Evaluation of different antigens in the complement-fixation test for diagnosis of Haemophilus pleuropneumoniae (parahaemolyticus) infections in swine

The identification of new serotypes of Haemophilus pleuropneumoniae (parahaemolyticus) and the frequency of pleural adhesions due to contagious pleuropneumonia in many fattening swine herds have prompted the study of the complement-fixation (CF) test as a diagnostic tool for use in swine. Whole cell antigens, mixed antigens, autoclaved antigens, and phenol-water-extracted antigens derived from different serotypes were prepared and tested with immunized-swine sera by the CF test. Mixed antigen consisting of whole cells from all known serotypes was the best screening antigen for routine use. This antigen gave positive titers with all sera in which a positive reaction against the separate serotype antigen was registered. The most highly serotype-specific reactions were obtained with antigens prepared by phenol-water extractions of whole cells. When whole-cell antigens were used in the CF test, antibodies to superficial serotype-specific and common species-specific antigens could be detected.     

Serological and immunological studies of pleuropneumonia of swine caused by Haemophilus parahaemolyticus

The development of circulating antibodies for H. parahaemolyticus was studied in experimentally infected SPF pigs and in-contact SPF pigs. Blood serum titers were determined by a modified complement fixation test with normal SPF swine serum as a source of supplementary complement factor, and by an indirect haemagglutination test.CF and IHA titers became positive within the first 2 weeks following exposure to H. parahaemolyticus, and reached peak values after 2 to 7 weeks (Figs. 1 to 3). The exposed pigs proved immune, in that they showed no clinical symptoms on challenge after resp. 6, 9 and 11 weeks.While distinct titers were thus obtained with both tests in SPF swine experimentally exposed to H. parahaemolyticus, the CF test proved more specific than the IHA test when the 2 tests were compared in a field outbreak of polyserositis (Glässers disease) caused by H. parasuis. The CF test would therefore seem to be preferable to the IHA test in field diagnostic work (Table 1).A noticeable finding was that challenge did not elicit an anamnestic antibody response in any of the immune pigs (Figs. 1 to 3). This fact together with negative bacteriological findings in the animals in question would seem to suggest that the challenge dose was unable to establish a permanent infection in the respiratory tract of the immune pigs.     

Evaluation of the cold complement fixation test for diagnosis of ovine brucellosis

SUMMARY A cold complement fixation test for ovine brucellosis in which fixation of complement occurs for 16 h at 4°C was compared with the conventional warm complement fixation test in which fixation takes place for 40 minutes at 37°C. The cold complement fixation test detected experimentally infected animals up to one week sooner than the warm complement fixation test and the titres were approximately one twofold dilution higher. Similar results were obtained when the 2 tests were compared using 11,922 serum samples collected from 700 ram flocks over a 2-year period. False positive reactions were observed in less than 0.1% of cases.     

Improved protection of swine from pleuropneumonia by vaccination with proteinase K-treated outer membrane of Actinobacillus (Haemophilus) pleuropneumoniae

The immunogenic and protective potentials of an outer membrane-enriched fraction (OM) from a serotype 5 strain of Actinobacillus (Haemophilus) pleuropneumoniae (APP) and the same OM degraded with proteinase K or periodate were evaluated in swine. Groups of pigs were vaccinated with two doses of OM, proteinase K-treated OM (P-OM), periodate-treated OM (PI-OM), or placebo vaccine and challenged intranasally with the homologous strain of APP. Results from triplicate experiments indicated that proteinase K treatment of OM resulted in an improved efficacy. This improved efficacy of P-OM vaccine over untreated OM vaccine was evidenced not only by less severe lung lesions in P-OM vaccinated pigs but also by significant reduction (P less than 0.05) in the number of P-OM vaccinated pigs which developed lung lesions upon challenge with APP. Assessment of sera from vaccinated animals by immunoblotting, complement fixation test, or ELISA indicated that the immunogenicity of some but not all protein or carbohydrate components were reduced (or eliminated) by proteinase K and periodate treatments respectively.     

Improving the specificity and yield of the contagious bovine pleuropneumonia complement fixation test antigen.

Several methods for increasing yield and specificity of the contagious bovine pleuropneumonia complement fixation test antigen which is derived from Mycoplasma mycoides subsp mycoides, strain V5, were examined. Changes in culture conditions that increased the cell mass per unit volume of culture did not result in comparable increase in antigen yield.Sixteen to 60-day-old cultures yielded more boiled cell antigen than younger cultures. Some antigen in young cultures appeared to be masked, probably by galactan. The yield of antigen extracted from boiled cells with ethonol was as much for two to eight-day-old cultures as for older cultures. The ethanol extract antigen was less reactive with false positive bovine sera than standard boiled antigen while reactivity with anti Mycoplasma mycoides sera was similar to that of standard antigen. Adsorbed gamma globulin was not detected in either boiled or ethanol extract antigen. The data suggest that several complement fixing antigens were present in antigens derived from older cultures.     

