Study of the virulence of Actinobacillus pleuropneumoniae in finishing pigs as a basis for vaccination development

For vaccine licensing data about efficiency and duration of protection are essential. Within the scope of the developement of a new subunit vaccine against Actinobacillus pleuropneumoniae (A.pp.) the protective efficiency over the whole length of the fattening period must be proven. This required infection experiments in finishing pigs. Eight pigs in the age of six months were infected experimentally into the trachea with an A.pp. serotyp 2 strain. To our knowledge data about the susceptibility of pigs of this age do not exist, so that the infectious dose for pigs of this age and this route of infection had to be determined. Two pigs each were infected with different doses of 10(10), 6 x 10(5), 8 x 10(3) and 2 x 10(3) CFU (colony forming units). The aim of the study was to produce a typical pleuropneumonia with fever and severe respiratory symptoms as well as characteristic pathomorphological lung alterations without loss of animals during the acute stage of infection. The pathogen should be cultivated from lung tissue. The recommended dose for testing the efficiency of vaccines turned out to be approximately 10(3) CFU A.pp. serotyp 2.     

Factors involved in immunity against Actinobacillus pleuropneumoniae in mice.

Active and passive immunization studies in mice were undertaken to examine the protective efficiency of vaccines prepared from different components of Actinobacillus pleuropneumoniae, or combinations thereof. Subcutaneous immunization using either washed formalinized whole cells, capsular polysaccharide, lipopolysaccharide or purified hemolysin I (105 kDa protein) partially protected mice against intranasal challenge with a lethal dose of homologous or heterologous A. pleuropneumoniae serotypes. However, full protection was obtained if the formalinized whole cells were supplemented with purified hemolysin. Similar protection was obtained when mice were immunized simultaneously with a sublethal dose of live cells by the intranasal route and with formalinized whole cells subcutaneously. Passive immunization using rabbit hyperimmune serum against formalinized whole cells provided almost total protection whereas hyperimmune serum against capsular polysaccharide, lipopolysaccharide or hemolysin alone provided only a partial protection. Cell mediated immunity as detected by the foot pad test may not be implicated significantly in the protein against acute A. pleuropneumoniae infection. However, humoral immune response seems to play an important role in protection. All the antigenic components examined may contribute to the protection to some extent. However, heat-labile components such as hemolysin and outer membrane proteins may play a crucial role in protection against acute challenge infection.     

Haemophilus pleuropneumoniae serotypes--cross protection experiments

Pigs vaccinated with a killed 6-hour culture of Haemophilus pleuropneumoniae serotype 2 with Freund's incomplete adjuvant were not protected against challenge with serotypes 1, 5, 6 or 8. Equivalent results were obtained when pigs were vaccinated with serotypes 4 or 5 and challenged with serotype 2. In earlier studies of immunity induced by intranasal immunization with live H. pleuropneumoniae organisms, it was clearly shown that intranasal inoculation with one serotype of H. pleuropneumoniae would induce a strong immunity to both homologous and heterologous serotypes (Nielsen 1979). The present study has shown that cross immunity is not obtained with parenteral immunization. The results strongly suggest that the immune response of the pig to parenteral vaccination is different from the response seen after natural infection, and indicate that an important part of the defence mechanism against H. pleuropneumoniae infection is a local immune-barrier which is effective in preventing the bacterium from penetrating the mucosa. In earlier vaccination experiments 90 per cent of vaccinates were protected against homologous challenge (Nielsen 1976). In the present work a vaccine containing serotypes 1 through 6 was fully protective against serotypes 2 and 3 and also against serotype 8, which shares antigenic determinants with serotypes 3 and 6. These results indicate that the protection obtained by parenteral immunization is serotype-specific. Vaccines must therefore contain the serotypes existing in the swine population.     

