Sperm viability, serological, molecular, and modified seminal plasma agglutination tests in the diagnosis of Leptospira in the semen and serum of bovine bulls.

The present study investigated the serum microscopic agglutination test (MAT) among 203 bovine bulls with reproduction by natural means, without apparent signs of orchitis or inflammation of accessory reproductive glands. Simultaneously, the semen of all bulls was subjected to sperm viability analysis and PCR based on the 16S rRNA gene. PCR-positive results of semen samples were confirmed by sequencing. A modified seminal plasma agglutination (MSPA) test, replacing the blood serum of all bulls in the MAT with seminal plasma was performed as well. Eight (8/203 = 3.9%) semen samples from bulls were considered nonviable (necrospermia and azoospermia) without relation to the PCR diagnosis. No agglutinin titers were identified in MSPA test. A high frequency (132/203 = 65%) of leptospiral agglutinin titers was identified in the MAT, particularly for the Sejroe serogroup (Hardjo CTG, 100/203 = 49.3%; Wolffi 74/203 = 36.4%; Guaricura 72/203 = 35.5%; and Hardjoprajitno 56/203 = 27.6%). Three (3/203 = 1.5%) semen samples of bulls were positive in the PCR, but these results were not confirmed by sequencing. The high frequency of serovars from the Sejroe serogroup typically adapted to bovines indicates the need for measures for the prophylaxis/control of the pathogen on the sampled farms. Discrepancies among the MAT, sperm viability, and molecular detection of leptospires in semen highlight the need for a combination of methods to diagnose leptospirosis in bovine bulls. To our knowledge, modified seminal plasma agglutination is described for the first time here to investigate anti-Leptospira antibodies produced locally in the genital tract in the diagnosis of bovine leptospirosis among bulls that reproduce by natural means.

Safety issues of raw milk: evaluation of bacteriological and physicochemical characteristics of human milk from a bank in a teaching hospital, focusing on Staphylococcus species.

Many infants are nurtured with milk supplied by human banks, whose bacteriological and physical-chemical profiles are a major issue. We investigated the bacteriological and physical-chemical characteristics, as well as genotypic and phenotypic and profiles of Staphylococcus species isolated from 240 samples of breast milk from a bank in a teaching hospital. Dornic acidity of milk revealed that 95.4% (229/240) had acceptable limits (< 8.0 oD). Caloric intake showed a wide variation in cream content (4%), fat (4%) and energy values (559.81 Kcal/L). Staphylococcus (105/186 or 56.5%) and Enterobacter (25/186 or 13.4%) were the most prevalent genera, although other microorganisms were identified, including Klebsiella pneumoniae and Pseudomonas aeruginosa. Amoxicillin/clavulanic acid (125/157 or 79.6%), vancomycin (115/157 or 73.2%), and cephalexin (112/157 or 71.3%) were the most effective antimicrobials. High resistance rates of isolates were found to penicillin G (141/157 or 89.8%), ampicillin (135/157 or 86%), and oxacillin (118/157 or 75.2%). Multidrug resistance to ≥ 3 antimicrobials occurred in 66.2% (123/186) of the isolates. Residues of microbial multiplication inhibitory substances were found in 85% (204/240) of samples. Among the coagulase-positive-CPS and negative-CoNS staphylococci, the mecA gene was detected in 53.3% (8/15) and 75% (30/40), respectively. Genes sea, seb and sec were detected in 20% (3/15) of CPS, while tsst-1 was detected in 13.34% (2/15). In addition, 13.3% (2/15) of S. aureus were toxin-producers. Genes sea, seb and sec were detected in 90% (36/40), 5% (2/40) and 15% (6/40) CoNS, respectively. Enterotoxin production was identified in 5% (2/40) of CoNS. The identification of multidrug-resistant bacteria, staphylococci species toxin-producers harboring methicillin-resistance genes, and residues of microbial multiplication inhibitory substances reinforce the need for a continuous vigilance of milk quality offered to infant consumption by human banks.

Bacteriological, cytological, and molecular investigation of Corynebacterium pseudotuberculosis, mycobacteria, and other bacteria in caseous lymphadenitis and healthy lymph nodes of slaughtered sheep.

