Carlos Pijoan

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.

Application of a Nested Polymerase Chain Reaction Assay to Detect Mycoplasma Hyopneumoniae from Nasal Swabs

The porcine respiratory disease complex (PRDC) is an increasingly important cause of decreased swine productivity and is characterized by slow growth, decreased feed efficiency, anorexia, cough, and dyspnea. Mycoplasma hyopneumoniae is among the most prevalent and important infectious agents associated with PRDC. Understanding of mycoplasmal pneumonia has been hindered by inadequate diagnostic methods. Many of the currently available tests are relatively insensitive or nonspecific when used in a diagnostic laboratory setting or are too costly or difficult for routine diagnostic use. Several polymerase chain reaction (PCR) assays have been described, but they are not sensitive enough to detect the microorganisms in live pigs, from either nasal or tracheal swabs. A nested PCR using 2 species-specific sets of primers from the 16S ribosomal DNA gave positive results with as little as 80 microorganisms and did not cross-react with other mycoplasma species or with other microorganisms commonly found in the respiratory tract of pigs. This assay was better suited for detection of M. hyopneumoniae from nasal swabs than was conventional PCR. Nasal swab samples were taken at different time periods following experimental challenge of 10 susceptible pigs. Only 2 of the 55 swabs examined gave a positive result with conventional PCR, whereas 30 of the 55 swabs gave a positive result using the nested PCR. Twenty of 40 (50%) nasal swabs from pigs experiencing a respiratory disease outbreak where M. hyopneumoniae had been diagnosed also gave a positive result with the nested PCR. To confirm that the amplified product was specific, 4 nested PCR products were purified, sequences were determined and aligned, and they were confirmed to be from M. hyopneumoniae.

Porcine reproductive and respiratory syndrome virus (PRRSv) interaction with Haemophilus parasuis

Abstract The interaction of bacteria and virus has been well demonstrated in the pathogenesis of respiratory disease in swine. The interaction between porcine respiratory and reproductive syndrome virus (PRRSv) and Haemophilus parasuis has not been studied. We initiated studies to evaluate a possible effect of the PRRSv on the pathogenesis of polyserositis caused by H. parasuis. A group of 30 three week old piglets were distributed in 4 groups. Group I (10 pigs) was inoculated with PRRSv and H. parasuis. Group II (10 pigs) was inoculated with H. parasuis alone. Group III (5 pigs) was inoculated with virus alone and group IV (5 pigs) was inoculated with culture media. Lesions consisted of a severe fibrinous polyserositis affecting 7 of 10 animals in group II and a mild fibrinous pleuritis in 1 of 10 animals of group I. Three of ten animals dually infected with the two agents died during the course of the study. These animals had pulmonary congestion and focal lung hemorrhages. No other animals died from other groups. Group III and IV had no macroscopic lesions. Microscopically group III had interstitial pneumonia. Immunomodulating virus effect may explain the differences in terms of lesions severity between groups I and II. Septic shock was suspected as cause of sudden death.

Evaluation of aerosol transmission of porcine reproductive and respiratory syndrome virus under controlled field conditions

The aim of this study was to determine whether porcine reproductive and respiratory syndrome virus (PRRSV) could be transmitted by aerosol under field conditions. A total of 210 five-month-old PRRSV-negative pigs were housed in a mechanically ventilated finishing facility containing 11 pens. Pen 1 contained 10 pigs (indirect contact controls) and pen 2 remained empty, providing a barrier of 2.5 m from the remaining pigs in pens 3 to 11. Fifteen or 16 of the pigs in each of pens 3 to 11 were infected experimentally with a field isolate of PRRSV and the other six or seven pigs served as direct contact controls. Five days after the pigs were infected, two trailers containing 10 five-week-old PRRSV-naive sentinel pigs were placed along each side of the building; one was placed 1 m from the exhaust fans on one side of the building, and the other was placed 30 m from the fans on the other side, and the sentinel pigs remained in the trailers for 72 hours. They were then moved to separate buildings on the same site, 30 and 80 m, respectively, from the infected barn, and their PRRSV status was monitored for 21 days. The direct and indirect contact control pigs became infected with PRRSV but the sentinel pigs did not.

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.

Passive Transfer of Maternal Mycoplasma hyopneumoniae-Specific Cellular Immunity to Piglets

ABSTRACT Immunity in the neonatal animal is primarily maternally derived, either by lymphocytes that pass into the newborn across the placenta or following colostrum ingestion. However, the effect of this passively transferred cellular maternal immunity on the newborns immune repertoire is not clearly understood. Various studies have shown that colostral lymphocytes are activated and possess functional abilities; however, no studies have shown the transfer of colostral antigen-specific T-cell-specific responses in a newborn. In this study we examined the transfer of vaccine-induced Mycoplasma hyopneumoniae cellular immunity from immune dams to newborn piglets. Newborn piglets from vaccinated and nonvaccinated dams were assessed in two ways for cellular immune responses specific to M. hyopneumoniae: (i) delayed-type hypersensitivity (DTH) testing and (ii) in vitro lymphocyte proliferation, assayed on piglet blood lymphocytes and sow colostral lymphocytes. DTH responses to M. hyopneumoniae were detected only for offspring of vaccinated sows, whereas DTH responses to the nonspecific mitogen phytohemagglutinin were seen for all piglets. M. hyopneumoniae-specific proliferation was seen for colostral lymphocytes from vaccinated sows and for blood lymphocytes from neonatal piglets of vaccinated dams but not for blood lymphocytes from piglets of nonvaccinated sows. Functional antigen-specific T cells were transferred to offspring from vaccinated sows and participated in the neonatal immune response upon stimulation. These data have implications for defining disease intervention strategies.

