Epinephrine and norepinephrine regulate the expression of virulence factors in Gallibacterium anatis.

Gallibacterium anatis is a member of the Pasteurellaceae family and is an opportunistic pathogen that causes gallibacteriosis in chickens. Stress plays a relevant role in promoting the development of pathogenicity in G. anatis. Epinephrine (E) and norepinephrine (NE) are relevant to stress; however, their effects on G. anatis have not been elucidated. In this work, we evaluated the effects of E and NE on the growth, biofilm formation, expression of adhesins, and proteases of two G. anatis strains, namely, the hemolytic 12656-12 and the nonhemolytic F149T biovars. E (10 µM/mL) and NE (30 and 50 µM/mL) increased the growth of G. anatis 12656-12 by 20 % and 25 %, respectively. E did not affect the growth of F149T, whereas 40 µM/mL NE decreased bacterial growth by 25 %. E and NE at a dose of 30-50 µM/mL upregulated five fibrinogen adhesins in the 12565-12 strain, whereas no effect was observed in the F149T strain. NE increased proteolytic activity in both strains, whereas E diminished proteolytic activity in the 12656-12 strain. E and NE reduced biofilm formation (30 %) and increased Congo red binding (15 %) in both strains. QseBC is the E and NE two-component detection system most common in bacteria. The qseC gene, which is the E and NE receptor in bacteria, was identified in the genomic DNA of the 12565-12 and F149TG. anatis strains via PCR amplification. Our results suggest that QseC can detect host changes in E and NE concentrations and that catecholamines can modulate the expression of several virulence factors in G. anatis.

Clinical-pathological findings induced by Histophilus somni isolated in subacute cardiac death in feedlot cattle.

The purpose of this report is to provide information about the different presentations of cardiac and extra-cardiac histophilosis and, to assess the antimicrobial (ATM) susceptibility of Histophilus somni isolated from these cardiac lesions to different ATM agents commonly used for treating bovine bacterial respiratory pathogens. Eight feedlot calves, which died after suffering from food rejection, apathy, hyperthermia, cough and nasal mucous discharge, and lack of response to ATM therapy, were studied. Cardiac lesions observed at necropsy included valvular/mural endocarditis, myocardial infarction, and necrotizing myocarditis, miliar non-suppurative myocarditis, myocardic necrotic sequestrum, and/or pericarditis. Histopathological, bacteriological and molecular studies confirmed the presence of a fastidious microorganism in the affected organs. H. somni showed no resistance to most ATM tested (ceftiofur, gamithromycin, enrofloxacin, florfenicol, tilmicosin). The results obtained in this study confirmed that H. somni was the main cause of the subacute cardiac lesions associated with hyperthermia, apathy and respiratory signs observed in cattle examined in this research. These presentations must be considered by veterinary practitioners in order to establish a rational therapeutic.

Identification, HPG2 Sequence Analysis, and Antimicrobial Susceptibility of Avibacterium paragallinarum Isolates Obtained from Outbreaks of Infectious Coryza in Commercial Layers in Sonora State, Mexico.

This is the first extensive report on the identification and characterization of Avibacterium paragallinarum (AVP) isolates obtained from outbreaks of infectious coryza (IC) in IC-vaccinated layer flocks from Sonora State in Mexico. Isolates obtained from IC outbreaks during the years 2007, 2014, 2015, 2017, and 2019 were identified by conventional PCR test and 16S rRNA gene analysis, serotyped by Page serotyping and genotyped by the recently described partial sequence analysis of the HPG2 region. Furthermore, antimicrobial susceptibility profiles were determined by a recently improved minimal inhibitory concentration (MIC) test. The conventional PCR test and the 16S rRNA analyses confirmed the isolates as AVP. Serotyping results showed the involvement of isolates belonging to serotypes A, B, and C in the IC outbreaks. Genotyping of the HPG2 region revealed the presence of sequence type (ST)1, ST4, and ST11, of which the latter has also been identified in Europe. The MIC susceptibility test showed that all tested isolates were susceptible for the majority of tested antimicrobials, including erythromycin and tetracycline, which are important antibiotics for the treatment of IC. The IC situation in Sonora State, Mexico, is complex because of the presence of serotypes A, B, and C. This finding emphasizes the importance of biosecurity in combination with the application of the most optimal vaccination programs in the control of IC in Sonora State, Mexico.

Nota de investigación­Análisis de secuencias de la región HPG2 y susceptibilidad antimicrobiana de aislamientos de Avibacterium paragallinarum obtenidos de brotes de coriza infecciosa en aves de postura comerciales en el estado de Sonora, México. Este es el primer informe extenso sobre la identificación y caracterización de aislamientos de Avibacterium paragallinarum (AVP) obtenidos de brotes de coriza infecciosa (IC) de parvadas de ponedoras vacunadas con coriza infecciosa en el estado de Sonora en México. Los aislamientos obtenidos de los brotes de coriza infecciosa durante los años 2007, 2014, 2015, 2017 y 2019 se identificaron mediante una prueba de PCR convencional y el análisis del gene de ARNr 16S, se serotipificaron mediante el método de Page y se genotipificaron mediante el análisis parcial de secuencias descrito recientemente de la región HPG2. Además, se determinaron los perfiles de susceptibilidad a los antimicrobianos mediante la prueba de concentración mínima inhibitoria (MIC) que ha sido mejorada recientemente. La prueba de PCR convencional y los análisis de secuencias del gene ARNr 16S confirmaron que los aislados eran A. paragallinarum. Los resultados de la serotipificación mostraron la participación de aislamientos pertenecientes a los serotipos A, B y C en los brotes de coriza infecciosa. La genotipificación de la región HPG2 reveló la presencia de secuencias del tipo (ST) 1, ST4 y ST11, de los cuales este último también ha sido identificada en Europa. La prueba de susceptibilidad por concentración mínima inhibitoria mostró que todos los aislados analizados eran susceptibles a la mayoría de los antimicrobianos analizados, incluida la eritromicina y la tetraciclina, que son antibióticos importantes para el tratamiento contra la coriza infecciosa. La situación de coriza infecciosa en el estado de Sonora, México, es compleja por la presencia de los serotipos A, B y C. Este hallazgo enfatiza la importancia de la bioseguridad en combinación con la aplicación de los programas de vacunación óptimos en el control de la coriza infecciosa en el estado de Sonora, México.

