Design and analysis of an Actinobacillus pleuropneumoniae transmission experiment.

This paper describes a methodology to quantify the transmission of Actinobacillus (A.) pleuropneumoniae from subclinically infected carrier pigs to susceptible contact pigs, and to test the effect of possible interventions on the transmission. The methodology includes the design of a transmission experiment, and a method with which A. pleuropneumoniae transmission can be quantified and with which the effect of an intervention on the transmission can be tested. The experimental design consists of two parts. First, subclinically infected carrier pigs are created by contact exposure of specific-pathogen-free pigs to endobronchially inoculated pigs. Second, transmission is observed from the group of carrier pigs to a second group of susceptible contact pigs after replacing the inoculated pigs by new contact pigs. The presented analytical method is a generalised linear model (GLM) with which the effect of an intervention on the susceptibility and infectivity can be tested separately, if the transmission is observed in heterogeneous populations. The concept of the experimental transmission model is illustrated by describing an A. pleuropneumoniae transmission experiment in which the effect of vaccination on the susceptibility is quantified. Although it could not be demonstrated that vaccination has an effect on the susceptibility of pigs, it was demonstrated that nasal excretion of A. pleuropneumoniae is related to the infectivity of pigs.

Transmission of Actinobacillus pleuropneumoniae in pigs is characterized by variation in infectivity.

Ten transmission trials with Actinobacillus pleuropneumoniae were carried out. The observed transmission was highly variable, which was surprising since the design of the trials was very similar. We investigated whether the variable transmission could be explained by variation in infectivity of A. pleuropneumoniae infected pigs. We looked for measurable characteristics, which could be indicative for infectious pigs or for the level of infectivity. The characteristic that appeared to be most indicative for a pig being infectious was an A. pleuropneumoniae positive tonsil at necropsy. The characteristic that was correlated to the level of infectivity was the number of A. pleuropneumoniae colonies isolated from the nasal swab, i.e. the probability for an infectious pig to infect a susceptible pig was tenfold higher on days where at least ten colonies were isolated. In this study it is shown that it is possible to measure the bacterial transmission of A. pleuropneumoniae under controlled circumstances if variation in infectivity is taken into account.

[The effect of a high dose bovine herpes virus 1 marker vaccine in pregnant heifers: virological, bacteriological, immunological, and pathological findings]. / De effecten van vaccinatie met een hoge dosis BHV1-markervaccin in hoogdrachtige vaarzen: virologische, bacteriologische, immunologische en pathologische bevindingen.

To determine a possible relationship between the compulsory vaccination against bovine herpesvirus 1 (BHV1) and cattle wasting disease, the effects of BHV1 vaccination on heifers were investigated. Twenty heifers in the third trimester of pregnancy were randomly allotted to a vaccine and a control group. The vaccine group was vaccinated twice with a 50-fold dose of BHV1 vaccine and the control group was inoculated with the diluent. The experiment was performed double blind. After vaccination, the cows were examined daily and condition scores were determined weekly. Blood, milk, and faeces samples were collected weekly for virological, bacteriological, and immunological investigation. The heifers were euthanized either 9 or 13 weeks after the first inoculation and pathological, virological, and bacteriological examination was performed. No differences were detected between the vaccine group and the control group. No concurrent infections were detected and there were no indications of immunosuppression after vaccination. No relationship between the BHV1 vaccination and wasting disease in cattle was detected.

A comparison of four serological tests for the diagnosis of caseous lymphadenitis in sheep and goats.

A double antibody sandwich ELISA (ELISA A) developed for the detection of Corynebacterium pseudotuberculosis infection in sheep and goats was modified to improve its sensitivity. To establish the sensitivity and specificity of this modified ELISA (ELISA B), sera from 183 sheep and 186 goats were tested using ELISAs A and B. Comparison was also made with two further ELISAs (C and D) developed in Australia that, respectively, detect antibodies to cell wall antigens or toxin.ELISA B had the best performance of the four tests. Its specificity was 98+/-1% for goats and 99+/-1% sheep. Its sensitivity was 94+/-3% for goats and 79+/-5% for sheep. ELISA B will now be tested for use in caseous lymphadenitis eradication and control programmes in The Netherlands. It will also be used in experimental studies of CL in Scotland.

Survey of infectious agents involved in acute respiratory disease in finishing pigs.

