A field evaluation of a new porcine circovirus type 2d and Mycoplasma hyopneumoniae bivalent vaccine in herds suffering from subclinical PCV2d infection and enzootic pneumonia.

BACKGROUND: This field efficacy study was designed to determine the efficacy of a new bivalent vaccine containing porcine circovirus type 2d (PCV2d) and Mycoplasma hyopneumoniae at three independent pig farms. METHODS: Three pig farms were selected based on their history of subclinical PCV2 infection and enzootic pneumonia. Each farm housed a total of 40, 18-day-old pigs that were randomly allocated to 1 of 2 treatment groups. Pigs were administered a 2.0 mL dose of the bivalent vaccine intramuscularly at 21 days of age in accordance with the manufacturer's recommendations, whereas unvaccinated pigs were administered a single dose of phosphate-buffered saline at the same age. RESULTS: Clinically, the average daily weight gain of vaccinated groups was significantly higher (p < 0.05) than those of unvaccinated animals during the growing (70-112 days of age), finishing (112-175 days of age) and overall (3-175 days of age) stages of production. Vaccinated animals elicited neutralizing anti-PCV2 antibodies and PCV2d-specific interferon-γ secreting cells (IFN-γ-SC), which reduced the amount of PCV2d genomic copies in blood and reduced lymphoid lesions severity when compared with unvaccinated animals. Similarly, vaccinated animals elicited M. hyopneumoniae-specific IFN-γ-SC, which reduced the amount of M. hyopneumoniae in the larynx and reduced lung lesions severity. CONCLUSIONS: The result of the field trial demonstrated that the bivalent vaccine was efficacious in the protection of swine herds suffering from subclinical PCV2d infection and enzootic pneumonia.

An evaluation of intradermal all-in-one vaccine based on an inactivated recombinant Mycoplasma hyopneumoniae strain expressing porcine circovirus type 2 (PCV2) capsid protein against Korean stains of PCV2d and M. hyopneumoniae challenge.

This study evaluates the efficacy of intradermal all-in-one vaccine (MHYOSPHERE® PCV ID, Laboratorios Hipra S.A. Amer, Spain) based on the strain Nexhyon, an inactivated recombinant M. hyopneumoniae strain with an embedded/integrated PCV2a capsid protein thereof, as the single active substance. Pigs were administered the vaccine intradermally at 21 days of age with 0.2 mL, then challenged at 49 days of age with either M. hyopneumoniae (intratracheal route), PCV2d (intranasal route), or both. Upon dual challenge, growth performance was improved when the intradermal all in one vaccine was administered compared to the unvaccinated group. In pigs receiving single or dual challenge, vaccination increased neutralizing antibodies against PCV2d and specific interferon-γ secreting cells for each pathogen. In contrast, viral load of PCV2d in the blood, M. hyopneumoniae load in the larynx, and the severity of pulmonary and lymphoid lesions were decreased. Vaccination provided good protection against challenge with M. hyopneumoniae and PCV2d.

A field efficacy trial of a trivalent vaccine containing porcine circovirus type 2a and 2b, and Mycoplasma hyopneumoniae in three herds.

BACKGROUND: This field trial was designed to evaluate the efficacy of a new trivalent vaccine containing porcine circovirus type 2a and 2b (PCV2a/b), and Mycoplasma hyopneumoniae at three independent locations. METHODS: Three farms were selected based on their history of PCV2 and M. hyopneumoniae co-infection. Each farm housed a total of 60, 3-day-old pigs that were randomly allocated to one of three treatment groups. Pigs were administered the trivalent vaccine intramuscularly with either a 1.0 ml dose at 3 and 24 days of age or a 2.0 ml dose at 21 days of age in accordance with the manufacturer's recommendations. RESULTS: Clinically, the average daily weight gain of the one-dose and two-dose vaccinated groups within all three farms was significantly higher (p < 0.05) than those of unvaccinated animals during the growing (70-112 days of age), finishing (112-175 days of age) and overall (3-175 days of age) stages of production. One-dose and two-dose vaccinated animals elicited neutralizing antibodies and interferon-γ-secreting cells (IFN-γ-SC), which reduced the amount of PCV2 in terms of blood load and reduced the severity of lymphoid lesions when compared with unvaccinated animals. Similarly, one-dose and two-dose vaccinated animals elicited IFN-γ-SC, which reduced the amount of M. hyopneumoniae in terms of laryngeal load and reduced the severity of lung lesions. CONCLUSIONS: The intramuscular administration of either one or two doses of trivalent vaccine was not significantly different in any of the evaluated parameters. The results of field trial demonstrated that the trivalent vaccine was efficacious in the protection of swine herds where PCV2d and M. hyopneumoniae were in active circulation.