Microbial diversity involved in the etiology of a bovine respiratory disease outbreak in a dairy calf rearing unit.

The etiological agents involved in a bovine respiratory disease (BRD) outbreak were investigated in a dairy heifer calf rearing unit from southern Brazil. A battery of PCR assays was performed to detect the most common viruses and bacteria associated with BRD, such as bovine viral diarrhea virus (BVDV), bovine respiratory syncytial virus (BRSV), bovine alphaherpesvirus 1 (BoHV-1), bovine coronavirus (BCoV), bovine parainfluenza virus 3 (BPIV-3), Mannheimia haemolytica, Pasteurella multocida, Histophilus somni, and Mycoplasma bovis. Bronchoalveolar lavage fluid (BALF) samples were taken from 21 heifer calves (symptomatic n = 15; asymptomatic n = 6) that, during the occurrence of the BDR outbreak, were aged between 6 and 90 days. At least one microorganism was detected in 85.7 % (18/21) of the BALF samples. Mixed infections were more frequent (72.2 %) than single infections (27.7 %). The interactions between viruses and bacteria were the most common in coinfections (55.5 %). The frequencies of BRD agents were 38.1 % for BRSV, 28.6 % for BVDV, 33.3 % for BCoV, 42.85 % for P. multocida, 33.3 % for M. bovis, and 19 % for H. somni. BoHV-1, BPIV-3, and M. haemolytica were not identified in any of the 21 BALF samples. Considering that BALF and not nasal swabs were analyzed, these results demonstrate the etiological multiplicity that may be involved in BRD outbreaks in dairy calves.

Diseases associated with bovine viral diarrhea virus subtypes 1a and 2b in beef and dairy cattle in Uruguay.

Bovine viral diarrhea virus (BVDV, Pestivirus) causes significant economic losses to the livestock industry worldwide. Although serological surveys show that BVDV exposure is widespread in cattle in Uruguay, BVDV-associated diseases are greatly underreported. The aim of this work is to describe the epidemiological, clinical, pathological, and virological findings from spontaneous outbreaks of BVDV-associated diseases in cattle in Uruguay. Diagnostic investigations were performed during 6 spontaneous disease outbreaks on beef and dairy cattle farms in the departments of Colonia, Rio Negro, and Soriano between November 2016 and April 2018. Carcasses of 8 naturally deceased cattle from these outbreaks were necropsied and subjected to histological examination and immunohistochemistry to detect BVDV antigen in the tissues. Reverse transcription real-time PCR and genomic sequencing were also performed to identify BVDV at the species and subtype levels. Other ancillary diagnostic tests, including bacterial cultures, were performed on a case-by-case basis to rule in/out differential diagnoses based on initial clinicopathological presumptive diagnoses. BVDV-associated conditions that were diagnosed in the 8 cases included mucosal disease, transient postnatal BVDV infections associated with digestive/septicemic salmonellosis by Salmonella serovar typhimurium, Histophilus somni bronchopneumonia, urinary tract coinfections with Escherichia coli and Streptococcus sp., enteric coinfection with coccidia, and transplacental fetal infections and abortions with Neospora caninum coinfection. BVDV-1a and BVDV-2b were each identified in four of the eight cases. We conclude that BVDV-1a and BVDV-2b contribute significantly to disease and mortality in cattle in Uruguay. Future research should estimate the economic impact of BVDV in the Uruguayan livestock sector.

Histophilus somni myocarditis and leptomeningitis in feedlot cattle: case report and occurrence in South America.

We investigated deaths in a group of feedlot steers in Argentina. The main findings in 3 steers autopsied were pulmonary congestion and edema, necrotizing myocarditis, pericarditis, suppurative leptomeningitis, and bronchopneumonia. Histophilus somni was detected by bacterial culture and immunohistochemistry in the hearts of the 3 animals. Partial sequences of the 16S rRNA gene of a H. somni isolate had 99% similarity with other H. somni sequences in GenBank. Most reports of H. somni septicemia in cattle originate from North America and western Europe. There is scant information about cardiac histophilosis in South America. A survey of diagnostic laboratory personnel in 7 South American countries documented various forms of bovine histophilosis in Argentina, Brazil, Uruguay, and Venezuela.

Potentiation of the humoral immune response elicited by a commercial vaccine against bovine respiratory disease by Enterococcus faecalis CECT7121.

