Virulence of Bordetella bronchiseptica in the porcine respiratory tract.

The virulence of Bordetella bronchiseptica in gnotobiotic piglets was studied by intranasal infection with 11 cultures derived from eight strains isolated from pigs (4), dogs (2), a human subject and a monkey. Six of the cultures contained organisms in phase I and five contained phenotypically different phase-III or -IV organisms. Of the phase-III and -IV cultures, four were derived from strains that had been isolated in phase I. Colonisation of the nasal cavity was investigated by counting bacteria in nasal swabs and washings. The toxigenicity of cell extracts from each strain and variant was determined by tests of lethality in mice or of cytopathogenicity in cell cultures. The results showed that two phase-I cultures from pigs colonised the nasal cavity and respiratory tract of gnotobiotic piglets better than did four phase-I cultures from other species. Phase-I organisms invariably produced capsules, fimbriae and mannose-resistant haemagglutination of guinea-pig erythrocytes. Four of five cultures in phases III and IV consisted of organisms that did not produce capsules, fimbriae or haemagglutination and colonised the nasal cavity poorly. Phase variation from I to III occurred in culture and in vivo, but variation from III to I occurred in vivo only and was accompanied by enhanced colonisation. Gnotobiotic piglets infected with porcine phase-I organisms exhibited atrophy of the nasal turbinate bones after 28 days; these organisms produced significantly more toxin than did bacteria in phase I from other species, or those in phases III and IV.(ABSTRACT TRUNCATED AT 250 WORDS)

Virulence of Bordetella bronchiseptica from pigs with or without atrophic rhinitis.

The virulence of 17 isolates of Bordetella bronchiseptica from 13 pig herds was compared by intranasal infection of gnotobiotic piglets and LD50 tests on mice. Of 59 piglets given 8.1-10-5 log 10 colony-forming units (cfu) of isolates from two herds with atrophic rhinitis (AR isolates) or isolates from six unaffected herds (non-AR isolates), 16 died of acute pneumonia; the survivors developed non-progressive turbinate hypoplasia and chronic pneumonia. Infection of 11 piglets with c. 3.0 log to cfu of three AR isolates or three non-AR isolates caused turbinate hypoplasia, but only slight pneumonia and no deaths. There were no significant differences between the virulence of AR and non-AR isolates in piglets. In LD50 tests in mice, there were no significant differences between the results from six AR isolates and six non-AR isolates, or from toxin prepared from two AR isolates and one non-AR isolates was fairly uniform, and that other factors must be responsible for the occurrence of progressive lesions of atrophic rhinitis in some but not all infected herds.

Adhesion of enteropathogenic Escherichia coli to pig intestinal brush borders: the existence of two pig phenotypes.

An in-vitro test that demonstrates adhesion of K88-positive Escherichia coli to brush borders prepared from the small intestine of the pig is described. K88-positive E. coli adhered to the brush borders from some pigs ("positive" pigs) but not others ("negative" pigs). The sires of the pigs tested could be placed into two groups, namely, those that sired only "positive" piglets, and those that sired a mixture of "positive" and "negative" piglets. The incidence of the two phenotypes in litters indicated that "positive" and "negative" piglets arose as a result of simple Mendelian inheritance. It is suggested that "negative" pigs could be bred and that they might have a natural resistance to neonatal infection with K88-positive E. coli.

Colonisation by Pasteurella multocida in atrophic rhinitis of pigs and immunity to the osteolytic toxin.

Gnotobiotic pig antisera to purified toxoid from a capsule type A or D strain of Pasteurella multocida contained large quantities of antitoxin but comparatively little antibody to a crude lysate of P. multocida. These sera given intraperitoneally to further pigs were almost completely protective against turbinate atrophy after intranasal inoculation of dilute acetic acid and infection with type D toxigenic P. multocida. In contrast, antisera to a crude lysate or bacterin of toxigenic P. multocida which contained large titres of antibody to P. multocida lysate, but no detectable antitoxin, were not protective. Colonisation by toxigenic P. multocida was significantly reduced in protected pigs and was similar to colonisation by nontoxigenic P. multocida in pigs untreated or treated with dilute acetic acid. These results indicated (1) that antitoxin was protective and cross protective between toxins from different capsule types; and (2) that the toxin was the main colonisation factor produced by toxigenic bacteria in the acetic acid model of infection and that immunity to it did not eliminate infection.