Effect of parity segregation on Mycoplasma hyopneumoniae infection dynamics and pneumonic lesions in pigs / Efeito da ordem de parto segregada sobre a dinâmica de infecção de Mycoplasma hyopneumoniae e lesões pulmonares ao abate

Gilts represent a group risk for Mycoplasma hyopneumoniae vertical transmission in swine herds. Therefore, parity segregation can be an alternative to control M. hyopneumoniae infections. The study evaluated the effect of parity segregation on M. hyopneumoniae infection dynamics and occurrence and severity of lung lesions at slaughter. For that, three multiple site herds were included in the study. Herd A consisted of the farm where gilts would have their first farrowing (parity order (PO) 1). After the first farrowing PO 1 sows were transferred to herd B (PO2-6). Herd C was a conventional herd with gilt replacement (PO1-6). Piglets born in each herd were raised in separated nursery and finishing units. Sows (n = 33 (A), 37 (B), 34 (C)) in all herds were sampled prior to farrowing and piglets (n = 54 (A), 71 (B), 66 (C)) were sampled longitudinally at 21, 63, 100, 140 days of age and at slaughter for M. hyopneumoniae detection by PCR and lung lesions scoring. M. hyopneumoniae prevalence in sows did not differ among herds. Prevalence of positive piglets was higher at weaning in the PO1 herd (A) (P < 0.05). However, prevalence of positive pigs from 100 days of age to slaughter age was higher in the PO2-6 herd (B) (P < 0.05). Lung lesion occurrence and severity were higher in herd B. The authors suggested that the lack of a proper gilt acclimation might have influenced the results, leading to sows being detected positive at farrowing, regardless of the parity.

As leitoas consistem em um grupo de risco na transmissão vertical de Mycoplasma hyopneumoniae dentro do sistema de produção de suínos. Dessa forma, a segregação de partos poderia ser utilizada como alternativa para controlar as infecções por M. hyopneumoniae. O objetivo deste estudo foi avaliar o efeito da segregação de partos sobre a dinâmica de infecção de M. hyopneumoniae e a ocorrência e severidade das lesões pulmonares ao abate. Para isso três sistemas de produção de suínos com três sítios cada foram incluídos no estudo. A granja A consistia da unidade onde as leitoas tem o primeiro parto, ou seja, alojava somente de fêmeas de ordem de parto 1 (Granja OP1). Após o primeiro parto as fêmeas OP1 foram transferidas para a granja B (Granja OP2-6), ou seja, consistia de fêmeas de ordem de parto 2 a 6, e a granja C consistiu em uma granja convencional com reposição de leitoas (Granja OP1-6), com fêmeas de ordem de parto 1 a 6. Os leitões nascidos de cada granja foram transferidos e criados em creches e terminações segregadas. As matrizes (n = 33 (A), 37 (B), 34 (C)) de todas as granjas do estudo foram amostradas previamente ao parto e os leitões (n = 54 (A), 71 (B), 66 (C)) foram amostrados longitudinalmente aos 21, 63, 100 e 140 dias de idade e ao abate. Em todos os momentos de coleta, as amostras foram avaliadas por PCR para detecção de M. hyopneumoniae. As lesões pulmonares foram avaliadas e escores de lesão foram atribuídos ao abate. A prevalência de matrizes positivas para M. hyopneumoniae não diferiu entre as granjas (P > 0,05). A prevalência ao desmame foi maior na granja A (OP1) (P < 0,05). No entanto, dos 100 dias de idade até o abate a prevalência de leitões positivos para M. hyopneumoniae foi maior na granja B (OP2-6) (P < 0,05). A ocorrência e severidade de lesões pulmonares foram maiores na granja B. Os autores sugerem que a falta de uma aclimatação adequada das leitoas pode ter influenciado nos resultados, levando à detecção de matrizes positivas ao parto, independente da ordem de parto.

