Bacterial pneumonia in captive wild boars in southern Brazil: etiological and pathological causes

Respiratory diseases are one of the major health issues described in intensive pig production, causing important economic losses. However, there is little information on the prevalence, etiology and clinical-pathological presentation of these diseases in wild boars. For this reason, this work investigated the presence in captive wild boars of pneumonic lesions and bacterial pathogens commonly detected and associated with respiratory diseases in domestic pigs. A total of 226 captive wild boar lungs from two farms were examined in a slaughterhouse in Southern Brazil. The pneumonic lesions were classified as cranioventral, dorsocaudal, and disseminated, and the quantification of lesions was calculated. From the total of 226 lungs, 121 were collected for laboratory examination. Lungs with macroscopic lesions suggestive of pneumonia were collected for histological, bacteriological and molecular analysis. The molecular analysis was performed to detect the presence of Actinobacillus (A.) pleuropneumoniae, Glaesserella (G.) parasuis, Mycoplasma (M.) hyopneumoniae, Mycoplasma (M.) hyorhinis and Streptococcus (S.) suis serotype 2. The percentages of histological lesions and bacterial agents and their association were calculated. Cranioventral consolidation (75.2%) was the most prevalent macroscopic lung lesion, followed by disseminated (21.5%) and dorsocaudal (3.3%) distribution. Microscopically, chronic lesions were the most prevalent, representing 70.2% of the lungs. Moreover, BALT hyperplasia was present in 86.5% of the lungs, suppurative bronchopneumonia in 65.7%, and alveoli infiltrate in 46.8%. Six bacterial pathogens commonly described as agents of pig pneumonia were identified by bacterial or molecular methods: Pasteurella (P.) multocida, S. suis, M. hyopneumoniae, A. pleuropneumoniae, G. parasuis and M. hyorhinis. Twenty-eight different combinations of pathogens were identified in 84 samples (69.4%). The most common combinations were: M. hyopneumoniae and A. pleuropneumoniae (13.1%), M. hyopneumoniae, G. parasuis and M. hyorhinis (10.7%), and M. hyopneumoniae, A. pleuropneumoniae and G. parasuis (8.3%). Additionally, M. hyopneumoniae was the most frequent pathogen detected in this study, representing 58.7% of the samples. The detection of M. hyopneumoniae and M. hyorhinis by PCR was associated with the presence of BALT hyperplasia (P < 0.05) and there was also an association between the detection of M. hyopneumoniae by PCR and suppurative bronchopneumonia (P < 0.05). In addition, a significant association (P < 0.05) between the detection of M. hyopneumoniae and A. pleuropneumoniae by PCR and the histological classification (acute, subacute or chronic lesions) was observed. The results of this study were similar to those observed in slaughtered domestic pigs, although, the detection of opportunist pathogens was less frequent than that usually described in pig pneumonia. The high prevalence of pneumonia in captive wild boars at slaughter and the similar characteristics of pneumonia in captive wild boars and domestic pigs suggest that the close phylogenetic relationship between pigs and wild boars could influence the susceptibility of both species to the colonization of the same pathogens, indicating that captive wild boars raised in confined conditions could be predisposed to respiratory diseases, similar to domestic pigs.(AU)

Characterization of Streptococcus suis through serotyping, SE-AFLP and virulence profile / Caracterização de Streptococcus suis por sorotipagem, SE-AFLP e perfil de virulência

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.(AU)

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.(AU)

Characterization of Streptococcus suis through serotyping, SE-AFLP and virulence profile / Caracterização de Streptococcus suis por sorotipagem, SE-AFLP e perfil de virulência

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.(AU)

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.(AU)