Vaccination against pathogens involved in bovine respiratory disease (BRD) is a useful tool to reduce the risk of this disease however, it has been observed that the commercially available vaccines only partially prevent the infections caused by Pasteurella multocida and Mannheimia haemolytica. Therefore, it is recommended to search for new adjuvant strategies to minimise the economic impact of this respiratory syndrome. A possibility to improve the conventional vaccine response is to modulate the immune system with probiotics, since there is accumulating evidence that certain immunomodulatory strains administered around the time of vaccination can potentiate the immune response. Considering veterinary vaccines are frequently tested in murine models, we have developed an immunisation schedule in BALB/c mice that allows us to study the immune response elicited by BRD vaccine. In order to evaluate a potential strategy to enhance vaccine efficacy, the adjuvant effect of Enterococcus faecalis CECT7121 on the murine specific humoral immune response elicited by a commercial vaccine against BRD was studied. Results indicate that the intragastric administration of E. faecalis CECT7121 was able to induce an increase in the specific antibody titres against the bacterial components of the BRD vaccines (P. multocida and M. haemolytica). The quality of the humoral immune response, in terms of antibody avidity, was also improved. Regarding the cellular immune response, although the BRD vaccination induced a low specific secretion of cytokines in the spleen cell culture supernatants, E. faecalis CECT7121-treated mice showed higher interferon-γ production than immunised control mice. Our results allowed us to conclude that the administration of E. faecalis CECT7121 could be employed as an adjuvant strategy to potentiate humoral immune responses.

Is Antimicrobial Photodynamic Therapy Effective for Microbial Load Reduction in Peri-implantitis Treatment? A Systematic Review and Meta-Analysis.

The systematic review and meta-analysis were undertaken to evaluate the effectiveness of antimicrobial photodynamic therapy (aPDT) in the microbiological alteration beneficial to peri-implantitis treatment. This study is registered with PROSPERO, number CRD42017064215. Bibliographic databases including Cochrane Library, Web of Science, Scopus and PubMed were searched from inception to 8 January 2017. The search strategy was assembled from the following MeSH Terms: "Photochemotherapy," "Dental Implants" and "Peri-Implantitis." Unspecific free-text words and related terms were also included. The Cochrane Collaboration's tool was used to evaluate the risk of bias of included studies. The random-effect model was chosen, and heterogeneity was evaluated using the I2 test. Three studies met the inclusion criteria. Meta-analysis demonstrated an association between aPDT and reduction in viable bacteria counts for: Aggregatibacter actinomycetemcomitans (OR = 1.31; confidence interval = 1.13, 1.49; P < 0.00001), Porphyromonas gingivalis (OR = 4.08; confidence interval = 3.22, 4.94; P < 0.00001) and Prevotella intermedia (OR = 1.66; confidence interval = 1.06, 2.26; P < 0.00001). A aPDT appears to be effective in bacterial load reduction in peri-implantitis and has a positive potential as an alternative therapy for peri-implantitis.

Gallibacterium elongation factor-Tu possesses amyloid-like protein characteristics, participates in cell adhesion, and is present in biofilms.

Gallibacterium, which is a bacterial pathogen in chickens, can form biofilms. Amyloid proteins present in biofilms bind Congo red dye. The aim of this study was to characterize the cell-surface amyloid-like protein expressed in biofilms formed by Gallibacterium strains and determine the relationship between this protein and curli, which is an amyloid protein that is commonly expressed by members of the Enterobacteriaceae family. The presence of amyloid-like proteins in outer membrane protein samples from three strains of G. anatis and one strain of Gallibacterium genomospecies 2 was evaluated. A protein identified as elongation factor-Tu (EF-Tu) by mass spectrometric analysis and in silico analysis was obtained from the G. anatis strain F149T. This protein bound Congo red dye, cross-reacted with anti-curli polyclonal serum, exhibited polymerizing properties and was present in biofilms. This protein also reacted with pooled serum from chickens that were experimentally infected with G. anatis, indicating the in vivo immunogenicity of this protein. The recombinant EF-Tu purified protein, which was prepared from G. anatis 12656-12, polymerizes under in vitro conditions, forms filaments and interacts with fibronectin and fibrinogen, all of which suggest that this protein functions as an adhesin. In summary, EF-Tu from G. anatis presents amyloid characteristics, is present in biofilms and could be relevant for the pathogenesis of G. anatis.

Bovine respiratory disease associated with Histophilus somni and bovine respiratory syncytial virus in a beef cattle feedlot from Southeastern Brazil / Doença respiratória bovina associada a Histophilus somni e vírus respiratório sincicial bovino em um confinamento de bovinos de corte na região sudeste do Brasil