Genetic diversity of Mycoplasma hyopneumoniae in Mendoza province / Diversidad genética de Mycoplasma hyopneumoniae en la provincia de Mendoza

En Argentina, la neumonía enzoótica porcina (NEP) es altamente prevalente y se han identificado diferentes tipos genéticos de Mycoplasma hyopneumoniae. Sin embargo, se carece de información acerca de la prevalencia de NEP y de otros aspectos epidemiológicos de esta entidad en la provincia de Mendoza. En esta investigación se usó un análisis multilocus de regiones repetidas en tándem (MLVA) de los loci P97 R1, P97 R1A y P146 R3 para evaluar la diversidad genética de M. hyopneumoniae a partir de muestras clínicas de cerdos de cinco granjas localizadas en diferentes distritos de la provincia de Mendoza. M. hyopneumoniae pudo ser tipificado a partir de 27 muestras de lavado broncoalveolar (LBA) y se identificaron 8 diferentes MLVA-tipos. Este es el primer informe acerca de la diversidad genética de M. hyopneumoniae en Mendoza. Los resultados obtenidos permiten describir de manera más acabada la diversidad genética de este agente en nuestro país.

Occurrence of Mycoplasma hyopneumoniae in slaughter pigs from Southern Brazil / Ocorrência de Mycoplasma hyopneumoniae em amostras de pulmão de suínos obtidas de frigorífico do sul do Brasil

Mycoplasma hyopneumoniae is the causative agent of enzootic pneumonia (EP), a disease that is highly prevalent and globally distributed, causing significant economic losses to the swine industry. Disease progression is characterized by reduced feed conversion and the development of lung lesions. Considering the limited information about the epidemiology of EP in Southern Brazil, the main objective of this study was to determine the occurrence of M. hyopneumoniae in swine lung samples and to evaluate the scores of lung lesions caused by local strains. A total of 120 samples was randomly collected and processed. DNA was extracted from lung tissue to perform nested-PCR and lungs were inspected to evaluate the presence of the pneumonia-like gross lesions of M. hyopneumoniae. The results showed 95.8% positive samples, while the lung lesion score analysis showed suggestive lesions in 60% of samples. The detection of positive samples in nested-PCR was associated with the presence of pneumonia-like gross lesions (P < 0.01). The results demonstrate a high occurrence of EP in slaughter pigs from southern Brazil.(AU)

O Mycoplasma hyopneumoniae é o agente causador da Pneumonia Enzoótica Suína (PES), doença altamente prevalente e mundialmente distribuída, causando grandes perdas econômicas para a indústria suinícola. A progressão da doença é caracterizada pela redução das taxas de conversão alimentar e o desenvolvimento de lesões pulmonares. Visto que há informação limitada sobre a epidemiologia da PES no sul do Brasil, o objetivo do presente trabalho foi determinar a prevalência de M. hyopneumoniae em amostras de pulmão suíno e avaliar o score de lesões pulmonares causadas pelas cepas locais. Um total de 120 amostras foram coletadas aleatoriamente, processadas e analisadas. O DNA foi extraído do tecido pulmonar para realização de Nested-PCR e os pulmões foram inspecionados para presença de lesões macroscópicas sugestivas de M. hyopneumoniae. Os resultados demonstraram 95,8% das amostras positivas para o patógeno. A análise do score pulmonar mostrou lesões sugestivas da PES em 60% das amostras. A detecção de amostras positivas no Nested-PCR foi associada com a presença de lesões sugestivas (P < 0.01). Os dados obtidos neste trabalho demonstram a alta prevalência da PES em granjas do RS.(AU)