DOENÇAS DO SISTEMA NERVOSO CENTRAL DE SUÍNOS

Doenças do sistema nervoso são frequentemente observadas na clínica de suínos e podem ser de origem infecciosa, não infecciosa ou multifatorial. As doenças infecciosas são a principal causa de morte em suínos e envolvem bactérias como Escherichia coli, Streptococcus suis (S. suis), Glaesserella (Haemophilus) parasuis e Salmonella choleraesuis. S. suis é um agente zoonótico, responsável por inflamação das meninges, válvulas cardíacas, pulmões e articulações de suínos e humanos. Nas lesões de suínos, o sorotipo 2 é o mais frequentemente isolado. Agentes virais como circovírus suíno apresentam elevado impacto na suinocultura e ocorre de forma endêmica nos rebanhos brasileiros. Foram realizados três estudos. No primeiro foi determinada, através de um estudo retrospectivo, a frequência, distribuição sazonal e anual das doenças que acometem o sistema nervoso central (SNC) de suínos no Sul do Brasil. Foram analisados 231 casos de suínos com lesão microscópica no SNC, distribuídos nas fases de maternidade, creche, crescimento, terminação e adultos. As doenças de origem infecciosa foram responsáveis por 87,01% das lesões no sistema nervoso de suínos. Leitões na fase de creche foram a categoria mais afetada (58,01%). A meningite bacteriana teve o maior número de observações no estudo, com tendência de ocorrência nos meses de outono/inverno e com caráter endêmico. Não foi observada associação sazonal ao número de diagnósticos nos casos de circovírus suíno, intoxicação por sal e abscessos encefálicos. Os casos de encefalomielite não supurativa e intoxicação por selênio tiveram maior número de observações no inverno e verão, respectivamente. No segundo estudo foram investigados agentes virais envolvidos em um surto de doença neurológica, na qual observou-se incoordenação motora, paresia e paralisia dos membros pélvicos. Nesse surto, ocorrido em uma granja no Sul do Brasil, 10 leitões com idade de 40 a 65 dias, foram submetidos ao exame post mortem. As lesões macroscópicas eram restritas a medula espinhal e caracterizadas por aumento de volume de raízes nervosas e gânglios, principalmente em região cervical e sacral. Microscopicamente as lesões eram caracterizadas por encefalomielite e ganglioneurite não supurativas. Amostras refrigeradas de SNC, líquido cefalorraquidiano e fezes foram analisadas molecularmente. A reação de polimerase em cadeia (PCR) de 10 amostras de SNC foram positivas para Teschovirus A, oito foram positivas para Sapelovirus A e sete para Enterovirus G. Mammastrovirus foi identificado em pool de fezes de leitões sadios. O terceiro manuscrito teve como objetivo descrever os aspectos clínicos, patológicos e moleculares de um surto de infecção por S. suis com elevada mortalidade. Os sinais clínicos iniciavam em torno de 10 dias pós-desmame e eram caracterizados por apatia, aumento de volume articular e relutância ao movimento. Em 24 horas, os sinais clínicos evoluíam para decúbito lateral, opistótono, nistagmo e movimentos de pedalagem. Macroscopicamente observava-se hiperemia dos vasos da leptomeninge e deposição de fibrina sobre a leptomeninge. Havia ainda espessamento de plexo coroide associado a dilatação de ventrículos laterais. Microscopicamente foi observada inflamação fibrinossupurativa em leptomeninges do cérebro e medula espinhal. Esse mesmo infiltrado era observado no plexo coroide, epêndima, raízes nervosas e no canal central da medula espinhal. A PCR identificou S. suis sorotipo 9 neste surto.

