RESUMO: O objetivo deste estudo foi avaliar a eficácia da antibioticoterapia associada à vacinação sobre a taxa de cura microbiológica de mastite subclínica causada por Staphylococcus aureus em vacas leiteiras em lactação. Foram selecionados 5 rebanhos, dos quais 72 vacas (120 quartos mamários, QM) foram diagnosticadas com mastite subclínica por S. aureus e foram alocadas aleatoriamente em um de três grupos de tratamento: a) Controle (sem tratamento); b) ATB (antibioticoterapia); e c) ATB+VAC (antibioticoterapia mais vacinação contra S. aureus). O tratamento intramamário consistiu em infusão de ampicilina 75 mg + cloxacilina 200 mg duas vezes ao dia, durante 5 dias. O tratamento parenteral foi feito por injeção de uma dose única (7,5 mg/kg) de enrofloxacina, no primeiro dia do protocolo de tratamento. As vacas vacinadas receberam três doses de uma vacina comercial, 14 dias antes do tratamento (d-14), no primeiro dia do protocolo de tratamento (d1) e 14 dias após o protocolo de tratamento (d+14). A taxa de cura das vacas não tratadas foi menor (0,06) do que das vacas tratadas com ATB (0,84) e ATB+VAC (0,85). Não foi observada diferença de taxa de cura entre vacas tratadas com ATB e ATB+VAC. Por outro lado, as vacas vacinadas apresentaram menor a contagem de células somáticas (CCS) após 28 dias de tratamento (4,76 log10) do que em vacas não tratadas (5,37 log10). Em conclusão, o tratamento com ampicilina e cloxacilina intramamária, associados à enrofloxacina intramuscular, apresentou alta taxa de cura para MSC causada por S. aureus durante a lactação. A utilização da vacinação contra S. aureus associada à antibioticoterapia não aumentou a taxa de cura dos QM durante a lactação, mas foi eficaz na redução do CCS quando comparada à QM não tratados. Entretanto, para ter certeza que a diminuição da CCS no grupo ATB+VAC estivesse associada à vacinação, o estudo deveria ter incluído um grupo adicional de apenas vacas vacinadas, sem terapia antimicrobiana, o que não foi feito no presente estudo e, portanto, é um das limitações do protocolo experimental utilizado.

ABSTRACT: The objective of this study was to evaluate the efficacy of the antibiotic therapy associated with vaccination on the microbiological cure rate of subclinical mastitis caused by Staphylococcus aureus in lactating dairy cows. A total of five herds, from which 72 cows (120 mammary quarters - MQ) were diagnosed with S. aureus subclinical mastitis, were included in this study. Cows were randomly allocated to one of three treatment groups: a) Control (no treatment); b) ATB (antibiotic therapy); and c) ATB+VAC (antibiotic therapy plus vaccination against S. aureus). Intramammary treatment consisted of twice-daily infusion of ampicillin 75mg + cloxacillin 200mg, for 5 days. Parenteral treatment was done by injection of a single dose (7.5mg/kg) of enrofloxacin, on the first day of the treatment protocol. Vaccinated cows received three doses of a commercial vaccine 14 days before treatment (d-14), on the first day of treatment protocol (d1), and 14 days after the treatment protocol (d+14). Non-treated cows had a lower cure rate (0.06) than cows treated with ATB (0.84) and ATB+VAC (0.85). No difference in cure rate was observed between cows treated with ATB and ATB+VAC. On the other hand, vaccinated cows had lower somatic cell count (SCC) after 28 days of the treatment protocols (4.76 log10) than non-treated cows (5.37 log10). In conclusion, treatment with intramammary ampicillin and cloxacillin, associated with intramuscular enrofloxacin presented a high cure rate for SCM caused by S. aureus during lactation. The use of vaccination against S. aureus in association with antibiotic therapy did not increase the cure rate of MQ during lactation, but it was effective in reducing the SCC when compared to non-treated MQ. Although to ensure that the decrease of the SCC in ATB+VAC group was associated with the vaccination, the study should have included an additional group of only vaccinated cows, without antimicrobial therapy, with was not done in the present study, and therefore is one of the limitations of the experimental protocol used.