Lesões pulmonares em suínos abatidos no matadouro Público Municipal de Esperança, Paraíba / Pulmonary lesions of slaughtered pigs at the municipal public slaughterhouse of Esperança, Paraíba

O objetivo do presente estudo foi avaliar as lesões macroscópicas e histológicas em pulmões de suínos abatidos no abatedouro público de Esperança, Paraíba. Foram inspecionados pulmões de 180 suínos entre julho e dezembro de 2013. Destes, 34 (18,8%) apresentaram lesões. Na análise anatomopatológica dos fragmentos coletados, 82,3% (28/34) exibiram lesões sugestivas de Pneumonia Enzoótica Suína (PES). Seis (17,7%) amostras apresentaram alterações causadas pela distribuição irregular de sangue. Casos sugestivos de PES crônica foram observados em 57,1% (16/28) dos fragmentos coletados. Em 42,9% (12/28) das amostras foram definidos como sugestivos de PES subaguda. Nenhum pulmão apresentou lesões sugestivas de PES aguda. A pesquisa demonstrou que lesões pulmonares em suínos são frequentemente detectadas no abatedouro de Esperança, Paraíba, sendo a maioria destas lesões sugestivas de PES.

The objective of the present study was to evaluate the macroscopic and histological lesions in the lungs of slaughtered pigs at the public slaughterhouse of Esperança, Paraíba. Lungs from 180 pigs were inspected between July and December 2013. Lesions were observed in 34 (18.8%) lungs. In the anatomopathological analysis of the collected fragments, 82.3% (28/34) presented suggestive lesions of Swine Enzootic Pneumonia (SEP). Six (17.7%) samples presented alterations caused by irregular blood distribution. Suggestive cases of chronic SEP were observed in 57.1% (16/28) of the collected fragments. In 42.9% (12/28) of the samples were defined as suggestive of subacute SEP. No lungs presented lesions suggestive of acute PES. The research showed that lung lesions in pigs are frequently detected in the Esperança, Paraíba slaughterhouse, with the majority of these lesions suggestive for SEP.

A novel chimeric protein composed of recombinant Mycoplasma hyopneumoniae antigens as a vaccine candidate evaluated in mice.

Enzootic Pneumonia (EP) is caused by the Mycoplasma hyopneumoniae pathogenic bacteria, and it represents a significant respiratory disease that is responsible for major economic losses within the pig industry throughout the world. The bacterins that are currently commercially available have been proven to offer only partial protection against M. hyopneumoniae, and the development of more efficient vaccines is required. Several recombinant antigens have been evaluated via different immunization strategies and have been found to be highly immunogenic. This work describes the construction and immunological characterization of a multi-antigen chimera composed of four M. hyopneumoniae antigens: P97R1, P46, P95, and P42. Immunogenic regions of each antigen were selected and combined to encode a single polypeptide. The gene was cloned and expressed in Escherichia coli, and the chimeric protein was recognized by specific antibodies against each subunit, as well as by convalescent pig sera. The immunogenic properties of the chimera were then evaluated in a mice model through two recombinant vaccines that were formulated as follows: (1) purified chimeric protein plus adjuvant or (2) recombinant Escherichia coli bacterin. The immune response induced in BALB/c mice immunized with each formulation was characterized in terms of total IgG levels, IgG1, and IgG2a isotypes against each antigen present in the chimera. The results of the study indicated that novel chimeric protein is a potential candidate for the future development of a more effective vaccine against EP.

