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1.
Vet Microbiol ; 235: 110-117, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31282368

RESUMO

Bovine Respiratory Disease (BRD) is a major threat to animal health and welfare in the cattle industry. Strains of Mannheimia haemolytica (Mh) that are resistant to multiple classes of antimicrobials are becoming a major concern in the beef industry, as the frequency of isolation of these strains has been increasing. Mobile genetic elements, such as integrative conjugative elements (ICE), are frequently implicated in this rapid increase in multi-drug resistance. The objectives of the current study were to determine the genetic relationship between the isolates collected at arrival before metaphylaxis and at revaccination after metaphylaxis, to identify which resistance genes might be present in these isolates, and to determine if they were carried on an ICE. Twenty calves culture positive for Mh at arrival and revaccination were identified, and a total of 48 isolates with unique susceptibility profiles (26 from arrival, and 22 from revaccination) were submitted for whole-genome sequencing (WGS). A phylogenetic tree was constructed, showing the arrival isolates falling into four clades, and all revaccination isolates within one clade. All revaccination isolates, and one arrival isolate, were positive for the presence of an ICE. Three different ICEs with resistance gene modules were identified. The resistance genes aphA1, strA, strB, sul2, floR, erm42, tetH/R, aadB, aadA25, blaOXA-2, msrE, mphE were all located within an ICE. The gene bla-ROB1 was also present in the isolates, but was not located within an ICE.


Assuntos
Antibacterianos/farmacologia , Bovinos/microbiologia , Farmacorresistência Bacteriana Múltipla/genética , Mannheimia haemolytica/efeitos dos fármacos , Mannheimia haemolytica/genética , Pasteurelose Pneumônica/microbiologia , Animais , Antibacterianos/uso terapêutico , Dissacarídeos/uso terapêutico , Variação Genética , Genoma Bacteriano , Compostos Heterocíclicos/uso terapêutico , Imunização Secundária , Sequências Repetitivas Dispersas , Mannheimia haemolytica/isolamento & purificação , Testes de Sensibilidade Microbiana , Pasteurelose Pneumônica/tratamento farmacológico , Filogenia , Vacinação , Sequenciamento Completo do Genoma
2.
BMC Microbiol ; 18(1): 205, 2018 12 05.
Artigo em Inglês | MEDLINE | ID: mdl-30518323

RESUMO

BACKGROUND: Mannheimia haemolytica has been recognized as the principal cause of pneumonic pasteurellosis in sheep and goats. It is one of the important diseases of small ruminants in Ethiopia. While annual vaccination using a monovalent vaccine (inactivated Pasteurella multocida biotype A) is common, respiratory diseases are still reported in various parts of Ethiopia. This suggests the need for further investigation into the species and strains responsible for the disease, which is vital information for development of a multivalent vaccine. The objective of the current study was to isolate M. heamolytica associated with pneumonic cases of sheep in selected areas of Central Ethiopia, determine its role and the strains/genotypes of the bacterium circulating in the study area. RESULTS: Bacteriological analysis of nasal swab samples collected from a total of 76 pneumonic cases of sheep showed that M. haemolytica was isolated from 26 of them while B.trehalosi from two cases. Further molecular analyses of the isolates using M. haemolytica species-specific and M.haemolytica serotype-1 antigen specific PCR assays revealed, 26 of the isolates were identified as M. haemolytica of which 21 of them were M. haemolytica serotype-1. Both M. haemolytica and B.trehalosi isolates were not detected in a PCR assay targeting capsular biosynthesis gene (capA) of P.multocida despite the non-specific products observed in M. haemolytica isolates. Phylogenetic analysis of M. haemolytica isolates included in this study in comparison with the reference strains with respect to PHSSA and Rpt2 genes revealed that the Ethiopian M. haemolytica isolates constituted three distinct genotypes consistent with site of origin. CONCLUSION: The study indicated that M.haemolytica is commonly associated with cases of pneumonia in sheep in the study areas of central Ethiopia although the remaining other pathogens responsible for majority of the cases are yet to be determined. Molecular characterization revealed the existence of three genotypes of M. haemolytica circulating in the study areas consistent to the site of isolation. The findings suggest further extensive work to determine all pathogens associated with sheep pneumonia and the strain distribution of M. heamolytica to understand its molecular epidemiology at national level and design cost effective prevention and control methods.


Assuntos
Mannheimia haemolytica/genética , Mannheimia haemolytica/isolamento & purificação , Pasteurelose Pneumônica/microbiologia , Doenças dos Ovinos/microbiologia , Animais , Etiópia , Genótipo , Mannheimia haemolytica/classificação , Filogenia , Ovinos , Especificidade da Espécie
3.
J Vet Diagn Invest ; 30(6): 837-847, 2018 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-30239324