Oral immunization using formalin-inactivated Actinobacillus pleuropneumoniae antigens entrapped in microspheres with aqueous dispersion polymers prepared using a co-spray drying process

Oral-vaccine microspheres based on formalin-inactivated Actinobacillus pleuropneumoniae serotype 1 (AP-1) antigens and enteric-coated polymers were prepared using a co-spray drying process. We evaluated using this for a peroral vaccine. We measured specific-antibody titers and protection from challenge in mouse and pig models. In mice (24 per group), a subcutaneous aluminum-adjuvant vaccine or oral vaccination with three doses of AQ6-AP microspheres provided similar protection against intranasal challenge with 5 x 10(8) colony-formation units (cfu) of AP-1 bacterial culture broth. Two weeks after four oral vaccinations with 600 mg of AQ6-AP microsphere acetate solution (containing formalin-inactivated AP-1 antigens of 1.0 x 10(10) cfu bacterial broth), pigs (9 per group) were challenged intranasally with 1 ml of AP-1 bacterial culture broth (5 x 10(9) cfu). The clinical signs, percentage of pig survival ratio, lung lesion areas, and microscopic examinations indicated that the oral AQ6-AP vaccine provided more protection than vaccinating pigs intramuscularly with AP-1 aluminum vaccine.     

Actinobacillus pleuropneumoniae serotype 1 carrying the defined aroA mutation is fully avirulent in the pig

The aroA gene from Actinobacillus pleuropneumoniae serotype 1 reference strain 4074 was isolated and sequenced. The gene complemented the aroA mutation in Escherichia coli AB2829. A kanamycin resistance cassette was inserted into the aroA gene and the mutant gene was reintroduced into A. pleuropneumoniae by allelic replacement. Intratracheal infection of susceptible pigs with A. pleuropneumoniae aroA caused no signs of respiratory disease or lung lesions in any of the animals at a dose 10(4) times the dose reliably known to induce acute pleuropneumonia; all animals infected with the unaltered control strain developed acute disease. The aroA mutant was rapidly eliminated from the lungs and tonsil of infected animals. The mutant may represent a safely attenuated strain for use in live bacterial vaccination or the delivery of antigen by the intranasal route. However, the residence time of the mutant in the respiratory tract of the pig may be too short for it to be useful in generating a protective immune response.     

Actinobacillus pleuropneumoniae serotype 5, subtypes a and b: cross protection experiments

Vaccination of pigs with a killed culture of A. pleuropneumoniae serotype 5, strain K17 (subtype a) afforded a high degree of protection against challenge with strains L20 and T928 (subtype b). The reverse experiment showed that strain L20 gave good protection against challenge with strain K17 whereas strain T928 did not afford an acceptable protection against challenge with this strain.The considerable cross immunity shown to exist between strains K17 and L20 indicates a high degree of homogeneity of the antigenic determinants of the two strains involved in induction of protective immunity and suggest that antibodies to capsular subtype specific determinants may not play a significant role in the specific defence against A. pleuropneumoniae strains belonging to serotype 5. The finding that a vaccine prepared from strain T928 did not afford an acceptable protection against challenge with strain K17 indicates a variable expression among serotype 5 strains of the antigenic determinants which induce protective immunity against A. pleuropneumoniae infection.     

Effect of endobronchial challenge with Actinobacillus pleuropneumoniae serotype 9 of pigs vaccinated with a vaccine containing Apx toxins and transferrin-binding proteins

The efficacy of a subunit vaccine containing the Apx toxins of Actinobacillus pleuropneumoniae and transferrin-binding proteins was determined. Ten pigs were vaccinated twice with the vaccine. Eight control animals were injected twice with a saline solution. Three weeks after the second vaccination, all pigs were endobronchially inoculated with 10(6.5) colony-forming units (CFU) of an A. pleuropneumoniae serotype 9 strain. In the vaccine group, none of the pigs died after inoculation. Only one pig of the control group survived challenge. Surviving pigs were killed at 7 days after challenge. The mean percentage of affected lung tissue was 64% in the control group and 17% in the vaccine group. Actinobacillus pleuropneumoniae was isolated from the lungs of all animals. The mean bacterial titres of the caudal lung lobes were 5.0 x 10(8) CFU/g in the control group and 3.0 x 10(6) CFU/g in the vaccine group. It was concluded that the vaccine induced partial protection against severe challenge.     

Experimental aerosol transmission of Actinobacillus pleuropneumoniae to pigs.