RESUMO: Enterococcus são reconhecidos mundialmente como significantes agentes nosocomiais, que têm continuamente se adaptado a diferentes nichos e adquirido resistência a várias classes de antibióticos. Cepas de E. faecalis e E. faecium vancomicina e gantamicina-resistentes têm sido associadas a infecções nosocomiais em humanos. Alguns estudos epidemiológicos sugerem a participação de aves como reservatórios de cepas de Enterococcus vancomicina e gentamicina-resistentes. Entretanto, a relação das aves de companhia como reservatórios destas cepas tem sido pouco estudada. Neste estudo, 126 psitacídeos foram avaliados, e 26,9% destes eram portadores de Enterococcus spp., incluindo as espécies E. faecalis, E. faecium, E. hirae, E. phoeniculicola, E. gallinarum e E. casseliflavus. O perfil de resistência antibiótica mostrou quatro cepas com alto nível de resistência a gentamicina (ANRG). Além de duas cepas com nível intermediário de resistência a vancomicina. As cepas resistentes foram isoladas de amostras fecais e de orofaringe de aves doentes e clinicamente saudáveis, sugerindo que psitacídeos podem estar atuando como reservatórios para Enterococcus spp. com ANRG. Contudo, Aves doentes parecem estar mais relacionadas à transmissão de enterococcus, do que aves saudáveis.

ABSTRACT: Enterococcus are recognized worldwide as significant nosocomial agents that have been continuously envolving to adapt to different niches and acquire resistance to several antibiotic classes. Vancomycin and gentamicin-resistant strains of E. faecalis and E. faecium have been associated with nosocomial human infections. Some epidemiological studies suggest the participation of pets as reservoirs of vancomycin and gentamicin-resistant Enterococcus strains. However, the role of companion birds as reservoirs of these strains has been poorly studied. In this study, 126 psittacine birds were evaluated and 26.9% carried Enterococcus spp., including the species E. faecalis, E. faecium, E. hirae, E. phoeniculicola, E. gallinarum and E. casseliflavus. The antibiotic resistance profile showed four high-level gentamicin-resistance (HLGR) strains. In addition, two strains presented intermediate levels of vancomycin resistance. Resistant strains were isolated from fecal and oropharynx samples of sick and clinically healthy birds, suggesting that psittacine birds may act as reservoirs of HLGR Enterococcus spp. However, sick birds appear to be more implicated in the enterococci transmission than healthy birds.

Leptospira interrogans serovar Canicola is one of the most important pathogenic serovars for the maintenance of urban leptospirosis. Even though it is considered highly adapted to dogs, serovar Canicola infection has already been described in other animals and even a few human cases. Here, we present the genomic characterisation of two Brazilian L. interrogans serovar Canicola strains isolated from slaughtered sows (L0-3 and L0-4) and their comparison with human strain Fiocruz LV133. It was observed that the porcine serovar Canicola strains present the genetic machinery to cause human infection and, therefore, represent a higher risk to public health. Both human and porcine serovar Canicola isolates also presented sequences with high identity to the Chinese serovar Canicola published plasmids pGui1 and pGui2. The plasmids identification in the Brazilian and Chinese serovar Canicola strains suggest that extra-chromosomal elements are one more feature of this serovar that was previously unnoticed.

Leptospira inadai is classified as a species of the Leptospira intermediate group that has been poorly studied due to its apparent insignificance to human and animal health. Nevertheless, over the last two decades the species has been described in human cases in India and in carrier animals in Ecuador. Here, we present the first identification and genomic characterisation of L. inadai serogroup Lyme isolated from captured rodent in Brazil. Even though the M34/99 strain was not pathogenic for hamsters, it was able to establish renal colonisation. The M34/99 strain presented high similarity with L. inadai serogroup Lyme human reference indicating that animal strain could also infect humans, although it does not represent high risk of severe disease. An extrachromosomal sequence was also identified in M34/99 strain and presented high identity with previously described L. inadai phage LinZ_10, suggesting that phage-like extrachromosomal sequence may be another feature of this understudied species.

Livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) has been mainly related with pig farming, in Europe and North America, with the ST398 as the most commonly identified type of LA-MRSA. Here we present the draft genome of the first vancomycin-intermediate MRSA ST398/t9538 isolated from a swine presenting exudative epidermitis in Brazil.

