Toxigenic type D Pasteurella multocida in New South Wales pig herds—prevalence and factors associated with infection

Between March and July 1987, a study was undertaken to determine the prevalence of and factors associated with toxigenic type D Pasteurella multocida infection in New South Wales pig herds. Toxigenic type D P. multocida was isolated from the nasal cavities of pigs in one (2%) of 50 randomly selected herds. Toxigenic isolates were also recovered from 2 (8%) of a separate group of 25 herds that had purchased pigs from a known infected piggery in South Australia (herd SA). Snout abnormalities were present in 9.4%, 3.2% and 1.8% of grower pigs in the 3 affected herds. Isolation of toxigenic P. multocida was significantly associated (p less than 0.0001) with the occurrence of clinically affected pigs in the herd. Purchase of at least 5 pigs from herd SA was associated with an elevated risk (p less than 0.05) of isolation of toxigenic P. multocida.     

Toxigenic Pasteurella multocida in rabbits with naturally occurring atrophic rhinitis

In a commercial rabbitry nasal swabs were taken from 36 animals with enzootic upper respiratory disease resembling porcine atrophic rhinitis. 35 Pasteurella multocida strains were isolated from 17 rabbits. Among 30 strains tested for dermonecrotic toxin production 3, derived from 3 animals, were positive in the guinea pig skin test. 15 Bordetella bronchiseptica strains were recovered from 14 rabbits. No toxigenic strains were found among 6 isolates tested using the same method.     

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.     

Vaccination against progressive atrophic rhinitis with a recombinant Pasteurella multocida toxin derivative.

Vaccination against progressive atrophic rhinitis using a purified recombinant derivative of the Pasteurella multocida toxin (PMT), was carried out. Ten pregnant gilts were vaccinated twice with the nontoxic derivative (dO) which apart from a lack of 121 amino acids had an amino acid sequence identical to PMT, while seven gilts were vaccinated with a purified, formaldehyde treated, native PMT and ten gilts served as non-vaccinated controls. The resulting piglets were inoculated intranasally with Bordetella bronchiseptica and toxigenic P. multocida. Among piglets from the nonvaccinated gilts all except one developed clinical atrophic rhinitis and 90% developed severe turbinate atrophy while only a few pigs in the vaccinated groups developed clinical or pathological signs of disease. Gilt colostra from the two vaccinated groups had similar mean anti-PMT titers and the mean titers in the offspring's sera from these groups were nearly identical throughout the study. No pigs born from unvaccinated gilts were seropositive until 8 wk of age (7 wk post-challenge) but 23% became seropositive at slaughter. The infection rate with toxigenic P. multocida in piglets and the total number of P. multocida colonies cultured from nasal swabs were significantly reduced at 5 wk and 8 wk of age in the vaccinated groups, when compared to controls. There was a significantly improved weight gain (greater than 9%) from birth to slaughter in offspring from vaccinated gilts. No significant differences in feed conversion rate or % lean meat were observed among the groups. The study showed the excellent immunoprotective properties of the nontoxic derivative of the PMT molecule.     

Bordetella bronchiseptica and toxigenic type D Pasteurella multocida as agents of severe atrophic rhinitis of swine

Bordetella bronchiseptica and toxigenic type-D Pasteurella multocida were cultured from pigs in each of five herds diagnosed as having severe atrophic rhinitis (AR). B. bronchiseptica alone, P. multocida alone, or both organisms isolated from four herds were inoculated intranasally into 1-week-old gnotobiotic pigs which were necropsied 4 weeks post-inoculation (PI). Nasal turbinate atrophy in B. bronchiseptica-inoculated pigs was moderate to severe, while P. multocida-inoculated pigs had slight to severe atrophy. Pigs inoculated with both organisms had moderate to complete turbinate atrophy. P. multocida was reisolated at necropsy from all pigs receiving the organism except those having no turbinate damage. B. bronchiseptica and P. multocida from a fifth herd were simultaneously inoculated into six naturally farrowed 6-day-old SPF pigs. Necropsy performed 4 weeks PI revealed severe to complete turbinate atrophy. Nasal turbinates were normal for control pigs in both experiments.     