Bovine respiratory disease (BRD) is a complex multifactorial and multi-etiological disease entity that is responsible for the morbidity and mortality particularly in feedlot cattle from North America. Information relative to the occurrence of BRD in Brazil and the associated infectious agents are lacking. This study investigated the participation of infectious agents of BRD in a beef cattle feedlot from Southeastern Brazil. Nasopharyngeal swabs of 11% (10/90) of cattle (n, 450) with clinical manifestations of respiratory distress were analyzed by targeting specific genes of the principal infectious pathogens of BRD. In addition, pulmonary fragments of one the animals that died were collected for histopathological and molecular diagnoses. The nucleic acids of Histophilus somni and bovine respiratory syncytial virus (BRSV) were identified in 20% (2/10) of the nasopharyngeal swabs of the animals with respiratory distress; another contained only BRSV RNA. Moreover, the nucleic acids of both infectious agents were amplified from the pulmonary fragments of the animal that died with histopathological evidence of bronchopneumonia and interstitial pneumonia; the nasopharyngeal swab of this animal also contained the nucleic acids of both pathogens. Additionally, all PCR and/or RT-PCR assays designed to detect the specific genes of Mannheimia haemolytica, Pasteurella multocida, Mycoplasma bovis, bovine viral diarrhea virus, bovine herpesvirus -1, bovine parainfluenza virus-3, and bovine coronavirus yielded negative results. Phylogenetic analyses suggest that the isolates of H. somni circulating in Brazil are similar to those identified elsewhere, while there seem to be diversity between the isolates of BRSV within cattle herds from different geographical locations of Brazil.(AU)

A Doença Respiratória Bovina (DRB) é uma infecção multifatorial e multietiológica que ocasiona aumento nas taxas de morbidade e mortalidade em confinamentos bovinos. As informações disponíveis com relação à ocorrência de DRB no Brasil e sua associação com agentes infecciosos são ainda ocasionais. Esse estudo avaliou a participação de agentes infecciosos relacionados à DRB em um confinamento de bovinos na região sudeste do Brasil. Swabs nasofaríngeos de 11% (n=10) dos bovinos (n=90) com sinais clínicos de DRB em um lote de 450 animais foram analisados por técnicas moleculares para amplificação de genes específicos dos principais patógenos relacionados à DRB. Adicionalmente, fragmentos de pulmão de um dos animais, que morreu por DRB foi coletado para diagnóstico histopatológico e molecular. O ácido nucleico de Histophilus somni e do vírus respiratório sincicial bovino (BRSV) foi identificado em 20% (2/10) dos swabs nasofaríngeos. Em outro animal foi possível amplificar apenas o RNA do BRSV. O ácido nucleico de ambos os patógenos foi amplificado a partir de fragmentos pulmonares, que na histopatologia apresentou evidências de broncopneumonia e pneumonia intersticial. A partir do swab nasofaríngeo desse animal também foi possível amplificar o ácido nucleico de ambos os patógenos. Adicionalmente, todas as reações de PCR e RT-PCR realizadas para amplificar genes específicos de Mannheimia haemolytica, Pasteurella multocida, Mycoplasma bovis, vírus da diarreia viral bovina, herpesvírus bovino 1, vírus parainfluenza bovino 3 e coronavírus bovino foram negativas. Esses resultados confirmam a participação do H. somni e do BRSV no desenvolvimento de DRB em bovinos confinados no Brasil e abrem a perspectiva da realização de estudos mais amplos com o objetivo de identificar os principais agentes etiológicos da DRB no Brasil.(AU)

Use of metaphylactic protocols based on the risk to develop bovine respiratory diseases in feedlot cattle / Efeito do uso de protocolos metafiláticos segundo o risco de doença respiratória bovina em confinamentos

ABSTRACT: Bovine respiratory diseases (BRD) affect production rates negatively because it compromise health and well-being of the affected animal. The hypothesis of this study was that the use of metaphylactic protocols based on the risk to develop BRD would reduce morbidity and pulmonary lesions. For this purpose, the aims of this study were to evaluate the effect of two metaphylactic protocols on the morbidity of feedlot cattle with a known sanitary history, occurrence of pulmonary lesions at slaughter, and the possible participation of Mannheimia haemolytica, Histophilus somni, Bovine alphaherpesvirus 1 (BoHV-1) and bovine respiratory syncytial virus (BRSV) in the development of BRD. An experimental study was designed in which 3,094 adult, male, cattle, were grouped according to the risk to develop BRD: a) group without metaphylaxis (n=2,104), low-risk animals; b) metaphylaxis group with oxytetracycline (n=789), moderate-risk animals; c) metaphylaxis group with tildipirosin (n=201), high-risk animals. All cattle were immunized against pathogens associated with BRD (BoHV-1, BVDV, BRSV, PI3). The morbidity for BRD was 8.2% (253/3,094); cattle within the moderate-risk group for BRD had the lowest frequency (6.1%), followed by high-risk animals with tildipirosin metaphylaxis (6.5%) and low-risk without metaphylaxis (9.1%) (P=0.019). At the abattoir, 1.2% of lungs with lesions were found. There was a difference (P=0.036) in the frequency of pulmonary lesions between healthy animals (1.1%) and those diagnosed with BRD (2.8%). Two agents associated with BRD were identified by PCR assays in the lungs (n=37) of cattle: M. haemolytica (16.2%) and H. somni (5.4%). In addition, concomitant infections involving these pathogens were identified in the lungs of two steers.