Nervous system diseases are frequently seen in the swine clinic and can be of infectious, non-infectious or multifactorial origin. Infectious diseases are the leading cause of death in pigs and involve bacteria such as Escherichia coli, Streptococcus suis (S. suis), Glaesserella (Haemophilus) parasuis and Salmonella choleraesuis. S. suis is a zoonotic agent, responsible for inflammation of the meninges, heart valves, lungs and joints of pigs and humans. In pig lesions, serotype 2 is the most frequently isolated. Viral agents such as porcine circovirus have a high impact on pig production and occur endemically in Brazilian herds. Three studies were carried out. In the first, it was determined, through a retrospective study, the frequency, seasonal and annual distribution of diseases that affect the central nervous system (CNS) of swine in southern Brazil. Two hundred thirty-one cases of pigs with lesions in the CNS were analyzed, distributed in the phases of suckling piglets, nursery, growing, finishing, and adults Infectious diseases were responsible for 87.01% of swine nervous system lesions. Piglets in the nursery phase were the most affected category (58.01%). Bacterial meningitis had the highest number of observations in the study, with a tendency to occur in the autumn/winter months and endemic character. There was no seasonal association with the number of diagnoses in cases of porcine circovirus, salt poisoning and brain abscesses. The cases of non-suppurative encephalomyelitis and selenium intoxication had the highest number of observations in winter and summer, respectively. In the second study, viral agents involved in an outbreak of neurological disease were investigated, which motor incoordination, paresis and paralysis of the posterior limbs were observed. In this outbreak, which occurred on a farm in southern Brazil, 10 piglets aged 40 to 65 days were submitted to post-mortem examination. Macroscopic lesions were restricted to the spinal cord and characterized by an increase in the volume of nerve roots and ganglia, mainly in the cervical and sacral regions. Microscopically, the lesions were characterized by encephalomyelitis and non-suppurative ganglioneuritis. Refrigerated samples of CNS, cerebrospinal fluid and feces were analyzed molecularly. The plymerase chain reaction (PCR) of 10 CNS samples were positive for Teschovirus A, eight were positive for Sapelovirus A and seven for Enterovirus G. Mammastrovirus was identified in a fecal pool of healthy piglets. The third manuscript aimed to describe the clinical, pathological and molecular aspects of an outbreak of S. suis infection with high mortality. The clinical signs started about 10 days after weaning and was characterized by acute nervous and locomotor disorders. Macroscopically, there was an increase in joint volume, hyperemia of the vessels of the leptomeninges and deposition of fibrin. There was also choroidal plexus thickening associated with dilation of lateral ventricles. Microscopically, fibrinosuppurative inflammation was observed in leptomeninges of brain and spinal cord. This same infiltrate was observed in choroid plexus, ependyma, nerve roots and in central canal of the spinal cord. PCR identified S. suis serotype 9 in this outbreak.

Análise comparativa de genomas de estirpes de Streptococcus suis isoladas de suínos no Brasil

Streptococcus suis é um patógeno de grande importância na indústria suína sendo capaz de provocar diferentes quadros clínicos em animais de várias idades e, além disso, também é considerado um agente zoonótico. Dessa forma, é de extrema importância a caracterização das estirpes que causam doença em suínos no Brasil. Os objetivos deste estudo foram a caracterização fenotípica e genotípica de 215 estirpes de S. suis isoladas entre os anos de 2001 e 2016, de suínos com sinais clínicos de infecção, provenientes de diferentes Estados do Brasil. Para tanto, as estirpes isoladas neste período e mantidas a 86C foram reativadas e submetidas à confirmação da identificação por PCR e espectrometria de massa MALDI TOF. Em seguida, foi realizada sorotipagem molecular e a identificação de genes de virulência pela PCR, e a foi realizada a genotipagem pelas técnicas de AFLP e PFGE. Para a identificação dos perfis de resistência aos antimicrobianos foi utilizada a técnica de microdiluição em caldo, obtendo os valores de concentração inibitória mínima (CIM). A partir dos resultados fenotípicos e genotípicos foram selecionadas 24 estirpes para o sequenciamento do genoma utilizando a plataforma Illumina® MiSeq. O sorotipo mais frequentemente identificado foi o 2 / ½ (82,8%); seguido em menor proporção pelos sorotipos 3, 4, 6, 7, 8, 9, 18, 27, 28, 1 / 14, sendo apenas seis estirpes não tipaveis (NT). Para caracterização do perfil de virulência foram avaliados quatro genes sly, arcA, epf e as variantes de mrp sendo identificados 11 perfis de virulência nas estirpes estudadas. A genotipagem por AFLP identificou 30 perfis genotípicos e a técnica de PFGE identificou 64 pulsotipos. Para ambas as técnicas não foi identificada uma correlação clara entre dados epidemiológicos e genotípicos, mas pode se observar uma tendência de agrupamento das estirpes por ano de isolamento. A partir dos resultados de CIM foram identificados nove perfis de susceptibilidade aos antimicrobianos com 72,1% das estirpes classificadas como multirresistentes. Foram detectadas altas taxas de resistência às tetraciclinas, macrolídeos, clindamicina e sulfadimetoxina, enquanto beta--lactâmicos e florfenicol foram os antimicrobianos mais eficazes. A partir da análise genômica não foram detectados marcadores de plasmídeos e apenas três genes de resistência foram identificados nos genomas brasileiros; destaca--se o gene ermB, que codifica resistência à macrolídeos e lincosamidas, que foi detectado em 79,2% das estirpes estudadas. Foram identificadas oito novas sequências de alelos na MLST e uma nova combinação alélica, de tal forma que foram identificadas seis novas sequências tipo (STs) dentre os 24 genomas analisados. A partir do mapeamento das nove estirpes do sorotipo 2 foi identificado um elemento genético móvel (CMGETZ080501), relacionado à resistência aos antimicrobianos, em sete destas; as duas estirpes Brasileiras do sorotipo 2 que se diferenciam das demais (45 e 47) foram as mais sensíveis. Em relação às cinco estirpes do sorotipo 3, foi observado no mapeamento que duas destas se distanciam das demais e apresentam maior similaridade ao genoma da referência do sorotipo 4. Este resultado também foi verificado tanto na wgSNP quanto na análise da MLST, confirmando que a caraterização capsular de S. suis não deve ser utilizada como um indicativo de similaridade genética. Tendo em vista a variabilidade genética e a alta frequência de multirresistência identificada nas estirpes de S. suis circulantes no país, é possível avaliar as razões para que o controle do agente continue sendo um grande desafio para a suinocultura intensiva nacional.