Caseous lymphadenitis (CL) in sheep is a chronic contagious disease caused by Corynebacterium pseudotuberculosis, commonly characterized by abscess formation in peripheral lymph nodes and disseminated infections. Nonetheless, other microorganisms, including with zoonotic relevance, can be isolated from CL-resembling lymph nodes. Currently, mycobacteria have been reported in visceral granulomatous lesions in small ruminants, a fact that poses a public health issue, particularly in slaughtered sheep intended for human consumption. Cytology using fine needle aspiration and microbiological culturing are suitable tests for routine diagnostic, whereas present drawbacks and molecular methods have been confirmatory. Data about the occurrence of mycobacteria in both lymph nodes with aspect of CL and apparently healthy visceral nodes of sheep slaughtered for human consumption are scarce. In this study, 197 visceral lymph nodes of sheep showed lymphadenitis and 202 healthy visceral lymph nodes of slaughtered sheep intended for human consumption were submitted to conventional bacteriological diagnosis, mycobacteria culturing, and cytological evaluation. Compatible Corynebacterium isolates were subjected to multiplex PCR targeting 16S rRNA, rpoB, and pld genes to detect C. pseudotuberculosis. Based on microbiological identification, C. pseudotuberculosis (86/197; 43.7%), streptococci γ-hemolytic (17/197; 8.6%), and Trueperella pyogenes (12/197; 6.1%) were prevalent in lymph nodes with abscesses, as opposed to staphylococci (53/202; 26.2%) in apparently healthy lymph nodes. No mycobacteria were isolated. Cytology identified 49.2% (97/197) Gram-positive pleomorphic organisms (coryneform aspect). Multiplex PCR confirmed genetic material of C. pseudotuberculosis in 74.4% (64/86) of the samples with C. pseudotuberculosis isolation and 66% (64/97) samples with cytological coryneform aspect (κ = 86.78%; 95% CI = 79.87-93.68%). These findings emphasize the prevalence of C. pseudotuberculosis in abscess formation among peripheral lymph nodes of sheep. Other bacteria were also identified in lymph nodes sampled that resembling C. pseudotuberculosis-induced infections that may difficult the diagnosis. Multiplex PCR revealed a valuable assay to detect C. pseudotuberculosis, in addition to routine methods applied to CL-diagnosis. No mycobacteria were identified in lymph nodes sampled, with and without apparent lesions. Nonetheless, due to public health impacts, this pathogen should be considered as a differential diagnosis of C. pseudotuberculosis-induced infections during inspection procedures of slaughtered sheep intended for human consumption.

Real-time quantitative PCR-based detection of Coxiella burnetii in unpasteurized cow's milk sold for human consumption.

Coxiella burnetii is a zoonotic pathogen with a worldwide distribution that is responsible for Q fever in humans. It is a highly infectious bacterium that can be transmitted from cattle to humans through the consumption of unpasteurized milk. We report the molecular identification of C. burnetii in raw cow's milk being sold directly for human consumption in Brazil without official inspection or pasteurization. One hundred and twelve samples of raw milk were analysed by real-time quantitative PCR (qPCR), and C. burnetii was detected in 3.57% (4/112) of the samples at a concentration ranging from 125 to 404 bacteria per millilitre. The identification of this zoonotic pathogen in raw milk sold directly for human consumption is a public health concern since C. burnetii can be transmitted through the oral route. This result indicates that health education and other preventive measures should be officially implemented in Brazil to prevent the spread of infection. To our knowledge, this is the first qPCR-based detection of C. burnetii in raw milk samples from cows sold in Brazil that do not undergo official inspection or pasteurization.