RESUMO

Bovine respiratory disease complex (BRDC) is one of the most significant diseases of cattle. Bacterial pathogens involved in BRDC include Mannheimia haemolytica, Mycoplasma bovis, Histophilus somni, and Pasteurella multocida. We developed and evaluated a multiplexed real-time hydrolysis probe (rtPCR) assay using block-based Peltier and rotary-based thermocycling on lung tissue, nasal swabs, and deep nasopharyngeal swabs. The rtPCR results were compared to culture or a gel-based M. bovis PCR using statistical analysis to determine optimum quantification cycle (Cq) cutoffs to maximize agreement. The limits of detection were 1.2-12 CFU/reaction for each pathogen. M. haemolytica was the most prevalent organism detected by rtPCR, and was most frequently found with P. multocida. The rtPCR assay enabled enhanced levels of detection over culture for all pathogens on both thermocycling platforms. The rotary-based thermocycler had significantly lower Cq cutoffs (35.2 vs. 39.7), which maximized agreement with gold standard culture or gel-based PCR results following receiver operating characteristic analysis to maximize sensitivity (Se) and specificity (Sp). However, overall assay Se and Sp were similar on both platforms (80.5% Se, 88.8% Sp vs. 80.1% Se, 88.3% Sp). Implementation of these tests could enhance the detection of these pathogens, and with high-throughput workflows could reduce assay time and provide more rapid results. The assays may be especially valuable in identifying coinfections, given that many more antemortem samples tested in our study were positive for 2 or more pathogens by rtPCR ( n = 125) than were detected using culture alone ( n = 25).


Assuntos
Complexo Respiratório Bovino/diagnóstico , Reação em Cadeia da Polimerase Multiplex/veterinária , Reação em Cadeia da Polimerase em Tempo Real/veterinária , Animais , Complexo Respiratório Bovino/classificação , Complexo Respiratório Bovino/microbiologia , Bovinos , Pulmão/microbiologia , Mannheimia haemolytica/isolamento & purificação , Reação em Cadeia da Polimerase Multiplex/instrumentação , Reação em Cadeia da Polimerase Multiplex/métodos , Infecções por Mycoplasma/classificação , Infecções por Mycoplasma/diagnóstico , Infecções por Mycoplasma/microbiologia , Infecções por Mycoplasma/veterinária , Mycoplasma bovis/isolamento & purificação , Nasofaringe/microbiologia , Pasteurella multocida/isolamento & purificação , Pasteurellaceae/isolamento & purificação , Infecções por Pasteurellaceae/classificação , Infecções por Pasteurellaceae/diagnóstico , Infecções por Pasteurellaceae/microbiologia , Infecções por Pasteurellaceae/veterinária , Reação em Cadeia da Polimerase em Tempo Real/instrumentação , Reação em Cadeia da Polimerase em Tempo Real/métodos
4.
J Dairy Sci ; 101(11): 10351-10360, 2018 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-30197142

RESUMO

Monitoring sickness behavior may improve identification, management, and welfare of sick animals. The objective of this study was to characterize components of sickness behavior in group-housed dairy calves, using an experimental disease challenge model with Mannheimia haemolytica (MH). Holstein bull calves (aged 3-7 weeks; 58.0 ± 12.0 kg of body weight) were group-housed based on age and body weight in sand-bedded pens (6 calves/pen, 6.6 m2/calf) and provided pasteurized waste milk (8 L/d) 2×/d and grain concentrate ad libitum. Within group, calves were randomly assigned to 1 of 2 treatments: (1) inoculation at the tracheal bifurcation with 3 × 109 cfu of MH suspended in 5 mL of sterile phosphate buffered saline (PBS) followed by a 120 mL wash PBS (MH; n = 12, 3/pen), or (2) inoculation with 5 mL + 120 mL of sterile PBS only (control; n = 12, 3/pen). Rectal temperature and health scores were collected from d 0 to +6 of the challenge. A range of behaviors, including feeding patterns and social interactions, were recorded from video from d 0 to +2. The challenge model resulted in calves experiencing a mild disease state: rectal temperatures of MH calves were elevated throughout the challenge compared with control calves, peaking at 12 h postinoculation (39.2 vs. 38.9°C; standard error = 0.14). Many behavioral responses were subject to treatment by day effects, with calves generally becoming less active following inoculation with MH and then returning to baseline. Affected behaviors surrounding feeding included milk feeding time, frequency of competitive displacements, and concentrate feeding time. Lying time was similarly subject to treatment by day effects, and MH calves also spent more time lying on their left side compared with their right (604 vs. 471 min/h; standard error = 32), whereas control calves expressed no laterality. Duration of social lying did not differ, but frequency of social lying bouts decreased in MH calves following inoculation (0.44 vs. 0.75 bouts/h; standard error = 0.04). Social grooming was initiated less by MH calves (0.78 vs. 1.96 min/h; standard error = 0.38), but they tended to receive more social grooming for a greater duration of time (1.59 vs. 1.25 min/h; standard error = 0.13). Overall, we found that infected calves exhibited reduced grooming, feeding, and social interactions, suggesting that these behavioral changes may be useful indicators of early stages of respiratory disease.


Assuntos
Comportamento Animal , Doenças dos Bovinos/fisiopatologia , Comportamento Alimentar , Mannheimia haemolytica/isolamento & purificação , Pneumonia Enzoótica dos Bezerros/fisiopatologia , Animais , Peso Corporal , Bovinos , Doenças dos Bovinos/microbiologia , Grão Comestível , Asseio Animal , Masculino , Pneumonia Enzoótica dos Bezerros/microbiologia , Distribuição Aleatória
5.
Antonie Van Leeuwenhoek ; 111(12): 2311-2321, 2018 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-29974354

RESUMO

Mannheimia haemolytica causes respiratory disease in cattle. Amyloid proteins are a major component of biofilms; they aid in adhesion and confer resistance against several environmental insults. The amyloid protein curli is highly resistant to protease digestion and physical and chemical denaturation and binds Congo red (CR) dye. The purpose of this study was to characterize an approximately 50-kDa CR-binding amyloid-like protein (ALP) expressed by M. haemolytica. This protein resisted boiling and formic acid digestion and was recognized by a polyclonal anti-Escherichia coli curli serum, suggesting its relationship with amyloid proteins. Immunolabeling and transmission electron microscopy showed that antibodies bound long, thin fibers attached to the bacterial surface. Mass spectrometry analysis indicated that these fibers are M. haemolytica OmpP2-like proteins. The purified protein formed filaments in vitro, and antiserum against it reacted positively with biofilms. An in silico analysis of its amino acid sequence indicated it has auto-aggregation properties and eight amyloid peptides. Rabbit polyclonal antibodies generated against this ALP diminished the adhesion of ATCC 31612 and BA1 M. haemolytica strains to A549 human epithelial cells, indicating its participation in cell adhesion. ALP expressed by M. haemolytica may be important in its pathogenicity and ability to form biofilms.


