Flavobacterium psychrophilum, the causative agent of bacterial cold-water disease (BCWD) in fresh... more Flavobacterium psychrophilum, the causative agent of bacterial cold-water disease (BCWD) in freshwater-reared salmonids, is also a common commensal organism of healthy fish. The virulence potential of F. psychrophilum isolates obtained from BCWD cases in Ontario between 1994 and 2009 was evaluated. In preliminary infection trials of rainbow trout juveniles, significant differences (0% to 63% mortality) in the virulence of the 22 isolates tested were noted following intraperitoneal injection with 108 cfu/fish. A highly virulent strain, FPG 101, was selected for further study. When fish were injected intraperitoneally with a 106 , 107 or 108 cfu/fish of F. psychrophilum FPG 101, the 108 cfu/fish dose produced significantly greater mortality (p < 0.05). The bacterial load in spleen samples collected from fish every 3 days after infection was determined using rpoC quantitative polymerase chain reaction amplification and by plate counting. Bacterial culture and rpoC qPCR were highly correlated (R2 = 0.92); however, culture was more sensitive than the qPCR assay for the detection of F. psychrophilum in spleen tissue. Ninety-seven per cent of the asymptomatic and the morbid fish had splenic bacterial loads of <2.8 log10 gene/copies and >3.0 log10 gene copies/reaction, respectively, following infection with 108 cfu/fish.
In recent years, Actinobacillus suis, Haemophilus parasuis, and Streptococcus suis have emerged a... more In recent years, Actinobacillus suis, Haemophilus parasuis, and Streptococcus suis have emerged as important pathogens of swine, particularly in high health status herds. Their association with a wide range of serious clinical conditions and has given rise to the moniker "suis-ide diseases." These organisms are early colonizers and, for that reason, are difficult to control by management procedures such as segregated early weaning. Vaccination, serodiagnostic testing, and even serotyping are complicated by the presence of multiple serotypes, cross-reactive antigens, and the absence of clear markers for virulence. In this review, we discuss our current understanding of the pathogenesis, epidemiology, and management of the causative agents of the "suis-ide diseases" of swine.
Flavobacterium psychrophilum is the etiological agent of bacterial coldwater disease (BCWD), an i... more Flavobacterium psychrophilum is the etiological agent of bacterial coldwater disease (BCWD), an important disease in the Ontario fish farming industry and in finfish aquaculture in temperate waters worldwide. The development of antimicrobial resistance by F. psychrophilum is a concern because management of outbreaks of BCWD often requires the use of antibiotics. Seventy‐two isolates of F. psychrophilum collected over a 16‐year period from farmed salmonids with clinical signs of BCWD were tested for susceptibility to 10 antimicrobial agents using cation‐adjusted Mueller–Hinton broth in custom Trek Sensititre susceptibility plates for aquaculture. The minimum inhibitory concentrations (MICs) for the isolates were determined by means of a broth microdilution antimicrobial susceptibility testing method established by the Clinical and Laboratory Standards Institute. Most of the F. psychrophilum isolates had decreased susceptibility to two of the four antibiotics licensed for use in Ontario (i.e., ormetoprim–sulfadimethoxine [≥0.5/9.5 μg/mL for 93% of isolates] and trimethoprim–sulfamethoxazole [≥0.25/4.8 μg/mL for 89% of isolates]). High MIC values (≥2 μg/mL) were obtained for florfenicol and oxytetracycline in 53% and 61% of the isolates, respectively, and 83% of the isolates were relatively susceptible (≤16 μg/mL) to erythromycin. The MIC values were also high for ampicillin, oxolinic acid, and gentamicin.
A real-time polymerase chain reaction (PCR) assay of the outer membrane protein (OMP) P2 gene was... more A real-time polymerase chain reaction (PCR) assay of the outer membrane protein (OMP) P2 gene was developed and used to test 97 putative Haemophilus parasuis pure cultures and 175 clinical tissue samples. With standard culture isolation as the gold standard, the diagnostic sensitivity and specificity of the PCR assay were determined to be 83% and 80%, respectively.