Anti-influenza A virus antibodies in Tayassuidae from commercial rearing farms in Brazil / Detecção de anticorpos contra vírus influenza A em Tayassuidae de criações comerciais no Brasil

ABSTRACT: Family Tayassuidae in the suborder Suina include two species of peccaries in Brazil: the white-lipped peccary (Tayassu pecari) and the collared peccary (Pecari tajacu). These animals share common pathogens with domestic swine (Sus scrofa); however, their role as potential carrier remains unclear. This study focused on detecting the prevalence of influenza A antibodies in Tayassu pecari and Pecari tajacu from commercial rearing farms from two states in Brazil. A set of 50 blood samples from Pecari tajacu and 55 from Tayassu pecari were analyzed using a commercial indirect ELISA in order to investigate anti influenza A antibodies. Pecari tajacu samples presented 22% (11/50) of seropositivity for the virus. Serological surveillance is an important tool to identify the presence and the spread of the influenza virus in feral pigs.

RESUMO: A família Tayassuidae pertencente a subordem Suina e compreende duas espécies presentes no Brasil: Queixada (Tayassu pecari) e o Caititu (Pecari tajacu). Ambas as espécies compartilham patógenos com o suíno doméstico (Sus scrofa), entretanto o papel destes animais como carreadores destas infecções permanece indefinido. O presente estudo teve como objetivo detectar a ocorrência de anticorpos contra vírus influenza A em amostras de soro de rebanhos comerciais de queixada e caititu, provenientes de dois estados do Brasil. Um total de 50 amostras de soro de Pecari tajacu e 55 amostras de Tayassu pecari foram testadas por meio de ELISA, sendo que 22% (11/50) das amostras de Pecari tajacu foram soropositivas para o agente. Estudos de vigilância sorológica são importantes para identificar a presença e a disseminação do vírus influenza em suínos selvagens.

Identification of Mycobacterium species and Rhodococcus equi in peccary lymph nodes.

Mycobacterium species and the virulence-associated proteins (vapA, vapB, and vapN genes) of Rhodococcus equi isolated from 330 lymph nodes of collared peccaries (Tayassu tajacu) and white-lipped peccaries (Tayassu pecari) intended for human consumption were investigated. Thirty-six (10.9%) R. equi strains were isolated; 3.3% (n = 11/330) were from white-lipped peccary lymph nodes, and 7.6% (25/330) were from collared peccary lymph nodes. Among the 11 isolates of R. equi from the white-lipped peccaries, 90.9% (n = 10/11) were obtained from the mesenteric lymph nodes, and only 9.1% (n = 1/10) were obtained from the mediastinal lymph nodes. In the 25 isolates of R. equi obtained from the collared peccaries, 40.0% (n = 10/25) were recovered from the mesenteric lymph nodes, 36% (n = 9/25) from the submandibular lymph nodes, and 24.0% (n = 6/25) from the mediastinal lymph nodes. No vapA, vapB, or vapN genes (plasmidless) or three host-associated types (pVAPA, pVAPB, and pVAPN) were identified among the R. equi isolates. Mycobacterium species were isolated in 3.03% (n = 10/330) of all the lymph nodes analyzed. Among the 10 mycobacterial isolates, 60% (n = 6/10) were from the white-lipped peccary lymph nodes, and 40% (n = 4/10) were from the collared peccary lymph nodes. Ten Mycobacterium species were detected by PCR-PRA with a predominance of M. avium type 1. Sequencing of the hsp65 and rpob genes revealed mycobacteria that were saprophytic (M. sinense and M. kumamotonense) and potentially pathogenic (M. colombiense and M. intracellulare) to humans and animals. To our knowledge, this is the first description of R. equi and/or mycobacterial species identified in the lymph nodes of peccary specimens. R. equi (plasmidless) and the mycobacterial species described here have been reported as causes of pulmonary and extrapulmonary infections in both immunocompetent and immunocompromised humans.

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.

Unusual caudal vena cava thrombosis in a cow, secondary to Trueperella (Arcanobacterium) pyogenes infection / Rara trombose da veia cava caudal em bovino, secundária à infecção por Trueperella (Arcanobacterium) pyogenes

