Simone Oliveira

Development of a PCR Test to Diagnose Haemophilus Parasuis Infections

A polymerase chain reaction (PCR) test was developed in order to improve the accuracy and speed of diagnosis of Haemophilus parasuis, an economically important respiratory pathogen that affects swine. The gene sequence of the 16S small subunit ribosomal RNA of H. parasuis (GenBank M75065) was compared with 56 16S sequences of related bacteria, including those frequently isolated from pig tissues. Two species-specific primers were designed: HPS forward and HPS reverse. The predicted size of the amplified PCR product was 821 bp. The PCR test could detect a minimum of 102 bacteria and 0.69 pg of DNA. Thirty-one H. parasuis isolates, including 12 different serovars and 19 field isolates, were positive using the PCR test. No amplification was observed when the test was run using DNA from 15 other bacterial species commonly isolated from swine tissues. A weak band was observed when the PCR test was performed using Actinobacillus indolicus DNA as template. Clinical samples tested by PCR included tissues and swabs from 5 animals naturally infected with H. parasuis and 1 experimentally infected animal. The PCR was positive in 26 of 30 clinical samples. Four samples showed weak bands, and these results were not considered positive. Haemophilus parasuis was isolated from 18 of 30 of these samples. Tissues from specific pathogen-free (SPF) pigs and from unrelated species were negative for H. parasuis isolation and PCR. The developed PCR was successfully used in the diagnosis of H. parasuis infection, especially when compared with traditional microbiology techniques.

Long-distance airborne transport of infectious PRRSV and Mycoplasma hyopneumoniae from a swine population infected with multiple viral variants.

Airborne transport of porcine reproductive and respiratory syndrome virus (PRRSV) and Mycoplasma hyopneumoniae (M hyo) has been reported out to 4.7 km. This study attempted to determine whether this event could occur over longer distances and across multiple viral variants. To accomplish this goal, a mixed infection of 3 PRRSV variants (1-8-4, 1-18-2 and 1-26-2) and M hyo 232 was established in a source population of growing pigs. Over 21-day period, air samples were collected from the source population and at designated distances from the herd. Samples were tested for PRRSV RNA and M hyo DNA by PCR and if positive, further characterized. In exhaust air from the source population, PRRSV and M hyo were detected in 21 of 21 and 8 of 21 air samples, respectively. Five of 114 (4.4%) long-distance air samples were positive for PRRSV and 6 of 114 (5.2%) were positive for M hyo. The 5 PRRSV-positive samples were collected at 2.3, 4.6, 6.6 and 9.1 km from the herd. All contained infectious virus and were >99.2% homologous to PRRSV 1-8-4. No evidence of PRRSV 1-18-2 or 1-26-2 was detected in long-distance samples. All 6 M hyo-positive samples were 99.9% homologous to M hyo 232 and 3 samples (collected at 3.5, 6.8 and 9.2km from the herd) were infectious. These results indicate that airborne transport of PRRSV 1-8-4 and M hyo 232 occurs over longer distances than previously reported and that both pathogens remained infectious.

Evidence of long distance airborne transport of porcine reproductive and respiratory syndrome virus and Mycoplasma hyopneumoniae

The ability of porcine reproductive and respiratory syndrome virus (PRRSV) and Mycoplasma hyopneumoniae to be transported over long distances via the airborne route was evaluated. A source population of 300 grow-finish pigs was experimentally inoculated with PRRSV MN-184 and M. hyopneumoniae 232 and over a 50-day period, air samples were collected at designated distances from the source herd using a liquid cyclonic collector. Samples were tested for the presence of PRRSV RNA and M. hyopneumoniae DNA by PCR and if positive, further characterized. Of the 306 samples collected, 4 (1.3%) were positive for PRRSV RNA and 6 (1.9%) were positive for M. hyopneumoniae DNA. The PRRSV-positive samples were recovered 4.7 km to the northwest (NW) of the source population. Four of the M. hyopneumoniae-positive samples were obtained at the NW sampling point; 2 samples at approximately 2.3 km and the other 2 samples approximately 4.7 km from the source population. Of the remaining 2 samples, one sample was obtained at the southeast sampling point and the other at the southwest sampling point, with both locations being approximately 4.7 km from the source. The four PRRSV-positive samples contained infectious virus and were ≥ 98.8% homologous to the MN-184 isolate used to inoculate the source population. All 6 of the M. hyopneumoniae-positive samples were 99.9% homologous to M. hyopneumoniae 232. These results support the hypothesis that long distance airborne transport of these important swine pathogens can occur.

Phenotypic and molecular characterization of a novel strongly hemolytic Brachyspira species, provisionally designated "Brachyspira hampsonii".