Outbreaks of respiratory disease constitute a major health problem in herds of finishing pigs and their aetiology often remains unclear. In this study, 16 outbreaks of respiratory disease with acute clinical signs in finishing pigs were investigated to determine which infectious agents were involved. From each herd four diseased and two clinically healthy pigs were examined pathologically and for the presence of viruses, bacteria and mycoplasmas. In addition, paired blood samples from 10 groupmates of the diseased pigs were tested for antibodies against commonly known causal agents of respiratory disease. A clear diagnosis was possible in 12 of the 16 outbreaks. Seven were due to an infection with influenza virus and five were due to an infection with Actinobacillus pleuropneumoniae. A combination of influenza virus and A pleuropneumoniae may have caused one other outbreak, but no clear cause could be established for the other three outbreaks.

Endobronchial inoculation with Apx toxins of Actinobacillus pleuropneumoniae leads to pleuropneumonia in pigs.

To establish the role of the Apx toxins in the pathogenesis of porcine pleuropneumonia, specific-pathogen-free pigs were inoculated deeply endobronchially with either culture filtrates of Actinobacillus pleuropneumoniae serotype 8 or 9, culture filtrates depleted of the Apx toxins by affinity chromatography, depleted culture filtrate supplemented with purified recombinant Apx toxins (rApx), or purified rApx toxins alone. Results of these experiments indicate that ApxI, ApxIII, and, to a lesser extent, ApxII are the bacterial factors that trigger the development of clinical symptoms and lung lesions typical for porcine pleuropneumonia.

Influence of Actinobacillus pleuropneumoniae and its metabolites on porcine alveolar epithelial cells.

The effect of Actinobacillus pleuropneumoniae and its metabolites on the viability of porcine alveolar epithelial cells was studied by using a neutral-red uptake test. Alveolar epithelial cells were obtained from 5-week-old colostrum-deprived pigs. The purity of these cells as assessed by the modified Papanicolaou stain was 90 to 95%. Incubation of these cells with 10(6) CFU of a biotype 1 serotype 1 strain resulted in death of the alveolar epithelial cells within 1.5 h. A cytotoxic effect was also seen when alveolar epithelial cells were incubated with sterile culture supernatants of biotype 1 serotype 1, biotype 1 serotype 10, and biotype 2 serotype 2 strains or with ApxI, ApxII, or ApxIII produced by recombinant Escherichia coli. Incubation of alveolar epithelial cells with a knockout mutant of the biotype 1 serotype 1 parent strain which is unable to secrete Apx toxins or with its supernatant did not result in death of these cells. These results indicate that cytotoxicity is at least in part due to production of Apx toxins.

A specific and sensitive PCR assay suitable for large-scale detection of toxigenic Pasteurella multocida in nasal and tonsillar swabs specimens of pigs.

A polymerase chain reaction (PCR) assay for the detection of toxigenic Pasteurella multocida in nasal and tonsillar swab specimens collected from pigs was developed. Target DNA was isolated with guanidine thiocyanate and diatomite, and 2 primer sets derived from sequences in the gene that encodes the dermonecrotic toxin of P. multocida were used simultaneously. The method was adapted to microtiter plate format allowing large-scale use of the PCR assay. To identify false-negative test results caused by failure of amplification, a positive control template was constructed that was spiked to each DNA sample. The PCR assay was evaluated with clinical samples and compared with 2 routinely used methods for detection of toxigenic P. multocida: isolation from a selective agar and direct detection of the toxin in extracts of primary cultures by an enzyme-linked immunosorbent assay (ELISA). The sensitivity of the PCR assay was tested with 346 nasal and tonsillar swabs specimens collected from pigs of 9 herds known to be infected with toxigenic P. multocida. Toxigenic P. multocida was isolated from 22 specimens, only 28 specimens tested positive in ELISA, but 40 tested positive in the PCR assay; thus the PCR assay is the most sensitive of the 3 methods. The specificity of the PCR assay was tested with 372 swab specimens collected from pigs of 6 herds certificated to be free from toxigenic P. multocida. Toxigenic P. multocida was not isolated from any of these specimens, all tested negative in ELISA, and 370 tested negative in PCR. The 2 positive specimens came from 2 pigs of 1 litter and tested only weakly positive in the PCR assay. From these results, it was concluded that the PCR assay is not only highly sensitive but also highly specific.

Susceptibility to Actinobacillus pleuropneumoniae infection in pigs from an endemically infected herd is related to the presence of toxin-neutralizing antibodies.