In order to demonstrate the possible role of aerosol in the transmission of Actinobacillus pleuropneumoniae, an experiment including 18 specific pathogen-free (SPF), 10-week-old piglets, randomly distributed into 2 adjacent units, was carried out. In these facilities, air was forced through absolute filters to prevent any contact with infectious agents. During the first 6 d post inoculation, the 2 units were connected by a rectangular opening and the air circulation was forced by the ventilation system from unit A (inoculated pigs) to unit B (non-inoculated pigs). The A. pleuropneumoniae strain (biovar 1 serovar 9) was isolated in France from an outbreak of porcine pleuropneumonia. Two different infecting doses, 10(7) cfu/animal and 10(8) cfu/animal, were inoculated by intranasal route in 6 pigs of unit A. The infection spread quickly from the inoculated pigs to the non-inoculated pigs. Clinical signs were acute during the 4 d post inoculation: hyperthermia, respiratory distress and, sometimes, death (6 pigs of the unit A and 2 pigs of the unit B). All pigs seroconverted against A. pleuropneumoniae serovar 9 within 2 weeks. Lung lesions were severe: fibrinous pleurisy and lung hemorrhages in the acute stage, pleural adherences and focal pulmonary necrosis in the chronic stage. Actinobacillus pleuropneumoniae was isolated from the tonsils and/or lungs in 16 animals. It could be also isolated from the air of the experimental unit. This study showed that A. pleuropneumoniae was readily transmitted through aerosol over a distance of at least 2.5 m.     

Blood lymphocyte subsets in pigs vaccinated and challenged with Actinobacillus pleuropneumoniae

Actinobacillus pleuropneumoniae bacterins do not induce protection in pigs while infection with low doses of the CM5 strain of A. pleuropneumoniae given by aerosol induces complete protection. To evaluate possible correlates of protection in blood lymphocyte subset phenotypes, pigs were treated with a commercial bacterin given intramuscularly, low dose (10(5)cfu/ml) aerosol infection with CM5 or control treatments of the bacterin adjuvant or phosphate buffered saline. All pigs were challenged with a high dose (10(7)cfu/ml) of A. pleuropneumoniae. Lymphocytes and sera were collected prior to and following primary and secondary immunizations and challenge, for evaluation of B- and T-cell markers and antibody to four A. pleuropneumoniae antigens. IgM(micro)+ B-cells were increased following primary exposure to antigen in the bacterin-vaccinated group only. An increase in CD4+ cells in the LD aerosol-infected group was apparent following secondary exposure to antigen. These early changes suggest little difference in lymphocyte populations between treatment groups, however, greater differences were observed following high-dose challenge; CD4+ lymphocytes were increased significantly in both bacterin and LD-challenged groups (p<0.05) while CD8+ cells decreased in the LD-group at this time period. Consequently, there were significant differences (p<0.05) in the CD4:CD8 ratio after high-dose challenge compared to earlier time points and control groups. Variation in cellular expression of SLA-DR and DQ was observed but trends correlating to treatment group were not evident. Complete protection or lack of protection associated with LD challenge or immunisation resulted in significant differences in B-cell frequencies and CD4:CD8 ratio phenotypes in pigs, but only changes in CD4:CD8 ratios appeared relevant to protection.     

Effect of endobronchial challenge with Actinobacillus pleuropneumoniae serotype 10 of pigs vaccinated with bacterins consisting of A. pleuropneumoniae serotype 10 grown under NAD-rich and NAD-restricted conditions