Protection against progressive atrophic rhinitis by vaccination with Pasteurella multocida toxin purified by monoclonal antibodies

Pasteurella multocida toxin was purified by affinity chromatography and inactivated by treatment with formaldehyde before use as a single component vaccine against progressive atrophic rhinitis in pigs. Twenty pregnant gilts which were vaccinated twice before farrowing with either low or high doses of the purified toxoid, developed dose-dependent positive serum and colostrum titres to the toxin and, unlike the progeny of 10 untreated control gilts, the offspring of the vaccinated gilts also had serum titres. These titres could be measured in blood samples taken for more than eight weeks from birth for most pigs born to gilts vaccinated with low doses and more than 12 weeks for pigs born to gilts vaccinated with high doses of the vaccine. All the piglets were inoculated intranasally with Bordetella bronchiseptica and toxigenic P multocida. The clinical and post mortem examinations of snouts revealed a significant reduction in the frequency and degree of conchal atrophy in the two groups of pigs from the vaccinated gilts compared with the pigs from control gilts. Clinically 90 per cent of the snouts of pigs born to vaccinated gilts appeared normal whereas only 28 per cent of the snouts of control pigs were not shortened or deviated at eight weeks of age. At slaughter 11 per cent of the pigs born to vaccinated gilts and 81 per cent of the control pigs had severe turbinate atrophy.(ABSTRACT TRUNCATED AT 250 WORDS)     

Pasteurella multocida and Bordetella bronchiseptica in atrophic rhinitis and pneumonia in swine.

A total of 163 pigs from nine farrow-to-finish herds representing various levels of atrophic rhinitis (AR) were selected for postslaughter examination of AR and pneumonia. Nasal swabs and lungs were cultured for detection of Bordetella bronchiseptica and Pasteurella multocida. Seventy-three pigs were examined at eight weeks of age and 90 contemporaries at six months of age. Mean AR scores were 1.21 and 1.11 for the eight week and six month old pigs, respectively (0 = normal, 3 = severe). In individual pigs increasing AR score was related to increasing pneumonia score in eight week old pigs but not in six month old hogs. In eight week old pigs, B. bronchiseptica and P. multocida were isolated more frequently from pigs with higher AR scores. From nasal swabs of six month old hogs, Bordetella was almost never recovered while Pasteurella was frequently isolated score. Toxigenic type DP. multocida was isolated from nasal cultures of only seven (4%) pigs and from lung cultures of only one pig. Pasteurella was never isolated from lungs of the eight week old pigs and Bordetella never from the six month old hogs. The isolation rate of P. multocida, predominantly type A, from lungs of six month old pigs increased from 11% in grossly normal lungs to 86% in lungs with severe pneumonia. Pigs from one herd free from lesions of AR and pneumonia were also examined; type AP. multocida was isolated from nasal cultures of one of six eight week old pigs. Somatic antigens of P. multocida were determined for 94 nasal and 20 lung isolates. Somatic serovar 3 was found in 93% of the nasal isolates and in all lung isolates.(ABSTRACT TRUNCATED AT 250 WORDS)     

Atrophic rhinitis caused by Pasteurella multocida type D: morphometric analysis.

In order to study the distribution and the extent of atrophy caused by Pasteurella multocida in the nasal conchae, experimental piglets were injected intramuscularly at seven days of age with either two or four 50% mouse lethal doses per kg body weight of P. multocida type D dermonecrotoxin. Experimental and control piglets were killed four, six and ten days postinjection. Serial transverse paraffin embedded sections of the noses were cut throughout the entire length of the nasal conchae. The area of the nasal ventral conchae was measured and the morphometric index of the nasal cavity was calculated. It was observed that P. multocida type D dermonecrotoxin induced severe atrophy of the nasal ventral conchae. This atrophy was present along the entire conchae. However, it was most severe at the level of the first and second premolar teeth.     

Experimental atrophic rhinitis in 2 and 4 month old pigs infected sequentially with Bordetella bronchiseptica and toxigenic type D Pasteurella multocida.