Streptococcus suis is a pathogen of great importance in the pig industry and can cause diverse clinical conditions in animals of different ages, in addition of this, is also considered a zoonotic agent. Thus, is essential the characterization of strains that causes disease in swine from Brazil. The current study aimed phenotypic and genotypic characterization of 215 S.suis strains isolated between 2001 and 2016 from diseased swine of different Brazilian states. Isolated strains were stored at 86 ° C and were posteriorly reactivated for confirmation through PCR and MALDI TOF mass spectrometry. Subsequently, molecular serotyping and virulence gene identification were performed by PCR, and genotyping was performed by AFLP and PFGE techniques. To identify the resistant profiles, the minimum inhibitory concentration (MIC) was determined using the broth microdilution technique. Based on the results obtained from the phenotypic and genotypic characterization, twenty four strains were selected for genome sequencing using the Illumina® MiSeq platform. The most frequently identified serotype was 2 / ½ (82.8%); followed to a lesser extent by serotypes 3, 4, 6, 7, 8, 9, 18, 27, 28, 1/14, with only six non typeable strains (NT). For virulence profile characterization, four genes sly, arcA, epf and mrp variants were screened, and were identified 11 virulence profiles in the studied strains. The genotyping by AFLP technique identified 30 genotypic profiles and the PFGE technique identified 64 pulsotypes. For both techniques, no clear correlation between epidemiological and genotypic data could be identified, but a tendency to group strains per year of isolation can be observed. The MIC results identified nine antimicrobial susceptibility profiles and 72.1% of the strains were classified as multidrug resistant. High rates of resistance to tetracyclines, macrolides, clindamycin and sulfadimethoxine were observed, while beta lactams and florfenicol were the most effective antimicrobials. In the genomic analysis, no plasmid markers were detected and only three resistance genes were identified in the S. suis Brazilian genomes; the ermB gene, which encodes resistance to macrolides and lincosamides, was detected in 79.2% of the 24 studied genomes. The MLST analysis identified eight new allele sequences and one new allelic combination, such that six new type sequences (STs) were identified among the 24 genomes analyzed. It was identified a mobile genetic element (CMGETZ080501) related to antimicrobial resistance in seven from nine strains of serotype 2; the two Brazilian serotype 2 strains that differ from the others (45 and 47) were the most susceptible strains to antimicrobials. Regarding genetic mapping of the strains from serotype 3, it was observed that two of them differ from the others and show greater similarity to the reference genotype of serotype 4 strains. This result was both verified in wgSNP and MLST analysis, confirming that the capsular characterization of S. suis should not be used as an indication of genetic similarity. Given the genetic variability and the high frequency of multidrug resistance identified in the S. suis strains circulating in the country, it is possible to evaluate the reasons for the control of the agent to remain a major challenge for the national intensive pig production systems.

Prevalência de Streptococcus suis sorotipo 2: discussão da literatura brasileira

Streptococcus suis is considered worldwide as one of the pathogens of biggest health and economic impact in the swine industry. Among the serotypes described as zoonotic, serotype 2 is the most frequently isolated from diseased animals and humans in most countries. The study of the epidemiology of S. suis infections in Brazil is important and may help in the development of effective control measures. The aim of this study was to conduct a critical review of Brazilian literature, with support of the world literature, addressing the diagnosis of the agent and its prevalence in clinically ill animals and healthy carriers, especially regarding to the prevalence of the serotype 2 in the country.