Since 2007, outbreaks of severe bloody diarrhea and hemorrhagic colitis have been reported in the United States and Canada. Though the primary causative agent of swine dysentery is Brachyspira hyodysenteriae, which is strongly hemolytic, the current report describes the isolation of a novel strongly hemolytic Brachyspira sp. This novel Brachyspira sp. was identified from clinical submissions at the Minnesota Veterinary Diagnostic Laboratory, and 40 of such isolates were obtained from 22 clinical submissions representing 5 states. Isolates were confirmed to be different from any known Brachyspira sp. on the basis of phylogenetic analysis of nucleotide sequences of nox and 16S ribosomal RNA (rRNA) genes. Phylogenetic analyses grouped all isolates into 2 clades (clades I and II), and grouping patterns were similar for both nox and 16S rRNA gene sequence analyses. Phenotypically, all isolates were indole and hippurate negative, and enzymatic profiling indicated 2 types of profiles, irrespective of the phylogenetic grouping, differing only in the production of β-glucosidase. The results suggest that a potentially virulent new species of Brachyspira sp., provisionally named “Brachyspira hampsonii ”, is circulating among swine herds in the United States.

Outer Membrane Proteins and DNA Profiles in Strains of Haemophilus parasuis Recovered from Systemic and Respiratory Sites

ABSTRACT Polyserositis caused by Haemophilus parasuis is an important disease that affects mostly weaned pigs. Recent studies have shown that virulence can differ among strains recovered from distinct body sites and also that it may be related to the presence of certain outer membrane proteins (OMPs). The objective of this study was to compare the OMP and DNA profiles of H. parasuis strains isolated from systemic and respiratory sites from diseased and healthy pigs. Strains evaluated in this study were processed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and repetitive-PCR techniques. Two experiments were conducted in order to better define the relationship among genotype, phenotype, and site of isolation. Experiment 1 included 53 H. parasuis isolates recovered from healthy and diseased pigs from unrelated herds. Experiment 2 included 31 isolates of H. parasuis obtained from diseased pigs involved in an outbreak in a large, multifarm system. Results showed that strains recovered from systemic sites had more homogeneous OMP and DNA profiles than those isolated from respiratory sites. Evaluation of isolates involved in the multifarm outbreak showed that only two H. parasuis strains were causing disease. These strains had homogeneous OMP and DNA profiles. However, it was noted that these two parameters were unrelated, since strains classified in the same genotype group expressed different OMP profiles. The homogeneity of OMP and DNA profiles of strains isolated from systemic sites strongly suggests the existence of clonal relationships between virulent strains and also suggests that expression of certain OMP profiles may be related to virulence.

Virulence-associated trimeric autotransporters of Haemophilus parasuis are antigenic proteins expressed in vivo

Glässer’s disease is a re-emerging swine disease characterized by a severe septicaemia. Vaccination has been widely used to control the disease, although there is a lack of extended cross-protection. Trimeric autotransporters, a family of surface exposed proteins implicated in host-pathogen interactions, are good vaccine candidates. Members of this family have been described in Haemophilus parasuis and designated as virulence-associated trimeric autotransporters (VtaA). In this work, we produced 15 recombinant VtaA passenger domains and looked for the presence of antibodies directed against them in immune sera by immunoblotting. After infection with a subclinical dose of H. parasuis Nagasaki, an IgG mediated antibody response against 6 (VtaA1, 5, 6, 8, 9 and 10) of the 13 VtaA of the Nagasaki strain was detected, indicating that they are expressed in vivo. IgA production against VtaA was detected in only one animal. VtaA were more likely to be late antigens when compared to early (Omp P5 and Omp P6) and late (YaeT) defined antigens. Antibody cross-reaction with two orthologs of Nagasaki’s VtaA5 and 6, VtaA15 and 16 of strain HP1319, was also detected. No antibodies against VtaA were detected in the sera of animals immunized with a bacterin of the Nagasaki strain, suggesting poor expression in the in vitro conditions used. Taken together, these results indicate that VtaA are good candidate immunogens that could be used to improve H. parasuis vaccines. However, their capacity to confer protective immunity needs to be further studied.

Aerobic bacteria from mucous membranes, ear canals, and skin wounds of feral cats in Grenada, and the antimicrobial drug susceptibility of major isolates

