Antimicrobial resistance in commensal Escherichia coli and Enterococcus spp. isolated from pigs subjected to different antimicrobial administration protocols.

The antimicrobial resistance (AMR) in human and animal pathogens is a global concern, and antimicrobial use (AMU) is considered the most important driver for its increase. The aim of this study was to assess AMR in Escherichia coli and Enterococcus spp. in faecal samples of pigs subjected to four different AMU protocols from birth to finishing: G1, no in-feed antimicrobials; G2: a total average dose 6018 mg antimicrobials/pig; G3: a total average dose 8127 mg antimicrobials/pig; and G4: a total average dose 15,678 mg antimicrobials/pig. Faecal samples were collected at six time points and AMR was assessed in both bacteria. The microbiota composition was assessed by 16S rRNA sequencing. Minor differences on the microbiota profile was observed among groups, but a lower Firmicutes:Bacteroidetes ratio was noted in G4. Escherichia coli and Enterococcus spp. strains isolated from all groups showed a high level of multi-drug resistance (MDR). The amount of antimicrobials used was significantly positively associated with the probability of MDR in both bacteria. Approximately 43% of the variation in MIC90 for colistin could be explained by AMU, and a one-day increase in administration of colistin increased MIC90 by 0.05 µg mL-1. In conclusion, the results suggest that the higher the use of antimicrobials in farms, the higher the MDR frequency and resistance to the highest priority critically important antimicrobials for humans in commensal gut bacteria of pigs.

Investigation of hemotropic Mycoplasmas in fetuses and sows with reproductive failure.

Swine eperythrozoonosis or porcine hemoplasmosis is an infectious disease caused mainly by Mycoplasma suis and is distributed worldwide. This study investigated the occurrence of porcine hemothropic mycoplasmas (PHMs) in fetuses and sows with reproductive failure. Two hundred and seventy-six samples (80 sows' blood and 196 fetal tissue samples) from 27 farms with reproductive disorders were evaluated. The PHMs DNA was detected in 15 out of 80 (18.7%) sows but it was not detected in the fetuses. The bacterial load ranged from 1.32 × 102 to 2.61 × 105 copies/µL. From the 27 tested herds, 11 (40.7%) showed at least one positive sow per farm. The majority of the reproductive problems observed in PMHs positive sows were stillborn fetuses (46.7%) and stillborn associated with fetal mummification (26.7%). So, we evidenced that porcine hemoplasmas circulate among sows in Brazilian herds, however, its real impact on reproductive problems remains unknown.

Inguinal Ring RNA Sequencing Reveals Downregulation of Muscular Genes Related to Scrotal Hernia in Pigs.

Scrotal hernias (SH) are common congenital defects in commercial pigs, characterized by the presence of abdominal contents in the scrotal sac, leading to considerable production and animal welfare losses. Since the etiology of SH remains obscure, we aimed to identify the biological and genetic mechanisms involved in its occurrence through the whole transcriptome analysis of SH affected and unaffected pigs' inguinal rings. From the 22,452 genes annotated in the pig reference genome, 13,498 were expressed in the inguinal canal tissue. Of those, 703 genes were differentially expressed (DE, FDR < 0.05) between the two groups analyzed being, respectively, 209 genes upregulated and 494 downregulated in the SH-affected group. Thirty-seven significantly overrepresented GO terms related to SH were enriched, and the most relevant biological processes were muscular system, cell differentiation, sarcome reorganization, and myofibril assembly. The calcium signaling, hypertrophic cardiomyopathy, dilated cardiomyopathy, and cardiac muscle contraction were the major pathways possibly involved in the occurrence of the scrotal hernias. The expression profile of the DE genes was associated with the reduction of smooth muscle differentiation, followed by low calcium content in the cell, which could lead to a decreased apoptosis ratio and diminished muscle contraction of the inguinal canal region. We have demonstrated that genes involved with musculature are closely linked to the physiological imbalance predisposing to scrotal hernia. According to our study, the genes MYBPC1, BOK, SLC25A4, SLC8A3, DES, TPM2, MAP1CL3C, and FGF1 were considered strong candidates for future evaluation.

High frequency and molecular characterization of porcine hemotrophic mycoplasmas in Brazil.

Mycoplasma suis and Mycoplasma parvum are the two hemotrophic mycoplasmas species described in pigs. M. suis is involved in infectious anemia, while M parvum infection is commonly subclinical. The objectives of this study were twofold: (i) to investigate the prevalence of porcine hemotrophic mycoplasmas in sows from the southern region of Brazil by quantitative real-time PCR (qPCR) and (ii) to genetically characterize a subset of the samples based on the 16S rRNA gene. A total of 429 blood samples were evaluated from 53 different farm sites. Porcine hemoplasmas was detected at all the 53 tested sites and in 79.72% of the samples (342/429). Two sequences were obtained for Mycoplasma spp. The phylogenetic analysis based on the 16S rRNA gene (900 bp) showed that the Mycoplasma sequences were closely related to the M. suis cluster and that one sequence was positioned in the M. parvum cluster. In conclusion, porcine hemoplasmas have a high rate of prevalence in sows from commercial farms in the southern region of Brazil. This study demonstrated the first molecular detection and characterization of partial 16S rRNA gene of M. parvum in Brazil.