Assuntos
Proteínas Amiloidogênicas/química , Proteínas da Membrana Bacteriana Externa/química , Biofilmes/crescimento & desenvolvimento , Mannheimia haemolytica/química , Células A549 , Sequência de Aminoácidos , Proteínas Amiloidogênicas/biossíntese , Proteínas Amiloidogênicas/genética , Proteínas Amiloidogênicas/isolamento & purificação , Animais , Aderência Bacteriana , Proteínas da Membrana Bacteriana Externa/biossíntese , Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/isolamento & purificação , Bovinos , Vermelho Congo/química , Expressão Gênica , Humanos , Soros Imunes/química , Soros Imunes/isolamento & purificação , Mannheimia haemolytica/genética , Mannheimia haemolytica/isolamento & purificação , Mannheimia haemolytica/metabolismo , Modelos Moleculares , Peso Molecular , Pasteurelose Pneumônica , Ligação Proteica , Estrutura Secundária de Proteína , Coelhos , Alinhamento de Sequência , Ovinos
6.
Sci Rep ; 8(1): 10553, 2018 Jul 12.
Artigo em Inglês | MEDLINE | ID: mdl-30002424

RESUMO

To design an antimicrobial treatment regimen for a bacterial disease, data on the drug pharmacodynamics (PD) against selected drug-susceptible strains of the pathogen are used. The regimen is applied across such strains in the field, assuming the PD parameter values remain the same. We used time-kill experiments and PD modeling to investigate the fluoroquinolone enrofloxacin PD against different isolates of two bovine respiratory disease pathogens: four Mannheimia haemolytica and three Pasteurella multocida isolates. The models were fitted as mixed-effects non-linear regression; the fixed-effects PD parameter values were estimated after accounting for random variation among experimental replicates. There was both inter- and intra- bacterial species variability in the PD parameters Hill-coefficient and Emax (maximal decline of bacterial growth rate), with more variable PD responses among M. haemolytica than among P. multocida isolates. Moreover, the Hill-coefficient was correlated to the isolate's maximal population growth rate in the absence of antimicrobial exposure (a.k.a. specific growth rate; Spearman's ρ = 0.98, p-value = 0.003, n = 6 isolates excluding one outlier). Thus, the strain's properties such as growth potential may impact its PD responses. This variability can have clinical implications. Modifying the treatment regimen depending on phenotypic properties of the pathogen strain causing disease may be a precision medicine approach.


Assuntos
Antibacterianos/farmacologia , Complexo Respiratório Bovino/tratamento farmacológico , Fluoroquinolonas/farmacologia , Mannheimia haemolytica/efeitos dos fármacos , Pasteurella multocida/efeitos dos fármacos , Animais , Antibacterianos/uso terapêutico , Complexo Respiratório Bovino/microbiologia , Bovinos , Fluoroquinolonas/uso terapêutico , Mannheimia haemolytica/genética , Mannheimia haemolytica/isolamento & purificação , Testes de Sensibilidade Microbiana , Modelos Biológicos , Pasteurella multocida/genética , Pasteurella multocida/isolamento & purificação , Fenótipo
7.
Vet Microbiol ; 219: 80-86, 2018 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-29778209

RESUMO

This study investigated the genetic characteristics of 121 ovine Mannheimia haemolytica isolates from lungs with (n = 75) and without pneumonic lesions (n = 46) using multilocus sequence typing (MLST), virulence-associated gene typing and pulsed-field gel electrophoresis (PFGE). Twelve STs were identified with most isolates (81%) belonged to ST16, ST28 and ST8. Analysis of the M. haemolytica MLST Database indicate a wide distribution of these genotypes in small ruminants, never reported in bovine isolates. This could suggest the adaptation of certain genetic lineages of M. haemolytica to small ruminants. e-BURST analysis grouped most STs into three clonal complexes (CC2, CC8 and CC28), consistent with a clonal population structure of M. haemolytica. Virulence-associated gene typing identified five virulence profiles in 64% and 65.1% of the M. haemolytica isolates from lungs with and without pneumonic lesions, respectively. These data suggest that M. haemolytica isolates from the lungs with and without pneumonic lesions are genetically homogeneous. By PGFE analysis a high level of genetic diversity was observed not only within isolates from lungs without pneumonic lesions but also among isolates from pneumonic lesions (GD 0.69 and GD 0.66, respectively; P > 0.05). These results indicate that multiple strains of M. haemolytica may be associated with individual cases of pneumonia in sheep.