In a 2-year period 54 feral cats were captured in Grenada, West Indies, and a total of 383 samples consisting of swabs from rectum, vagina, ears, eyes, mouth, nose and wounds/abscesses, were cultured for aerobic bacteria and campylobacters. A total of 251 bacterial isolates were obtained, of which 205 were identified to species level and 46 to genus level. A commercial bacterial identification system (API/Biomerieux), was used for this purpose. The most common species was Escherichia coli (N=60), followed by Staphylococcus felis/simulans (40), S. hominis (16), S. haemolyticus (12), Streptococcus canis (9), Proteus mirabilis (8), Pasteurella multocida (7), Streptococcus mitis (7), Staphylococcus xylosus (7), S. capitis (6), S. chromogenes (4), S. sciuri (3), S. auricularis (2), S. lentus (2), S. hyicus (2), Streptococcus suis (2) and Pseudomonas argentinensis (2). Sixteen other isolates were identified to species level. A molecular method using 16S rRNA sequencing was used to confirm/identify 22 isolates. Salmonella or campylobacters were not isolated from rectal swabs. E. coli and S. felis/simulans together constituted 50% of isolates from vagina. S. felis/simulans was the most common species from culture positive ear and eye samples. P. multocida was isolated from 15% of mouth samples. Coagulase-negative staphylococci were the most common isolates from nose and wound swabs. Staphylococcus aureus, or S. intemedius/S. pseudintermedius were not isolated from any sample. Antimicrobial drug resistance was minimal, most isolates being susceptible to all drugs tested against, including tetracycline.

Identification of potentially virulent strains of Haemophilus parasuis using a multiplex PCR for virulence-associated autotransporters (vtaA)

Haemophilus parasuis is the aetiological agent of Glässers disease and is also a commensal of the upper respiratory tract of pigs. Trimeric autotransporter (vtaA) genes have been identified in H. parasuis and divided into three groups on the basis of the translocator domain sequence. In this study, group 3 vtaA genes were demonstrated by PCR in all 157 H. parasuis isolates tested. Group 1 vtaA genes were associated with virulent strains; 52/54 (96%) group 1 vtaA negative field isolates were isolated from the nasal passages of healthy animals, whereas no group 1 vtaA negative field isolates were isolated from cases of Glässers disease. There was an association between absence of group 1 vtaA, sensitivity to phagocytosis and serum and classification of isolates into nasal cluster C by multilocus sequence typing. A multiplex PCR was developed for diagnosis of H. parasuis at the species level (group 3 vtaA positive) and to differentiate putative non-virulent strains (group 1 vtaA negative). When applied to field samples, the PCR confirmed a high prevalence of H. parasuis in conventionally farmed pigs and demonstrated that almost half of the animals carried potentially virulent strains.

Characterisation of ceftiofur resistance in swine bacterial pathogens.

A PCR based method was developed for the identification of ceftiofur resistance genes (bla(CMY-2), bla(TEM-1), and ampC) in swine bacterial pathogens. Using this method, the ceftiofur resistant (n=76) and susceptible (n=45) strains of Bordetella bronchiseptica, Salmonella spp., Escherichia coli, and Pasteurella multocida were screened for the presence of these three genes. The resistant genes were detected in 70% (bla(TEM-1)), 68% (bla(CMY-2)) and 45% (ampC) of the resistant isolates and in 18% (bla(TEM-1)), 27% (bla(CMY-2)), and 36% (ampC) of the susceptible isolates. Results obtained in the present study showed widespread distribution of these three resistance genes in ceftiofur-resistant swine pathogens. It was also observed that more pathogens are acquiring these resistance genes.

Evaluation of Actinobacillus pleuropneumoniae diagnostic tests using samples derived from experimentally infected pigs.

New serological tests have recently been introduced for Actinobacillus pleuropneumoniae diagnosis. No information is currently available on how these tests compare regarding the detection of antibodies from subclinically infected pigs. To answer this question, 80 pigs were randomly assigned to experimental groups infected with A. pleuropneumoniae serovars 1, 3, 5, 7, 10, 12, 15 and a non-inoculated control group. Blood samples and oropharyngeal swabs were collected prior to infection and for 7 consecutive weeks thereafter. Serum samples were tested using the Swinecheck(®) APP ELISA and the Multi-APP ELISA (University of Montreal). All pigs were euthanized at 49 days post-inoculation. Tonsil and lung samples were cultured for isolation and tested by PCR. The Multi-APP ELISA detected seroconversion 1 week earlier than the Swinecheck(®) APP ELISA with the earliest seroconversion detected at 1 week post-infection (serovar 10) and the latest at 3 weeks post-infection (serovar 1). Seroconversion at day 49 was serovar-dependent and varied from 4 (44%) positives detected in the serovar 10 group to 9 positives (100%) detected in the serovar 15 group. Thirty-one pigs were serologically positive for A. pleuropneumoniae at 49 days post-infection and only 15 still carried A. pleuropneumoniae on their tonsils based on PCR results. No cross-reactions were observed when serum samples were cross-tested using the Swinecheck(®) APP ELISA. A. pleuropneumoniae was successfully isolated from the lung of 2 pigs that developed pleuropneumonia, but was not isolated from tonsils due to heavy contamination by the resident flora. This study offers a comprehensive evaluation of the diagnostic tools currently available for detection of A. pleuropneumoniae subclinical infection.