The family Pasteurellaceae is comprised of three genera: Haemophilus, Actinobacillus, and Pasteur... more The family Pasteurellaceae is comprised of three genera: Haemophilus, Actinobacillus, and Pasteurella (HAP). These bacteria cause important diseases in humans and animals and are of great medical and economical importance. Recently, the HAP bacteria have been the subject of intensive research in the fields of molecular mechanisms of pathogenicity, immunogenicity, and vaccine development. A considerable amount of work has been done in the molecular analysis of several virulence components including capsule, lipopolysaccharide, outer membrane proteins, and exotoxins. While cloning and expression of genes from the HAP bacteria in established hosts like E. coli K12 strains appears to be easy, very little work has been described on the genetic engineering of the HAP bacteria themselves.
Porcine reproductive and respiratory syndrome virus (PRRSV) is a major cause of economic losses i... more Porcine reproductive and respiratory syndrome virus (PRRSV) is a major cause of economic losses in the swine industry. The disease is widespread worldwide, and so PRRSV-negative pigs are often difficult to find for the study of PRRSV in vivo. To determine if a small animal model could be developed for PRRSV, 3 strains of laboratory rodent were examined for their susceptibility to the virus. No virus replication was detected in BALB/c or SCID (severe combined immunodeficiency) mice after intraperitoneal inoculation. Moderate replication of PRRSV was detected in primary cotton rat lung cell cultures, but no viral replication was detected following intranasal or intraperitoneal inoculation. Following intratracheal inoculation, viral transcripts were detected in the lungs of cotton rats, but only for 1 day. This study indicates that PRRSV replication in common laboratory rodent species is inefficient, and suggests that a rodent model for this virus is not appropriate.
Haemophilus parasuis is an important opportunistic pathogen in swine of high health status, but t... more Haemophilus parasuis is an important opportunistic pathogen in swine of high health status, but to date no proven virulence factors have been described. As virulence factors are known to be regulated during disease, the objective of this study was to identify genes of a virulent serovar 5 strain with altered expression after iron restriction or in the presence of porcine cerebrospinal fluid (CSF), conditions that reflect in vivo growth conditions. Using differential-display reverse-transcriptase-mediated polymerase chain reaction, we found that homologues of genes encoding fructose bisphosphate aldolase (fba), adenylosuccinate synthetase (purA), 2',3'-cyclic nucleotide phosphodiesterase (cpdB), lipoprotein signal peptidase (lspA), pyrophosphate reductase (lytB), superoxide dismutase (sodC), tyrosyl t-RNA synthetase (tyrS), cysteine synthetase (cysK), an unknown protein, and a homologue of a hydrolase of the haloacid dehydrogenase superfamily were upregulated in response to iron restriction. In addition, the purA, cpdB, lspA, lytB, and sodC homologues, cDNAs homologous with a Na+/alanine symporter, fatty acid ligase (fadD), diadenosine tetraphosphatase (apaH), and an unknown protein were upregulated in response to CSF. In screening for the presence of these differentially expressed genes to assess their usefulness as diagnostic markers of high virulence potential, we detected homologues of all of these genes in all of the reference strains of the 15 established serovars. The hydrolase homologue, however, was expressed only in representative H. parasuis strains associated with a high virulence potential, suggesting that this enzyme may play a role in pathogenesis.
The purpose of the study was to identify the serotypes of Streptococcus suis from tonsil swabs in... more The purpose of the study was to identify the serotypes of Streptococcus suis from tonsil swabs in clinically ill and healthy pigs in Ontario using a multiplex polymerase chain reaction (PCR) method. Although 22 different serotypes were identified, most isolates were S. suis-like bacteria or untypable.
Isolates of Actinobacillus (Haemophilus) pleuropheumoniae were studied by restriction endonucleas... more Isolates of Actinobacillus (Haemophilus) pleuropheumoniae were studied by restriction endonuclease fingerprinting (REF) analysis using the enzymes BamHI and HindIII. Restriction fragments were resolved by polyacrylamide gel electrophoresis and visualized by silver staining. Except for serotypes 1 and 9, reference strains of A. pleuropneumoniae serotypes 1 to 10 had clearly distinguishable REF profiles. Analysis of REF profiles of southern Ontario field isolates revealed limited heterogeneity amongst isolates of serotype 1 or serotype 5. The REF profiles of the serotype 7 isolates studied showed greater variation. Heterogeneity could not be correlated with the presence of plasmids nor with antibiotic resistance. Limited heterogeneity could also be detected amongst REF profiles of A. pleuropneumoniae isolates recovered from a closed herd suggesting that there is a small amount of genetic variation within clonal populations.