Antimicrobial susceptibility profile of historical and recent Brazilian pig isolates of Pasteurella multocida / Perfil de suscetibilidade a antimicrobianos de isolados históricos e contemporâneos de Pasteurella multocida de suínos no Brasil

Pasteurella (P.) multocida is the causative agent of pneumonic pasteurellosis in swine, which is commonly associated with the final stages of enzootic pneumonia or porcine respiratory disease complex. Although this syndrome is one of the most common and important diseases of pigs, data on antimicrobial susceptibility of P. multocida isolates are uncommon in Brazil. Therefore, the present study was carried out to determine and to compare antimicrobial susceptibility profile of Brazilian P. multocida isolated from pigs with lesions of pneumonia or pleuritis during two-time periods. Historical isolates (period of 1981 to 1997; n=44) and recent isolates (period of 2011 to 2012; n=50) were used to determine the MIC of amoxicillin, enrofloxacin, florfenicol and tetracycline by microbroth dilution. Florfenicol had the lowest level of resistance for both historical and recent isolates (0% and 6%, respectively), while tetracycline had the highest (20.5% and 34%, respectively). Multi-drug resistance (MDR) to amoxicillin/florfenicol/tetracycline was observed in 6% of recent isolates. There was a significant increase (p˂0.05) in resistance for amoxicillin and enrofloxacin in recent isolates compared with historic isolates (3.8% and 18%, respectively), most likely due to the selective pressure of antimicrobial usage to treat and prevent P. multocida infections. The results of this study showed an increase of isolates resistant to important drugs used in treatment of P. multocida infections in pigs, demonstrating the need for the implementation of rational use of antimicrobials in Brazilian swine industry.(AU)

Pasteurella (P.) multocida é o agente da pasteurelose pneumônica em suínos, a qual é comumente associada com o estágio final da pneumonia enzoótica suína ou complexo das doenças respiratórias dos suínos. Apesar de ser uma das doenças mais comuns e importantes na suinocultura, dados sobre suscetibilidade antimicrobiana de isolados de P. multocida são raros no Brasil. Dessa forma, o presente estudo foi realizado para determinar e comparar o perfil de suscetibilidade de isolados de P. multocida de suínos com lesões de pneumonia ou pleurite no Brasil durante dois períodos. Isolados históricos (período de 1981 a 1997; n=44) e contemporâneos (período de 2011 a 2012; n=50) foram usados para determinar a concentração inibitória mínima (CIM) de amoxicilina, enrofloxacina, florfenicol e tetraciclina através do teste de microdiluição em caldo. Florfenicol apresentou o menor nível de resistência para ambos os isolados históricos e contemporâneos (0% e 6%, respectivamente), enquanto que tetraciclina apresentou o maior nível de resistência (20.5% e 34%, respectivamente). Resistência a múltiplos antimicrobianos (amoxicilina, florfenicol e tetraciclina) foi observada em 6% dos isolados recentes. Foi observado aumento significativo (p˂0.05) na resistência a amoxicilina e enrofloxacina em isolados recentes comparado com isolados históricos (3.8% e 18%, respectivamente), provavelmente devido à pressão de seleção de antimicrobianos usados no tratamento e prevenção de infecções causadas por P. multocida. Os resultados deste trabalho demostraram o aumento de isolados resistentes a importantes drogas utilizadas no tratamento de infecções causadas por P. multocida em suínos, evidenciando a necessidade da implementação do uso racional de antimicrobianos na suinocultura brasileira.(AU)

Protection efficacy of the rLTB-R1 chimera against experimental swine mycoplasmal pneumonia

Background: Mycoplasma hyopneumoniae is the etiological agent of the Swine Mycoplasmal Pneumonia (SMP), one of the most economically significant diseases in the swine industry worldwide. Commonly used vaccines for SMP control consist of inactivated whole cells (bacterins). These vaccines are efficacious against M. hyopneumoniae challenge, but do not prevent colonization by the pathogen or completely eliminate pneumonia. P97 adhesin is conserved in the M. pneumoniae virulent strains, therefore it is an attractive target to be used in recombinant vaccines against M. hyopneumoniae. The aim of the present study was to evaluate protection afforded by rLTB-R1, a recombinant chimera composed by LTB fused with the R1 repeat region of P97 adhesin of M. hyopneumoniae, in specific-pathogen-free (SPF) piglets vaccinated by intranasal or intramuscular route and challenged with a pathogenic strain of M. hyopneumoniae. Materials, Methods & Results: PCR products of the LTB and R1 coding sequences were fused, then cloned into pETDEST42™ expression vector. The rLTB-R1 was expressed in Escherichia coli BL21 (DE3) Salt induction (SI). The piglets were divided into three groups: four piglets were intranasally vaccinated with 1 mg of rLTB-R1 solubilized in 1 mL of PBS at 0 and 14 days (IN rLTB-R1 group); four piglets were intramuscularly vaccinated with 1 mg of rLTB-R1 solubilized in 1 mL of PBS at 0 and 14 days (IM rLTB-R1 group); three piglets were intranasally and intramuscularly inoculated with 1 mL of PBS (control group). Two weeks after the last immunization (28 day), piglets were intratracheally challenged with 10 mL of a suspension containing 109 color-changing unit (CCU) of pathogenic M. hyopneumoniae 7448 strain on three consecutive days. Until the challenge (28 days), intranasal and intramuscular vaccination with rLTB-R1 induced seroconversions of antiR1 systemic antibodies of 1.6 and 4.6 ×, respectively. The IN rLTB-R1 group had no pulmonary lesion, rLTB-R1 conferred protection against experimental SMP. On the other hand, IM rLTB-R1 and control groups had on average 7.24% and 8.46% of pulmonary lesion, respectively, showing that intramuscular vaccination with rLTB-R1 did not confer protection. Discussion: The rLTB-R1, when intranasally administrated to mice, elicited production of anti-R1 IgA in trachea and bronchi as well as specific Th1 response, suggesting an adequate stimulation of the mucosal immune system. We believe that rLTB-R1 induced a similar immune response in piglets intranasally vaccinated, conferring protection against experimental SMP. The present study, the rLTB-R1 alone, without any chemical adjuvant, stimulated a significant seroconversion of anti-R1 systemic antibodies in pigs intramuscularly vaccinated, showing the potential of LTB as a parenteral adjuvant in swine vaccination. Previous work has shown that the intramuscular administration route was evaluated in pigs because mice intramuscularly vaccinated with rLTB-R1 presented significant levels of anti-R1 IgA in trachea and bronchi, suggesting that rLTB can stimulate some degree of mucosal immunity even if not delivered by a mucosal route. However, in the present study, piglets intramuscularly vaccinated with rLTB-R1 presented high levels of anti-R1 systemic antibodies, they were not protected. On the other hand, intranasal vaccination of piglets with rLTB-R1 elicited low levels of antiR1 systemic antibodies (1.6 × at 28 days), but it conferred full protection against experimental SMP. The present study demonstrated that intranasal vaccination of piglets with rLTB-R1 conferred protection against experimental SMP. A more detailed analysis of the protective immune response induced by rLTB-R1 in pigs is currently being performed.