Resumo: Streptococcus suis é um dos patógenos de maior importância para indústria suinícola mundial. Apesar de sua importância, a caracterização de isolados de S. suis na América do Sul ainda é pouco descrita. O presente estudo descreve a avaliação de isolados de S. suis provenientes de diferentes Estados brasileiros, e sua caracterização sorológica e molecular. Foram avaliados 174 isolados de S. suis e os mesmos foram submetidos a SE-AFLP e pesquisa de marcadores de virulência. Os sorotipos 1, 2, 3, 4, 7, 18, 22 e 32 foram identificados dentre os isolados estudados e apenas 4% foram caracterizados como não tipáveis. O perfil de virulência mrp+/epf+/sly+ foi o mais frequente. A análise do SE-AFLP resultou em 29 perfis distribuídos em três grupos principais com mais de 65% de similaridade genética. Os isolados apresentaram tendência de se agrupar segundo origem e sorotipo; no entanto, não foi observada correlação entre os grupamentos e os perfis de virulência.

Abstract: Streptococcus suis is one of most important pathogens in the swine industry worldwide. Despite its importance, studies of S. suis characterization in South America are still rare. This study evaluates S. suis isolates from distinct Brazilian states, from 1999 to 2004, and its molecular and serological characterization. A total of 174 isolates were studied. S. suis identification was confirmed by PCR and isolates were further serotyped and genotyped by SE-AFLP and amplification of virulence markers. Serotype 1, 2, 3, 4, 7, 18, 22 and 32 were identified among the studied isolates, and only 4% were characterized as non-typeable. The mrp+/epf+/sly+ genotype was the most frequent. The SE-AFLP analysis resulted in 29 patterns distributed in three main clusters with over 65% of genetic similarity. Isolates presented a slight tendency to cluster according to serotype and origin; however, no further correlation with virulence genotypes was observed.

Leptospira kirschneri is one of the pathogenic species of the Leptospira genus. Human and animal infection from L. kirschneri gained further attention over the last few decades. Here we present the isolation and characterisation of Brazilian L. kirschneri serogroup Pomona serovar Mozdok strain M36/05 and the comparative genomic analysis with Brazilian human strain 61H. The M36/05 strain caused pulmonary hemorrhagic lesions in the hamster model, showing high virulence. The studied genomes presented high symmetrical identity and the in silico multilocus sequence typing analysis resulted in a new allelic profile (ST101) that so far has only been associated with the Brazilian L. kirschneri serogroup Pomona serovar Mozdok strains. Considering the environmental conditions and high genomic similarity observed between strains, we suggest the existence of a Brazilian L. kirschneri serogroup Pomona serovar Mozdok lineage that could represent a high public health risk; further studies are necessary to confirm the lineage significance and distribution.

In the present paper, we announce new draft genomes of four Leptospira interrogans strains named Acegua, RCA, Prea, and Capivara. These strains were isolated in the state of Rio Grande do Sul, Brazil, from cattle, dog, Brazilian guinea pig, and capybara, respectively.

O período de desmame nos leitões é caracterizado por alterações fisiológicas como menor ganho de peso, aumento na produção de espécies reativas de oxigênio (EROs) e aumento na concentração plasmática de cortisol com possíveis implicações para a resposta imune. Foi avaliado o efeito da administração parenteral das vitaminas A, D e E sobre o desempenho produtivo, o metabolismo oxidativo e a função de leucócitos polimorfonucleares (PMNLs) em suínos durante esta fase de crescimento. Foram utilizados 20 leitões, machos, com 20 dias de idade que receberam ADE injetável (135.000 UI vitamina A, 40.000 UI vitamina D e 40mg vitamina E/animal), aos 20 e 40 dias de idade. Foi determinado o ganho de peso e as concentrações de glutationa reduzida (GSH), malondialdeído (MDA) e superóxido dismutase (SOD) e a capacidade microbicida e fagocítica dos PMNLs. Não houve diferença entre o peso vivo médio durante o experimento, porém maior ganho de peso percentual foi observado 20 dias após o desmame para o grupo tratado. As concentrações de GSH e SOD não diferiram entre os grupos, porém a lipoperoxidação foi maior no grupo controle aos 60 dias de idade. As correlações entre as variáveis do metabolismo oxidativo foram -0,41 para GSH e o MDA, -0,54 para GSH e SOD e 0,34 para MDA e SOD. A intensidade da produção intracelular de EROs, a porcentagem de PMNLs que produziram EROs e a intensidade de fagocitose dos PMNLs não diferiram entre os tratamentos. A administração de ADE injetável melhorou o ganho de peso percentual no período de 20 a 40 dias de idade, diminuiu o estresse oxidativo aos 60 dias de idade e não influenciou função dos PMNLs dos leitões.