Experiences with vaccination against Haemophilus pleuropneumonia of swine

In a Haemophilus pleuropneumonia problem herd with piglet production and fattening, sows and weaned pigs were vaccinated with a Danish vaccine (Pleurinord). Due to consequent vaccination of the sows the health of the piglets could be improved decisively, resulting in an increased number of raised piglets per sow and year from 16.8 to 20.3. In the vaccinated fattening pigs compared to the unvaccinated ones the following effects were observed: markedly reduced expenses for medication and a markedly reduced frequency of characteristic lesions in the respiratory tract; the improvement of the daily weight gains and the reduction of losses remained behind the expectations. Decisive causes for this were respiratory and enteric diseases unrelated to Haemophilus, which were favoured by serious mismanagement and inadequacies in the feeding regimen and barn climate. On the example of the vaccinated herd it is shown how important the analysis of a multifactorial disease situation is, in order to be able to objectify better or at all the influence of a vaccination program under field conditions. Within a concept of prevention and control of Haemophilus pleuropneumonia the vaccination is a helpful part.     

Prevalence of pig herds affected by pleuropneumonia associated with Haemophilus pleuropneumoniae in eastern England

A survey for the macroscopic lesions indicative of pneumonic infection in the pig with Haemophilus pleuropneumoniae was made in an abattoir in eastern England. A total of 78 herds located in 11 counties of eastern or central England were seen between December 1982 and August 1983. Lesions were noted in the batches submitted by 44 (56 per cent) of the 78 herds. A further 16 herds (21 per cent) submitted batches containing pigs affected by pleurisy principally of the caudal lobes but without the pneumonic lesions. Lesions suggestive of enzootic pneumonia were also seen in 61 herds (78 per cent). Circumstances restricted corroborative bacteriological examinations to 53 and serological examinations to 33 herds. Strains of H pleuropneumoniae (predominantly serotype 3 but also serotype 2) were isolated from 26 herds. These comprised 22 out of 42 (51 per cent) of those where typically affected plucks, or plucks with caudal lobe pleurisy, were encountered, and four out of 11 (36 per cent) in which there was either no observable thoracic disease or enzootic pneumonia only. Complement fixing antibodies to serotype 3 or 2 antigens occurred in 26 out of 33 herds (79 per cent). These comprised 25 (83 per cent) of 30 herds with batches exhibiting either typical pulmonary lesions and, or, caudal lobe pleurisy and one of three herds without such lesions. Collectively these data indicate that herds containing pigs with pleuropneumonia are common at least in the more easterly parts of England and that H pleuropneumoniae, usually but not always associated with disease, is also widespread.     

Role of haemophilus pleuropneumoniae lipopolysaccharide endotoxin in the pathogenesis of porcine Haemophilus pleuropneumonia

Intact Haemophilus pleuropneumoniae cells (strain Shope 1, serotype 1), highly purified lipopolysaccharide (LPS) obtained from this strain of H pleuropneumoniae, as well as from Escherichia coli O111:B4, filter-sterilized H pleuropneumoniae cell-free culture supernatant fluid, and heat-inactivated supernatant fluid were given intranasally to CF1 mice and intratracheally to pigs. Pulmonary lesions induced by H pleuropneumoniae in mice were similar to those induced by H pleuropneumoniae in pigs. Histologically, lungs of mice and pigs killed 1 or 2 days after inoculation with 200 micrograms of highly purified H pleuropneumoniae LPS had lesions similar to one another and were similar to those in mice and pigs given intact H pleuropneumoniae, except that little or no necrosis or hemorrhage was observed. In mice killed 1 or 2 days after inoculation of 200 micrograms of E coli O111:B4 LPS, pulmonary lesions were similar to those in mice given H pleuropneumoniae LPS. Pulmonary lesions in mice given cell-free culture supernatant fluid obtained from a midlog-phase growth culture of H pleuropneumoniae cultivated in a chemically defined medium were severe and consisted of neutrophil infiltration and extensive necrosis. In mice, the heat-inactivated supernatant fluid produced mild lesions that consisted of foci of neutrophil aggregation and no necrosis. Extensive necrosis observed in lesions caused by cell-free culture supernatant fluid could be attributed to the action of a heat-labile component, perhaps by the extracellular heat-labile hemolysin produced by H pleuropneumoniae cultivated in chemically defined medium. A LPS endotoxin and a heat-labile factor may be involved in the pulmonary lesion development in the acute phase of porcine Haemophilus pleuropneumonia.     

