RESUMO
AIM OF STUDY: The objective of this study was isolation and morphological characterization of temperate bacteriophages obtained from M. haemolytica strains and evaluation of their lytic properties in vitro against M. haemolytica isolated from the respiratory tract of calves. MATERIAL AND METHODS: The material for the study consisted of the reference strain M. haemolytica serotype 1 (ATCC®) BAA-410™, reference serotypes A1, A2, A5, A6, A7, A9 and A11, and wild-type isolates of M. haemolytica. Bacteriophages were induced from an overnight bacterial starter culture of all examined M. haemolytica strains treated with mitomycin C. The lytic properties and host ranges were determined by plaque assays. The morphology of the bacteriophages was examined in negative-stained smears with 5% uranyl acetate solution using a transmission electron microscope. The genetic analysis of the bacteriophages was followed by restriction analysis of bacteriophage DNA. This was followed by analysis of genetic material by polymerase chain reaction (PCR). RESULTS: Eight bacteriophages were obtained, like typical of the families Myoviridae, Siphoviridae and Podoviridae. Most of the bacteriophages exhibited lytic properties against the M. haemolytica strains. Restriction analysis revealed similarities to the P2-like phage obtained from the strain M. haemolytica BAA-410. The most similar profiles were observed in the case of bacteriophages φA1 and φA5. All of the bacteriophages obtained were characterized by the presence of additional fragments in the restriction profiles with respect to the P2-like reference phage. In the analysis of PCR products for the P2-like reference phage phi-MhaA1-PHL101 (DQ426904) and the phages of the M. haemolytica serotypes, a 734-bp phage PCR product was obtained. The primers were programmed in Primer-Blast software using the structure of the sequence DQ426904 of reference phage PHL101. CONCLUSIONS: The results obtained indicate the need for further research aimed at isolating and characterizing bacteriophages, including sequence analysis of selected fragments. Moreover, standardization of methods for obtaining them in order to eliminate M. haemolytica bacteria involved in the etiopathogenesis of BRDC is essential.
Assuntos
Bacteriófagos/isolamento & purificação , Extratos Celulares/isolamento & purificação , Mannheimia haemolytica/isolamento & purificação , Mannheimia haemolytica/virologia , Animais , Bacteriófagos/genética , Bacteriófagos/fisiologia , Bovinos , Especificidade de Hospedeiro , Sistema Respiratório/microbiologiaRESUMO
BACKGROUND: Mannheimia haemolytica is a commensal bacterium that resides in the upper respiratory tract of cattle that can play a role in bovine respiratory disease. Prophages are common in the M. haemolytica genome and contribute significantly to host diversity. The objective of this research was to undertake comparative genomic analysis of phages induced from strains of M. haemolytica serotype A1 (535A and 2256A), A2 (587A and 1127A) and A6 (1152A and 3927A). RESULTS: Overall, four P2-like (535AP1, 587AP1, 1127AP1 and 2256AP1; genomes: 34.9-35.7 kb; G+C content: 41.5-42.1 %; genes: 51-53 coding sequences, CDSs), four λ-like (535AP2, 587AP2, 1152AP2 and 3927AP1; genomes: 48.6-52.1 kb; 41.1-41.4 % mol G+C; genes: 77-83 CDSs and 2 tRNAs) and one Mu-like (3927AP2; genome: 33.8 kb; 43.1 % mol G+C; encoding 50 CDSs) phages were identified. All P2-like phages are collinear with the temperate phage φMhaA1-PHL101 with 535AP1, 2256AP1 and 1152AP1 being most closely related, followed by 587AP1 and 1127AP1. Lambdoid phages are not collinear with any other known λ-type phages, with 587AP2 being distinct from 535AP2, 3927AP1 and 1152AP2. All λ-like phages contain genes encoding a toxin-antitoxin (TA) system and cell-associated haemolysin XhlA. The Mu-like phage induced from 3927A is closely related to the phage remnant φMhaMu2 from M. haemolytica PHL21, with similar Mu-like phages existing in the genomes of M. haemolytica 535A and 587A. CONCLUSIONS: This is among the first reports of both λ- and Mu-type phages being induced from M. haemolytica. Compared to phages induced from commensal strains of M. haemolytica serotype A2, those induced from the more virulent A1 and A6 serotypes are more closely related. Moreover, when P2-, λ- and Mu-like phages co-existed in the M. haemolytica genome, only P2- and λ-like phages were detected upon induction, suggesting that Mu-type phages may be more resistant to induction. Toxin-antitoxin gene cassettes in λ-like phages may contribute to their genomic persistence or the establishment of persister subpopulations of M. haemolytica. Further work is required to determine if the cell-associated haemolysin XhlA encoded by λ-like phages contributes to the pathogenicity and ecological fitness of M. haemolytica.
Assuntos
Mannheimia haemolytica/virologia , Prófagos/genética , Prófagos/isolamento & purificação , Ativação Viral , Composição de Bases , DNA Viral/química , DNA Viral/genética , Dados de Sequência Molecular , Prófagos/fisiologia , Análise de Sequência de DNA , Homologia de Sequência , SinteniaRESUMO
AIMS: This study aimed to characterize the impact of lytic and temperate bacteriophages on the genetic and phenotypic diversity of Mannheimia haemolytica from feedlot cattle. METHODS AND RESULTS: Strictly lytic phages were not detected from bovine nasopharyngeal (n = 689) or water trough (n = 30) samples, but Myoviridae- or Siphoviridae-like phages were induced from 54 of 72 M. haemolytica strains by mitomycin C, occasionally from the same strain. Phages with similar restriction fragment length polymorphism profiles (RFLP ≥70% relatedness) shared common host serotypes 1 or 2 (P < 0·0001). Likewise, phages with similar RFLP tended to occur in genetically related host bacteria (70-79% similarity). Host range assays showed that seven phages from host serotypes 1, 2 and 6 lysed representative strains of serotypes 1, 2 or 8. The genome of vB_MhM_1152AP from serotype 6 was found to be collinear with P2-like phage φMhaA1-PHL101. CONCLUSIONS: Prophages are a significant component of the genome of M. haemolytica and contribute significantly to host diversity. Further characterization of the role of prophage in virulence and persistence of M. haemolytica in cattle could provide insight into approaches to control this potential respiratory pathogen. SIGNIFICANCE AND IMPACT OF THE STUDY: This study demonstrated that prophages are widespread within the genome of M. haemolytica isolates and emphasized the challenge of isolating lytic phage as a therapeutic against this pathogen.
Assuntos
Bacteriófagos/isolamento & purificação , Especificidade de Hospedeiro , Mannheimia haemolytica/virologia , Animais , Bacteriófagos/classificação , Bacteriófagos/genética , Bovinos , Enrofloxacina , Fluoroquinolonas/farmacologia , Variação Genética , Mannheimia haemolytica/classificação , Mannheimia haemolytica/genética , Prófagos/isolamento & purificaçãoRESUMO
The diversity of temperate bacteriophages was examined in 32 Mannheimia haemolytica, six Mannheimia glucosida and four Pasteurella trehalosi isolates. Phage particles were induced and identified by electron microscopy in 24 (75%) M. haemolytica isolates, but in only one (17%) M. glucosida and one (25%) P. trehalosi isolate. The M. haemolytica phages were relatively diverse as seven Siphoviridae, 15 Myoviridae and two Podoviridae-like phages were identified; the Myoviridae-type phages also exhibited structural variation of their tails. The bacteriophages induced in M. glucosida and P. trehalosi were of the Myoviridae type. Restriction endonuclease (RE) analysis identified nine distinct RE types among the M. haemolytica bacteriophages, providing further evidence of their relative diversity. A limited number of phages caused plaques on indicator strains and the phages exhibited a narrow host range. A subgroup of 11 bovine serotype A1 and A6 isolates contained Myoviridae-type phages of the same RE type (type A), but these differed in their abilities to infect and form plaques on the same panel of indicator strains. A P2-like phage (phiPHL213.1), representative of the RE type A phages, was identified from the incomplete M. haemolytica genome sequence. The phiPHL213.1 genome contains previously unidentified genes and represents a new member of the P2 phage family.
Assuntos
Doenças dos Bovinos/microbiologia , Caudovirales/classificação , Caudovirales/fisiologia , Variação Genética , Mannheimia haemolytica/virologia , Pasteurelose Pneumônica/microbiologia , Doenças dos Ovinos/microbiologia , Animais , Bacteriófago P2/classificação , Bacteriófago P2/genética , Bacteriófago P2/fisiologia , Bacteriófago P2/ultraestrutura , Bovinos , Caudovirales/genética , Caudovirales/ultraestrutura , DNA Viral/análise , Mannheimia haemolytica/isolamento & purificação , Microscopia Eletrônica , Mapeamento por Restrição , Ovinos , Ativação ViralRESUMO
The 34,525 nucleotide sequence of a double-stranded DNA bacteriophage (phiMhaA1-PHL101) from Mannheimia haemolytica serotype A1 has been determined. The phage encodes 50 open reading frames. Twenty-three of the proteins are similar to proteins of the P2 family of phages. Other protein sequences are most similar to possible prophage sequences from the draft genome of Histophilus somni 2336. Fourteen open reading frames encode proteins with no known homolog. The P2 orthologues are collinear in phiMhaA1-PHL101, with the exception of the phage tail protein gene T, which maps in a unique location between the S and V genes. The phage ORFs can be arranged into 17 possible transcriptional units and many of the genes are predicted to be translationally coupled. Southern blot analysis revealed phiMhaA1-PHL101 sequences in other A1 isolates as well as in serotype A5, A6, A9, and A12 strains of M. haemolytica, but not in the related organisms, Mannheimia glucosida or Pasteurella trehalosi.
Assuntos
Bacteriófagos/classificação , Bacteriófagos/genética , Mannheimia haemolytica/virologia , Bacteriófagos/isolamento & purificação , Composição de Bases , Sequência de Bases , Replicação do DNA , Vírus de DNA/genética , DNA Viral/metabolismo , Regulação Viral da Expressão Gênica , Transcrição Gênica , Proteínas Virais/genética , Proteínas Virais/metabolismoRESUMO
Pasteurella multocida is an important animal pathogen. Bacterial ghosts produced by the expression of phage PhiX174 lysis gene E are empty cells devoid of cytoplasmic and genomic material. Lysis of P. multocida 7A and P. haemolytica A1 carrying Pasteurella-specific lysis vectors (pSR2 and pSON2) occurred 140 min after induction of gene E expression induced by temperature upshift. The E-mediated cell lysis and killing activity was the same in both Pasteurella species and no viable cells could be detected after lysis of P. multocida and P. haemolytica. Pasteurella ghosts were used for immunization of rabbits and mice. Rabbits immunized subcutaneously with either P. multocida- or P. haemolytica-ghosts developed antibodies reacting with the immunizating strain, as well as with other Pasteurella strains. The number of proteins in whole cell protein extracts recognized by the sera constantly increased during the observation period of 51 days. In addition, dose-dependent protection against homologous challenge was observed in mice immunized with P. multocida-ghosts. Animals which received 1.15 x 10(8) ghosts and a challenge dose of up to 60 cfu (LD90), showed 100% protection. According to these results, we suggest ghosts of P. multocida and P. haemolytica as new vaccine candidates.
Assuntos
Vacinas Bacterianas/imunologia , Mannheimia haemolytica/imunologia , Pasteurella multocida/imunologia , Animais , Bacteriófago phi X 174/genética , Western Blotting , DNA Bacteriano/genética , DNA Recombinante/genética , Relação Dose-Resposta Imunológica , Eletroforese em Gel de Poliacrilamida , Mannheimia haemolytica/ultraestrutura , Mannheimia haemolytica/virologia , Camundongos , Microscopia Eletrônica de Varredura , Pasteurella multocida/ultraestrutura , Pasteurella multocida/virologia , Plasmídeos/genética , Coelhos , Temperatura , Transformação BacterianaRESUMO
Con la finalidad de medir la protección contra pasteurelosis pulmonar que otorgan diferentes inmunógenos de Pasteurella haemolytica A1, éstos se evaluaron en 2 etapas: en condiciones de desafío experimental, y de desafío natural. En la primera, 42 corderos fueron distribuidos en 6 grupos: el Grupo A recibió cultivo vivo, el B sirvió como testigo, al C se le trató con una bacterina comercial, el D con leucotoxina (sobrenadante de cultivo), al E se le administró extracto solubre capsular (ESC) con leucotoxina y el F fue tratado con ESC con leucotoxina más adyuvante. En el día 35 los ovinos fueron expuestos al virus de PI3 y 7 días después fueron desafiados con P. haemolytica por vía transtorácica. Los corderos sobrevivientes fueron sacrificados al día 49, para el registro de lesiones y estudio bacteriológico. Se les tomó diariamente la temperatura rectal y muestras de suero cada 7 días, con el fin de medir anticuerpos anticápsula y antilecotoxina. En la evaluación de campo se trabajó con 4 grupos de 80 animales cada uno. Se midieron niveles de anticuerpos y se registraron parámetros de morbilidad y mortalidad. En la primera fase los títulos de anticuerpos anticápsula y antileucotoxina fueron estadísticamente significativos (P<0.05) en los grupos tratados con bacteria viva, leucotoxina y bacteriana comercial en comparación con el resto de los grupos; las lesiones fueron más severas en los grupos B, E y F. En la fase de campo en los grupos 1,2 y 4 se enfermaron la neumonía clínica 3 (3.75 por ciento), 5 (6.25 por ciento) y 4 (5 por ciento) animales, respectivamente, y en el grupo 3 (testigo) fueron 23 (28.75 por ciento). Con respecto a la mortalidad y los niveles de anticuerpos se encontró diferencia (P<0.05) entre los grupos 1 y 3. Los resultados obtenidos en ambas fases sugieren que la leucotoxina (sobrenadante de cultivo) y la bacteria viva pueden ser una opción importante en la prevención de las neumonías en ovinos
Assuntos
Animais , Ovinos/imunologia , Indicadores de Morbimortalidade , Mannheimia haemolytica/virologia , Pneumonia/imunologiaRESUMO
Danofloxacin (CP-76,136) is in a class of agents that inhibit DNA gyrase and trigger induction of the SOS response and temperate bacteriophages. Killing studies against the bovine pathogen Pasteurella haemolytica demonstrated that danofloxacin exhibits particularly rapid killing kinetics. Here, lysogenic Escherichia coli bearing lambda is found to be more sensitive to danofloxacin than nonlysogenic E. coli. Danofloxacin exposure also induced a prophage in P. haemolytica. The potency of danofloxacin against lysogens in likely enhanced by this prophage induction.