RESUMO: As doenças respiratórias dos bovinos (DRB) afetam negativamente as taxas de produção por comprometer a saúde e o bem-estar animal. A hipótese deste estudo foi que o uso de protocolos metafiláticos com base no risco de desenvolvimento de DRB reduziria a morbidade e as lesões pulmonares ao abate. Para tanto, objetivou-se avaliar o efeito de dois protocolos metafiláticos sobre a morbidade de bovinos confinados com histórico sanitário conhecido, ocorrência de lesões pulmonares no abate e possível participação de Mannheimia haemolytica, Histophilus somni, Alphaherpesvírus bovino 1 (BoHV-1) e Vírus respiratório sincicial bovino (BRSV) na DRB. Foram avaliados 3.094 bovinos adultos, machos, agrupados de acordo com o risco de desenvolvimento de DRB: a) grupo sem metafilaxia (n=2.104), animais de baixo risco; b) grupo metafilaxia com oxitetraciclina (n=789), animais de risco moderado; c) grupo metafilaxia com tildipirosina (n=201), animais de alto risco. Todos os animais foram previamente vacinados contra os principais agentes virais causadores das DRB (BoHV-1, BVDV, BRSV e PI3). A morbidade para DRB foi de 8,2% com menor frequência em animais com risco moderado e metafilaxia com oxitetraciclina (6,1%), seguido por animais com alto risco com metafilaxia com tildipirosina (6,5%) e animais com baixo risco sem metafilaxia (9,1%) (P=0,019). No frigorífico, foram encontrados 1,2% 1,2% de animais com lesões pulmonares. Houve diferença (P=0,036) na frequência de lesões pulmonares entre animais saudáveis (1,1%) e aqueles diagnosticados com DRB (2,8%). Foram identificados dois agentes associados à DRB pela técnica de PCR nos pulmões (n=37) dos bovinos: M. haemolytica (16,2%) e H. somni (5,4%). Adicionalmente, infecções concomitantes envolvendo estes potenciais patógenos foram identificadas nos pulmões de dois animais.

Bovine respiratory disease associated with Histophilus somni and bovine respiratory syncytial virus in a beef cattle feedlot from Southeastern Brazil / Doença respiratória bovina associada a Histophilus somni e vírus respiratório sincicial bovino em um confinamento de bovinos de corte na região sudeste do Brasil

Bovine respiratory disease (BRD) is a complex multifactorial and multi-etiological disease entity that is responsible for the morbidity and mortality particularly in feedlot cattle from North America. Information relative to the occurrence of BRD in Brazil and the associated infectious agents are lacking. This study investigated the participation of infectious agents of BRD in a beef cattle feedlot from Southeastern Brazil. Nasopharyngeal swabs of 11% (10/90) of cattle (n, 450) with clinical manifestations of respiratory distress were analyzed by targeting specific genes of the principal infectious pathogens of BRD. In addition, pulmonary fragments of one the animals that died were collected for histopathological and molecular diagnoses. The nucleic acids of Histophilus somni and bovine respiratory syncytial virus (BRSV) were identified in 20% (2/10) of the nasopharyngeal swabs of the animals with respiratory distress; another contained only BRSV RNA. Moreover, the nucleic acids of both infectious agents were amplified from the pulmonary fragments of the animal that died with histopathological evidence of bronchopneumonia and interstitial pneumonia; the nasopharyngeal swab of this animal also contained the nucleic acids of both pathogens. Additionally, all PCR and/or RT-PCR assays designed to detect the specific genes of Mannheimia haemolytica, Pasteurella multocida, Mycoplasma bovis, bovine viral diarrhea virus, bovine herpesvirus -1, bovine parainfluenza virus-3, and bovine coronavirus yielded negative results. Phylogenetic analyses suggest that the isolates of H. somni circulating in Brazil are similar to those identified elsewhere, while there seem to be diversity between the isolates of BRSV within cattle herds from different geographical locations of Brazil.

A Doença Respiratória Bovina (DRB) é uma infecção multifatorial e multietiológica que ocasiona aumento nas taxas de morbidade e mortalidade em confinamentos bovinos. As informações disponíveis com relação à ocorrência de DRB no Brasil e sua associação com agentes infecciosos são ainda ocasionais. Esse estudo avaliou a participação de agentes infecciosos relacionados à DRB em um confinamento de bovinos na região sudeste do Brasil. Swabs nasofaríngeos de 11% (n=10) dos bovinos (n=90) com sinais clínicos de DRB em um lote de 450 animais foram analisados por técnicas moleculares para amplificação de genes específicos dos principais patógenos relacionados à DRB. Adicionalmente, fragmentos de pulmão de um dos animais, que morreu por DRB foi coletado para diagnóstico histopatológico e molecular. O ácido nucleico de Histophilus somni e do vírus respiratório sincicial bovino (BRSV) foi identificado em 20% (2/10) dos swabs nasofaríngeos. Em outro animal foi possível amplificar apenas o RNA do BRSV. O ácido nucleico de ambos os patógenos foi amplificado a partir de fragmentos pulmonares, que na histopatologia apresentou evidências de broncopneumonia e pneumonia intersticial. A partir do swab nasofaríngeo desse animal também foi possível amplificar o ácido nucleico de ambos os patógenos. Adicionalmente, todas as reações de PCR e RT-PCR realizadas para amplificar genes específicos de Mannheimia haemolytica, Pasteurella multocida, Mycoplasma bovis, vírus da diarreia viral bovina, herpesvírus bovino 1, vírus parainfluenza bovino 3 e coronavírus bovino foram negativas. Esses resultados confirmam a participação do H. somni e do BRSV no desenvolvimento de DRB em bovinos confinados no Brasil e abrem a perspectiva da realização de estudos mais amplos com o objetivo de identificar os principais agentes etiológicos da DRB no Brasil.