Assuntos
Genótipo , Pulmão/microbiologia , Mannheimia haemolytica/genética , Mannheimia haemolytica/isolamento & purificação , Pasteurelose Pneumônica/microbiologia , Animais , Variação Genética , Pulmão/patologia , Mannheimia haemolytica/classificação , Mannheimia haemolytica/patogenicidade , Tipagem de Sequências Multilocus , Ovinos/microbiologia , Doenças dos Ovinos/epidemiologia , Doenças dos Ovinos/microbiologia , Especificidade da Espécie , Virulência/genética
8.
Toxins (Basel) ; 10(5)2018 04 25.
Artigo em Inglês | MEDLINE | ID: mdl-29693562

RESUMO

Mannheimia (Pasteurella) haemolytica causes bronchopneumonia in domestic and wild ruminants. Leukotoxin is the critical virulence factor of M. haemolytica. Since β-hemolysis is caused by a large number of leukotoxin-positive M. haemolytica isolates, all β-hemolytic M. haemolytica isolates are considered to be leukotoxic as well. However, conflicting reports exist in literature as to the leukotoxic and hemolytic properties of M. haemolytica. One group of researchers reported their leukotoxin-deletion mutants to be hemolytic while another reported their mutants to be non-hemolytic. The objective of this study was to determine whether β-hemolysis is a reliable indicator of leukotoxicity of M. haemolytica isolates. Ninety-five isolates of M. haemolytica were first confirmed for presence of leukotoxin gene (lktA) by a leukotoxin-specific PCR assay. Culture supernatant fluids from these isolates were then tested for presence of leukotoxin protein by an ELISA, and for leukotoxic activity by a cytotoxicity assay. All isolates were tested for β-hemolysis by culture on blood agar plates. Sixty-two isolates (65%) produced leukotoxin protein while 33 isolates (35%) did not. Surprisingly, 18 of the 33 isolates (55%), that did not produce leukotoxin protein, were hemolytic. Of the 62 isolates that produced leukotoxin, 55 (89%) were leukotoxic while 7 (11%) were not. All except one of the 55 leukotoxic isolates (98%) were also hemolytic. All seven isolates that were not leukotoxic were hemolytic. Taken together, these results suggest that β-hemolysis may not be a reliable indicator of leukotoxicity of M. haemolytica isolates. Furthermore, all M. haemolytica isolates that possess lktA gene may not secrete active leukotoxin.


Assuntos
Exotoxinas , Hemólise , Leucócitos , Mannheimia haemolytica , Fatores de Virulência , Animais , Bovinos/microbiologia , Eritrócitos , Exotoxinas/genética , Exotoxinas/metabolismo , Mannheimia haemolytica/genética , Mannheimia haemolytica/isolamento & purificação , Mannheimia haemolytica/metabolismo , Ovinos/microbiologia , Fatores de Virulência/genética , Fatores de Virulência/metabolismo
9.
Braz J Microbiol ; 49(3): 591-600, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29551641

RESUMO

Histophilus somni is a Gram-negative bacterium that is associated with a disease complex (termed histophilosis) that can produce several clinical syndromes predominantly in cattle, but also in sheep. Histophilosis is well described in North America, Canada, and in some European countries. In Brazil, histophilosis has been described in cattle with respiratory, reproductive, and systemic disease, with only one case described in sheep. This report describes the occurrence of Histophilus somni-associated disease in sheep from Southern Brazil. Eight sheep with different clinical manifestations from five farms were investigated by a combination of pathological and molecular diagnostic methods to identify additional cases of histophilosis in sheep from Brazil. The principal pathological lesions were thrombotic meningoencephalitis, fibrinous bronchopneumonia, pulmonary abscesses, and necrotizing myocarditis. The main clinical syndromes associated with H. somni were thrombotic meningoencephalitis (n=4), septicemia (n=4), bronchopneumonia (n=4), and myocarditis (n=3). H. somni DNA was amplified from multiple tissues of all sheep with clinical syndromes of histophilosis; sequencing confirmed the PCR results. Further, PCR assays to detect Pasteurella multocida and Mannheimia haemolytica were negative. These findings confirmed the participation of H. somni in the clinical syndromes investigated during this study, and adds to the previous report of histophilosis in sheep from Brazil.


Assuntos
Mannheimia haemolytica/isolamento & purificação , Infecções por Pasteurellaceae/veterinária , Doenças dos Ovinos/microbiologia , Animais , Brasil , Mannheimia haemolytica/genética , Infecções por Pasteurellaceae/microbiologia , Reação em Cadeia da Polimerase , Ovinos
10.
J Vet Diagn Invest ; 30(2): 249-251, 2018 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-29105570

RESUMO

We investigated the occurrence of infectious pathogens during an outbreak of bovine respiratory disease (BRD) in a beef cattle feedlot in southern Brazil that has a high risk of developing BRD. Nasopharyngeal swabs were randomly collected from steers ( n = 23) and assessed for the presence of infectious agents of BRD by PCR and/or RT-PCR assays. These included: Histophilus somni, Mannheimia haemolytica, Pasteurella multocida, Mycoplasma bovis, bovine respiratory syncytial virus (BRSV), bovine coronavirus (BCoV), bovine viral diarrhea virus (BVDV), bovine alphaherpesvirus 1 (BoHV-1), and bovine parainfluenza virus 3 (BPIV-3). Pulmonary sections of one steer that died with clinical BRD were submitted for pathology and molecular testing. The frequencies of the pathogens identified from the nasopharyngeal swabs were: H. somni 39% (9 of 23), BRSV 35% (8 of 23), BCoV 22% (5 of 23), and M. haemolytica 13% (3 of 23). PCR or RT-PCR assays did not identify P. multocida, M. bovis, BoHV-1, BVDV, or BPIV-3 from the nasopharyngeal swabs. Single and concomitant associations of infectious agents of BRD were identified. Fibrinous bronchopneumonia was diagnosed in one steer that died; samples were positive for H. somni and M. haemolytica by PCR. H. somni, BRSV, and BCoV are important disease pathogens of BRD in feedlot cattle in Brazil, but H. somni and BCoV are probably under-reported.