The weaning period of piglets is characterized by physiological alterations, such as decreased weight gain, increased reactive oxygen species (ROS) and increased serum cortisol levels with possible effects on the immune response. The effect of parenteral administration of vitamins A, D and E on production performance, oxidative metabolism, and the function of polymorphonuclear leukocytes (PMNLs) was assessed in piglets during the weaning period. The sample was comprised of 20 male piglets that were given an injectable ADE vitamin combination (135,000 IU vitamin A, 40,000 IU vitamin D and 40mg vitamin E/ animal) at 20 and 40 days of age. Weight gain, concentration of reduced glutathione (GSH), malondialdehyde (MDA), superoxide dismutase (SOD) and the microbicidal and phagocytic activity of PMNLs were assessed. No difference was observed in the average piglet weight during the study; however, a greater percentage of weight gain was observed after weaning in the treated group. The concentrations of GSH and SOD did not differ between groups, although lipid peroxidation was greater in the control group at 60 days of age. The investigated variables of oxidative metabolism were correlated as follows: -0.41 for GSH and MDA, -0.54 for GSH and SOD and 0.34 for MDA and SOD. The intensity of intracellular ROS production, the percentage of ROS-producing PMNLs and the intensity of phagocytosis by PMNLs did not differ between treatment groups. Administration of the injectable ADE combination improved the percentage of weight gain between 20 and 40 days of age, decreased oxidative stress at 60 days of age and did not influence the function of PMNLs in piglets.

Amostras de Escherichia coli isoladas de 24 psitacídeos doentes foram sorogrupadas e investigadas para a presença de genes que codificam os seguintes fatores de virulência: attaching e effacing (eae), plasmídeo EAF (EAF), pili associado à pielonefrite (pap), fímbria S (sfa), adesina afimbrial (afa), cápsula K1 (neu), curli (crl, csgA), hemaglutinina termosensível (tsh), enterotoxina termo-estável 1 de E. coli enteroagregativa (astA), toxina termolábil (LT) e toxina termoestável (STa e STb), Shiga-like toxinas (stx1 e stx2), fator citotóxico necrotizante 1 (cnf1), hemolisina (hly), produção de aerobactina (iuc) e resistência sérica (iss). Os resultados mostraram que os isolados pertenciam a 12 sorogrupos: O7; O15; O21; O23; O54; O64; O76; O84; O88; O128; O152 e O166. Os genes de virulência encontrados foram: crl em todos os isolados, pap em 10 isolados, iss em sete isolados, csgA em cinco isolados, iuc e tsh em três isolados e eae em dois isolados. A combinação dos genes de virulência revelou 11 perfis genotípicos distintos. Todas as amostras foram negativas para os genes que codificam EAF, EAEC, K1, sfa, afa, hly, cnf, LT, STa, STb, stx1 e stx2. Estes resultados demonstraram que algumas amostras de E. coli isoladas de psitacídeos apresentam os mesmos fatores de virulência presentes nos patotipos de E. coli patogênicas para aves (APEC), uropatogênicas (UPEC) e E. coli enteropatogênicas (EPEC).