Experimental infections with Bordetella bronchiseptica and/or toxigenic type D Pasteurella multocida were studied in 2- and 4-month-old primary specific-pathogen-free pigs. None of the 2-month-old pigs inoculated with B. bronchiseptica or P. multocida alone developed turbinate atrophy. All the pigs inoculated with B. bronchiseptica (10(7) CFU/head) and P. multocida (10(9) CFU/head for 5 consecutive days) together, however, developed clinical and post-mortem signs of atrophic rhinitis (AR) similar to the naturally occurring disease. Slight to severe turbinate atrophy was observed in the 4-month-old pigs inoculated with B. bronchiseptica and P. multocida (at the same concentration as above) at necropsy.     

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)     

Detection of neutralizing antibodies against Pasteurella multocida toxin in swine with atrophic rhinitis

Blood sera of 1171 pigs, not vaccinated against PAR, were tested for antibodies against P. multocida toxin in a neutralization test with EBL cell cultures. 277 sera were from 18 herds with clinical PAR; 104 of them (37.5%) had neutralizing antibody titres from 1:2 to 1:1024. No antitoxin was detected in the sera of 866 pigs in 25 PAR nonsuspicious herds. In one nonsuspicious herd, however, 11 of 28 sera were neutralizing in the 1:2 or 1:4 dilution. 30 sows had been vaccinated with inactivated toxigenic P. multocida and/or with the P. multocida toxoid. 21 of these sows had neutralizing sera with titres between 1:8 and 1:4096. The neutralization test presented can be applied for herd diagnosis of PAR and for demonstration of vaccination effects.     

Isolation of toxigenic Pasteurella multocida from an outbreak of atrophic rhinitis in pigs

An outbreak of severe atrophic rhinitis is described. A toxigenic strain of P. multocida was isolated from one affected pig. Severe turbinate atrophy was not associated with significantly reduced bodyweight gain in a batch of 34 pigs examined at slaughter. The history of the outbreak suggested that pigs recently introduced from overseas may have been the source of the disease.     

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.     

Use of ELISA to detect toxigenic Pasteurella multocida in atrophic rhinitis in swine.

The use of an enzyme-linked immunosorbent assay (ELISA) as a means of detecting dermonecrotoxin-producing strains of Pasteurella multocida was investigated. The assay was evaluated as a means to identify toxigenic P. multocida isolates recovered from nasal secretions of swine with atrophic rhinitis. The sensitivity and specificity of the ELISA for detecting dermonecrotoxin-producing P. multocida strains were compared to those of mouse-inoculation and cytotoxicity assays. The ELISA was highly sensitive and more specific than animal inoculation or tissue culture assay and is thus a more effective method for screening swine herds for the presence of toxigenic strains of P. multocida. The ELISA is a rapid, effective, economical way to identify toxigenic P. multocida isolates.     

Identification of toxigenic Pasteurella multocida in atrophic rhinitis of pigs by in vitro characterisation

Toxigenic strains of Pasteurella multocida were readily differentiated from non-toxigenic strains by an agarose overlay method using bovine turbinate cells or bovine lung cells. Cells which were young and densely confluent were best suited to this assay. The incubation period required to distinguish toxigenic strains was dependent on the confluence of the monolayers, which was affected by the seeding rate, cell passage level and growth time prior to overlay. The agarose overlay method correctly identified 11 of 11 reference strains of Pasteurella multocida, and visible cytotoxic changes were present in the monolayers after 48 to 65 h. Outbreaks of the enzootic form of atrophic rhinitis in 2 New South Wales piggeries were associated with the isolation of toxigenic type D strains of P. multocida.     

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.     

Development of a genetically modified nontoxigenic Pasteurella multocida toxin as a candidate for use in vaccines against progressive atrophic rhinitis in pigs

OBJECTIVE: To construct a genetically modified nontoxigenic Pasteurella multocida toxin (PMT) and examine its immunoprotective activity against challenge exposure with wild-type PMT in pigs. ANIMALS: 5 healthy pigs. PROCEDURE: A nontoxigenic PMT was created by replacing the serine at position 1164 with alanine (S1164A) and the cysteine at position 1165 with serine (C1165S). