised by: E. coli and C. perfringens A (21%), E coli and C. perfringens A cpb2 + (8%), E. coli and R. equi (4%), and E. coli and C. difficile tcdA-/tcdB-/cdtB-. However, no statistical association (p>0.05) was observed to co-infections and different environments of cats. The identification of E. coli and Clostridium sp. (containing virulent markers) and R. equi in normal feces of healthy cats from urban and farm areas, highlight the potential of this domestic species as reservoir of enteric pathogens to humans, because increase of close contact of owners and their companion animals, particularly cats.Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq

Platinosomíase em felinos domésticos: um diferencial para obstrução biliar

The platinosomiase is a disease the hepatobiliary system of domestic felines being caused by trematode Platynosomum fastosum. The life cycle of the parasite includes the presence of three intermediate hosts (snails, terrestrial isopodes, lizards or frogs). The cat is considered definitive host of the parasite and acquires the infection eating a vertebrate intermediate host containing metacercariae. The adult parasites generally inhabit the liver, gallbladder and bile ducts of cats. The severity of clinical manifestations depends mainly on the number of flukes in the biliary tract. Many animals are asymptomatic or exhibit nonspecific clinical manifestations such as anorexia, lethargy, weight loss, vomiting and diarrhea. In large infestations can occur biliary obstruction resulting in jaundice, cirrhosis, hepatitis cholangiohepatitis and even death. The definitive diagnosis is accomplished through by the detection of fluke eggs in feces or through laparotomy and liver biopsy. Treatment should be based on the use of anthelmintic for the elimination of the parasite and supportive therapy for the animal. Prevention is difficult due to predatory nature of the cat. The control can be accomplished through periodic fecal examinationsA platinosomíase é uma doença que acomete o sistema hepatobiliar dos felinos domésticos, sendo causada pelo trematódeo Platynosomum fastosum. O ciclo de vida do parasita inclui a presença de três hospedeiros intermediários (caracóis, isópodos terrestres, lagartixas ou sapos). O gato é considerado hospedeiro definitivo do parasita e adquire a infecção ao ingerir um hospedeiro intermediário vertebrado contendo metacercárias. Os parasitas adultos geralmente habitam o fígado, vesícula biliar e ductos biliares dos felinos. A gravidade das manifestações clínicas depende, principalmente, do número de trematódeos no trato biliar. Muitos animais são assintomáticos ou exibem manifestações clínicas inespecíficas como anorexia, letargia, emagrecimento, êmese e diarréia. Em grandes infestações pode ocorrer obstrução biliar, resultando em icterícia, cirrose, colangio-hepatite e até morte. O diagnóstico definitivo é realizado pela detecção dos ovos dos trematódeos nas fezes ou através de laparotomia exploratória e biópsia hepática. O tratamento deve se baseado no uso de anti-helmínticos para a eliminação do parasita e na terapia de suporte para o animal. A prevenção é difícil devido à natureza predatória do gato. O controle pode ser realizado através de exames parasitológicos de fezes periódico

Detecção de Brucella spp. em leite bovino não pasteurizado através da Reação de Cadeia pela Polimerase (PCR)

A brucelose é uma importante zoonose causada por bactérias do gênero Brucella. O homem é infectado pelo contato com as secreções reprodutivas como placenta, lóquios placentários, sêmen e secreções penianas de animais infectados ou pelo consumo de leite e derivados não pasteurizados. Com o objetivo de pesquisar a presença da bactéria no leite, foram avaliadas, através da técnica da reação em cadeia da polimerase (PCR), 30 amostras de leite cru comercializadas clandestinamente na região de Botucatu, São Paulo, bem como 50 amostras de leite entregues em laticínio, previamente à pasteurização. Das 80 amostras analisadas pela técnica de PCR, 10 (12,5%) foram positivas e 70 (87,5%) negativas. Dentre as amostras positivas, 5 amostras (16,6%) eram provenientes de comerciantes ilegais e outras 5 amostras (10%) foram obtidas no laticínio. A positividade para Brucella spp. demonstra que o patógeno se encontra presente de forma importante na região de Botucatu, São Paulo, e que o risco associado à saúde pública devido à comercialização de produtos clandestinos sem prévia pasteurização é real

Índice de resistência múltipla aos antimicrobianos, concentração inibitória mínima e ESBL fenotípica em linhagens de Proteus mirabilis e Proteus vulgaris isoladas de diferentes afecções em animais domésticos

Proteus spp. are opportunistic multidrug resistant enterobacteria associated with diverse clinical diseases in domestic animals. However, Proteus infections in domestic animals are often misdiagnosed or considered contaminants in microbiological cultures rather than a primary agent of disease. Descriptions of Proteus infections in domestic animals are typically restricted to case reports, retrospective studies, or surveillance of other microorganisms. The present study investigated multiple antibiotic resistance indices, minimum inhibitory concentrations (MICs), and ESBL production in 73 strains of Proteus mirabilis (n = 69) and Proteus vulgaris (n = 4) isolated from domestic animals with various clinical manifestations. In dogs, the pathogen was most commonly associated with cystitis (48.21), enteritis (21.42%), otitis (14.29%), and conjunctivitis (3.57%). In bovines, the microorganism was predominant in cases of enteritis (22.22%), abscess (11.11%), otitis (11.11%), omphalitis (11.11%), and peritonitis (11.11%), and in organ fragments (11.11%). In equines (50.0%) and cats (100.0%), diarrhea was the main clinical sign. In vitro standard disk diffusion assay showed that the most effective antimicrobials against the isolates were imipenem (98.63), norfloxacin (95.89), amikacin (95.89), levofloxacin (90.41), ceftriaxone (87.64), and florfenicol (87.67). In contrast, the isolates commonly showed resistance to novobiocin (95.89), azithromycin (57.53), and trimethropim/sulfamethoxazole (39.73). Among the 73 isolates, the efficacy of amoxicillin/clavulanic acid, gentamicin, ceftriaxone, and ciprofloxacin according to MICs was 87.67%, 86.30%, 84.93%, and 82.19%, respectively. The MIC50 values of amoxicillin/clavulanic acid, ceftriaxone, ciprofloxacin, and gentamicin were, respectively, 1.0, 0.004, 0.03, and 1.0 µg/mL. Thirty-three strains (45.21%) showed a antimicrobial multiple resistance index of ? 0.3. Multidrug resistance profiles of isolates were observed most frequently in dogs (n = 25; 75.76%), particularly in those with cystitis (n = 13; 52.0%), followed by bovines (n = 4; 12.12%), equines (n = 2; 6.06%), and cats (n = 2; 6.06%). Two (2.7%) strains, obtained from canine skin and feces, were diagnosed phenotypically as ESBL-producers. Here, we observed the diversity of Proteus infections in domestic animals. The presence of multidrug-resistant isolates and ESBL-producers reinforces the need for appropriate antimicrobial use and in vitro antimicrobial tests to support therapy.Proteus spp. são enterobactérias oportunistas, multirresistentes aos antimicrobianos, associadas a diversas infecções em animais domésticos. No entanto, as infecções por Proteus em animais de produção e de companhia são negligenciadas ou, por vezes, o patógeno é considerado “contaminante”, ainda que em infecções como agente primário. Os registros de infecções por Proteus sp. em animais domésticos estão restritos aos relatos de casos, estudos retrospectivos ou compondo estudos com outros micro-organismos. O presente estudo investigou o índice de resistência múltipla (IRMA) e a concentração inibitória mínima (CIM) de 73 Proteus mirabilis (n=69) e Proteus vulgaris (n=4) a diferentes antimicrobianos, bem com a produção fenotípica de ESBL, em isolados obtidos de várias manifestações clínicas em animais domésticos. Em cães, o micro-organismo foi identificado predominantemente em casos de cistite (48,21%), enterite (21,42%), otite (14,29%) e conjuntivite (3,57%). Nos bovinos, o agente foi isolado predominantemente de casos enterite (22,22%), abscesso (11,11%), otite (11,11%), onfalite (11,11%), peritonite (11,11%), metrite (11,11%) e em fragmento de órgão (11,11%). Nos equinos (50,0%) e felinos (100,0%) o micro-organismo foi isolado principalmente de enterite. A maior sensibilidade dos isolados no teste “in vitro” de difusão com discos foi observada para imipeném (98,63%), norfloxacino (95,89%), amicacina (95,89%), levofloxacino (90,41%), ceftriaxona (87,64%) e florfenicol (87,67%), enquanto a maior resistência das linhagens foi observada para novobiocina (95,89%), azitromicina (57,53%) e sulfametoxazole-trimetropim (39,73%). Dentre as 73 linhagens, a eficácia da amoxicilina/ácido clavulânico, gentamicina, ceftriaxona e ciprofloxacino utilizando o teste de CIM foi, respectivamente, 87,67%, 86,30%, 84,93% e 82,19%. A CIM50 para amoxicilina/ácido clavulânico, ceftriaxona, ciprofloxaicno e gentamicina foi, respectivamente, 1,0 µg/mL, 0,004 µg/mL, 0,03 µg/mL e 1,0 µg/mL. O índice de resistência múltipla aos antimicrobianos (IRMA ? 0,3) foi observado em 33 (45,21%) linhagens, variando entre 0,1 a 1. Entre os isolados com perfis de multirresistência, a maior ocorrência foi observada em cães (n=25; 75,76%), particularmente em animais com cistite (n=13; 52,0%), seguido pelos bovinos (n=4; 12,12%), equinos (n=2; 6,06%) e felinos (n=2; 6,06%). A presença fenotípica de ESBL foi identificada em dois (2,7%) isolados obtidos de dermatite e fezes de cães. Infere-se a diversidade de infecções causadas por Proteus em animais domésticos, a presença de isolados ESBL-positivos e multirresistentes aos antimicrobianos, reforçando a importância da instituição do tratamento do patógeno com respaldo em testes “in vitro” de sensibilidade microbiana e do uso racional de antimicrobianos no tratamento de infecções em animais domésticos