The pathogenesis of turbinate atrophy in pigs caused by Bordetella bronchiseptica.

The pathogenicity of 3 strains of Bordetella bronchiseptica designated B58, PV6 and B65 was compared by intranasal infection of gnotobiotic piglets. Strain B58 was a phase 1 isolate that produced haemolysin, an adhesin for calf erythrocytes, adenylate cyclase, mouse lethal factor, dermonecrotic factor and cytotoxin. B65 was a variant of B58 that produced no detectable haemolysin, adhesin or adenylate cyclase and 10-fold smaller amounts than B58 of mouse lethal factor, dermonecrotic factor and cytotoxin. Strain PV6 was a phase 1 isolate that produced only haemolysin, adhesin and adenylate cyclase. After nasal infection of gnotobiotic pigs, 10(3.2)-10(6.2) colony forming units ml-1 (cfu ml-1) of strains B58 and PV6 were cultured from nasal washings during the next 25 days. In contrast, only 10(1.0)-10(2.8) cfu ml-1 of strain B65 were recovered during the same period. Only pigs infected with strain B58 had turbinate atrophy when they were slaughtered 25 days after infection and neutralising antibody to cytotoxin was detected only in these pigs. These results suggested that the cytotoxin, which may be the same as the mouse lethal and dermonecrotic factors, was the cause of turbinate atrophy. They also support the view that the adhesin for calf erythrocytes is required for colonisation of the nasal cavity in vivo.

Virulence of Pasteurella multocida in atrophic rhinitis of gnotobiotic pigs infected with Bordetella bronchiseptica.

Bordetella bronchiseptica and Pasteurella multocida have been impLicated in the aetiology of atrophic rhinitis of pigs but the precise cause and pathogenesis of field outbreaks have still to be clarified. The virulence of 11 strains of P multocida was investigated by intraperitoneal injection of culture filtrates in BALB/C mice, or by infection of gnotobiotic piglets given B bronchiseptica five days previously. Three of four type D strains of P multocida were lethal for mice and caused severe turbinate lesions and shortening of the snout with B bronchiseptica in gnotobiotic pigs; large numbers of P multocida and B bronchiseptica persisted for 64 days in the nasal cavity of these pigs. The fourth strain caused moderately severe turbinate lesions in gnotobiotic pigs infected with B bronchiseptica; small numbers of P multocida were found in these pigs and the lesions were attributed mainly to B bronchiseptica. Filtrates from seven strains of P multocida (four type A and three type D) were not lethal for mice and these strains with B bronchiseptica caused moderately severe turbinate lesions in gnotobiotic pigs; five of them colonised the nasal cavity reasonably well for 35 days but the lesions were attributed mainly to B bronchiseptica. The turbinate bones had regenerated by 64 days in pigs given type A strains of P multocida whereas the lesions persisted in pigs given type D strains. Antibodies to P multocida were detected in sera from infected gnotobiotic pigs by acid agglutination but not by indirect haemagglutination tests; neutralising activity to the mouse lethal toxin was detected in serum from one of five piglets at 64 days. The lethal toxin was inactivated at 56 degrees C for 30 minutes, by incubation with protease K for two hours and by 0.2 per cent formalin for 18 hours at 37 degrees C but not by trypsin; it was precipitated by 30 to 40 per cent saturation with ammonium sulphate and remained in the supernatant after centrifugation at 150,000 g. It was concluded that infection with virulent, type D strains of P multocida and B bronchiseptica could explain severe outbreaks of atrophic rhinitis; large numbers of both organisms persisted in the nasal cavity of gnotobiotic pigs with severe lesions; and that a soluble, heat-labile toxin may be an important virulence determinant in the type D strains of P multocida that cause severe atrophic rhinitis.

Comparison of methods for the sampling and isolation of toxigenic Pasteurella multocida from the nasal cavity of pigs.