Streptococcus suis é mundialmente considerado um dos patógenos de maior impacto sanitário e econômico na indústria suinícola. Dentre os sorotipos descritos como zoonóticos, o sorotipo 2 é o mais frequentemente isolado de animais e humanos doentes na maioria dos países. O estudo da epidemiologia das infecções por S. suis no Brasil é importante para a implantação de medidas efetivas de controle. O objetivo do presente trabalho foi realizar uma revisão crítica da literatura brasileira, com suporte da literatura mundial, abordando o diagnóstico do agente e sua prevalência em animais clinicamente doentes e portadores sadios, com destaque para a prevalência do sorotipo 2 no país.

Prevalence of Streptococcus suis serotype 2: discussion of the Brazilian literature / Prevalência de Streptococcus suis sorotipo 2: discussão da literatura brasileira

Streptococcus suis is considered worldwide as one of the pathogens of biggest health and economic impact in the swine industry. Among the serotypes described as zoonotic, serotype 2 is the most frequently isolated from diseased animals and humans in most countries. The study of the epidemiology of S. suis infections in Brazil is important and may help in the development of effective control measures. The aim of this study was to conduct a critical review of Brazilian literature, with support of the world literature, addressing the diagnosis of the agent and its prevalence in clinically ill animals and healthy carriers, especially regarding to the prevalence of the serotype 2 in the country.

Streptococcus suis é mundialmente considerado um dos patógenos de maior impacto sanitário e econômico na indústria suinícola. Dentre os sorotipos descritos como zoonóticos, o sorotipo 2 é o mais frequentemente isolado de animais e humanos doentes na maioria dos países. O estudo da epidemiologia das infecções por S. suis no Brasil é importante para a implantação de medidas efetivas de controle. O objetivo do presente trabalho foi realizar uma revisão crítica da literatura brasileira, com suporte da literatura mundial, abordando o diagnóstico do agente e sua prevalência em animais clinicamente doentes e portadores sadios, com destaque para a prevalência do sorotipo 2 no país.

Prevalence of Streptococcus suis serotype 2: discussion of the Brazilian literature / Prevalência de Streptococcus suis sorotipo 2: discussão da literatura brasileira

Streptococcus suis is considered worldwide as one of the pathogens of biggest health and economic impact in the swine industry. Among the serotypes described as zoonotic, serotype 2 is the most frequently isolated from diseased animals and humans in most countries. The study of the epidemiology of S. suis infections in Brazil is important and may help in the development of effective control measures. The aim of this study was to conduct a critical review of Brazilian literature, with support of the world literature, addressing the diagnosis of the agent and its prevalence in clinically ill animals and healthy carriers, especially regarding to the prevalence of the serotype 2 in the country. (AU)

Streptococcus suis é mundialmente considerado um dos patógenos de maior impacto sanitário e econômico na indústria suinícola. Dentre os sorotipos descritos como zoonóticos, o sorotipo 2 é o mais frequentemente isolado de animais e humanos doentes na maioria dos países. O estudo da epidemiologia das infecções por S. suis no Brasil é importante para a implantação de medidas efetivas de controle. O objetivo do presente trabalho foi realizar uma revisão crítica da literatura brasileira, com suporte da literatura mundial, abordando o diagnóstico do agente e sua prevalência em animais clinicamente doentes e portadores sadios, com destaque para a prevalência do sorotipo 2 no país. (AU)

In vitro biofilm forming potential of Streptococcus suis isolated from human and swine in China

Streptococcus suis is a swine pathogen and also a zoonotic agent. The formation of biolms allows S. suis to become persistent colonizers and resist clearance by the host immune system and antibiotics. In this study, biofilm forming potentials of various S. suis strains were characterized by confocal laser scanning microscopy (CLSM), scanning electron microscopy (SEM) and tissue culture plates stained with crystal violet. In addition, the effects of five antimicrobial agents on biofilm formation were assayed in this study. S. suis produced biofilms on smooth and rough surface. The nutritional contents including glucose and NaCl in the growth medium modulated biofilm formation. There was a significant difference in their biofilm-forming ability among all 46 S. suis strains. The biofilm-forming potential of S. suis serotype 9 was stronger than type 2 and all other types. However, biofilm formation was inhibited by five commonly used antimicrobial agents, penicillin, erythromycin, azithromycin, ciprofloxacin, and ofloxacin at subinhibitory concentrations, among which inhibition of ciprofloxacin and ofloxacin was stronger than that of other three antimicrobial agents.Our study provides a detailed analysis of biofilm formation potential in S. suis, which is a step towards understanding its role in pathogenesis, and eventually lead to a better understanding of how to eradicate S. suis growing as biofilms with antibiotic therapy.