The efficacy of two bacterins containing an Actinobacillus pleuropneumoniae serotype 10 strain was evaluated. The bacterial cells constituting bacterin 1 and 2 were grown under nicotinamide adenine dinucleotide (NAD)-rich (low-adherence capacity to alveolar epithelial cell cultures) and NAD-restricted (high-adherence capacity to alveolar epithelial cell cultures) conditions, respectively. Ten pigs were vaccinated twice with the bacterin 1 and nine pigs with the bacterin 2. Ten control animals were injected twice with a saline solution. Three weeks after the second vaccination, all pigs were endobronchially inoculated with 106.5 colony-forming units (CFU) of an A. pleuropneumoniae serotype 10 strain. In the bacterin 1 and 2 group, three and two pigs died after inoculation, respectively. Only two pigs of the control group survived challenge. Surviving pigs were killed at 7 days after challenge. The percentage of pigs with severe lung lesions (> 10% of the lung affected) was 100% in the control group, 70% in the bacterin 1 group and 22% in the bacterin 2 group. Actinobacillus pleuropneumoniae was isolated from the lungs of all animals. The mean bacterial titres of the caudal lung lobes were 7.0 x 10(6) CFU/g in the control group, 6.3 x 10(5) CFU/g in the bacterin 1 group and 1.3 x 10(6) CFU/g in the bacterin 2 group. It was concluded that both bacterins induced partial protection against severe challenge. Furthermore, there are indications that the bacterin 2, containing A. pleuropneumoniae bacteria grown under conditions resulting in high in vitro adhesin, induced better protection than the bacterin 1.     

Efficacy of an intranasal immunization with gEgC and gEgI double-deletion mutants of Aujeszky's disease virus in maternally immune pigs and the effects of a successive intramuscular booster with commercial vaccines

In this study, an intranasal immunization strategy was set up in maternally immune pigs in order to protect them not only clinically but also virologically. Two genetically engineered Aujeszky's disease virus (ADV) strains, Kaplan gE-gI- and Kaplan gE-gC-, were used for intranasal immunization. Both strains were safe for 4-week-old pigs. A single intranasal inoculation of 10(6.0) TCID50 of Kaplan gE-gI- and Kaplan gE-gC- at 4 weeks of age in the presence of moderate titres of maternally derived antibodies (SN titres: 12-16) reduced the amount of weight loss, fever and virus excretion upon challenge 6 weeks later. In a second experiment, the effect of an additional intramuscular booster with three different commercial vaccines (containing attenuated Bartha or NIA3-783 or inactivated Phylaxia; all suspended in an oil-in-water emulsion) at 10 weeks of age was evaluated. One month after the last intramuscular booster, between five and seven pigs from each group were selected for challenge. All intranasally/intramuscularly immunized pigs showed a significantly better clinical and virological protection after challenge than the single intranasally immunized pigs. In the double immunized group, the protection was better when Kaplan gE-gC- was used for the intranasal priming (only two of 14 pigs excreted virus with a duration of 4 days) than when Kaplan gE-gI- was used (13 of 18 pigs excreted virus with a duration ranging from 1 to 4 days). The virological protection was not influenced by the type of vaccine used for booster vaccination. Because the intranasal/intramuscular immunization approach is very compatible with current pig movements on farms and pigs with moderate levels of maternally derived antibodies can effectively be immunized, it can be considered as a good alternative to intramuscular/intramuscular vaccinations especially in regions with a high ADV prevalence.     

Pulmonary lesions in guinea pigs experimentally infected with Actinobacillus pleuropneumoniae (A.p.) serovar 1

Pathological studies were carried out on the lungs of guinea pigs intratracheally inoculated with 4.6 x 10(6-8) colony forming units (CFU)/head of Actinobacillus pleuropneumoniae serovar 1. All animals in the highest dose group died within 24 hr post inoculation (hpi) and showed pulmonary lesions being hemorrhagic in nature while all animals in the lowest dose group were killed as scheduled at 11 days post inoculation (dpi) and showed only hyperplasia of peribronchial lymphoid tissues. In the middle dose group, two died within 24 hpi, two died at 9 dpi, and the remaining one was killed at 11 dpi. Two guinea pigs which died at 9 dpi showed fibrinonecrotic pleuropneumonia which is the most characteristic acute pulmonary lesion in swine, and has not yet been reproduced in laboratory animals up to the present time. This suggests that guinea pigs may be a useful laboratory animal for studying the pathogenesis of Actinobacillus pleuropneumoniae infection in swine.     

Partial protection against tissue cysts formation in pigs vaccinated with crude rhoptry proteins of Toxoplasma gondii

A vaccine containing crude Toxoplasma gondii rhoptry proteins incorporated in the immunostimulating complexes (ISCOM) adjuvant was tested in pigs for protecting against tissue cyst formation. For this, 38 mixed breed pigs were divided into four groups, G1 (vaccinated challenged, n=10) received two doses (100 microg/dose) of the rhoptry vaccine at days 0 and 21, G2 (vaccinated challenged, n=10) received viable tachyzoites (7 x 10(7)) of the RH strain at day 0, G3 (unvaccinated challenged, n=10) and G4 (unvaccinated unchallenged, n=8). Pigs were challenged with 4 x 10(4) VEG strain oocysts 57 days later. The G1 pigs produced high IgG antibody levels in the indirect enzyme-linked immunosorbent assay (ELISA) after the second dose of rhoptry vaccine, but were not clinically protected against a high dose oocyst challenge. Partial protection was observed in G1 at the chronic phase of infection, when compared with G3. Pigs in group 2 developed high antibody levels and were protected against clinic signs. T. gondii was not detected in two (G1) and three (G2) pigs by mouse bioassay. The results indicate partial protection in pigs vaccinated with a rhoptry vaccine.     

The comparative pathogenicity of four serovars of Actinobacillus (Haemophilus) pleuropneumoniae

The pathogenicity of 2 isolates of each of serovars 7, 3, 1 and 2 of Actinobacillus pleuropneumoniae was tested by intranasal inoculation into 60, 6-week-old large white pigs. Four dose rates varying from 0.27 to 560 x 10(6) organisms per pig with 10-fold serial dilutions were used. Surviving pigs were necropsied 7 days after inoculation. The proportion of pigs dying and developing gross lesions following infection was significantly greater for pigs given serotype 1 than for each of the other 3 serotypes, which did not differ significantly from each other. Twelve of 16 pigs given either of the 2 isolates of serovar 1 died after acute illness and 1 of 44 pigs given either of the 2 isolates each of serovars 7, 3 and 2 died. Pigs given serovar 1 showed high temperatures, severe respiratory distress, frothy haemorrhagic nasal discharge and weight loss. Lung lesions were produced in all 16 pigs given serovar 1, in 7 of 14 pigs given serovar 7, 7 of 14 pigs receiving serovar 3 and in 5 of 16 pigs given serovar 2. The lethal infections were characterised by a severe acute fibrinohaemorrhagic necrotising pleuropneumonia, whereas non-lethal cases had lung lesions ranging from necrotising purulent pleuropneumonia to abscessation. Significant differences between isolates in proportions of tissues culture positive for A. pleuropneumoniae for serovars 7 and 2, but not for serovars 3 and 1 suggested that isolates may vary in virulence within serovars, but more detailed studies are needed to clarify this point.     

Effect of challenge dose and route on porcine reproductive and respiratory syndrome virus (PRRSV) infection in young swine

Porcine reproductive and respiratory syndrome virus (PRRSV) is perceived to be highly infectious because of the rapid spread of the virus through populations of domestic swine throughout the world. However, no information has been published on the minimum infectious dose of PRRSV and the effect of challenge dose on clinical response. In this experiment, ten groups of pigs (n = 3 per group) were inoculated with one of five different quantities (10(1)-10(5) fluorescent foci units per millilitre) of PRRSV (isolate ISU-P) by either intramuscular or intranasal routes. Clinical signs and body temperature were monitored for 21 days. Serum was collected periodically throughout the study period to monitor the presence of virus in serum and the early immune response of pigs. A 2-mL inoculum containing 10(1) fluorescent foci units of virus per millilitre was found sufficient to achieve infection by either route. Time to onset of clinical signs was highly associated with challenge dose (P < 0.01), regardless of route of exposure. However, no dose- or route-dependent differences in the severity of clinical manifestation were observed. No significant differences in the time of onset or degree of humoural immune response to PRRSV infection were observed between different treatment groups. However, intramuscular exposure appeared to induce a more uniform antibody response compared to intranasal exposure. These results confirmed that PRRSV is highly infectious; a fact that should be taken into consideration when designing strategies for the prevention and control of PRRSV.     