Protection efficacy of the rLTB-R1 chimera against experimental swine mycoplasmal pneumonia

Background: Mycoplasma hyopneumoniae is the etiological agent of the Swine Mycoplasmal Pneumonia (SMP), one of the most economically significant diseases in the swine industry worldwide. Commonly used vaccines for SMP control consist of inactivated whole cells (bacterins). These vaccines are efficacious against M. hyopneumoniae challenge, but do not prevent colonization by the pathogen or completely eliminate pneumonia. P97 adhesin is conserved in the M. pneumoniae virulent strains, therefore it is an attractive target to be used in recombinant vaccines against M. hyopneumoniae. The aim of the present study was to evaluate protection afforded by rLTB-R1, a recombinant chimera composed by LTB fused with the R1 repeat region of P97 adhesin of M. hyopneumoniae, in specific-pathogen-free (SPF) piglets vaccinated by intranasal or intramuscular route and challenged with a pathogenic strain of M. hyopneumoniae. Materials, Methods & Results: PCR products of the LTB and R1 coding sequences were fused, then cloned into pETDEST42™ expression vector. The rLTB-R1 was expressed in Escherichia coli BL21 (DE3) Salt induction (SI). The piglets were divided into three groups: four piglets were intranasally vaccinated with 1 mg of rLTB-R1 solubilized in 1 mL of PBS at 0 and 14 days (IN rLTB-R1 group); four piglets were intramuscularly vaccinated with 1 mg of rLTB-R1 solubilized in 1 mL of PBS at 0 and 14 days (IM rLTB-R1 group); three piglets were intranasally and intramuscularly inoculated with 1 mL of PBS (control group). Two weeks after the last immunization (28 day), piglets were intratracheally challenged with 10 mL of a suspension containing 109 color-changing unit (CCU) of pathogenic M. hyopneumoniae 7448 strain on three consecutive days. Until the challenge (28 days), intranasal and intramuscular vaccination with rLTB-R1 induced seroconversions of antiR1 systemic antibodies of 1.6 and 4.6 ×, respectively. The IN rLTB-R1 group had no pulmonary lesion, rLTB-R1 conferred protection against experimental SMP. On the other hand, IM rLTB-R1 and control groups had on average 7.24% and 8.46% of pulmonary lesion, respectively, showing that intramuscular vaccination with rLTB-R1 did not confer protection. Discussion: The rLTB-R1, when intranasally administrated to mice, elicited production of anti-R1 IgA in trachea and bronchi as well as specific Th1 response, suggesting an adequate stimulation of the mucosal immune system. We believe that rLTB-R1 induced a similar immune response in piglets intranasally vaccinated, conferring protection against experimental SMP. The present study, the rLTB-R1 alone, without any chemical adjuvant, stimulated a significant seroconversion of anti-R1 systemic antibodies in pigs intramuscularly vaccinated, showing the potential of LTB as a parenteral adjuvant in swine vaccination. Previous work has shown that the intramuscular administration route was evaluated in pigs because mice intramuscularly vaccinated with rLTB-R1 presented significant levels of anti-R1 IgA in trachea and bronchi, suggesting that rLTB can stimulate some degree of mucosal immunity even if not delivered by a mucosal route. However, in the present study, piglets intramuscularly vaccinated with rLTB-R1 presented high levels of anti-R1 systemic antibodies, they were not protected. On the other hand, intranasal vaccination of piglets with rLTB-R1 elicited low levels of antiR1 systemic antibodies (1.6 × at 28 days), but it conferred full protection against experimental SMP. The present study demonstrated that intranasal vaccination of piglets with rLTB-R1 conferred protection against experimental SMP. A more detailed analysis of the protective immune response induced by rLTB-R1 in pigs is currently being performed.

Pathogenic variability among Pasteurella multocida type A isolates from Brazilian pig farms.

BACKGROUND: Pasteurella multocida type A (PmA) is considered a secondary agent of pneumonia in pigs. The role of PmA as a primary pathogen was investigated by challenging pigs with eight field strains isolated from pneumonia and serositis in six Brazilian states. Eight groups of eight pigs each were intranasally inoculated with different strains of PmA (1.5 mL/nostril of 10e7 CFU/mL). The control group (n = 12) received sterile PBS. The pigs were euthanized by electrocution and necropsied by 5 dpi. Macroscopic lesions were recorded, and swabs and fragments of thoracic and abdominal organs were analyzed by bacteriological and pathological assays. The PmA strains were analyzed for four virulence genes (toxA: toxin; pfhA: adhesion; tbpA and hgbB: iron acquisition) by PCR and sequencing and submitted to multilocus sequence typing (MLST). RESULTS: The eight PmA strains were classified as follows: five as highly pathogenic (HP) for causing necrotic bronchopneumonia and diffuse fibrinous pleuritis and pericarditis; one as low pathogenic for causing only focal bronchopneumonia; and two as nonpathogenic because they did not cause injury to any pig. PCR for the gene pfhA was positive for all five HP isolates. Sequencing demonstrated that the pfhA region of the HP strains comprised four genes: tpsB1, pfhA1, tpsB2 and pfhA2. The low and nonpathogenic strains did not contain the genes tpsB2 and pfhA2. A deletion of four bases was observed in the pfhA gene in the low pathogenic strain, and an insertion of 37 kb of phage DNA was observed in the nonpathogenic strains. MLST clustered the HP isolates in one group and the low and nonpathogenic isolates in another. Only the nonpathogenic isolates matched sequence type 10; the other isolates did not match any type available in the MLST database. CONCLUSIONS: The hypothesis that some PmA strains are primary pathogens and cause disease in pigs without any co-factor was confirmed. The pfhA region, comprising the genes tpsB1, tpsB2, pfhA1 and pfhA2, is related to the pathogenicity of PmA. The HP strains can cause necrotic bronchopneumonia, fibrinous pleuritis and pericarditis in pigs and can be identified by PCR amplification of the gene pfhA2.