The efficacy of detecting toxigenic Pasteurella multocida from nasal swabs of slaughtered and live pigs was assessed. The isolation of toxigenic P multocida from nasal cavities of slaughtered bacon pigs from two herds with atrophic rhinitis was reduced by immersion in the hot water tank by 25 per cent and 75 per cent. Individual sows from one of the infected herds were repeatedly swabbed to find the best method of isolating toxigenic P multocida. Toxigenic P multocida were isolated from 50 per cent of cotton swabs inoculated on to selective medium the same day. After 24 hours in the post, 45 per cent of cotton swabs placed in transport medium, 38 per cent of alginate swabs dissolved in transport medium and inoculated into mice, and 36 per cent of the dissolved swabs inoculated directly on to selective medium yielded toxigenic P multocida. These bacteria were isolated from only 25 per cent of cotton swabs held in transport medium at 10 degrees C for 48 hours to simulate prolonged postage times; from slaughtered pigs a similar reduction in isolation was seen with swabs kept for 24 or 48 hours. The reduced isolation caused by a delay before culture was associated with an overgrowth of other flora. The development of this flora was prevented by storage of swabs at 4 degrees C in the laboratory or by the use of cool boxes for postage.

Interactions between Bordetella bronchiseptica and toxigenic Pasteurella multocida in atrophic rhinitis of pigs.

Three strains of Bordetella bronchiseptica were compared for their ability to assist colonisation of the nasal cavity of gnotobiotic pigs by toxigenic Pasteurella multocida. Toxigenic P multocida (counted in nasal washings) colonised the cavity in large numbers in pigs previously infected with a cytotoxic phase I strain of B bronchiseptica (B58), whereas it colonised only in small numbers in those previously infected with B65, a phenotypic phase III variant of B58. Toxigenic P multocida colonised pigs infected with a non-cytotoxic phase I strain of B bronchiseptica (PV6) in fewer numbers than were seen in pigs infected with the cytotoxic phase I strain but in greater numbers than in pigs infected with the phase III strain. The turbinates of pigs infected with the cytotoxic phase I strain of B bronchiseptica and toxigenic P multocida were most severely affected and those in pigs infected with the non-cytotoxic phase I strain and toxigenic P multocida were moderately reduced in size. The turbinates of pigs infected with the phase III strain and toxigenic P multocida were slightly reduced in size except for one piglet whose turbinates were severely affected. Pigs infected with the non-cytotoxic phase I strain of B bronchiseptica alone showed no signs of atrophy and their turbinates were used to calculate reductions (per cent) in those infected with P multocida. The reduction (per cent) in size of turbinates and total numbers of P multocida isolated from the nasal washings of each pig were linearly related.(ABSTRACT TRUNCATED AT 250 WORDS)

Quantitative observations on Bordetella bronchiseptica infection in atrophic rhinitis of pigs.

Clinical atrophic rhinitis in seven pig herds could not be associated with the infection rate or higher numbers of B bronchiseptica in nasal swabs when compared with unaffected herds. B bronchiseptica isolates from herds with atrophic rhinitis and receiving sulphonamide medication were resistant to sulphonamides in vitro and there was a beneficial clinical response after changing to oxytetracycline medication. In an unaffected herd three piglets naturally infected with B bronchiseptica but possessing low levels of passive antibody showed marked turbinate hypoplasia when killed at seven weeks, the lesions had resolved in four of six litter mates by 21 weeks and did not occur in another litter of nine piglets which had a high level of passive antibody. The results indicate that although B bronchiseptica can produce non-progressive turbinate changes in pigs that have inadequate antibody protection, the relationship between these lesions and the chronic progressive field disease needs further investigation.

Cell culture assay for toxigenic Pasteurella multocida from atrophic rhinitis of pigs.

A toxin produced by strains of Pasteurella multocida isolated from pigs with atrophic rhinitis caused a cytopathic effect in cell cultures derived from embryonic bovine lung. The toxin was produced during the late logarithmic phase of bacterial growth and inactivated by heating for 30 minutes at 56 degrees C. The cell culture assay was reproducible and 10(3) to 10(4) times more sensitive than a lethal assay in BALB/c mice. There was complete agreement between results in the two tests with 76 isolates of P multocida. Neutralising activity was demonstrated in both assays with sera from infected gnotobiotic piglets. It was concluded that embryonic bovine lung cell cultures provided a sensitive in vitro test for the differentiation of toxigenic from non toxigenic isolates of P multocida. The assay could be used in diagnostic laboratories and for characterisation of the toxin.

Detection and distribution of toxigenic Pasteurella multocida in pig herds with different degrees of atrophic rhinitis.