Comparison of the efficacy of a subunit and a live streptomycin-dependent porcine pleuropneumonia vaccine

Objective To evaluate the efficacy of two new-generation porcine pleuropneumonia vaccines when challenged with Australian isolates of Actinobacillus pleuropneumoniae of serovars 1 and 15.
Design The Porcilis APP vaccine and an experimental streptomycin-dependent strain of A pleuropneumoniae were evaluated in a standardised pen trial. Each vaccine/challenge group consisted of 10 pigs.
Results With the serovar 1 challenge, the Porcilis APP vaccine and the live vaccine, compared with the control group, gave significant protection in terms of clinical signs, lung lesions, re-isolation scores and average daily gain (ADG) postchallenge. Only the Porcilis APP vaccine provided significant protection against mortality. In the serovar 15 challenged pigs, the only significant difference detected was that the Porcilis APP vaccinated pigs had a better postchallenge ADG than the controls. None of the Porcilis APP vaccinated pigs showed signs of depression postvaccination and none were euthanased after challenge with either serovar 1 or 15. The pigs vaccinated with the live vaccine showed obvious depression after each vaccination and a total of 3 pigs were euthanased after challenge (one with serovar 1 and two with serovar 15).
Conclusions Both of the vaccines provided significant protection against a severe challenge with serovar 1 A pleuropneumoniae. Neither vaccine was effective against a serovar 15 A pleuropneumoniae challenge. There was evidence that the Porcilis APP vaccine did provide some protection against the serovar 15 challenge because the ADG, after challenge of pigs given this vaccine, was greater than the control pigs.     

Mortality in swine herds endemically infected with Haemophilus pleuropneumoniae: effect of immunization with cross-reacting lipopolysaccharide core antigens of Escherichia coli

The benefit of increased immunity to cross-reacting lipopolysaccharide core antigens of gram-negative bacteria induced by vaccination with an Rc mutant of Escherichia coli 0111:B4 (strain J5) was evaluated in commercial swine herds endemically infected with Haemophilus pleuropneumoniae. Weanling pigs were vaccinated IM with E coli J5 (group 1) before the expected time of H pleuropneumoniae infection. Clinical signs, antibiotic treatment frequency, mortality, growth performance (days to market weight), and serologic responses of the pigs were monitored for approximately 5 months after vaccination. The results were compared with those of pigs vaccinated IM with a commercial H pleuropneumoniae bacterin (group 2) and with those of nonvaccinated control pigs of the same age (group 3). The treatment frequency and growth performance were similar in the 3 groups. However, vaccination with E coli J5 or with the H pleuropneumoniae bacterin lowered mortality, compared with mortality in the controls. Serum titers against E coli J5 increased after vaccination with the E coli J5 bacterin, but were not increased by vaccination with the H pleuropneumoniae. In contrast, serum titer to E coli J5 increased in all treatment groups as a result of H pleuropneumoniae infection or exposure. The protection against lethal H pleuropneumoniae infections in swine that was provided by vaccination with the E coli J5 and the H pleuropneumoniae bacterin appeared to be immunologically distinct on the basis of serologic analysis, indicating the possibility of different mechanisms of protection.     

Efficacy of an Intranasal Immunization with gEgC and gEgI Double-deletion Mutants of Aujeszky’s Disease Virus in Maternally Immune Pigs and the Effects of a Successive Intramuscular Booster with Commercial Vaccines

In this study, an intranasal immunization strategy was set up in maternally immune pigs in order to protect them not only clinically but also virologically. Two genetically engineered Aujeszky’s disease virus (ADV) strains, Kaplan gE^?gI^? and Kaplan gE^?gC^?, were used for intranasal immunization. Both strains were safe for 4-week-old pigs. A single intranasal inoculation of 10^6.0 TCID₅₀ of Kaplan gE^?gI^? and Kaplan gE^?gC^? at 4 weeks of age in the presence of moderate titres of maternally derived antibodies (SN titres: 12–16) reduced the amount of weight loss, fever and virus excretion upon challenge 6 weeks later. In a second experiment, the effect of an additional intramuscular booster with three different commercial vaccines (containing attenuated Bartha or NIA₃-783 or inactivated Phylaxia; all suspended in an oil-in-water emulsion) at 10 weeks of age was evaluated. One month after the last intramuscular booster, between five and seven pigs from each group were selected for challenge. All intranasally/intramuscularly immunized pigs showed a significantly better clinical and virological protection after challenge than the single intranasally immunized pigs. In the double immunized group, the protection was better when Kaplan gE^?gC^? was used for the intranasal priming (only two of 14 pigs excreted virus with a duration of 4 days) than when Kaplan gE^?gI^? was used (13 of 18 pigs excreted virus with a duration ranging from 1 to 4 days). The virological protection was not influenced by the type of vaccine used for booster vaccination. Because the intranasal/intramuscular immunization approach is very compatible with current pig movements on farms and pigs with moderate levels of maternally derived antibodies can effectively be immunized, it can be considered as a good alternative to intramuscular/intramuscular vaccinations especially in regions with a high ADV prevalence.     