Identification of Pasteurella multocida transcribed genes in porcine lungs through RNAseq.

Pasteurella multocida is one of the most important pathogen that causes pneumonia in swine. Although several virulence factors are known, the pathogenesis of this bacterium is not well-studied. Therefore, to study the pathogenesis of P. multocida infection in porcine lung, next-generation RNA sequencing was used to compare the transcriptomes of P. multocida grown in vivo and in vitro, respectively. After P. multocida infection a total of 704 genes were expressed in vitro, 1422 genes were expressed in vivo, and 237 genes were differentially expressed based on statistical analyses, padj of ≤0.1. Genes encoding ribosomal proteins or other products that function in the regulation of transcription and translation were downregulated, whereas genes whose products affected cellular processes (protein transport and RNA degradation) and metabolic pathways, such as those of amino acid metabolism and nucleotide metabolism, were upregulated in vitro compared with in vivo. This study shows that differentially expressed genes in P. multocida regulate pathways that operate during stress, iron capture, heat shock, and nitrogen regulation. However, extensive investigation of the pathogenic mechanism of P. multocida is still required.

Structure analysis of capsid protein of Porcine circovirus type 2 from pigs with systemic disease

Abstract Economic losses with high mortality rate associated with Porcine circovirus type 2 (PCV2) is reported worldwide. PCV2 commercial vaccine was introduced in 2006 in U.S. and in 2008 in Brazil. Although PCV2 vaccines have been widely used, cases of PCV2 systemic disease have been reported in the last years. Eleven nursery or fattening pigs suffering from PCV2 systemic disease were selected from eight PCV2-vaccinated farms with historical records of PCV2 systemic disease in Southern Brazil. PCV2 genomes were amplified and sequenced from lymph node samples of selected pigs. The comparison among the ORF2 amino acid sequences of PCV2 isolates revealed three amino acid substitutions in the positions F57I, N178S and A190T, respectively. Using molecular modeling, a structural model for the capsid protein of PCV2 was built. Afterwards, the mutated residues positions were identified in the model. The structural analysis of the mutated residues showed that the external residue 190 is close to an important predicted region for antibodies recognition. Therefore, changes in the viral protein conformation might lead to an inefficient antibody binding and this could be a relevant mechanism underlying the recent vaccine failures observed in swine farms in Brazil.

Structure analysis of capsid protein of Porcine circovirus type 2 from pigs with systemic disease.

Economic losses with high mortality rate associated with Porcine circovirus type 2 (PCV2) is reported worldwide. PCV2 commercial vaccine was introduced in 2006 in U.S. and in 2008 in Brazil. Although PCV2 vaccines have been widely used, cases of PCV2 systemic disease have been reported in the last years. Eleven nursery or fattening pigs suffering from PCV2 systemic disease were selected from eight PCV2-vaccinated farms with historical records of PCV2 systemic disease in Southern Brazil. PCV2 genomes were amplified and sequenced from lymph node samples of selected pigs. The comparison among the ORF2 amino acid sequences of PCV2 isolates revealed three amino acid substitutions in the positions F57I, N178S and A190T, respectively. Using molecular modeling, a structural model for the capsid protein of PCV2 was built. Afterwards, the mutated residues positions were identified in the model. The structural analysis of the mutated residues showed that the external residue 190 is close to an important predicted region for antibodies recognition. Therefore, changes in the viral protein conformation might lead to an inefficient antibody binding and this could be a relevant mechanism underlying the recent vaccine failures observed in swine farms in Brazil.

Anatomopathological pneumonic aspects associated with highly pathogenic Pasteurella multocida in finishing pigs / Aspectos pneumônicos anatomo-patológicos associados a Pasteurella multocida de alta patogenicidade em suínos de terminação

The bacterium Pasteurella multocida is a frequent cause of porcine respiratory disease complex in finishing pigs. Historically, the bacterium is recognized as an opportunistic agent, causing secondary bacterial pneumonia in pigs. Several Brazilian reports have suggested the ability of P. multocida to cause primary pulmonary infection that leads to the death of finishing pigs prior to slaughter. The aim of this study was to evaluate anatomopathological pulmonary findings associated with P. multocida infection that were obtained from animals with clinical respiratory disease and from animals at slaughter. Twenty-five lung samples from 14 herds of finishing pigs with acute clinical respiratory disease and 19 lungs collected at slaughter from a different set of 14 herds were studied. In all lung samples, bacterial isolation was performed, and only samples with pure P. multocida growth were included in the study. Gross and histopathological lesions were evaluated, as well as Influenza A, porcine circovirus type 2 (PCV2) and Mycoplasma hyopneumoniae co-infections. Pleuritis and pericarditis were more often observed in clinical samples (P<0.05). Moreover, there was a numerical trend indicating that pericarditis, lymphadenomegaly and cavity exudates were more often present in clinical samples. Thirteen lung samples were negative to M. hyopneumoniae, Influenza A and PCV2 by immunohistochemistry (IHC), with only P. multocida identified. In these cases, gross lesions such as pleuritis, pericarditis and lymphadenomegaly were always present, and no histologic lesions indicative of other agents such as Actinobacillus pleuropneumoniae, Actinobacillus suis or Haemophilus parasuis were observed. These findings suggest the ability of some P. multocida isolates to cause primary respiratory and systemic infection. However, in this study, it was not possible to determine specific virulence markers to explain these findings.