Our objective was to identify pigs of an endemically infected herd that were susceptible to pleuropneumonia due to Actinobacillus pleuropneumoniae. The presence of toxin-neutralizing antibodies was studied in serum of 36 pigs from birth until 24 weeks of age. Titers gradually declined during the first twelve weeks of life and increased thereafter. Sera from one-hundred 3-weeks-old piglets and one-hundred 20-weeks-old pigs were sampled and neutralization titers were determined. From each group we selected 5 pigs with the lowest titers and 5 pigs with the highest titers. These selected pigs (n = 20) were inoculated endobronchially with A. pleuropneumoniae. Pigs that survived from infection were necropsied after 48 h. Pigs with low neutralization titers had severe lung lesions, whereas pigs with high titers had no or minor lung lesion. These differences were significant (P < 0.05). From this field study we conclude that susceptibility to Actinobacillus pleuropneumoniae can be predicted by absence of toxin-neutralizing antibodies.

Prevalence and development of antibodies neutralizing the haemolysin and cytotoxin of Actinobacillus pleuropneumoniae in three infected pig herds.

Sero-epidemiological studies were carried out in pigs aged 1 to 24 weeks in three herds in which Actinobacillus pleuropneumoniae was endemic. The sera were tested in the complement-fixation test and for their ability to neutralize the haemolytic and cytotoxic activities of the A. pleuropneumoniae serotypes isolated from the herds. Almost all (98%) sera from 1-week-old piglets neutralized the haemolytic and cytotoxic activities but only 21% fixed complement. At the end of the finishing period, most pigs (82%) had sera that neutralized haemolytic and cytotoxic activities and only 22% fixed complement. In longitudinal studies the neutralization titres decreased during the first 12-13 weeks of age. Thereafter, 75% of the pigs had increased titres in the haemolysin- and cytotoxin-neutralization tests and only 5% of the pigs had increased titres in the complement-fixation test. In none of these pigs were clinical signs of pleuropneumonia seen. Thus in these endemically infected herds the prevalence of complement-fixing antibodies was low, whereas the prevalence of neutralizing antibodies was high. The fact that serum-neutralizing titres are low in 12-week-old pigs might be the reason that pigs of this age are the most vulnerable to the disease.

Use of Multiscreen plates for the preparation of bacterial DNA suitable for PCR.

A rapid and inexpensive method for isolating bacterial DNA for use in PCR is described. The method is based on the guanidinium thiocyanate (GuSCN)-lysis method of Boom et al. (J. Clin. Microbiol. 28:495-503, 1990) and enables a multiple of 96 samples to be prepared in only one hour. We use Multiscreen plates and a vacuum manifold from Millipore. Clinical samples are lysed and washed in the wells of a Multiscreen plate, and DNA is eluted in a standard microplate. Purified DNA was recovered with high yields (over 25%). The method allows multichannel or robotic pipetting for both the sample preparation as well as for the PCR step. The method has been applied successfully to detect pathogenic Streptococcus suis type 2 in nasal and tonsil swab specimens of pigs.

The CAMP effect of Actinobacillus pleuropneumoniae is caused by Apx toxins.

Actinobacillus pleuropneumoniae shows synergistic haemolysis when cocultured with Staphylococcus aureus on blood agar plates. This CAMP effect has been attributed to a discrete CAMP factor, but also to the A. pleuropneumoniae-RTX-toxins I, II, and III. We examined the CAMP effect of recombinant Escherichia coli strains that secreted each of these toxins, and of A. pleuropneumoniae mutant strains that were devoid of one or more these toxins. We found that the E. coli strains were CAMP positive, whereas the A. pleuropneumoniae strain devoid of functional toxin genes was CAMP negative. This demonstrated that the CAMP effect of A. pleuropneumoniae is caused by the toxins and that no CAMP factor per se exists.

Knockout mutants of Actinobacillus pleuropneumoniae serotype 1 that are devoid of RTX toxins do not activate or kill porcine neutrophils.

The Actinobacillus pleuropneumoniae RTX-toxins ApxI, ApxII, and ApxIII are important virulence factors of this swine pathogen. It is hypothesized that the Apx toxins are deleterious to defense cells of the host, enabling the bacterium to infect the host. To confirm this, we studied the effect on porcine polymorphonuclear neutrophils of mutant strains of A. pleuropneumoniae that were devoid of Apx toxins. For this purpose, we developed a system for targeted mutagenesis of A. pleuropneumoniae based on the conditionally replicating plasmid pVE6063 and insertional mutagenesis by homologous recombination. Employing this system on the reference strain of serotype 1, a strain that secretes ApxI and ApxII, we generated mutant strains that were devoid of ApxI and/or ApxII. We compared the ability of the parent strain and the mutant strains to provoke an oxidative burst in porcine neutrophils and to kill these cells. The parent strain and mutants that secreted either ApxI or ApxII provoked an oxidative burst and killed the neutrophils, whereas mutant strains that were devoid of ApxI and ApxII did not. These experiments indicate the importance of ApxI and ApxII to these profound effects on neutrophils and emphasize the importance of ApxI and ApxII in pathogenesis.