Association of number of tandem repeats in two important adhesins in Mycoplasma hyopneumoniae / Associação entre os números de repetições em tandem em duas importantes adesinas de Mycoplasma hyopneumoniae

Diversidade genética de Mycoplasma hyopneumoniae tem sido relatada em análise múltipla de repetições em tandem em número variável (MLVA). O objetivo deste estudo foi descrever a distribuição espacial e a heterogeneidade genética de tipos de M. hyopneumoniae no Brasil, bem como investigar a correlação entre regiões de repetição 1 (RR1) e 3 (RR3) de duas adesinas importantes (P97 e P146). Foram identificados 39 tipos de MLVA baseados no número de repetições em tandem em P97 RR1 e RR3 P146. A correlação negativa significativa (Spearman's rho = -0,26; P = 0,022) entre P97 RR1 e RR3 P146 foi observada, o que sugere um possível mecanismo compensatório que permitiria a bactéria manter a sua capacidade de adesão. Os resultados contribuem para compreender a epidemiologia das M. hyopneumoniae no quarto maior país produtor de suínos do mundo.(AU)

Detection, quantification and genetic variability of Mycoplasma hyopneumoniae from apparently healthy and pneumonic swine / Detecção, quantificação e variabilidade genética de Mycoplasma hyopneumoniae a partir de suínos pneumônicos e aparentemente saudáveis

Molecular differences among Mycoplasma hyopneumoniae strains present in pneumonic lungs of swine have been largely studied. However, no comparative studies concerning the strains present in apparently healthy pigs have been carried out. This study aimed to detect, quantify and perform molecular analysis of M. hyopneumoniae strains in pig lungs with and without pneumonic lesions. The detection of M. hyopneumoniae was performed using multiplex PCR (YAMAGUTI, 2008), real-time PCR (STRAIT et al., 2008) and multiple VNTR amplification (VRANCKX et al., 2011). Molecular characterization of the strains was achieved by analysis of the VNTR copy number in P97R1, P146R3, H2R1 and H4. M. hyopneumoniae was detected in samples from healthy and pneumonic pigs and the amount of M. hyopneumoniae positive samples detected varied with the type of assay. The greater number of positive samples was identified by the multiple VNTR amplification combined with capillary electrophoresis. Using real-time PCR, 4.9*104 M. hyopneumoniae genome copies/mL was detected in apparently healthy lungs. A mean quantity of 3.9*106 M. hyopneumoniae genome copies/mL was detected in pneumonic lungs. The analysis of VNTR copy number demonstrated a high genetic variability of the M. hyopneumoniae strains present in apparently healthy and pneumonic lungs. Strains having 3 VNTR copy number in P97R1, were detected only in pneumonic lungs and strains having 40 and 43 VNTR copy number in P146R3 were detected only in apparently healthy lungs. Despite the genetic variability of M. hyopneumoniae, predominant strains in the swine farms could be identified...

As diferenças moleculares entre as estirpes de Mycoplasma hyopneumoniae presentes em pulmões de suínos com pneumonia tem sido estudadas. Porém, estudos comparativos relativos as estirpes presentes nos suínos aparentemente saudáveis não foram levados a cabo. O objetivo do estudo foi a detecção, quantificação e analise molecular de M. hyopneumoniae nos pulmões suínos com e sem lesões pneumônicas. Para a detecção de M. hyopneumoniae usaramse o PCR Multiplo (YAMAGUTI, 2008), o PCR a Tempo Real (STRAIT et al., 2008) e a amplificação de múltiplo VNTR (VRANCKX et al., 2011). A caracterização molecular das estirpes foi realizada mediante a análise do número de copias de VNTR em P97R1, P146R3, H2R1 e H4. O M. hyopneumoniae foi detectado em amostras de suínos saudáveis e pneumônicos e a quantidade de M. hyopneumoniae nas amostras positivas variou com o tipo de ensaio. O maior número de amostras positivas foi identificado pela amplificação de múltiplas VNTR combinado com a eletroforese de capilares. Usando o PCR a Tempo Real, 4.9*104 copias de genoma/mL de M. hyopneumoniae foram detectadas em pulmões aparentemente saudáveis. Uma quantidade média de 3.9*106 copias de genoma/mL de M. hyopneumoniae foi detectada em pulmões pneumônicos. A análise do número de copias de VNTR demonstrou uma elevada variabilidade...