Use of metaphylactic protocols based on the risk to develop bovine respiratory diseases in feedlot cattle / Efeito do uso de protocolos metafiláticos segundo o risco de doença respiratória bovina em confinamentos

ABSTRACT: Bovine respiratory diseases (BRD) affect production rates negatively because it compromise health and well-being of the affected animal. The hypothesis of this study was that the use of metaphylactic protocols based on the risk to develop BRD would reduce morbidity and pulmonary lesions. For this purpose, the aims of this study were to evaluate the effect of two metaphylactic protocols on the morbidity of feedlot cattle with a known sanitary history, occurrence of pulmonary lesions at slaughter, and the possible participation of Mannheimia haemolytica, Histophilus somni, Bovine alphaherpesvirus 1 (BoHV-1) and bovine respiratory syncytial virus (BRSV) in the development of BRD. An experimental study was designed in which 3,094 adult, male, cattle, were grouped according to the risk to develop BRD: a) group without metaphylaxis (n=2,104), low-risk animals; b) metaphylaxis group with oxytetracycline (n=789), moderate-risk animals; c) metaphylaxis group with tildipirosin (n=201), high-risk animals. All cattle were immunized against pathogens associated with BRD (BoHV-1, BVDV, BRSV, PI3). The morbidity for BRD was 8.2% (253/3,094); cattle within the moderate-risk group for BRD had the lowest frequency (6.1%), followed by high-risk animals with tildipirosin metaphylaxis (6.5%) and low-risk without metaphylaxis (9.1%) (P=0.019). At the abattoir, 1.2% of lungs with lesions were found. There was a difference (P=0.036) in the frequency of pulmonary lesions between healthy animals (1.1%) and those diagnosed with BRD (2.8%). Two agents associated with BRD were identified by PCR assays in the lungs (n=37) of cattle: M. haemolytica (16.2%) and H. somni (5.4%). In addition, concomitant infections involving these pathogens were identified in the lungs of two steers.(AU)

RESUMO: As doenças respiratórias dos bovinos (DRB) afetam negativamente as taxas de produção por comprometer a saúde e o bem-estar animal. A hipótese deste estudo foi que o uso de protocolos metafiláticos com base no risco de desenvolvimento de DRB reduziria a morbidade e as lesões pulmonares ao abate. Para tanto, objetivou-se avaliar o efeito de dois protocolos metafiláticos sobre a morbidade de bovinos confinados com histórico sanitário conhecido, ocorrência de lesões pulmonares no abate e possível participação de Mannheimia haemolytica, Histophilus somni, Alphaherpesvírus bovino 1 (BoHV-1) e Vírus respiratório sincicial bovino (BRSV) na DRB. Foram avaliados 3.094 bovinos adultos, machos, agrupados de acordo com o risco de desenvolvimento de DRB: a) grupo sem metafilaxia (n=2.104), animais de baixo risco; b) grupo metafilaxia com oxitetraciclina (n=789), animais de risco moderado; c) grupo metafilaxia com tildipirosina (n=201), animais de alto risco. Todos os animais foram previamente vacinados contra os principais agentes virais causadores das DRB (BoHV-1, BVDV, BRSV e PI3). A morbidade para DRB foi de 8,2% com menor frequência em animais com risco moderado e metafilaxia com oxitetraciclina (6,1%), seguido por animais com alto risco com metafilaxia com tildipirosina (6,5%) e animais com baixo risco sem metafilaxia (9,1%) (P=0,019). No frigorífico, foram encontrados 1,2% 1,2% de animais com lesões pulmonares. Houve diferença (P=0,036) na frequência de lesões pulmonares entre animais saudáveis (1,1%) e aqueles diagnosticados com DRB (2,8%). Foram identificados dois agentes associados à DRB pela técnica de PCR nos pulmões (n=37) dos bovinos: M. haemolytica (16,2%) e H. somni (5,4%). Adicionalmente, infecções concomitantes envolvendo estes potenciais patógenos foram identificadas nos pulmões de dois animais.(AU)

Identification of a Hemagglutinin from Gallibacterium anatis.

Gallibacterium anatis has the ability to hemagglutinate rabbit erythrocytes; however, no bacterial component has yet been associated with this function. In the present work, a protein of approximately 65 kDa with hemagglutinating activity for glutaraldehyde-fixed chicken erythrocytes was purified by ion interchange chromatography from G. anatis F149(T) secreted proteins. The protein was recognized by a rabbit polyclonal serum against a hemagglutinin from Avibacterium paragallinarum. The 65 kDa purified protein presented identity with a G. anatis filamentous hemagglutinin by mass spectrometric analysis. As well, the bacterial surface of G. anatis was labeled by immune gold assays using a polyclonal serum against the 65-kDa protein. A similar protein was recognized in four other G. anatis strains by immunoblots using the same antiserum. The protein binds sheep or pig biotinylated fibrinogen, suggesting an interaction with basement membrane eukaryotic cells components, and the protein is present in G. anatis biofilms. Overall, the results suggest that the 65 kDa hemagglutinin is a common antigen and a potential virulence factor in G. anatis.