Análise de polimorfismos em genes relacionados à virulência e resistência de Streptococcus suis em uma população suína / Analysis of genetic polymorphisms related to virulence and resistance in a population ofStreptococcus suis

Background: Streptococcus suis is a bacterium that causes several diseases in swines and can infect humans; in addition, it represents an important risk factor for professionals from this area. In cases of outbreak, despite some animals may remain as asymptomatic disease carriers, mortality can vary between 4 and 14%. Virulence of this bacterium may be associated with different serotypes exist, as well as with cellular proteins present in the capsule, or by mutations in the genomes of different serotypes, or even between samples of the same serotype. The strains of serotype 2 are considered to be the most virulent, although they can have moderate virulence, or even being less virulent. The objective of this work is to analyze genetic variability in S. suis samples. Materials, Methods & Results: Forty S. suis samples provided by the Laboratório Microvet (Minas Gerais, Brazil) and isolated between 1995 and 2003 on a farm located in Ponte Nova, MG, have been used. The lyophilized samples were reactivated in BHI, for 24 h, at 37°C; after that, they were cultivated and isolated in Todd Hewitt nutrient medium plus agar-agar and 10% defibrinated sheep blood and kept at 37°C, for 18 h. Serotyping of samples was conducted by Microvet and the data was compiled for comparison with the results. Genomic DNA was extracted from 3x106 cells, using DNAzol kit (Invitrogen®), under the protocol proposed by the manufacturer, followed by PCR individually for each primer pair - dpr and aroA, using 200 µM dNTP, 3.4 mM MgCl2, 2 U of Taq DNA polymerase (Invitrogen®), 1.6x amplification buffer and 0.20 µM of the primers in a final volume of 25 µL. The annealing temperature was 59°C, for aroA gene, and 60°C, for dpr gene. The amplified fragments of genes were digested with 0.2 µL of the restriction enzymes Alu I, AflIII, EcoRI and MPS I, at 37°C, for 2 h. All products were analyzed by separation on 3% agarose gel. Eight in the forty samples analyzed showed polymorphisms in the dpr gene for cleavage site of Alu I enzyme, thus generating samples with two and three DNA fragments. Analysis with the other endonucleases does not show differences; besides, no cleavage has occurred in the fragment. All samples analyzed for the aroA gene were observed to be identical for the cleavage sites of the used enzymes. During the analysis of serotypes, 39 (97.5%) samples were identified as type 2 and one (2.5%) as type 3. Discussion: The dpr gene showed different patterns between the assessed S. suis colonies, what indicates the existence of polymorphisms in the restriction sites. These data partially corroborate those already reported in the literature, where the percentage of amino acid polymorphisms was 17.9% in the dpr gene. In this study, changes identified in the dpr gene are not conclusive; still, modifications in nucleotide bases of certain genes may be related to expressions of differential proteins, associated with pathogenicity and virulence of pathogens. Even with the use of antibiotics and hygienic-sanitary control measures, records of difficult-to-control outbreaks are constant in the swine populations from which the samples were isolated. The sample of serotype 3 was collected from healthy animal, whereas the serotype 2 samples were collected from sick animals. These factors reinforce the idea that alterations in genomes of bacteria may be establishing new mechanisms to escape, hence favoring the survival of pathogens.