Doenças respiratórias em bezerros na região sul do Rio Grande do Sul: estudo retrospectivo de 33 surtos / Respiratory diseases in calves in southern Rio Grande do Sul: study of 33 outbreaks

Foram estudados 33 surtos de pneumonia em bovinos jovens na área de influência do Laboratório Regional de Diagnóstico (LRD) da Faculdade de Veterinária da Universidade Federal de Pelotas (UFPel) entre os anos de 2000 e 2011. Foram diagnosticados 18 surtos de pneumonia (54,54%) em bovinos de raças leiteiras, Holandês ou Jersey e 13 surtos (39,39%) em gado de corte ou cruzas de gado de corte. A morbidade variou entre 0,06%-100% e a mortalidade foi de 0,06%-34,6%. A doença ocorreu igualmente em todas as estações do ano e foi mais frequente em bezerros de 1-3 meses totalizando 13 surtos. Sete surtos ocorreram em bovinos entre 4-6 meses, sete entre 7-12 meses e seis surtos ocorreram em bezerros de 1-29 dias. Pneumonia enzoótica pela infecção pelo vírus sincicial respiratório bovino (BRSV) com lesões histológicas de broncopneumonia, pneumonia intersticial e presença de células sinciciais foi mais frequente em bovinos de raças de corte com dez surtos (58,8%); seis surtos dessa enfermidade ocorreram em raças de leite (35,2%). O diagnóstico foi confirmado por imuno-histoquímica em sete casos. Os sinais clínicos da maioria dos casos de pneumonia observados caracterizaram-se por dispneia, emagrecimento, apatia, tremores, bruxismo, desidratação, respiração ruidosa, tosse, corrimento nasal seroso ou mucopurulento, decúbito e morte. As lesões macroscópicas caracterizaram-se por presença de áreas de consolidação vermelho-escuras, edema e enfisema nas regiões crânio-ventrais dos lobos pulmonares cardíaco e apical ou pneumonia intersticial com distribuição difusa, edema e enfisema. Histologicamente, as lesões pulmonares eram variáveis. Broncopneumonia necrossupurativa difusa acentuada com hiperplasia de pneumócitos tipo II e edema intersticial e alveolar foi observada em 15 casos. Os resultados deste trabalho demonstram que as pneumonias são importantes causas de perdas econômicas em bovinos jovens na região de influência do LRD. Deve ser destacado que a pneumonia enzoótica devido a infecção pelo BRSV é importante tanto em bovinos de corte como de leite independente da forma de criação.

Thirty-three outbreaks involving pneumonia in young cattle diagnosed in the area of influence of the Regional Diagnostic Laboratory (LRD) of the Veterinary School of the Federal University of Pelotas (UFPel) between 2000 and 2011 were studied. Eighteen outbreaks (54.54%) were diagnosed in dairy breeds and Jersey or Holstein and 13 outbreaks (39.39%) in beef cattle or crossbred beef cattle. Morbidity ranged from 0.06% -100% and mortality was 0.06% to 34.61%. The disease occurred in all seasons of year and was more frequent in calves aged 1-3 months (11 outbreaks). Seven outbreaks occurred in cattle between 4-6 months, seven between 6-12 months and six outbreaks occurred in calves 1-30 days. Bovine respiratory disease due to infection with bovine respiratory syncytial virus (BRSV) was more frequent in beef cattle breeds with eight outbreaks (44.44%); six outbreaks of this disease occurred in calves of dairy breeds (33.33%). The diagnosis was confirmed by immunohistochemistry in seven cases. The main clinical signs were characterized by dyspnea, weight loss, lethargy, tremors, bruxism, dehydration, rapid and noisy breathing, coughing, serous or mucopurulent nasal discharge, recumbency and death. Gross lesions were characterized by dark red areas of consolidation, edema and emphysema in cranio-ventral regions of the apical and cardiac lung lobes or diffuse interstitial pneumonia, edema and emphysema. Histologically, lung lesions were variable. Diffuse bronchopneumonia necrotizing with marked hyperplasia of type II pneumocytes and alveolar and interstitial edema was observed in 15 cases. The results of this study demonstrate that pneumonia is an important cause of economic loss in young cattle in the area of influence of the LRD and that enzootic pneumonia due to BRSV infection is important in both beef cattle and milk cattle regardless of husbandry systems.