Real-time PCR for detection of Brucella ovis and Histophilus somni in ovine urine and semen / PCR em tempo real para detecção de Brucella ovis e Histophilus somni em urina e sêmen ovino

A epididimite infecciosa ovina é uma das principais enfermidades reprodutivas de carneiros. O presente estudo teve por objetivo desenvolver protocolos de PCR em tempo real para B. ovis e H. somni e avaliar sua aplicabilidade em amostras de sêmen e urina de carneiros. Delinearam-se primers e sondas espécie-específicos para cada agente. As sondas foram delineadas com o sistema TaqMan incorporando um marcador FAM para B. ovis e Cy5 para H. somni na extremidade 5' e um quencher na extremidade 3'. A PCR em tempo real para B. ovis e H. somni foi altamente sensível, uma vez que a amplificação de DNA ocorreu com até 0,2ng de DNA/reação. A especificidade dos iniciadores e sondas foi avaliada com amostras de DNA de outros agentes causadores de epididimite ovina e nenhuma amplificação inespecífica foi observada. A aplicabilidade da técnica em amostras biológicas também foi confirmada, pois não houve perda de eficácia (P>0,05) quando comparada à PCR convencional com amostras de sêmen e urina de carneiros experimentalmente infectados.

Real-time PCR for detection of Brucella ovis and Histophilus somni in ovine urine and semen / PCR em tempo real para detecção de Brucella ovis e Histophilus somni em urina e sêmen ovino

A epididimite infecciosa ovina é uma das principais enfermidades reprodutivas de carneiros. O presente estudo teve por objetivo desenvolver protocolos de PCR em tempo real para B. ovis e H. somni e avaliar sua aplicabilidade em amostras de sêmen e urina de carneiros. Delinearam-se primers e sondas espécie-específicos para cada agente. As sondas foram delineadas com o sistema TaqMan incorporando um marcador FAM para B. ovis e Cy5 para H. somni na extremidade 5' e um quencher na extremidade 3'. A PCR em tempo real para B. ovis e H. somni foi altamente sensível, uma vez que a amplificação de DNA ocorreu com até 0,2ng de DNA/reação. A especificidade dos iniciadores e sondas foi avaliada com amostras de DNA de outros agentes causadores de epididimite ovina e nenhuma amplificação inespecífica foi observada. A aplicabilidade da técnica em amostras biológicas também foi confirmada, pois não houve perda de eficácia (P>0,05) quando comparada à PCR convencional com amostras de sêmen e urina de carneiros experimentalmente infectados(AU)

Naturally Occurring ß-Nicotinamide Adenine Dinucleotide-Independent Avibacterium paragallinarum Isolate in Peru.

The ß-nicotinamide adenine dinucleotide (NAD) requirement has been considered to be essential for the isolation of the causal agent of infectious coryza, Avibacterium paragallinarum. Nevertheless, NAD-independent reports from South Africa and Mexico dismissed this paradigm. It is now accepted that both NAD-dependent and NAD-independent agents are able to cause infectious coryza and thus belong to the species A. paragallinarum. Here, we report for the first time in Peru a NAD-independent isolate from broiler chickens with typical signs of infectious coryza that have received a trivalent inactivated vaccine against infectious coryza. The isolate was identified based on its morphology, biochemical and serologic tests, and PCR results. Partial 16S rRNA gene sequence analysis confirmed the isolate as A. paragallinarum. There have been no cases of NAD-independent A. paragallinarum isolates reported in South America. Increasing reports around the world highlight not only the need to reconsider the in vitro nutritional requirements of this species for its correct isolation but also the cross-protection conferred by commercial infectious coryza vaccines against NAD-independent isolates.

Histophilus somni-induced thrombotic meningoencephalitis in cattle from northern Paraná, Brazil / Meningoencefalite trombótica-induzida por Histophilus somni em bovinos da região norte do Paraná

Thrombotic meningoencephalitis (TME) is a fatal neurological disease of cattle, predominantly from North America, that is caused by Histophilus somni with sporadic descriptions from other countries. This manuscript describes the occurrence of spontaneous TME in cattle from northern Paraná, Brazil. Most cattle had acute neurological manifestations characteristic of brain dysfunction. Hematological and cerebrospinal fluid analyses were not suggestive of bacterial infections of the brain. Histopathology revealed meningoencephalitis with vasculitis and thrombosis of small vessels that contained discrete neutrophilic and/or lymphocytic infiltrates admixed with fibrin at the brainstem, cerebral cortex, and trigeminal nerve ganglion of all animals. All tissues from the central nervous system used during this study were previously characterized as negative for rabies virus by the direct immunofluorescence assay. PCR and RT-PCR assays investigated the participation of infectious agents associated with bovine neurological disease by targeting specific genes of H. somni, Listeria monocytogenes, bovine herpesvirus -1 and -5, bovine viral diarrhea virus, and ovine herpesvirus-2. PCR and subsequent sequencing resulted in partial fragments of the 16S rRNA gene of H. somni from brain sections of all animals with histopathological diagnosis of TME; all other PCR/RT-PCR assays were negative. These findings confirmed the participation of H. somni in the neuropathological disease observed in these animals, extend the geographical distribution of this disease, and support previous findings of H. somni from Brazil.(AU)

Meningoencefalite trombótica (Thrombotic meningoencephalitis- TME) é uma doença neurológica fatal de bovinos ocasionada por Histophilus somni. A infecção tem sido descrita predominantemente na América do Norte e de forma esporádica em outros países. O objetivo deste estudo é relatar a ocorrência de TME em bovinos da região norte do estado do Paraná, Brasil. A maioria dos animais apresentaram sinais clínicos neurológicos característicos de disfunção cerebral aguda. Análises hematológicas e do fluido cerebrospinal não foram sugestivas de infecção bacteriana do cérebro. A histopatologia revelou meningoencefalite com vasculite e trombose de pequenos vasos com discreto infiltrado neutrofílico e/ou linfocítico mesclada com fibrina no tronco e córtex cerebral e no gânglio do nervo trigêmio de todos os animais. As amostras de sistema nervoso central incluídas nesse estudo foram previamente caracterizadas como negativas para raiva por meio de técnica de imunofluorescência direta. A participação de agentes infecciosos associados à doença neurológica em bovinos foi avaliada por técnicas moleculares como PCR e RT-PCR para amplificação parcial de genes de H. somni, Listeria monocytogenes, herpesvírus bovino 1 e 5, vírus da diarreia viral bovina e herpesvírus ovino 2. As seções do cérebro de todos os animais com diagnóstico histopatológico de TME foram positivas em PCR para a detecção do gene 16S rRNA de H. somni. O sequenciamento dos produtos amplificados confirmou a presença de DNA de H. somni nos fragmentos de cérebro avaliados. As reações de PCR/RT-PCR para todos os outros micro-organismos avaliados resultaram negativas. Os resultados desse estudo confirmaram a participação do H. somni nos episódios de doença neurológica observada nos animais avaliados, amplia a distribuição geográfica da TME e ratifica estudos prévios realizados no Brasil que demonstraram a presença de H. somni em outras formas de manifestação clínica das infecções por essa bactéria.(AU)

Identificação de Avibacterium paragallinarum em frangos de corte e poedeiras comerciais no Estado de Pernambuco / Avibacterium paragallinarum identification in broiler and commercial laying hens in the state of Pernambuco, Brazil

As aves da indústria avícola brasileira são suscetíveis a surtos de doenças infecciosas relacionadas a perdas econômicas, principalmente por enfermidades infecciosas causadas por Mycoplasma, Avibacterium paragallinarum e vírus que acometem o trato respiratório. Objetivou-se neste estudo pesquisar o envolvimento de A. paragallinarum em surto de doença respiratória aviária. Foram coletados 18 swabs da região infraorbitária dos seios nasais de aves, sendo seis amostras de frangos de corte com sinais clínicos e 12 de poedeiras com sinais clínicos. As amostras foram cultivadas em meios específicos para o agente e também testadas com a técnica molecular Reação em Cadeia de Polimerase. Das amostras analisadas no isolamento, 100% foram negativas. Na PCR, duas (16,66%) foram positivas. A detecção de A. paragallinarum na PCR em galinhas poedeiras com sinais clínicos respiratótios indica que esta bactéria está associada à etiologia da síndrome respiratória das aves nas granjas no estado de Pernambuco...

Pullets of the Brazilian poultry industry are susceptible to outbreaks of infectious diseases with economic losses, mainly caused by Mycoplasma, Avibacterium paragallinarum and viruses affecting the respiratory tract. The objective of this study was to verify the involvement of A. paragallinarum in an outbreak of avian respiratory disease. Eighteen swabs from the infra-orbital sinuses were collected, six samples from broilers and twelve from hens with clinical signs. The samples were cultured in specific media for the agent and also tested with the molecular technique of Polymerase Chain Reaction (PCR). All samples cultured in specific media were negative; PCR revealed two samples (16.66%) as positive. The detection of A. paragallinarum by PCR in laying hens with clinical respiratory signs indicates that this bacterium is associated with the etiology of respiratory syndrome in poultry farms in the state of Pernambuco...

Identificação de Avibacterium paragallinarum em frangos de corte e poedeiras comerciais no Estado de Pernambuco / Avibacterium paragallinarum identification in broiler and commercial laying hens in the state of Pernambuco, Brazil

As aves da indústria avícola brasileira são suscetíveis a surtos de doenças infecciosas relacionadas a perdas econômicas, principalmente por enfermidades infecciosas causadas por Mycoplasma, Avibacterium paragallinarum e vírus que acometem o trato respiratório. Objetivou-se neste estudo pesquisar o envolvimento de A. paragallinarum em surto de doença respiratória aviária. Foram coletados 18 swabs da região infraorbitária dos seios nasais de aves, sendo seis amostras de frangos de corte com sinais clínicos e 12 de poedeiras com sinais clínicos. As amostras foram cultivadas em meios específicos para o agente e também testadas com a técnica molecular Reação em Cadeia de Polimerase. Das amostras analisadas no isolamento, 100% foram negativas. Na PCR, duas (16,66%) foram positivas. A detecção de A. paragallinarum na PCR em galinhas poedeiras com sinais clínicos respiratótios indica que esta bactéria está associada à etiologia da síndrome respiratória das aves nas granjas no estado de Pernambuco.(AU)

Pullets of the Brazilian poultry industry are susceptible to outbreaks of infectious diseases with economic losses, mainly caused by Mycoplasma, Avibacterium paragallinarum and viruses affecting the respiratory tract. The objective of this study was to verify the involvement of A. paragallinarum in an outbreak of avian respiratory disease. Eighteen swabs from the infra-orbital sinuses were collected, six samples from broilers and twelve from hens with clinical signs. The samples were cultured in specific media for the agent and also tested with the molecular technique of Polymerase Chain Reaction (PCR). All samples cultured in specific media were negative; PCR revealed two samples (16.66%) as positive. The detection of A. paragallinarum by PCR in laying hens with clinical respiratory signs indicates that this bacterium is associated with the etiology of respiratory syndrome in poultry farms in the state of Pernambuco.(AU)