Assuntos
Doenças dos Bovinos/epidemiologia , Surtos de Doenças/veterinária , Doenças Respiratórias/veterinária , Animais , Derrame de Bactérias , Brasil/epidemiologia , Bovinos , Doenças dos Bovinos/microbiologia , Doenças dos Bovinos/prevenção & controle , Ensaio de Imunoadsorção Enzimática/veterinária , Herpesvirus Bovino 1/isolamento & purificação , Masculino , Mannheimia haemolytica/isolamento & purificação , Nariz/microbiologia , Vírus da Parainfluenza 3 Bovina/isolamento & purificação , Carne Vermelha , Vírus Sincicial Respiratório Bovino/isolamento & purificação , Doenças Respiratórias/epidemiologia , Doenças Respiratórias/microbiologia , Eliminação de Partículas Virais
11.
Microb Pathog ; 112: 176-181, 2017 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-28970175

RESUMO

Mannheimia haemolytica is causative agent of pneumonic pasteurellosis (mannheimiosis) that causes huge economic losses to livestock farmers. We investigated the microbial and clinico-pathological patterns associated with ovine pneumonic pasturellosis during an outbreak. Prior to death, infected sheep revealed clinical signs including dyspnoea, salivation, pyrexia and mucopurulent nasal discharge. Mortality was significantly (p < 0.05) high in young sheep as compared to adults. Necropsy findings revealed presence of froth in trachea, congestion and consolidation of lungs, pulmonary edema, severe pleural adhesions, pericarditis, hemorrhages on mucosa of jejunum and kidneys. Histopathological examination revealed circumscribed and centrally calcified necrotic areas punctuated with chronic inflammatory cells and interstitial pneumonia. Moreover, bronchial epithelial hyperplasia, edema, congestion, mononuclear cell infiltration, thick interlobular septae and peri-vascular cuffing were the striking changes in lungs. Furthermore, lungs showed severe fibrin depositions along with abundant amount of fibrin meshwork on pleura infiltrated with chronic inflammatory cells. Histologically, liver, kidneys and lymph nodes showed degenerative changes. Mannheimia haemolytica and Pasteurella multocida were differentially identified on the basis of culture characteristics and biochemical tests. M. haemolytica was further confirmed by using polymerase chain reaction. From the findings of current study, it is concluded that M. haemolytica is a major respiratory threat in small ruminants that causes severe pneumonic changes in infected animals.


Assuntos
Bactérias/patogenicidade , Pulmão/microbiologia , Mannheimia haemolytica/patogenicidade , Pasteurella multocida/patogenicidade , Pasteurelose Pneumônica/diagnóstico , Reação em Cadeia da Polimerase/veterinária , Doenças dos Ovinos/diagnóstico , Animais , Calcinose/patologia , Calcinose/veterinária , Clima , DNA Bacteriano/análise , Surtos de Doenças/veterinária , Células Epiteliais/patologia , Feminino , Hiperplasia/veterinária , Rim/microbiologia , Rim/patologia , Pulmão/patologia , Linfonodos/microbiologia , Linfonodos/patologia , Masculino , Mannheimia haemolytica/genética , Mannheimia haemolytica/isolamento & purificação , Mortalidade , Necrose/patologia , Paquistão/epidemiologia , Pasteurella multocida/genética , Pasteurella multocida/isolamento & purificação , Pasteurelose Pneumônica/epidemiologia , Pasteurelose Pneumônica/microbiologia , Pasteurelose Pneumônica/patologia , Patologia Molecular , Reação em Cadeia da Polimerase/métodos , Ovinos , Doenças dos Ovinos/epidemiologia , Doenças dos Ovinos/microbiologia , Doenças dos Ovinos/patologia
12.
Acta Vet Hung ; 65(3): 327-339, 2017 09.
Artigo em Inglês | MEDLINE | ID: mdl-28956490

RESUMO

Pasteurella multocida, Mannheimia haemolytica and Trueperella pyogenes are three bacterial pathogens closely associated with the bovine respiratory disease complex (BRDC). In the current study, a multiplex PCR for the simultaneous detection of these three bacteria in cultures was established. After serial optimisation, the detection limit of the method for the genomic DNA of the three bacteria was 40 pg/µl. The method could detect the genomic DNA of these three bacteria but not the genomic DNA of seven other bacterial strains. Together with the bacterial enrichment technology, the multiplex PCR could be used for detecting the three bacteria in animal tissues. This method might be valuable for speeding up laboratory diagnosis and directing the treatment of BRDC to these three bacterial pathogens.