Passive haemagglutination and complement fixation as diagnostic tests for contagious caprine pleuropneumonia caused by the F-38 strain of mycoplasma

The protective effects of high levels of O115 antibodies and K88 antibodies in the sera of hyperimmunized rabbits on the intestinal loop dilation elicited by live cultures of Escherichia coli O115: K88:H39 and its enterotoxins were examined. K88 antibodies did not prevent fluid accumulation when live E coli O115:K88:H39 or its enterotoxins were injected into ligated loops in rabbits actively immunised with K12:K88 or when K88 antiserum was mixed directly with the challenge shortly before injection into the loops. In two of six rabbits immunised with heat-killed O115 E coli the live strain failed to elicit a fluid response and in two others the response was reduced. In all cases the O115 antiserum inhibited the fluid response evoked by the live, homologous bacteria, but not by enterotoxin preparations, when the serum was mixed directly with the challenge before injection.     

Evaluation of a selective medium for isolation of Haemophilus pleuropneumoniae.

Crystal violet, lincomycin, spectinomycin and bacitracin were evaluated as selective agents in media for isolation of Haemophilus pleuropneumoniae. No single antimicrobial agent or combination of two or more inhibited all non-Haemophilus strains (Escherichia coli, Pasteurella haemolytica, Pasteurella multocida, Streptococcus faecalis, Streptococcus equisimilis and Staphylococcus aureus) without marked suppression of 16 H. pleuropneumoniae strains. A medium containing 1 micrograms/mL of crystal violet, 1 microgram/mL of lincomycin, 8 micrograms/mL of spectinomycin and 128 micrograms/mL of bacitracin inhibited one E. coli strain and the Gram-positive strains while H. pleuropneumoniae strains were suppressed to a minor degree only. Haemophilus pleuropneumoniae was isolated on the selective medium on three occasions from the nose or pharynx of two out of eight experimentally inoculated pigs. Haemophilus pleuropneumoniae was recovered from the nose of only two pigs at necropsy and from tonsil of one, whereas the lower airways in most pigs and the lung lesions in all pigs were positive. There was no advantage to using the selective medium for the recovery of H. pleuropneumoniae at necropsy from these eight experimentally infected pigs, probably because other bacteria were absent or present in very low numbers in the tissues with H. pleuropneumoniae. The isolation rate on selective medium was higher than the rate on non-selective medium (p less than or equal to 0.1; chi 2 test) when the airways of slaughtered pigs were cultured. This was likely due to a high degree of contamination. Dry swabs placed in tryptone yeast extract with nicotinamide-adenine-dinucleotide gave a significantly higher recovery rate than commercial Culturette swabs in modified Stuart's transport medium.(ABSTRACT TRUNCATED AT 250 WORDS)     

Quantitative morphology of peracute pulmonary lesions in swine induced by Haemophilus pleuropneumoniae

Twenty-seven six-week-old cesarean-derived, colostrum-deprived pigs were inoculated intratracheally with an isolate of Haemophilus pleuropneumoniae serotype 5 (principles) of high virulence (I-200) or low virulence (B-8) or phosphate buffered saline (controls). Pigs given I-200 had severe serofibrinous pleuropneumonia at three hours after inoculation; two of three pigs were dead by 24 hours after inoculation. Interalveolar septa in the caudal lung lobes were 41% thicker than septa from control pigs at three hours after inoculation and 79% thicker by 24 hours after inoculation. Interalveolar septal capillaries in caudal lung lobes were 10.2% larger than control capillaries at three hours after inoculation and 25.6% larger by 24 hours after inoculation. Interalveolar septal capillary platelet volume was greater than the platelet volume of controls; 70% of these platelets were aggregated. There was severe diffuse alveolar, interalveolar septal, and interlobular septal edema at three hours after inoculation with fibrin, neutrophils, and macrophages present in later samples. Thirty-three percent of the lung parenchyma was necrotic at 24 hours after inoculation. Endothelial cell degeneration was generally mild, but necrotic in regions of pulmonary infarction. Pigs inoculated with the B-8 isolate did not develop marked macroscopic lesions at any sampling time. Interalveolar septa were 18% thicker than controls nine hours after inoculation and 5% thicker at six and 24 hours after inoculation. Capillary platelet volume was greatest at nine hours after inoculation with 50% of these platelets aggregated; 30% of the platelet volume was aggregated at the 24-hour sample period. Moderate diffuse pulmonary and interlobular septal edema was present at three, six, and nine hours after inoculation, but absent 24 hours after inoculation. Intravascular macrophages were present in the six, nine, and 24-hour lung samples in both B-8 and I-200 inoculated pigs. These cells were adherent to interalveolar septal capillary endothelial cells and contained phagocytized cellular debris and fibrin. These results indicate the early effects of H. pleuropneumoniae infection involve macrophage and platelet activation, and a marked increase in interalveolar septal capillary permeability.     

Evaluation of enzyme-linked immunosorbent assay in comparison with complement fixation test for the diagnosis of subclinical paratuberculosis in cattle.

An enzyme-linked immunosorbent assay (ELISA) was evaluated and compared in parallel with the standard complement fixation test (CFT) for the diagnosis of bovine subclinical paratuberculosis. Bovine sera preabsorbed with the mixture of Mycobacterium phlei and kaolin suspension were assayed for antibody activities to the crude protoplasmic antigen of Mycobacterium paratuberculosis in the ELISA. ELISA antibody titer was expressed as ELISA antibody index (EAI) value: EAI = (At-An)/(Ap-An), where At, Ap and An are the absorbance values of a 1:200 dilution of unknown test sera, a 1:400 dilution of positive control serum, and a 1:200 dilution of negative control serum. An EAI of 0.6 or greater was established as a reasonable cutoff point for a positive antibody titer by ELISA. Of the 156 sera from cattle with subclinical M. paratuberculosis-infection, 106 (67.9%) were positive by ELISA and 41 (26.3%) by CFT. Of the 3,880 sera from cattle in the herds which had no history or evidence of paratuberculosis, 3,875 (99.9%) were negative by ELISA, and 3,787 (97.6%) by CFT. Positive ELISA titers were detectable 1 to 5 months earlier than positive CFT titers in experimentally infected cattle, and 7 to 10 months earlier in naturally infected cattle. These results indicate that the ELISA should replace the CFT as the routine test of choice for the diagnosis of bovine paratuberculosis.     

Serodiagnosis of swine pleuropneumonia due to Actinobacillus pleuropneumoniae serotypes 7 and 4 using long-chain lipopolysaccharides.

A saline boiled extract (SBE), capsular polysaccharides (CPS) and long-chain lipopolysaccharides (LC-LPS) of Actinobacillus pleuropneumoniae serotype 7 have been evaluated in ELISA for the serodiagnosis of swine pleuropneumonia caused by this serotype. Mean optical densities (ODs) obtained with the 3 antigens using sera from negative herds as well as from animals experimentally and naturally exposed to A. pleuropneumoniae serotypes 7 or 4 were not statistically different. The positive ELISA reaction with anti-serotype 4 sera was unexpected with the CPS, which are supposed to be serotype-specific; LPS traces present in the CPS appeared to be responsible for this reaction. In addition, sera from animals exposed to A. pleuropneumoniae serotypes 5 or 10 presented cross-reactions with the SBE and the CPS, but not with the LC-LPS. Cross-reactions were mainly due to rough LPS, as shown by immunoblotting. The LC-LPS is easily obtainable and can be used for the detection of antibodies in animals infected with A. pleuropneumoniae serotypes 7 and 4.     

Survey of Actinobacillus (Haemophilus) pleuropneumoniae infection in swine by different methods

Lung and serum samples from pigs that died or were emergency-slaughtered in a pooled, conventional fattening herd were examined to survey Actinobacillus pleuro-pneumoniae infection and to compare the sensitivity of different testing methods. A total of 110 lungs were used for cultural isolation of the agent and direct immunofluorescence (IF) of impression smears. Boiled lung suspensions were tested by coagglutination (Co-A) and agar gel precipitation (AGP). Eighty-seven sera were tested along with lung samples from the same pigs. The lungs yielded a varied bacterial flora most often containing Pasteurella multocida and less frequently Actinomyces (Corynebacterium) pyogenes, E. coli and Salmonella. A. pleuropneumoniae was isolated from 30 lungs: from 22 lungs it grew out in pure culture, from 7 as mixed culture with P. multocida and from 1 as mixed culture with A. pyogenes. The number of positive samples obtained by the different methods was as follows: coagglutination test (with boiled lung suspensions): 63 (57.3%); immunofluorescence: 43 (39.2%); AGP test (with serum): 31 (35.6%); AFP test (with boiled lung suspension): 25 (22.7%). A total of 23 samples (20.7%) were negative by all serological tests and by cultural isolation. Most samples gave positive results by two or more tests while 26 samples only by one test (most often, on 13 occasions, by the Co-A test). The Co-A test detected antigenic components of serotypes that have not been isolated in Hungary so far. This indicates that it is not enough to test one strain from a given lung sample: several colonies must be cultured and serotyped.(ABSTRACT TRUNCATED AT 250 WORDS)