An assessment of the duration of Mycoplasma hyopneumoniae infection in an experimentally infected population of pigs.

Mycoplasma hyopneumoniae (M. hyopneumoniae) is the primary pathogen of enzootic pneumonia (EP), a highly prevalent respiratory disease that affects pigs worldwide. Previous studies have demonstrated that M. hyopneumoniae infection can be longer than 185 days; however, the total duration of infection has not been determined yet. Therefore, the objective of this study was to determine the duration of M. hyopneumoniae infection in asymptomatic carriers. To achieve our goal, 60 pigs were inoculated with M. hyopneumoniae strain 232 and the persistence of M. hyopneumoniae in the respiratory tract was assessed by detection of the bacterial DNA in bronchial swabs and the ability of the infected pigs to transmit the pathogen to sentinels. Infection of the inoculated animals was demonstrated by the detection of M. hyopneumoniae DNA in nasal swabs, seroconversion to the bacteria and the onset of dry coughing. Experimentally infected pigs shed M. hyopneumoniae prior to and after the cough was observed. M. hyopneumoniae DNA was detected in 100% of experimentally infected pigs at 94 days post infection (dpi), 61% at 214dpi and 0% at 254dpi. Experimentally infected pigs transmitted the bacteria to sentinels at 80 and 200dpi. Results of this study have demonstrated that M. hyopneumoniae infected pigs can be incubatory as well as convalescent carriers of the pathogen and that convalescent carriers can remain infectious for up to 200 days. Total clearance of M. hyopneumoniae in the group was evidenced, demonstrating that duration of M. hyopneumoniae infection lasts less than 254 days.

Interactions of Pseudorabies virus with swine alveolar macrophages I: virus replication

SummaryThe replication of Pseudorabies virus (PRV) in cultured swine alveolar macrophages (AM) was studied using 6 different virus strains. AM were highly permissive to PRV infection, with progeny virus titres of 107 TCID50/ml from some strains. Virus progeny titres were higher in cultures infected with the field strains S-62 and 4892 than in cultures infected with the strains Bartha or PRV-C. Virus replication, viral DNA synthesis and the concomitant cell damage were dependent upon virus input m.o.i.s. and virus strain. Furthermore, cells from 7 day old pigs yielded higher virus progeny titres than cells of 6 week old pigs. The results from this study provide support to the premise that PRV infection may predispose anials to respiratory disease.

Immunohistochemical detection of Haemophilus parasuis serovar 5 in formalin-fixed, paraffin-embedded tissues of experimentally infected swine

An avidin-biotin complex immunohistochemistry technique was developed to detect Haemophilus parasuis serovar 5 in experimentally infected 18-21-day-old conventional pigs, using a rabbit polyclonal antiserum. Seven of 10 intratracheally inoculated animals developed a low to medium degree of fibrinous polyserositis; meninges and pleura were the most severely affected areas. Haemophilus parasuis was recovered from 9 of 10 pigs; in 2 of them H. parasuis was isolated from tracheal swabs only. Positive immunohistochemistry results, mainly observed as free bacteria or bacteria within inflammatory cell cytoplasm in the fibrinopurulent exudate, were observed in 8 of 10 animals. Cross-reactivity with Actinobacillus pleuropneumoniae was detected but not with other gram-positive and gram-negative bacteria tested. This immunohistochemistry technique seemed to be at least as sensitive as microbiologic cultures and could be useful in studies of pathogenesis and retrospective diagnosis. However, cross-reactivity with A. pleuropneumoniae means that positive immunohistochemistry results in lung tissue from field cases would be dubious.

Effect of porcine reproductive and respiratory syndrome virus on subsequent Pasteurella multocida challenge in pigs

Abstract This trial was conducted to evaluate the effect of Porcine reproductive and respiratory syndrome virus (PRRSv) on a subsequent challenge with Pasteurella multocida in pigs. Sixteen, 3–4 week-old piglets, from a PRRSv and Aujeszky disease virus (ADV) free herd were used. Animals were equally and randomly allocated in four groups which were treated according the following schedule: Group I: negative controls; Group II: inoculation with only PRRSV; Group III: inoculation with PRRSV and P. multocida; Group IV: inoculation with ADV and P. multocida (positive controls). PRRSV and ADV were inoculated intranasally, at the doses of 104.6 and 104.5 TCID50/ml, respectively. Five days later, pigs from groups III and IV were inoculated intranasally, with two ml of a 109 CFU/ml suspension of equal parts of P. multocida, strains A52 and A24. No lesions were observed in piglets of group I. Microscopically, interstitial pneumonia was identified in all piglets of groups II and III and 3 4 piglets from group IV. Bronchopneumonia was detected in 3 4 of the piglets from group III and in all animals of group IV which, additionally, showed meningo-encephalitis and purulent rhinitis. Macroscopically, only piglets of groups III and IV had lung consolidation. However, much lower pneumonic scores (2.3%) were observed in group III, where 3 of 4 piglets were affected. On the other hand, all piglets of group IV showed some degree of pulmonary consolidation, with a mean score of 13.7%. Based on these results, it appears that the role of PRRSV as a initiator of secondary diseases is still undefined, but is probably mild. There was no clear interaction between PRRSv and Pasteurella multocida under the conditions and strains tested here.