Assuntos
Actinomycetaceae/isolamento & purificação , Mannheimia haemolytica/isolamento & purificação , Reação em Cadeia da Polimerase Multiplex/métodos , Pasteurella multocida/isolamento & purificação , Sensibilidade e Especificidade
13.
Vet Microbiol ; 208: 118-125, 2017 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-28888626

RESUMO

Current information on prevalence and antimicrobial resistance (AMR) of bacterial respiratory pathogens is crucial to guide antimicrobial choice for control and treatment of bovine respiratory disease (BRD). The objectives were to describe the prevalence of three BRD-associated bacteria (Mannheimia haemolytica, Pasteurella multocida, and Histophilus somni) in the lower airways of feedlot cattle, and to analyze AMR in these bacteria. Cattle with (n=210) and without (n=107) BRD were sampled by trans-tracheal aspiration at four feedlots (Nov. 15-Jan. 16). These cattle had received 2.5mg/kg of tulathromycin on arrival at the feedlot for BRD control and two in-feed pulses of chlortetracycline (5g/animal/day for 5days) within the first 21days on feed to prevent histophilosis. Bacteria were detected by culture and AMR was tested by microdilution. Pasteurella multocida was the most frequent bacterium isolated in cattle with BRD (54.8%), followed by M. haemolytica (30.5%) and H. somni (22.9%). Compared to those with BRD, healthy cattle were less likely to be positive for P. multocida (OR=0.27), M. haemolytica (OR=0.32), or H. somni (OR=0.25). There were high levels of resistance (>70%) against tulathromycin and oxytetracycline in M. haemolytica and P. multocida isolates and high levels of resistance against oxytetracycline (67%) and penicillin (52%) in H. somni isolates. None or few isolates were resistant to florfenicol, enrofloxacin and ceftiofur. The high prevalence of resistance against tulathromycin and oxytetracycline suggests that these antimicrobials should not be repeatedly used for both control and treatment of BRD and/or histophilosis.


Assuntos
Antibacterianos/farmacologia , Complexo Respiratório Bovino/microbiologia , Infecções por Pasteurellaceae/veterinária , Pasteurellaceae/isolamento & purificação , Animais , Bovinos , Feminino , Abrigo para Animais , Masculino , Mannheimia haemolytica/classificação , Mannheimia haemolytica/isolamento & purificação , Testes de Sensibilidade Microbiana , Pasteurella multocida/isolamento & purificação , Infecções por Pasteurellaceae/microbiologia , Prevalência , Sorogrupo , Traqueia/microbiologia
14.
BMC Vet Res ; 13(1): 221, 2017 Jul 13.
Artigo em Inglês | MEDLINE | ID: mdl-28705198

RESUMO

BACKGROUND: Pasteurella multocida, Mannheimia haemolytica, Histophilus somni and Trueperella pyogenes are four bacterial agents commonly associated with bovine respiratory disease (BRD). In this study a bacterial multiplex real-time PCR (the RespoCheck PCR) was evaluated for the detection in bronchoalveolar lavage fluid (BALF) of these four bacterial agents. RESULTS: The analytical sensitivity of the multiplex real-time PCR assay determined on purified DNA and on bacterial cells of the four target pathogens was one to ten fg DNA/assay and 4 × 10-1 to 2 × 100 CFU/assay. The analytical specificity of the test was, as evaluated on a collection of 118 bacterial isolates, 98.3% for M. haemolytica and 100% for the other three target bacteria. A set of 160 BALF samples of calves originating from ten different herds with health problems related to BRD was examined with bacteriological methods and with the RespoCheck PCR. Using bacteriological examination as the gold standard, the diagnostic sensitivities and specificities of the four bacterial agents were respectively between 0.72 and 1.00 and between 0.70 and 0.99. Kappa values for agreement between results of bacteriological examination and PCRs were low for H. somni (0.17), moderate for P. multocida (0.52) and M. haemolytica (0.57), and good for T. pyogenes (0.79). The low and moderate kappa values seemed to be related to limitations of the bacteriological examination, this was especially the case for H. somni. CONCLUSION: It was concluded that the RespoCheck PCR assay is a valuable diagnostic tool for the simultaneous detection of the four bacterial agents in BALF of calves.


Assuntos
Complexo Respiratório Bovino/microbiologia , Líquido da Lavagem Broncoalveolar/microbiologia , Reação em Cadeia da Polimerase em Tempo Real/veterinária , Actinomycetaceae/isolamento & purificação , Infecções por Actinomycetales/veterinária , Animais , Técnicas Bacteriológicas/veterinária , Complexo Respiratório Bovino/diagnóstico , Bovinos , Mannheimia haemolytica/isolamento & purificação , Pasteurella multocida/isolamento & purificação , Pasteurellaceae/isolamento & purificação , Infecções por Pasteurellaceae/veterinária , Sensibilidade e Especificidade
15.
Vet Microbiol ; 203: 73-80, 2017 May.
Artigo em Inglês | MEDLINE | ID: mdl-28619171

RESUMO

Mannheimia haemolytica is a major bacterial contributor to bovine respiratory disease complex that costs the livestock industry a billion dollars a year in USA. Commercial vaccines are only partially efficacious under field conditions. Earlier studies found that outer membrane protein preparations and culture supernatants can induce immune responses that enhance resistance to challenge by M. haemolytica strains. The objective of this study was to characterize secretome of two M. haemolytica stains grown under two different media. Bacteria-free concentrated supernatants from M. haemolytica culture was subjected to LC-MS/MS. The secretome of M. haemolytica from both strains yielded 923 proteins. Using bioinformatic tools, 283 were identified as secreted proteins. Further breakdown of 283 proteins showed that 114 (40.2%), 184 (65.0%), 138(48.7%), 151 (53.3%) and 172 (60.7%) were characterized as secreted proteins by SignalP 4.1, SecretomeP 2.0, LipoP, Phobius, and PRED-TAT, respectively. A total of 95 (33.56%) proteins were characterized as being secreted via non-classical pathway as opposed to the majority that were secreted in signal peptide dependent pathway. The demonstrated proteins include all previously immunologically characterized M. haemolytica proteins. The potential of using secretome analysis in the design and development of a multivalent vaccine is discussed.