A bactéria Pasteurella multocida é causa frequente do Complexo de Doenças Respiratórias dos suínos em animais de terminação. Historicamente, a bactéria é reconhecida como agente oportunista, causando pneumonia bacteriana secundária. Diversos relatos brasileiros sugerem a habilidade da P. multocida de causar infecção pulmonar primária que leva a mortalidade de animais de terminação antes do abate. O objetivo deste estudo foi avaliar achados anatomopatológicos pulmonares associados com infecção por P. multocida, obtidas de animais acometidos clinicamente por doença respiratória e de animais ao abate. Avaliou-se 25 amostras de pulmão de 14 rebanhos obtidas de animais de terminação com sinais clínicos de doença respiratória aguda, e 19 pulmões coletados ao abate de 14 rebanhos diferentes. Em todos os pulmões, realizou-se isolamento bacteriano, e apenas amostras com crescimento puro de P. multocida foram incluídas no trabalho. Avaliou-se as lesões macro e microscopicamente, assim como co-infecções por Influenza A, Circovirus suíno tipo 2 (PCV2) e Mycoplasma hyopneumoniae. Pleurite e pericardite foram mais frequentemente observadas em amostras clinicas (P<0,05). Ainda, houve tendência numérica indicando a ocorrência de linfadenomegalia e exsudação cavitária, mais presentes em amostras clínicas. Treze amostras de pulmão foram negativas para M. hyopneumoniae, Influenza A e PCV2 por imunoistoquímica (IHQ), com identificação de apenas P. multocida. Nestes casos, lesões macroscópicas como pleurite, pericardite e linfadenomegalia foram sempre presentes, sem lesões histológicas indicativas de outros agentes como Actinobacillus pleuropneumoniae, Actinobacillus suis ou Haemophilus parasuis. Estes achados sugerem a habilidade de alguns isolados de P. multocida de causarem quadro respiratório primário e infecção sistêmica. No entanto, neste estudo, não foi possível determinar marcadores de virulência específicos para justificar tais achados.

Mycoplasma hyorhinis infection in early cases of mycoplasmal pneumonia in swine and evaluation of diagnostic assays / Infecção por Mycoplasma hyorhinis em casos precoces de pneumonia micoplásmica em suínos e comparação entre técnicas diagnósticas

Mycoplasmal pneumonia is an important disease in the pig industry. Due to the controversial role of Mycoplasma hyorhinis in this disease, confirmation of the presence of this bacterium and the identification of its roles in respiratory disease remain major challenges. The objectives of this study were to evaluate the presence of M. hyorhinis in early cases of mycoplasmal pneumonia and to determine the usefulness of fluorescent in situ hybridization (FISH) for the diagnosis of respiratory mycoplasmosis in naturally infected pigs. Ninety M. hyopneumoniae and/or M. hyorhinis-infected lung tissue samples based on diagnostic mosaic (DM) were used. The average age of the animals was 116 and 57 days (P<0.01) for groups 1 (positive-M. hyopneumoniae only) and 2 (positive-M. hyorhinis only), respectively. These findings suggest that development of lesions caused by M. hyorhinis occurs earlier than for M. hyopneumoniae. Using the DM as the gold standard, the sensitivity and specificity of FISH for M. hyopneumoniae were 75 and 100%, respectively, and were 40 and 73.3% for the immunohistochemistry (IHC). The sensitivity and specificity of FISH for M. hyorhinis were 76.7 and 100%, respectively. These findings demonstrate that FISH can be a useful tool for diagnosing mycoplasmosis. Viral antigens (PCV2 or influenza A) were detected in 53.3% (16/30) of the samples in group 2 (M. hyorhinis-PCR positive) and 13.3% (4/30) of the samples in group 1 (M. hyopneumoniae-PCR positive). This finding indicates that the association of M. hyorhinis and viral infection in nursery pigs likely starts due to a viral immunosuppressive condition.(AU)

A pneumonia micoplásmica causada por bactérias do gênero Mycoplasma é uma enfermidade de grande importância para indústria suinícola, sendo ainda controverso o papel desempenhado por Mycoplasma hyorhinis nessa doença. A confirmação da presença dessas bactérias bem como a identificação de seus papéis em doenças respiratórias continua sendo um grande desafio. Os objetivos desse estudo foram comparar diferentes técnicas, em especial a de hibridização fluorescente in situ (FISH), para diagnóstico de micoplasmoses respiratória em suínos naturalmente infectados e avaliar a presença do M. hyorhinis em casos precoces de pneumonia micoplásmica. Foram utilizadas 90 amostras de tecido pulmonar infectado para cada um ou ambos os agentes (M. hyopneumoniae e M. hyorhinis) determinados pelo mosaico de diagnóstico (sinais clínicos, lesões macroscópicas e microscópicas e pela PCR). No grupo de animais positivos pela PCR apenas para M. hyorhinis (Grupo 2) a média da idade foi de 57,32 dias e no grupo apenas positivo para M. hyopneumoniae (Grupo 1) a média foi de 116,31 dias (P<0,01). Estes achados sugerem que a colonização e o aparecimento de lesões causadas pelo M. hyorhinis seja mais precoce do que aquelas causadas pelo M. hyopneumoniae. As alterações microscópicas foram estatisticamente (P<0,01) mais intensas no grupo 1 do que no grupo 2. Usando o mosaico de diagnóstico como padrão ouro, a sensibilidade e especificidade na FISH para M. hyopneumoniae foi de 75 e 100%, respectivamente, e 40 e 73,3%, na imuno-histoquímica. A sensibilidade e especificidade da FISH para M. hyorhinis foi de 76,7 e 100%. Esses valores demonstram que a FISH pode ser uma ferramenta útil para diagnóstico de micoplasmoses. Foi detectada a presença de agentes virais (PCV2 ou influenza) em 53,3% das amostras do grupo 2 (M. hyorhinis) e em 13,3% das amostras do grupo 1 (M. hyopneumoniae).(AU)

Identificação de transcritos diferencialmente expressos por Pasteurella multocida em condições de privação de ferro / Identification of differentially expressed transcripts by Pasteurella multocida iron-starved condition