Flavobacterium psychrophilum, the causative agent of bacterial cold-water disease (BCWD) in fresh... more Flavobacterium psychrophilum, the causative agent of bacterial cold-water disease (BCWD) in freshwater-reared salmonids, is also a common commensal organism of healthy fish. The virulence potential of F. psychrophilum isolates obtained from BCWD cases in Ontario between 1994 and 2009 was evaluated. In preliminary infection trials of rainbow trout juveniles, significant differences (0% to 63% mortality) in the virulence of the 22 isolates tested were noted following intraperitoneal injection with 108 cfu/fish. A highly virulent strain, FPG 101, was selected for further study. When fish were injected intraperitoneally with a 106 , 107 or 108 cfu/fish of F. psychrophilum FPG 101, the 108 cfu/fish dose produced significantly greater mortality (p < 0.05). The bacterial load in spleen samples collected from fish every 3 days after infection was determined using rpoC quantitative polymerase chain reaction amplification and by plate counting. Bacterial culture and rpoC qPCR were highly correlated (R2 = 0.92); however, culture was more sensitive than the qPCR assay for the detection of F. psychrophilum in spleen tissue. Ninety-seven per cent of the asymptomatic and the morbid fish had splenic bacterial loads of <2.8 log10 gene/copies and >3.0 log10 gene copies/reaction, respectively, following infection with 108 cfu/fish.
In recent years, Actinobacillus suis, Haemophilus parasuis, and Streptococcus suis have emerged a... more In recent years, Actinobacillus suis, Haemophilus parasuis, and Streptococcus suis have emerged as important pathogens of swine, particularly in high health status herds. Their association with a wide range of serious clinical conditions and has given rise to the moniker "suis-ide diseases." These organisms are early colonizers and, for that reason, are difficult to control by management procedures such as segregated early weaning. Vaccination, serodiagnostic testing, and even serotyping are complicated by the presence of multiple serotypes, cross-reactive antigens, and the absence of clear markers for virulence. In this review, we discuss our current understanding of the pathogenesis, epidemiology, and management of the causative agents of the "suis-ide diseases" of swine.
Flavobacterium psychrophilum is the etiological agent of bacterial coldwater disease (BCWD), an i... more Flavobacterium psychrophilum is the etiological agent of bacterial coldwater disease (BCWD), an important disease in the Ontario fish farming industry and in finfish aquaculture in temperate waters worldwide. The development of antimicrobial resistance by F. psychrophilum is a concern because management of outbreaks of BCWD often requires the use of antibiotics. Seventy‐two isolates of F. psychrophilum collected over a 16‐year period from farmed salmonids with clinical signs of BCWD were tested for susceptibility to 10 antimicrobial agents using cation‐adjusted Mueller–Hinton broth in custom Trek Sensititre susceptibility plates for aquaculture. The minimum inhibitory concentrations (MICs) for the isolates were determined by means of a broth microdilution antimicrobial susceptibility testing method established by the Clinical and Laboratory Standards Institute. Most of the F. psychrophilum isolates had decreased susceptibility to two of the four antibiotics licensed for use in Ontario (i.e., ormetoprim–sulfadimethoxine [≥0.5/9.5 μg/mL for 93% of isolates] and trimethoprim–sulfamethoxazole [≥0.25/4.8 μg/mL for 89% of isolates]). High MIC values (≥2 μg/mL) were obtained for florfenicol and oxytetracycline in 53% and 61% of the isolates, respectively, and 83% of the isolates were relatively susceptible (≤16 μg/mL) to erythromycin. The MIC values were also high for ampicillin, oxolinic acid, and gentamicin.
A real-time polymerase chain reaction (PCR) assay of the outer membrane protein (OMP) P2 gene was... more A real-time polymerase chain reaction (PCR) assay of the outer membrane protein (OMP) P2 gene was developed and used to test 97 putative Haemophilus parasuis pure cultures and 175 clinical tissue samples. With standard culture isolation as the gold standard, the diagnostic sensitivity and specificity of the PCR assay were determined to be 83% and 80%, respectively.