Assuntos
Complexo Respiratório Bovino/diagnóstico , Biologia Computacional , Mannheimia haemolytica/isolamento & purificação , Infecções por Pasteurellaceae/veterinária , Proteômica , Animais , Complexo Respiratório Bovino/microbiologia , Bovinos , Cromatografia Líquida/veterinária , Infecções por Pasteurellaceae/diagnóstico , Infecções por Pasteurellaceae/microbiologia , Espectrometria de Massas em Tandem/veterinária
16.
J Microbiol Methods ; 139: 155-160, 2017 08.
Artigo em Inglês | MEDLINE | ID: mdl-28551457

RESUMO

Mannheimia haemolytica is an important respiratory pathogen of ruminants. Of the 12 capsular serovars identified, 1 and 6 are most frequently associated with disease in cattle, while 2 is largely a commensal. Comparative analysis of 24 M. haemolytica genomes was used to identify unique genes associated with capsular polysaccharide synthesis as amplification targets in a multiplex PCR assay to discriminate between serotype 1, 2, and 6 strains. The specificity of serotype specific gene targets was evaluated against 47 reference strains representing 12 known serovars of M. haemolytica and 101 field isolates identified through antisera agglutination as serotypes 1, 2, or 6. The results suggest this simple and cost-effective serotype specific PCR assay can be used as an alternative to agglutination based techniques to serotype the majority of M. haemolytica collected from bovines, thus averting the need to use animals and invest in expensive sera development for agglutination assays. In addition, the gene targets identified in this study can be used in silico to identify serotype 1, 2, and 6 strains in sequenced M. haemolytica isolates without the need for culture based analysis.


Assuntos
Cápsulas Bacterianas/genética , Doenças dos Bovinos/microbiologia , Mannheimia haemolytica/classificação , Reação em Cadeia da Polimerase Multiplex/métodos , Infecções por Pasteurellaceae/veterinária , Sorotipagem/métodos , Animais , Cápsulas Bacterianas/imunologia , Bovinos , Genes Bacterianos/genética , Genoma Bacteriano , Mannheimia haemolytica/genética , Mannheimia haemolytica/isolamento & purificação , Infecções por Pasteurellaceae/microbiologia , Sorogrupo , Sorotipagem/economia
17.
J Microbiol Methods ; 136: 30-33, 2017 05.
Artigo em Inglês | MEDLINE | ID: mdl-28267571

RESUMO

Genotype 2M. haemolytica predominantly associate over genotype 1 with the lungs of cattle with respiratory disease and ICEs containing antimicrobial resistance genes. Distinct protein masses were detected by MALDI-TOF MS between genotype 1 and 2 strains. MALDI-TOF MS could rapidly differentiate genotype 2 strains in veterinary diagnostic laboratories.


Assuntos
Genótipo , Mannheimia haemolytica/genética , Mannheimia haemolytica/isolamento & purificação , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Animais , Proteínas de Bactérias/genética , Bovinos , Doenças dos Bovinos/microbiologia , DNA Bacteriano/genética , Genes MDR/genética , Pulmão/microbiologia , Mannheimia haemolytica/classificação , Mannheimia haemolytica/patogenicidade , Infecções por Pasteurellaceae/diagnóstico , Infecções por Pasteurellaceae/microbiologia
18.
Am J Vet Res ; 78(3): 350-358, 2017 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-28240958

RESUMO

OBJECTIVE To compare predictive values, extent of agreement, and gamithromycin susceptibility between bacterial culture results of nasopharyngeal swab (NPS) and bronchoalveolar lavage fluid (BALF) samples obtained from calves with bovine respiratory disease (BRD). ANIMALS 28 beef calves with clinical BRD. PROCEDURES Pooled bilateral NPS samples and BALF samples were obtained for bacterial culture from calves immediately before and at various times during the 5 days after gamithromycin (6 mg/kg, SC, once) administration. For each culture-positive sample, up to 12 Mannheimia haemolytica, 6 Pasteurella multocida, and 6 Histophilus somni colonies underwent gamithromycin susceptibility testing. Whole-genome sequencing was performed on all M haemolytica isolates. For paired NPS and BALF samples collected 5 days after gamithromycin administration, the positive and negative predictive values for culture results of NPS samples relative to those of BALF samples and the extent of agreement between the sampling methods were determined. RESULTS Positive and negative predictive values of NPS samples were 67% and 100% for M haemolytica, 75% and 100% for P multocida, and 100% and 96% for H somni. Extent of agreement between results for NPS and BALF samples was substantial for M haemolytica (κ, 0.71) and H somni (κ, 0.78) and almost perfect for P multocida (κ, 0.81). Gamithromycin susceptibility varied within the same sample and between paired NPS and BALF samples. CONCLUSIONS AND CLINICAL RELEVANCE Results indicated culture results of NPS and BALF samples from calves with BRD should be interpreted cautiously considering disease prevalence within the population, sample collection relative to antimicrobial administration, and limitations of diagnostic testing methods.