Ferro (Fe) é um elemento essencial e a capacidade de adquiri-lo in vivo têm sido descrita em diversos agentes patogênicos através de fatores de virulência. Análises de transcritos durante a privação de Fe tem sido descritos através da técnica de "microarray", entretanto a técnica de RNA-seq recentemente tem demonstrado resultados superiores. Neste trabalho, o isolado de Pasteurella multocida (Pm 16759) altamente patogênico em suínos foi cultivado em duas condições com diferentes concentrações de Fe (controle e privação) com o objetivo de analisar transcritos diferencialmente expressos. O RNA total das duas condições foi extraído e sequenciado através da plataforma de nova geração Ion Torrent. Os dados foram analisados no Software Ion Reporter(tm) e processados no programa Rockhopper. Foram obtidas 1.341.615 leituras com tamanho médio de 81pb, com 96% de alinhamento com o genoma de Pasteurella multocida subsp. multocida 3480 e 98,8% de acurácia. No mapeamento das leituras das duas condições, observou-se 2,652 transcritos e destes, 177 (6,7%) foram diferencialmente expressos, sendo 93 na condição controle (Fe+) e 84 na condição de privação (Fe-). Na condição de privação de Fe, o perfil de transcritos foram associados a função de transporte celular (fbp ABC, permease de alta afinidade com Fe2+/Pb2+ e proteína periplasmática de alta afinidade com Fe2+ ), reguladores transcricionais e proteínas hipotéticas. O perfil na condição controle (Fe+) apresentou transcritos diferencialmente expressos associados ao RNAs anti-sense (asRNA) e genes do metabolismo energético (fructose-1,6-bisfosfatase). O estudo comprovou que a restrição de Fe aumenta a expressão de genes envolvidos no transporte celular, reguladores transcricionais, proteínas hipotéticas e desconhecidas e permitiu ainda a identificação de novos genes como a permease de alta afinidade com Fe2+/Pb2+ e proteina periplasmática de alta afinidade com Fe2+ , que configuram uma possível via alternativa de absorção de Fe.(AU)

Iron (Fe) is an essential element and the ability to acquire it in vivo has been described in several pathogens as virulence factors. Global analyses of transcripts during iron deprivation have been described by microarray studies, however recently RNA-seq analysis showed superior results. The high pathogenic swine strain of Pasteurella multocida (BRMSA 1113) was grown in two conditions with different concentrations of Fe (control and deprivation) in order to analyze the differentially expressed transcripts. The total RNA of the two conditions was extracted and sequenced by new generation Ion Torrent plataform. Data were analyzed in Ion Reporter(tm) Software and processed in Rockhopper software. Sequence analysis shows 1,341,615 readings with median length of 81pb, with 96% of alignment to the reference genome Pasteurella multocida strain 3489, and 98.8% accuracy. Reads mapping to genome of P. multocida in these two conditions detected 2,652 transcripts, which 177 (6.7%) were differentially expressed, with 93 in the control condition (Fe+) and 84 provided with iron deprivation condition (Fe-). In condition (Fe-), differential expressed transcript profile were associated to function of cellular transport (fbpABC, high-affinity Fe2+/Pb2+ permease and periplasmic protein probably involved in hight-affinity Fe2+ ), transcptional regulators and hypothetical proteins. The control condition (Fe+) shows differential expressed transcripts profile associated to RNA anti-sense (asRNA) energetic metabolism genes (fructose-1,6-bisphosphatase). The study showed that the Fe restriction increases the expression of genes involved in cellular transport, transcriptional regulators, hypothetical and unknown proteins, and also allowed the identification of High-affinity Fe2+/Pb2+ permease e Periplasmic protein probably involved in high-affinity Fe2+, that constitute a possible alternative route for Fe absorption.(AU)

Pasteurella multocida type A as the primary agent of pneumonia and septicaemia in pigs / Pasteurella multocida tipo A como agente primário de pneumonia e septicemia em suínos

In order to understand better the pathological aspects and spread of Pasteurella multocida type A as the primary cause of pneumonia in pigs, was made an experiment with intranasal inoculation of different concentrations of inocula [Group (G1): 108 Colony Forming Units (CFU)/ml; G2: 107 CFU/ml; G3: 106 CFU/ml and G4: 105 CFU/ml], using two pigs per group. The pigs were obtained from a high health status herd. Pigs were monitored clinically for 4 days and subsequently necropsied. All pigs had clinical signs and lesions associated with respiratory disease. Dyspnoea and hyperthermia were the main clinical signs observed. Suppurative cranioventral bronchopneumonia, in some cases associated with necrosuppurative pleuropneumonia, fibrinous pericarditis and pleuritic, were the most frequent types of lesion found. The disease evolved with septicaemia, characterized by septic infarctions in the liver and spleen, with the detection of P. multocida type A. In this study, P. multocida type A strain #11246 was the primary agent of fibrinous pleuritis and suppurative cranioventral bronchopneumonia, pericarditis and septicaemia in the pigs. All concentrations of inoculum used (105-108 CFU/ml) were able to produce clinical and pathological changes of pneumonia, pleuritis, pericarditis and septicemia in challenged animals...

Para entender melhor os aspectos patológicos e disseminação de Pasteurella multocida tipo A como causa primária de pneumonia em suínos foi realizado um experimento com inoculação intranasal de diferentes concentrações de inóculos [Grupo (G1): 108 Unidades Formadoras de Colônias (UFC)/ml; G2: 107 UFC/ml; G3: 106 UFC/ml e G4: 105 UFC/ml], usando dois suínos por grupo. Esses suínos foram obtidos de um rebanho de alto status sanitário. Os animais foram monitorados clinicamente por quarto dias e subsequentemente necropsiados. Todos os suínos apresentaram sinais clínicos e lesões associadas com doença respiratória. Dispneia e hipertermia foram os principais sinais clínicos observados. Broncopneumonia cranioventral supurativa, em alguns casos associados com pleuropneumonia necrossupurativa, pleurites e pericardite fibrinosa foram mais frequentes. A doença evoluiu com septicemia, caracterizada por infartos sépticos no fígado e baço, com detecção de P. multocida. Neste estudo, P. multocida tipo A isolado 11246 foi agente primário de pleurite fibrinosa e broncopneumonia cranioventral supurativa, pericardite fibrinosa e septicemia em suínos. Todas as concentrações de inóculo utilizado (105-108 UFC/ml) foram capazes de produzir sinais clínicos e patológicos de alterações de pneumonia, pleurites, pericardites e septicemia nos animais...