Escherichia coli isolates from 24 sick psittacine birds were serogrouped and investigated for the presence of genes encoding the following virulence factors: attaching and effacing (eae), enteropathogenic E. coli EAF plasmid (EAF), pili associated with pyelonephritis (pap), S fimbriae (sfa), afimbrial adhesin (afa), capsule K1 (neu), curli (crl, csgA), temperature-sensitive hemagglutinin (tsh), enteroaggregative heat-stable enterotoxin-1 (astA), heat-stable enterotoxin -1 heat labile (LT) and heat stable (STa and STb) enterotoxins, Shiga-like toxins (stx1 and stx2), cytotoxic necrotizing factor 1 (cnf1), haemolysin (hly), aerobactin production (iuc) and serum resistance (iss). The results showed that the isolates belonged to 12 serogroups: O7; O15; O21; O23; O54; O64; O76; O84; O88; O128; O152 and O166. The virulence genes found were: crl in all isolates, pap in 10 isolates, iss in seven isolates, csgA in five isolates, iuc and tsh in three isolates and eae in two isolates. The combination of virulence genes revealed 11 different genotypic patterns. All strains were negative for genes encoding for EAF, EAEC, K1, sfa, afa, hly, cnf, LT, STa, STb, stx1 and stx2. Our findings showed that some E. coli isolated from psittacine birds present the same virulence factors as avian pathogenic E. coli (APEC), uropathogenic E. coli (UPEC) and Enteropathogenic E. coli (EPEC) pathotypes.

Um estudo de caso-controle em leitegadas de um a sete dias de idade foi realizado com o objetivo de identificar os principais agentes infecciosos envolvidos na diarreia neonatal de leitões. As amostras de fezes (n=276) foram coletadas em granjas de suínos no Estado do Rio Grande do Sul, Brasil, no período de maio a setembro de 2007. Leitegadas com diarreia foram consideradas casos (n=147) e leitegadas normais, controles (n=129). As amostras foram examinadas através do teste de aglutinação em látex, PAGE, cultivo, ELISA, PCR e métodos microscópicos para a excreção dos principais agentes de diarreia: virais (rotavirus), bacterianos (Escherichia coli, Clostridium perfringens tipos A e tipo C e Clostridium difficile) e parasitários (coccídeos e Cryptosporidium spp.). Durante o período do estudo não foram observados surtos e a diarréia, quando presente, apresentou-se leve. Pelo menos um agente foi identificado nas amostras fecais de 25 entre 28 granjas (89,3%) analisadas e em 16 granjas (57,1%) mais de um agente foi detectado. Os principais agentes encontrados nas granjas foram coccídeos (42,86%) e rotavírus (39,29%). Os principais agentes detectados nas leitegadas com diarreia foram Clostridium difficile (10,6%), Clostridium perfringens tipo A (8,8%) e rotavírus (7,5%). Por outro lado, nas leitegadas controle os agentes mais prevalentes foram Clostridium difficile (16,6%) e coccídeos (8,5%). E. coli Beta hemolítica e Clostridium perfringens tipo C não foram detectados. O presente estudo de caso-controle demonstrou que nenhum agente infeccioso esteve associado significativamente com diarreia (p>0.05). Esses achados reforçam a necessidade de que haja cuidado na interpretação de resultados de exames laboratoriais em materiais coletados de leitões com diarreia neonatal leve, pois a detecção isolada de um agente infeccioso não indica necessariamente que o mesmo seja a causa do problema.

A case-control study was carried out in litters of 1 to 7-day-old piglets to identify the main infectious agents involved with neonatal diarrhea in pigs. Fecal samples (n=276) from piglets were collected on pig farms in the State of Rio Grande do Sul, Brazil, from May to September 2007. Litters with diarrhea were considered cases (n=129) and normal litters (n=147) controls. The samples were examined by latex agglutination test, PAGE, conventional isolating techniques, ELISA, PCR, and microscopic methods in order to detect rotavirus, bacterial pathogens (Escherichia coli, Clostridium perfringens type A and C, and Clostridium difficile), and parasites (Coccidian and Cryptosporidium spp.). Outbreaks of diarrhea were not observed during sampling. At least one agent was detected in fecal samples on 25 out of 28 farms (89.3%) and in 16 farms (57.1%) more than one agent was found. The main agents diagnosed were Coccidia (42.86%) and rotavirus (39.29%). The main agents identified in litters with diarrhea were Clostridium difficile (10.6%), Clostridium perfringens type A (8.8%) and rotavirus (7.5%); in control litters, Clostridium difficile (16.6%) and Coccidian (8.5%). Beta hemolytic Escherichia coli and Clostridium perfringens type C were not detected. When compared with controls, no agent was significantly associated with diarrhea in case litters. These findings stress the need for caution in the interpretation of laboratorial diagnosis of mild diarrhea in neonatal pigs, as the sole detection of an agent does not necessarily indicate that it is the cause of the problem.