The family Pasteurellaceae is comprised of three genera: Haemophilus, Actinobacillus, and Pasteur... more The family Pasteurellaceae is comprised of three genera: Haemophilus, Actinobacillus, and Pasteurella (HAP). These bacteria cause important diseases in humans and animals and are of great medical and economical importance. Recently, the HAP bacteria have been the subject of intensive research in the fields of molecular mechanisms of pathogenicity, immunogenicity, and vaccine development. A considerable amount of work has been done in the molecular analysis of several virulence components including capsule, lipopolysaccharide, outer membrane proteins, and exotoxins. While cloning and expression of genes from the HAP bacteria in established hosts like E. coli K12 strains appears to be easy, very little work has been described on the genetic engineering of the HAP bacteria themselves.
Porcine reproductive and respiratory syndrome virus (PRRSV) is a major cause of economic losses i... more Porcine reproductive and respiratory syndrome virus (PRRSV) is a major cause of economic losses in the swine industry. The disease is widespread worldwide, and so PRRSV-negative pigs are often difficult to find for the study of PRRSV in vivo. To determine if a small animal model could be developed for PRRSV, 3 strains of laboratory rodent were examined for their susceptibility to the virus. No virus replication was detected in BALB/c or SCID (severe combined immunodeficiency) mice after intraperitoneal inoculation. Moderate replication of PRRSV was detected in primary cotton rat lung cell cultures, but no viral replication was detected following intranasal or intraperitoneal inoculation. Following intratracheal inoculation, viral transcripts were detected in the lungs of cotton rats, but only for 1 day. This study indicates that PRRSV replication in common laboratory rodent species is inefficient, and suggests that a rodent model for this virus is not appropriate.
Haemophilus parasuis is an important opportunistic pathogen in swine of high health status, but t... more Haemophilus parasuis is an important opportunistic pathogen in swine of high health status, but to date no proven virulence factors have been described. As virulence factors are known to be regulated during disease, the objective of this study was to identify genes of a virulent serovar 5 strain with altered expression after iron restriction or in the presence of porcine cerebrospinal fluid (CSF), conditions that reflect in vivo growth conditions. Using differential-display reverse-transcriptase-mediated polymerase chain reaction, we found that homologues of genes encoding fructose bisphosphate aldolase (fba), adenylosuccinate synthetase (purA), 2',3'-cyclic nucleotide phosphodiesterase (cpdB), lipoprotein signal peptidase (lspA), pyrophosphate reductase (lytB), superoxide dismutase (sodC), tyrosyl t-RNA synthetase (tyrS), cysteine synthetase (cysK), an unknown protein, and a homologue of a hydrolase of the haloacid dehydrogenase superfamily were upregulated in response to iron restriction. In addition, the purA, cpdB, lspA, lytB, and sodC homologues, cDNAs homologous with a Na+/alanine symporter, fatty acid ligase (fadD), diadenosine tetraphosphatase (apaH), and an unknown protein were upregulated in response to CSF. In screening for the presence of these differentially expressed genes to assess their usefulness as diagnostic markers of high virulence potential, we detected homologues of all of these genes in all of the reference strains of the 15 established serovars. The hydrolase homologue, however, was expressed only in representative H. parasuis strains associated with a high virulence potential, suggesting that this enzyme may play a role in pathogenesis.
The purpose of the study was to identify the serotypes of Streptococcus suis from tonsil swabs in... more The purpose of the study was to identify the serotypes of Streptococcus suis from tonsil swabs in clinically ill and healthy pigs in Ontario using a multiplex polymerase chain reaction (PCR) method. Although 22 different serotypes were identified, most isolates were S. suis-like bacteria or untypable.
Isolates of Actinobacillus (Haemophilus) pleuropheumoniae were studied by restriction endonucleas... more Isolates of Actinobacillus (Haemophilus) pleuropheumoniae were studied by restriction endonuclease fingerprinting (REF) analysis using the enzymes BamHI and HindIII. Restriction fragments were resolved by polyacrylamide gel electrophoresis and visualized by silver staining. Except for serotypes 1 and 9, reference strains of A. pleuropneumoniae serotypes 1 to 10 had clearly distinguishable REF profiles. Analysis of REF profiles of southern Ontario field isolates revealed limited heterogeneity amongst isolates of serotype 1 or serotype 5. The REF profiles of the serotype 7 isolates studied showed greater variation. Heterogeneity could not be correlated with the presence of plasmids nor with antibiotic resistance. Limited heterogeneity could also be detected amongst REF profiles of A. pleuropneumoniae isolates recovered from a closed herd suggesting that there is a small amount of genetic variation within clonal populations.
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