Mycoplasmas hyorhinis in different regions of cuba: diagnosis

M. hyorhinis is considered one of the etiological agents of arthritis in sucking pigs, but recently as seen, some strains can produce pneumonia that could not be distinguished from the mycoplasmosis caused by M. hyopneumoniae. The study was conducted to research the presence of Mycoplasma hyorhinis (M. hyorhinis ) in different regions of the country from exudates of pig lungs with typical EP lesions. Exudates from 280 pig lungs with typical EP lesions were studied using molecular techniques such as PCR, real time PCR and amplification of the 16S-23S rRNA. It was detected that the 66 percent of the samples studied resulted positive to M. hyorhinis, and the presence of this species was detected in all the provinces. Amplification and studies on the intergenic region 16S-23S of M. hyorhinis rRNA demonstrated the existing variability among strains of a same species. This study is the first report on M. hyorhinis detection in Cuba.

Monitoramento da presença de Mycoplasma hyopneumoniae em granjas de suínos durante a implementação de programas de erradicação / Monitoring the presence of Mycoplasma hyopneumoniae in swine farms during the implementation of eradication programs

O objetivo deste estudo foi monitorar a presença de M. hyopneumoniae em granjas suínas durante a implementação de programas de erradicação utilizando diferentes técnicas de diagnóstico focalizando no PCR. Trabalhou-se com uma empresa que possuía três granjas, uma parto-terminação (390 matrizes), uma múltiplo-sítio (4100 matrizes) e uma nova granja que povoava suas novas instalações. Nas duas primeiras, foi desenvolvido um programa de despovoamento parcial para erradicar a pneumonia enzoótica suína, a última foi povoada pelos suínos dos anteriores após a erradicação. Nos três rebanhos, os suínos foram monitorados por: sorologia (ELISA), PCR, lesões pulmonares macro e microscópicas e a presença de tosse não produtiva. A ausência de tosse, a baixa porcentagem de suínos soropositivos na fase de terminação e a baixa proporção de lesões pulmonares no abate sugerem que a pneumonia enzoótica suína foi erradicada, mas não o agente causativo -M. hyopneumoniae- cujo DNA foi detectado pela PCR, mostrando diferentes comportamentos de acordo com o rebanho.

The aim of this study was to monitor the presence of M. hyopneumoniae in pig farms during the implementation of eradication programs using different diagnostic techniques focusing on PCR. They worked with a company owner of three farms, a farrow-to-finish (390 sows), a multiple-site (4100 sows) and a new one that was populated its new facility. In the first two were developed a partial depopulation program to eradicate swine enzootic pneumonia, the latter one was populated with pigs after the previous eradication. In the three farms, the pigs were monitored by: serology (ELISA), PCR, macroscopic and microscopic lung lesions and the presence of non-productive cough. The absence of cough, low percentage of seropositive pigs in the finishing stage and the low proportion of lung lesions at slaughter suggest that swine enzootic pneumonia was eradicated, but not the causative agent -M. hyopneumoniae- whose DNA was detected by PCR showing different behaviors according to the herd.