Genetic map of the Actinobacillus pleuropneumoniae RTX-toxin (Apx) operons: characterization of the ApxIII operons.

Actinobacillus pleuropneumoniae RTX-toxin III (ApxIII) is implicated as an important virulence factor of A. pleuropneumoniae, the causative agent of porcine pleuropneumonia. Recently, the genes coding for ApxIII (apxIIICA) of serotype 8 were cloned and characterized. The toxin appeared to be a member of the RTX-toxin family, as are the other two secreted toxins of A. pleuropneumoniae, i.e., ApxI and ApxII. In this report, we describe the cloning and sequencing of the remaining part of the ApxIII operon of serotype 8. This sequence coded for the RTX secretion proteins ApxIIIB and ApxIIID, which showed 86 and 63% similarity to ApxIB and ApxID, respectively, and 83 and 63% similarity to HlyB and HlyD of Escherichia coli, respectively. Potential functional domains, such as eight transmembrane regions and an ATP-binding cassette, were present in ApxIIIB. We examined the presence of apxIIICABD sequences in the 12 serotypes of A. pleuropneumoniae and found that these sequences were present only in serotypes 2, 3, 4, 6, and 8, the serotypes that secrete ApxIII. Comparison of the apxIIICABD gene sequences of the serotypes revealed very few serotype-specific differences. Only the C terminus of ApxIIIA of serotype 2 differed from ApxIIIA of the other serotypes. The differences were located between the glycine-rich repeats and the secretion signal. The analysis of the apxIIICABD genes completed our efforts to characterize the ApxI, ApxII, and ApxIII operons of the reference strains of the 12 serotypes of A. pleuropneumoniae. We present a complete map of the ApxI, ApxII, and ApxIII operons and discuss this in terms of gene expression and complementation and the role of the toxins in pathogenesis.

Production of Apx toxins by field strains of Actinobacillus pleuropneumoniae and Actinobacillus suis.

The three Apx toxins of Actinobacillus pleuropneumoniae have potential value for use in vaccines and diagnostic tests which will be species specific instead of serotype specific, provided that the Apx toxins are species specific and all field strains produce these toxins. We examined 114 A. pleuropneumoniae field strains and found that they secreted either ApxI, ApxII, ApxI and ApxII, or ApxII and ApxIII and secreted no other cytolytic activities. However, proteins similar to ApxI and ApxII were also produced by Actinobacillus suis.

NAD-independent Actinobacillus pleuropneumoniae strains: production of RTX toxins and interactions with porcine phagocytes.

Actinobacillus pleuropneumoniae RTX toxin (Apx) production by A. pleuropneumoniae biotype 2 (NAD-independent) serotype 2 strains was studied. Western blot analysis of culture supernatants of all biotype 2 strains tested revealed the presence of a 103 kDa protein which reacted with a monoclonal antibody against ApxIIA. This protein was also recognized by sera of pigs infected with a biotype 2-serotype 2 strain. Furthermore, antibodies that could neutralize ApxIIA were present in these sera. Proteins corresponding to ApxIA or ApxIIIA were not detected. The effects of a biotype 1-serotype 2 and a biotype 2-serotype 2 strain and their metabolites on the oxidative activity of porcine pulmonary alveolar macrophages (PAM) and polymorphonuclear cells (PMN) were compared using a chemiluminescence (CL) technique. Viable bacteria of both biotypes stimulated the production of oxygen radicals by phagocytes. CL responses were higher for the biotype 1 than for the biotype 2 strain. After having reached a peak value, the oxidative activity decreased until a total inhibition was achieved. Inactivated washed bacteria had no influence on the oxidative activity of phagocytes. In contrast, heat labile factors in culture supernatants of both biotypes stimulated and inhibited the oxidative activity of PAM in a dose-dependent manner. Dilutions of supernatant up to 1/32 of the biotype 2 strain and up to 1/512 of the biotype 1 strain were toxic for PAM, while dilutions from 1/64 to 1/128 of the biotype 2 strain and from 1/1024 to 1/4096 of the biotype 1 strain stimulated the oxidative activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Structural analysis of the Actinobacillus pleuropneumoniae-RTX-toxin I (ApxI) operon.