Prevention of pleuropneumonia in pigs by in-feed medication with sulphadimethoxine and sulphamethoxazole in combination with trimethoprim

The prophylactic effect of in-feed medication of conventional pigs with sulphadimethoxine (SDM), sulphamethoxazole (SMX), and trimethoprim (TMP) was tested by using an Actinobacillus pleuropneumoniae infection model. In each of five experiments, six pigs were given medicated feed twice daily and three pigs received antibiotic-free feed and served as positive (unmedicated, infected) controls. The following drugs or drug combinations were tested (in mg per kg feed): 500 SDM + 100 TMP, 500 SMX + 100 TMP, 125 SMX + 25 TMP, 125 SMX (alone) and 25 TMP (alone). After six days of feed medication, all animals were endobronchially inoculated with A. pleuropneumoniae in a dose of 1-3.10(4) colony-forming units (CFU). The response to the challenge in all control pigs was characterized by fever, lethargy, anorexia, reduced water consumption, and laboured breathing. At autopsy all controls manifested a fibrinous haemorrhagic pleuropneumonia. In-feed medication with 500 SDM + 100 TMP, 500 SMX + 100 TMP as well as 125 SMX + 25 TMP resulted in an effective protection against the challenge in all treated animals. After consumption of feed medicated with 125 mg per kg SMX or 25 mg per kg TMP, pleuropneumonia was evident in all challenged pigs. The results of this study indicate an in vivo potentiation of SMX and TMP in pigs against this respiratory tract pathogen.     

Identification and detection of Actinobacillus pleuropneumoniae by PCR based on the gene apxIVA

The apxIVA gene, a recently discovered RTX determinant of Actinobacillus pleuropneumoniae, was shown to be species-specific. DNA hybridization experiments using probes for various regions of apxIVA revealed that the 3'-terminus of this gene was present in all 14 serotypes of A. pleuropneumoniae but absent from phylogenetically related species. A primer pair spanning this region specifically amplified a 422bp fragment in PCR experiments with DNA from the reference strains of the 14 serotypes and 194 field strains isolated from various geographic locations worldwide. DNA sequence analysis of PCR products derived from all serotypes were identical except in serotypes 3, 8, and 10, which showed minor differences. The PCR did not amplify any product when DNA from 17 different bacterial species closely related to A. pleuropneumoniae was used as template. In addition, the PCR was negative with DNA of several Actinobacillus sp. which were initially characterized as A. pleuropneumoniae using routine phenotypic and serological analyses but which were subsequently shown by 16S rRNA sequence analysis to belong to yet undefined Actinobacillus species. The sensitivity of the PCR was determined to be 10pg of A. pleuropneumoniae DNA. A set of nested primers amplified a 377bp fragment specifically with A. pleuropneumoniae DNA. DNA titration experiments using the flanking and nested primer pairs showed an improved level of sensitivity to approximately 10fg of genomic DNA. The nested PCR was used to monitor the spread of A. pleuropneumoniae in pigs experimentally infected with a virulent serotype 1 strain and housed in a controlled environment facility. A. pleuropneumoniae DNA could be detected by nested PCR in nasal swab samples of infected pigs receiving either a high dose (5x10(5)) or a low dose (1x10(4)) challenge and in unchallenged cohorts that were contact-infected by the inoculated animals. Furthermore, PCR confirmed the presence of A. pleuropneumoniae in 16/17 homogenates from necrotic lung lesions, while the bacterium was successfully recovered from 13 of these lesions by culture.