Detection of Mycoplasma hyopneumoniae in lungs and nasal swabs of pigs by nested PCR / Detecção de Mycoplasma hyopneumoniae em pulmões e suabes nasais de suínos por nested PCR

Fifty-four samples were collected from growing and finishing pigs for the molecular diagnosis of enzootic porcine pneumonia. Nineteen lung fragments were obtained from pigs that showed signs of respiratory disease and 35 nasal swabs were obtained from clinically healthy pigs. For the detection of the bacterial genome in the samples, the nested PCR technique was used to amplify a fragment of 706bp. This fragment was subsequently cloned and sequenced. The sequence of obtained nucleotides was compared with six other sequences of Mycoplasma hyopneumoniae and 11 sequences of other bacteria available in the Genbank. To measure the sensitivity of the nested PCR, serial dilutions (10-1 to 10-15) of cloned fragments were conducted based on the concentration of 300ng. Ten lung fragments and eight nasal swabs showed positive for M. hyopneumoniae and the limit of detection was estimated to be 0.3fg DNA cloned. The sequence of nucleotides obtained showed 99.1 percent homology with the other sequences of M. hyopneumoniae, demonstrating that the nested PCR used in this study may provide an important diagnostic tool for the detection of this agent.

Foram coletadas 54 amostras de animais em fase de crescimento e terminação para o diagnóstico molecular da pneumonia enzoótica suína. Dezenove fragmentos de pulmão foram obtidos de suínos que apresentavam sinais de doença respiratória e 35 suabes nasais foram obtidas de suínos clinicamente saudáveis. Para a detecção do genoma bacteriano nas amostras, foi utilizada a técnica de nested PCR que originou um fragmento de 706pb, o qual foi, posteriormente, clonado e sequenciado. A sequência de nucleotídeos obtida foi comparada com outras seis sequências de Mycoplasma hyopneumoniae e 11 sequências de outras bactérias disponíveis no Genbank. Para medir a sensibilidade da nested PCR, foram realizadas diluições seriadas (10-1 a 10-15) do fragmento clonado, partindo da concentração de 300ng. Dez fragmentos de pulmões e oito suabes nasais apresentaram resultado positivo para M. hyopneumoniae e o limite de detecção foi estimado em 0,3fg de DNA clonado. A sequência de nucleotídeos obtida foi de 99.1 por cento de homologia com as outras sequências de M. hyopneumoniae, demonstrando que a nested PCR utilizada neste estudo pode ser uma importante ferramenta de diagnóstico para a detecção desse agente.

Detection of Mycoplasma hyopneumoniae by polymerase chain reaction in swine presenting respiratory problems / Detecção do mycoplasma hyopneumoniae pela reação em cadeia da polimerase em suínos apresentando problemas respiratórios

Since Mycoplasma hyopneumoniae isolation in appropriate media is a difficult task and impractical for daily routine diagnostics, Nested-PCR (N-PCR) techniques are currently used to improve the direct diagnostic sensitivity of Swine Enzootic Pneumonia. In a first experiment, this paper describes a N-PCR technique optimization based on three variables: different sampling sites, sample transport media, and DNA extraction methods, using eight pigs. Based on the optimization results, a second experiment was conducted for testing validity using 40 animals. In conclusion, the obtained results of the N-PCR optimization and validation allow us to recommend this test as a routine monitoring diagnostic method for Mycoplasma hyopneumoniae infection in swine herds.

A Nested-PCR (N-PCR) tem como objetivo melhorar a sensibilidade do diagnóstico direto da Pneumonia Enzoótica Suína, pois o isolamento do Mycoplasma hyopneumoniae é trabalhoso tornando-se inviável na rotina. Neste trabalho, foi realizado um projeto piloto para a otimização da técnica de N-PCR, utilizando três variáveis: tipo de amostra biológica, meio de transporte da amostra e método de extração do DNA, utilizando oito animais. Os resultados obtidos foram empregados no segundo experimento para a validação do teste utilizando 40 animais. Os resultados obtidos, pela otimização da N-PCR, neste trabalho, permite sugerir esta prova como método de diagnóstico de rotina no monitoramento das infecções por Mycoplasma hyopneumoniae em granjas de suínos.