Assuntos
Líquido da Lavagem Broncoalveolar/microbiologia , Doenças dos Bovinos/diagnóstico , Nasofaringe/microbiologia , Doenças Respiratórias/veterinária , Animais , Bovinos , Doenças dos Bovinos/microbiologia , Genoma Bacteriano/genética , Macrolídeos/farmacologia , Masculino , Mannheimia haemolytica/efeitos dos fármacos , Mannheimia haemolytica/genética , Mannheimia haemolytica/isolamento & purificação , Pasteurella multocida/efeitos dos fármacos , Pasteurella multocida/genética , Pasteurella multocida/isolamento & purificação , Pasteurellaceae/efeitos dos fármacos , Pasteurellaceae/genética , Pasteurellaceae/isolamento & purificação , Valor Preditivo dos Testes , Doenças Respiratórias/diagnóstico , Doenças Respiratórias/microbiologia
19.
J Vet Diagn Invest ; 29(1): 20-34, 2017 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-28074713

RESUMO

We examined the pathogens, morphologic patterns, and risk factors associated with bovine respiratory disease (BRD) in 136 recently weaned cattle ("weanlings"), 6-12 mo of age, that were submitted for postmortem examination to regional veterinary laboratories in Ireland. A standardized sampling protocol included routine microbiologic investigations as well as polymerase chain reaction and immunohistochemistry. Lungs with histologic lesions were categorized into 1 of 5 morphologic patterns of pneumonia. Fibrinosuppurative bronchopneumonia (49%) and interstitial pneumonia (48%) were the morphologic patterns recorded most frequently. The various morphologic patterns of pulmonary lesions suggest the involvement of variable combinations of initiating and compounding infectious agents that hindered any simple classification of the etiopathogenesis of the pneumonias. Dual infections were detected in 58% of lungs, with Mannheimia haemolytica and Histophilus somni most frequently recorded in concert. M. haemolytica (43%) was the most frequently detected respiratory pathogen; H. somni was also shown to be frequently implicated in pneumonia in this age group of cattle. Bovine parainfluenza virus 3 (BPIV-3) and Bovine respiratory syncytial virus (16% each) were the viral agents detected most frequently. Potential respiratory pathogens (particularly Pasteurella multocida, BPIV-3, and H. somni) were frequently detected (64%) in lungs that had neither gross nor histologic pulmonary lesions, raising questions regarding their role in the pathogenesis of BRD. The breadth of respiratory pathogens detected in bovine lungs by various detection methods highlights the diagnostic value of parallel analyses in respiratory disease postmortem investigation.


Assuntos
Broncopneumonia/veterinária , Doenças dos Bovinos/epidemiologia , Animais , Animais Lactentes , Autopsia/veterinária , Broncopneumonia/epidemiologia , Bovinos , Doenças dos Bovinos/diagnóstico , Doenças dos Bovinos/microbiologia , Imuno-Histoquímica/veterinária , Irlanda/epidemiologia , Mannheimia haemolytica/isolamento & purificação , Vírus da Parainfluenza 3 Bovina/isolamento & purificação , Pasteurella multocida/isolamento & purificação , Reação em Cadeia da Polimerase/veterinária , Vírus Sincicial Respiratório Bovino/isolamento & purificação
20.
J Med Microbiol ; 65(10): 1137-1142, 2016 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-27582268

RESUMO

This study aimed to determine the occurrence of Mannheimiahaemolytica, Pasteurella multocida and Mycoplasma spp., in relation to clinical signs of respiratory disease. Tracheobronchial lavage samples were collected from 96 (healthy and unhealthy) cattle in the State of São Paulo, Brazil. Mycoplasma spp. (12.5 %) and Pasteurellamultocida (15.50 %) were the most prevalent species. Bacillus sp., Staphylococcus sp., Escherichia coli, Klebsiella oxytoca, Pseudomonas aeruginosa and Klebsiella pneumoniae were also isolated. Mollicutes (70.83 %), Mycoplasmabovis (2.94 %) and Mycoplasma dispar (38.23 %) were identified using conventional PCR. Submassive sound on acoustic percussion of the thorax was associated with the absence of Mollicutes (P=0.025). Whistling (P=0.076) and coarse crackle (P=0.046) were associated with the absence of Mycoplasma dispar. Clear sound on acoustic percussion of the thorax was associated with the absence of Mycoplasmabovis (P=0.007). Coughing was associated with the presence of Pasteurellamultocida [P=0.035; confidence interval (CI), 1.12-26.89], but its absence was associated with mucopurulent (P=0.0215; CI, 1.55-34.5) and mucoid nasal discharge (P=0.068; CI, 19-28.5), submassive sound (P=0.031; CI, 1.23-75.5), fine crackle (P=0.058; CI, 1.23-20.1) and coarse crackle (P=0.046; CI, 2.38-70.8). The high prevalence of Pasteurella multocida and Mycoplasma spp. in unhealthy calves increases the importance of these micro-organisms in the pathogenesis of respiratory diseases. This study increases the information about the role of Mycoplasma dispar in respiratory diseases. Differences in some species in relation to clinical signs can be applied as a presumptive diagnosis.


Assuntos
Líquido da Lavagem Broncoalveolar/microbiologia , Doenças dos Bovinos/microbiologia , Mannheimia haemolytica/isolamento & purificação , Mycoplasma/isolamento & purificação , Pasteurella multocida/isolamento & purificação , Infecções Respiratórias/veterinária , Animais , Brasil , Bovinos , Doenças dos Bovinos/diagnóstico , Estudos Transversais , Feminino , Masculino , Mannheimia haemolytica/genética , Mycoplasma/genética , Pasteurella multocida/genética , Infecções Respiratórias/diagnóstico , Infecções Respiratórias/microbiologia
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