The caudal vena cava thrombosis, or pulmonary thromboembolism, in cattle is correlated with lactic acidosis, caused by diets rich in grains and highly fermentable, associated or not to septic situations, used in feedlots of beef or high-producing dairy cattle. This paper reports an unusual caudal vena cava thrombosis in a cow, secondary to Trueperella (Arcanobacterium) pyogenes infection, resulting in reduced milk production, anorexia, pale mucous membranes, ruminal atony, sternal decubitus and autoauscultation position. The heart was enlarged at necropsy, presence of clots distributed along the thoracic cavity, adherence between lung and pleura, abscesses, emphysema, petechiae, suffusions and ecchymosis in lungs, thickening of the caudal vena cava wall, hepatomegaly with chronic passive congestion ("nutmeg" aspect), and rumenitis. In lab, the actinomycete Trueperella (Arcanobacterium) pyogenes was isolated from liver and lung samples, probably resulting through dissemination of the bacteria of the rumen content, what reaffirms the opportunistic behavior of this actinomycete.(AU)

A síndrome da veia cava caudal ou tromboembolismo pulmonar bovino está relacionada à acidose láctica causada por dietas ricas em grãos e altamente fermentáveis, associados ou não a quadros sépticos, usadas em confinamentos de bovinos de corte ou para vacas leiteiras de alta produção. O presente artigo reporta caso raro de trombose da veia cava caudal em uma vaca, secundária a infecção por Trueperella (Arcanobacterium) pyogenes, apresentando reduzida produção de leite, anorexia, palidez de mucosas, atonia ruminal, decúbito esternal e posição de autoauscultação. À necrópsia observou-se coração aumentado de tamanho, coágulos distribuídos por toda cavidade torácica, aderência entre os pulmões e pleura, abscessos, enfisema, petéquias, sufusões, equimoses nos pulmões, espessamento da parede da veia cava caudal com trombo, hepatomegalia com congestão passiva crônica (aspecto de "noz moscada"), e ruminite. Em laboratório isolou-se o actinomiceto Trueperella (Arcanobacterium) pyogenes a partir de amostras de fígado e pulmão, provavelmente resultando da disseminação da bactéria proveniente do conteúdo ruminal, e reafirma o comportamento oportunista deste actinomiceto.(AU)

Occurrence of mycobacteria in bovine milk samples from both individual and collective bulk tanks at farms and informal markets in the southeast region of Sao Paulo, Brazil.

BACKGROUND: Mycobacterium spp. is one of the most important species of zoonotic pathogens that can be transmitted from cattle to humans. The presence of these opportunistic, pathogenic bacteria in bovine milk has emerged as a public-health concern, especially among individuals who consume raw milk and related dairy products. To address this concern, the Brazilian control and eradication program focusing on bovine tuberculosis, was established in 2001. However, bovine tuberculosis continues to afflict approximately 1,3 percent of the cattle in Brazil. In the present study, 300 samples of milk from bovine herds, obtained from both individual and collective bulk tanks and informal points of sale, were cultured on Löwenstein-Jensen and Stonebrink media. Polymerase chain reaction (PCR)-based tests and restriction-enzyme pattern analysis were then performed on the colonies exhibiting phenotypes suggestive of Mycobacterium spp., which were characterized as acid-fast bacilli. RESULTS: Of the 300 bovine milk samples that were processed, 24 were positively identified as Mycobacterium spp.Molecular identification detected 15 unique mycobacterial species: Mycobacterium bovis, M. gordonae, M. fortuitum, M. intracellulare, M. flavescens, M. duvalii, M. haemophilum, M. immunogenum, M. lentiflavum, M. mucogenicum, M. novocastrense, M. parafortuitum, M. smegmatis, M. terrae and M. vaccae. The isolation of bacteria from the various locations occurred in the following proportions: 9 percent of the individual bulk-tank samples, 7 percent of the collective bulk-tank samples and 8 percent of the informal-trade samples. No statistically significant difference was observed between the presence of Mycobacterium spp. in the three types of samples collected, the milk production profiles, the presence of veterinary assistance and the reported concerns about bovine tuberculosis prevention in the herds. CONCLUSION: The microbiological cultures associated with PCR-based identification tests are possible tools for the investigation of the presence of Mycobacterium spp. in milk samples. Using these methods, we found that the Brazilian population may be regularly exposed to mycobacteria by consuming raw bovine milk and related dairy products. These evidences reinforces the need to optimize quality programs of dairy products, to intensify the sanitary inspection of these products and the necessity of further studies on the presence of Mycobacterium spp. in milk and milk-based products.