An atypical Toxoplasma gondii genotype in a rural Brazilian dog co-infected with Leishmania (Viannia) braziliensis

Toxoplasmosis and leishmaniasis are two worldwide zoonoses caused by the protozoan parasites Toxoplasma gondii and Leishmania spp., respectively. This report describes the clinical and laboratorial findings of a co-infection with both parasites in a 4-year-old female dog suspected of ehrlichiosis that presented anemia, thrombocytopenia, hypoalbuminemia, hyperglobulinemia, tachyzoite-like structures to the lung imprints, and polymerase chain reaction (PCR) results positive for T. gondii (kidney, lung, and liver) and Leishmania spp. Co-infection with Toxoplasma gondii and Leishmania braziliensis was confirmed by sequencing; restriction fragment length polymorphism-polymerase chain reaction (RFLP-PCR) confirmed an atypical T. gondii genotype circulating in dogs that has been reported to cause human congenital toxoplasmosis

Successful therapy in unusual generalized Dermatophilus congolensis infection in a calf based on modified in vitro disk diffusion test

<div><p>ABSTRACT: Bovine dermatophilosis is a dermatitis characterized by typical focal or localized lesions with “paintbrush” aspect and occasionally as disseminated cutaneous disease. We report the case of a one-year-old Nelore female with history of chronic cutaneous disseminated lesions that appeared immediately after a rainfall period. Serous to purulent exudates, hair with tufted appearance, hyperkeratotic, non-pruritic, hardened, yellowish to brown, and coalescent crusty lesions were observed distributed all over its body. Removal of the crusts revealed ulcerated or hemorrhagic areas, with irregular elevated crusts like “paintbrush”. Microbiological diagnosis enabled the identification of a microorganism, the Dermatophilus congolensis. Despite disseminated and chronic lesions, we obtained a successful therapy with parenteral therapy using long-acting tetracycline based on modified in vitro disk diffusion test. The present report highlights success therapy in uncommon generalized bovine dermatophilosis with selection of first-choice drugs based on modified in vitro susceptibility test, and need of responsible use of antimicrobials in livestock.</p></div