Actinobacillus pleuropneumoniae-RTX-toxin I (ApxI), an important virulence factor, is secreted by serotypes 1, 5, 9, 10, and 11 of A. pleuropneumoniae. However, sequences homologous to the secretion genes apxIBD of the ApxI operon are present in all 12 serotypes except serotype 3. The purpose of this study was to determine and compare the structures of the ApxI operons of the 12 A. pleuropneumoniae serotypes. We focused on the nucleotide sequence comparison of the ApxI-coding genes, the structures of the ApxI operons, and the transcription of the ApxI operons. We determined the nucleotide sequences of the toxin-encoding apxICA genes of serotype 9 and found that the gene for the structural toxin, apxIA, was almost identical to the apxIA gene of serotype 1. The toxin-encoding genes of the other serotypes are also similar for the main part; nevertheless, two variants were identified, one in serotypes 1, 9, and 11 and one in serotypes 5 and 10. The two apxIA variants differ mainly within the distal 110 nucleotides. Structural analysis demonstrated that intact ApxI operons, consisting of the four contiguous genes apxICABD, are present in serotypes 1, 5, 9, 10, and 11. ApxI operons with a major deletion in the apxICA genes are present in serotypes 2, 4, 6, 7, 8, and 12. Serotype 3 does not contain ApxI operon sequences. We found that all ApxI operons are transcriptionally active despite the partial deletion of the operon in some serotypes. The implications of these data for the expression and secretion of ApxI and the other Apx-toxins, ApxII and ApxIII, as well as for the development of a subunit vaccine against A. pleuropneumoniae will be discussed.

Serological characterisation of Actinobacillus pleuropneumoniae strains of serotypes 1, 9 and 11.

Various serological tests such as agglutination, coagglutination, indirect haemagglutination, immunodiffusion and counterimmunoelectrophoresis were used to characterise serologically Actinobacillus pleuropneumoniae isolates of serotypes 1, 9 and 11 using rabbit hyperimmune sera against serotypes 1 to 12. The rapid slide agglutination test using whole-cell suspension and the indirect haemagglutination test using whole-cell saline extract as antigens gave type specific reactions for serotypes 1 and 9. Antigens comprising saline extracts of boiled or autoclaved cells demonstrated common epitopes among the isolates of all three serotypes in the indirect haemagglutination test. Quantification of the type and group-specific antigens by the coagglutination test. Quantification of the type and group-specific antigens by the coagglutination test distinguished serotypes 1 and 9 strains from those of serotype 11. Results of absorption studies in immunodiffusion tests indicated that the cross-reactivity encountered among strains of serotypes 1, 9 and 11 might be due to common epitopes associated with cell-wall antigens. However, they certainly also have type-specific epitopes, possibly associated with superficially located, capsular antigens.

Cloning and characterization of the Actinobacillus pleuropneumoniae-RTX-toxin III (ApxIII) gene.

To study the role of Actinobacillus pleuropneumoniae-RTX-toxin III (ApxIII) in the pathogenesis of porcine pleuropneumonia, we cloned and characterized the gene encoding this toxin. For that purpose, we screened an expression library of genomic DNA of serotype 8 with an ApxIII-specific monoclonal antibody and isolated a 425-bp fragment of an immunoreactive clone. Using this fragment as a probe, we identified and cloned an overlapping chromosomal NsiI restriction fragment of 5.0 kbp. Escherichia coli cells that contained this fragment produced a protein similar to ApxIII. Like ApxIII, the protein had a molecular mass of approximately 120 kDa, was recognized by an ApxIII-specific antibody, killed porcine lung macrophages, and was not lytic for sheep erythrocytes. We concluded from these data that the 5.0-kbp NsiI fragment contained the ApxIII-coding gene. Nucleotide sequence analysis of the 5.0-kbp NsiI fragment revealed the presence of two genes, apxIIIC and apxIIIA. These genes coded for proteins ApxIIIC and ApxIIIA, respectively, which were 53 and 50% identical to the prototypic RTX proteins HlyC and HlyA of E. coli. We assumed that the apxIIIA gene coded for the structural RTX toxin and that the apxIIIC gene coded for its activator. In addition, we found that ApxIII could be secreted from E. coli by the heterologous RTX transporter proteins HlyB and HlyD. The deduced amino acid sequence of ApxIIIA was 50% identical to that of ApxIA and 41% identical to that of ApxIIA. We concluded that, beside ApxI and ApxII, ApxIII is the third RTX toxin produced by A. pleuropneumoniae.