Aspectos patológicos e microbiológicos das doenças respiratórias em suínos de terminação no Brasil / Pathological and microbiological aspects of respiratory disease in fattening pigs in Brazil

Para avaliação dos aspectos patológicos e microbiológicos de casos clínicos de doenças respiratórias em suínos de terminação foram analisados 75 suínos doentes oriundos de 36 lotes. Suínos que apresentavam sinais clínicos respiratórios evidentes foram necropsiados para avaliação macroscópica e colheita de amostras para análise histopatológica e microbiológica. Foram realizados testes de isolamento bacteriano para as principais bactérias do sistema respiratório dos suínos, PCR para Mycoplasma hyorhinis, imuno-histoquímica para Influenza A, Circovirus suíno tipo 2 e Mycoplasma hyopneumoniae. A sensibilidade antimicrobiana de 24 amostras de Pasteurella multocida tipo A foi avaliada por testes de concentração inibitória mínima para os principais antimicrobianos utilizados em suinocultura. Mycoplasma hyopneumoniae e Pasteurella multocida tipo A foram os agentes infecciosos mais prevalentes. Broncopneumonia supurativa e pleurite foram as principais lesões respiratórias encontradas. Pasteurella multocida tipo A, quando presente, aumentou a extensão das lesões pulmonares. Todas as amostras de Pasteurella multocida testadas foram sensíveis aos antimicrobianos Doxiciclina, Enrofloxacina e Tilmicosina. Em 58% das amostras foi identificado mais de um agente infeccioso, evidenciando a alta prevalência da associação de agentes nas doenças respiratórias de suínos em terminação...

For pathological and microbiological evaluation of porcine respiratory disease in fattening pigs, seventy five animals showing respiratory distress, fever and/or cough were analyzed. These pigs were necropsied and samples were collected for histological and microbiological analysis. Bacterial isolation procedures were performed aiming to detect major swine bacterial respiratory pathogens. Also, PCR for Mycoplasma hyorhinis, and immunohistochemistry for Influenza A, porcine circovirus type 2, and Mycoplasma hyopneumoniae were carried out. Mycoplasma hyopneumoniae and Pasteurella multocida type A were the most prevalent infectious agents. The antimicrobial sensitivity of 24 samples of P. multocida type A was evaluated by minimum inhibitory concentration tests and all these samples were sensitive to doxycycline, tilmicosin and enrofloxacin. Suppurative bronchopneumonia and pleuritis were main respiratory lesions found. When P. multocida type A was present, the extension of lung lesions was increased. In 58% of the samples more than one infectious agent was identified, suggesting a high prevalence of infectious agents associations in porcine respiratory disease in Brazil...

Ocorrência de genes tad associados à formação de biofilme em isolados de Pasteurella multocida de pulmões de suínos com pneumonia / Occurrence of tad genes associated with biofilm formation in isolates of Pasteurella multocida from lungs of pigs with pneumonia

Nowadays pig intensive production systems increase microbial selection pressure favoring spread of respiratory diseases. The bacteria Pasteurella multocida is associated with various respiratory diseases in pigs under farming conditions causing high economic losses. In vitro biofilm formation has been described in P. multocida which role is described in tissue colonization, enhancing resistance to host defenses and antibiotics. The aims of this study were to analyze the occurrence of P. multocida in pig pneumonia and health lung microbiota, and occurrence of tad locus genes in these isolates. Seventy isolates of P. multocida were analyzed which sixty seven were from lungs with lesion and three from healthy lungs. Serotype A occurred mainly in lung lesion (85.71%), in healthy lung, instead, only serotype D was observed. Genes tadA, tadB, tadC, tadD, tadE, tadF and tadG were present in 89.55% isolates from lesion lung. Genes tadA, tadB and tadC were present in all isolates from healthy lungs but, tadD, tadE tadF and tadG were present in 0%, 33.3%, 33,3% and 66.6%, respectively. This work associated tadD, tadE and tadF gene positive isolates of P. multocida to lung pneumonic lesions.

Os atuais sistemas de criação intensiva de suínos aumentam a pressão de seleção microbiana propiciando a disseminação de doenças respiratórias. A bactéria Pasteurella multocida é associada a diversas patologias respiratórias em animais submetidos a esse tipo de criação, causando grandes perdas econômicas. A formação de biofilme foi descrita in vitro em P. multocida e fatores analisados indicaram a facilitação na colonização dos tecidos, aumentando a resistência às defesas do hospedeiro e aos antibióticos. Os objetivos deste trabalho foram analisar a ocorrência de P. multocida em pneumonias de suínos e na microbiota de pulmões sem lesão e a ocorrência dos genes do lócus tad nestes isolados. Foram analisados 70 isolados de P. multocida de pulmões, sendo sessenta e sete com lesão e três sem lesão. Isolados do sorotipo A ocorreram principalmente em pulmões com lesões (85,71%), enquanto em pulmões sem lesão observou-se somente o sorotipo D. Os genes tadA, tadB, tadC, tadD, tadE tadF e tadG estavam presentes em 89,55% dos isolados de pulmões com lesões. Os genes tadA, tadB e tadC estavam presentes em todos os isolados de pulmões sem lesão, porém os genes tadD, tadE, tadF e tadG estavam presentes em 0%, 33,3%, 33,3% e 66,6%, dos isolados sem lesão, respectivamente. Neste trabalho observou-se a associação da ocorrência dos genes tadD, tadE e tadF em isolados de P. multocida e a presença de lesões em pulmões.

Interstitial nephritis of slaughtered pigs in the State of Mato Grosso, Brazil / Nefrite intersticial em suínos abatidos no Estado de Mato Grosso

This study evaluated histological lesions in kidney samples from pigs with nephritis in two slaughterhouses in the State of Mato Grosso, Brazil. Four hundred samples were subjected to histology, anti-porcine circovirus type 2 (PCV2) immunohistochemistry (IHC), anti-Leptospira sp. immunofluorescence (IF), and polymerase chain reaction (PCR) for PCV2, porcine parvovirus (PPV), and Torque teno virus type 1 and 2 (TTV1, TTV2) detection. Histological lesions were found in 81% of the samples, and mononuclear interstitial nephritis was the most frequent lesion (77.50%). A follicular pattern was observed in 40.97% of the interstitial nephritis lesions. PCV2, PPV, TTV1, and TTV2 were identified in the kidneys by PCR in 27.25%, 28.50%, 94%, and 87.5% of the samples, respectively. Leptospira sp. was not detected through IF. Infection by PCV2 (PCR) and the presence of histological lesions (P=0.008) and giant cells (P=0.0016) were significantly associated. An association was observed between the TTV2-TTV1 co-infection (P<0.0001) and the risk for pathogenesis. These findings indicated that PCV2, PPV, TTV1, and TTV2 were widely distributed among pigs in the local farms and that the presence of these agents should be considered in the differential diagnosis of kidneys with interstitial nephritis in pigs.