Four herds of pigs were selected which had different degrees of clinical atrophic rhinitis and used different specific counter-measures. In two of them, the clinical signs occurred spasmodically and were slight. The sows, suckling pigs and growing pigs in all the herds were sampled for toxigenic Pasteurella multocida. In one of the slightly affected herds (herd D), the weaners were moved to a second farm for finishing. No toxigenic P multocida were found at the breeding farm, but 50 per cent of the large growing pigs were positive. It seemed that the organism had entered only at the finishing farm and that the mild clinical signs were due to the infection starting in older pigs than usual. In the second mildly affected herd, 47 per cent of the sows and 42 per cent of the growers were infected. Three toxigenic isolates from this herd produced as severe turbinate damage experimentally in specific-pathogen-free pigs as a stock pathogenic strain. Except in herd D, toxigenic P multocida were found in all the age groups of pigs sampled. However, the pattern of distribution of the organism within the herds was not obviously correlated with the severity of the disease. In a fifth herd there were obvious cases of clinical atrophic rhinitis, with marked turbinate atrophy, from which toxi-genic P multocida were recovered in abundance. Subsequently, the clinical disease disappeared and, despite extensive and repeated sampling, the organism was not found again.

Screening pig herds for toxigenic Pasteurella multocida and turbinate damage in a health scheme for atrophic rhinitis.

The Pig Health Control Association launched a health scheme for atrophic rhinitis in 1978. For several years pig herds were monitored by scoring the degree of turbinate damage and by clinical inspections. When laboratory facilities became available for detecting toxigenic Pasteurella multocida, nasal swabs were taken from pigs in Association herds during 1988 and 1989 to determine whether the organism was present. Sows were screened routinely and in the worst affected herds, sucklers and weaners were also swabbed. In 12 of 19 herds with consistently low snout scores toxigenic P multocida were not isolated, and in 15 herds which developed higher snout scores with time toxigenic P multocida were also not found. Eleven herds had never been listed by the Association, either because their snout scores were consistently high or because they had received importations of stock from herds with high snout scores; of six of these herds with the most persistently high snout scores five showed varying degrees of the clinical signs of atrophic rhinitis, but none of the six showed evidence of infection with toxigenic P multocida, and the organism was not found in the other five herds in the group. There seems to be an overlap between the clinical and gross pathological signs of atrophic rhinitis seen in some herds not infected with toxigenic P multocida and the mild and spasmodic signs of atrophic rhinitis seen in some herds which are substantially infected with the organism.

Epidemiological study of Pasteurella multocida and Bordetella bronchiseptica in atrophic rhinitis.

An epidemiological study of atrophic rhinitis was carried out in four pig herds. Observations were made of (i) infection with Bordetella bronchiseptica and Pasteurella multocida, (ii) the presence of brachygnathia superior (BS score), (iii) the extent (grade) of turbinate atrophy and pneumonia at slaughter and (iv) growth rates from two to 16 weeks of age and average daily weight gains to slaughter. In two of the herds with no history of atrophic rhinitis, B bronchiseptica and non-toxigenic strains of P multocida were isolated; only one of 47 pigs (2 per cent) had a BS score greater than +10 mm and the most severe turbinate atrophy observed in 21 pigs at slaughter was grade 3. In contrast, from two herds with atrophic rhinitis, toxigenic strains of P multocida were isolated as well as B bronchiseptica and non-toxigenic P multocida. BS scores of greater than +10 mm were present in six of 47 pigs (13 per cent) of which five were infected with toxigenic P multocida and had severe turbinate atrophy of grade 4 or 5. There was no significant reduction in growth rates in the affected compared with the unaffected herds nor in the affected compared with the unaffected pigs in the same herd. Neither was there a correlation between progressive disease and the extent of pneumonia found at slaughter. It was concluded that in field cases of the disease, high BS scores plus severe turbinate atrophy were associated with infection by toxigenic type-D strains of P multocida.

Attachment of Moraxella bovis to calf corneal cells and inhibition by antiserum.

An in vitro assay was developed using calf corneal cells to assess the importance of fimbriae in the colonisation of the bovine ocular surface by Moraxella bovis, and the role of fimbrial antibodies in the bovine immune response and resistance to infectious bovine keratoconjunctivitis (IBK). Fimbriae promoted adherence of M. bovis to calf corneal cells in culture; 15 fimbriate isolates, representative of 6 fimbrial serogroups of M. bovis, adhered to the cells whereas 4 non-fimbriate isolates failed to do so. Fimbrial antibodies in hyperimmune rabbit serum inhibited attachment of all fimbriate strains of the homologous fimbrial serogroup but not those of 5 heterologous serogroups. The relevance of these results to the use of a polyvalent fimbrial vaccine in the control of IBK is discussed.