Análise de polimorfismos em genes relacionados à virulência e resistência de Streptococcus suis em uma população suína / Analysis of genetic polymorphisms related to virulence and resistance in a population ofStreptococcus suis

Background: Streptococcus suis is a bacterium that causes several diseases in swines and can infect humans; in addition, it represents an important risk factor for professionals from this area. In cases of outbreak, despite some animals may remain as asymptomatic disease carriers, mortality can vary between 4 and 14%. Virulence of this bacterium may be associated with different serotypes exist, as well as with cellular proteins present in the capsule, or by mutations in the genomes of different serotypes, or even between samples of the same serotype. The strains of serotype 2 are considered to be the most virulent, although they can have moderate virulence, or even being less virulent. The objective of this work is to analyze genetic variability in S. suis samples. Materials, Methods & Results: Forty S. suis samples provided by the Laboratório Microvet (Minas Gerais, Brazil) and isolated between 1995 and 2003 on a farm located in Ponte Nova, MG, have been used. The lyophilized samples were reactivated in BHI, for 24 h, at 37°C; after that, they were cultivated and isolated in Todd Hewitt nutrient medium plus agar-agar and 10% defibrinated sheep blood and kept at 37°C, for 18 h. Serotyping of samples was conducted by Microvet and the data was compiled for comparison with the results. Genomic DNA was extracted from 3x106 cells, using DNAzol kit (Invitrogen®), under the protocol proposed by the manufacturer, followed by PCR individually for each primer pair - dpr and aroA, using 200 µM dNTP, 3.4 mM MgCl2, 2 U of Taq DNA polymerase (Invitrogen®), 1.6x amplification buffer and 0.20 µM of the primers in a final volume of 25 µL. The annealing temperature was 59°C, for aroA gene, and 60°C, for dpr gene. The amplified fragments of genes were digested with 0.2 µL of the restriction enzymes Alu I, AflIII, EcoRI and MPS I, at 37°C, for 2 h. All products were analyzed by separation on 3% agarose gel. Eight in the forty samples analyzed showed polymorphisms in the dpr gene for cleavage site of Alu I enzyme, thus generating samples with two and three DNA fragments. Analysis with the other endonucleases does not show differences; besides, no cleavage has occurred in the fragment. All samples analyzed for the aroA gene were observed to be identical for the cleavage sites of the used enzymes. During the analysis of serotypes, 39 (97.5%) samples were identified as type 2 and one (2.5%) as type 3. Discussion: The dpr gene showed different patterns between the assessed S. suis colonies, what indicates the existence of polymorphisms in the restriction sites. These data partially corroborate those already reported in the literature, where the percentage of amino acid polymorphisms was 17.9% in the dpr gene. In this study, changes identified in the dpr gene are not conclusive; still, modifications in nucleotide bases of certain genes may be related to expressions of differential proteins, associated with pathogenicity and virulence of pathogens. Even with the use of antibiotics and hygienic-sanitary control measures, records of difficult-to-control outbreaks are constant in the swine populations from which the samples were isolated. The sample of serotype 3 was collected from healthy animal, whereas the serotype 2 samples were collected from sick animals. These factors reinforce the idea that alterations in genomes of bacteria may be establishing new mechanisms to escape, hence favoring the survival of pathogens.(AU)

Swine infection by Streptococcus suis: a retrospective study / Infecção em suínos por Streptococcus suis: estudo retrospectivo

The epidemic aspects of swine infections caused by Streptococcus suis were studied, focusing mainly on the occurrence of several serotypes. A total of 323 samples of S. suis were isolated from clinically ill animals, serotyped according to the co-agglutination procedure, and analyzed. The serotyping revealed that S. suis was present in several Brazilian states. The largest number was isolated from the states of Minas Gerais (62.5 percent), São Paulo (10.8 percent), and Paraná (9.3 percent). Serotype 2 was the most frequent (61.0 percent), followed by the serotypes 1, 3, 4, 7, and 8. The largest number of isolations was obtained from the brain (60.1 percent), followed by the lungs (10.4 percent). About 9.4 percent of the cases were due to septicemia.(AU)

Estudaram-se os aspectos epidêmicos das infecções de suínos causadas por Streptococcus suis, enfocando, principalmente, a ocorrência de diferentes sorotipos. Foram analisadas 323 amostras isoladas de animais clinicamente doentes, as quais foram sorotipadas de acordo com o procedimento de co-aglutinação. Foi verificado que S. suis está presente em vários estados brasileiros e o maior número de isolados originou-se dos estados de Minas Gerais (62,5 por cento), São Paulo (10,8 por cento) e Paraná (9,3 por cento). O sorotipo 2 foi o mais freqüente (61.0 por cento), seguido pelos sorotipos 1, 3, 4, 7 e 8. Os isolamentos foram obtidos principalmente de cérebro (60,1 por cento) e pulmões (10,4 por cento). Os casos de septicemia representaram 9,4 por cento.(AU)