O propósito desse estudo foi avaliar as lesões histológicas observadas em rins condenados por nefrite pelo Serviço de Inspeção Federal, em dois frigoríficos de Mato Grosso, Brasil. Foram coletados 400 rins condenados por nefrite e submetidos aos exames de histologia, imuno-histoquímica (IHC) para Circovirus suíno Tipo 2 (PCV2), imunofluorescência direta (IF) para Leptospira sp. e reação em cadeia pela polimerase (PCR) para detecção de PCV2, Parvovirus suíno (PPV) e Torque teno vírus Tipo 1 e 2 (TTV1 e TTV2). Foram observadas lesões histológicas em 81% das amostras, sendo nefrite intersticial mononuclear a mais freqüente (77,50%). Das lesões de nefrite intersticial encontradas, 40,97% apresentaram padrão folicular. Através da PCR foi observada ampla distribuição dos agentes (PCV2, PPV, TTV1 e TTV2) nas propriedades e municípios, com ocorrência de 27,25%, 28,50%, 94% e 87,50%, respectivamente. Leptospira sp. não foi detectada através da IF. Houve associação significativa da infecção do PCV2 com presença de lesão histológica (P=0,008) e de células gigantes (P=0,0016). Também houve associação entre a co-infecção TTV2 e TTV1 (P<0,0001). Esses achados indicam que os vírus PCV2, PPV, TTV1 e TTV2 devem ser considerados no diagnóstico diferencial de rins com nefrite intersticial em suínos.

Efeito de probiótico na infecção e excreção fecal de Salmonella em suínos / Effect of probiotic on the Salmonella infection and fecal excretion in pigs

A transmissão de Salmonella na cadeia produtiva de suínos é um problema de difícil controle. O objetivo do estudo foi avaliar o efeito da administração oral de probiótico sobre a ocorrência de infecção e excreção fecal de Salmonella em suínos em fase de crescimento. Os tratamentos consistiram de ração basal sem aditivos (controle) ou adicionada de probiótico (10(7)ufc g-1 de células viáveis dos gêneros Bifidobacterium, Enterococcus, Lactobacillus e Saccharomyces). Foram alocados seis leitões de 50 dias em cada tratamento, com duas repetições por tratamento. Todos os animais foram inoculados com Salmonella Typhimurium (10(6)ufc mL-1) após 14 dias do alojamento. Semanalmente, foram coletadas amostras de sangue e fezes e no dia 35 pós-inoculação os animais foram sacrificados e necropsiados. Os animais de ambos os tratamentos foram infectados por Salmonella e soroconverteram. Não houve diferença (P>0,05) entre os grupos nas médias de Salmonella, Enterococcus, Lactobacillus e coliformes totais nas fezes, porém a administração de probiótico resultou em menor frequência de isolamento de Salmonella a partir de fígado (P=0,04), linfonodos mesentéricos (P=0,04), pulmão (P=0,03) e baço (P=0,01). Conclui-se que os microrganismos probióticos testados não foram capazes de impedir a infecção ou a excreção fecal de Salmonella em suínos de crescimento, mas diminuíram o número de portadores em linfonodos mesentéricos.

Control of Salmonella transmission has been a challenge for the pork production companies. The aim of this study was to evaluate the effect of oral administration of probiotics on the occurrence of infection and fecal excretion of Salmonella in growers. The treatments consisted of basal diet without additives (control) or added of probiotic (10(7)cfu g-1 of viable cells of the genera Bifidobacterium, Enterococcus, Lactobacillus and Saccharomyces). Six 50 days-old pigs were allocated into each treatment, with two replicates per treatment. All animals were inoculated with Salmonella Typhimurium (10(6)cfu mL-1) after 14 days of housing. Afterwards, blood and feces samples were taken weekly and on day 35 post-inoculation the animals were euthanized and necropsied. The animals in both treatment groups were infected by Salmonella and seroconverted. There was no difference (P>0.05) between groups in mean counts of Salmonella, Enterococcus, Lactobacillus and coliforms in the feces samples, but the probiotic administration resulted in a lower frequency of isolation of Salmonella from liver (P=0.04), mesenteric lymph nodes (P=0.04), lung (P=0.03) and spleen (P=0.01). It was concluded that the probiotic microorganisms tested in this study were not able to protect against the infection or to decrease the fecal excretion of Salmonella in growing pigs, but were able to decrease the number of carriers in the mesenteric lymph nodes.

Natural co-infection of torque teno virus and porcine circovirus 2 in the reproductive apparatus of swine.

This work aimed to detect and study natural co-infection of Circoviridae torque teno virus (TTV) and porcine circovirus 2 (PCV2) in the swine reproductive apparatus. Semen and organs from 17 boars were tested by nested and real-time PCR. PCV2 was amplified from semen (47%), lymph nodes (84.6%) and testicles (35.3%). TTV2 was amplified from 16/17 testis and 13/13 lymph nodes. TTV1 DNA was detected in fewer testicle samples (2/17), which were also TTV2 positive. Analyzed ovaries, follicular fluid and uteri of 83 culled sows showed TTV2, TTV1 and PCV2 from 49.3%, 30.1% and 6.0% of the sows, respectively. Sperm analysis indicated insignificant differences between PCV2 and TTVs positive and negative boars. The most frequent pathologic lesion in sows was endometritis (28.9%), but this was unassociated with PCV2 or TTVs detection. These findings question the importance of PCV2 and TTV2 natural co-infection in the pathology of porcine reproductive failures.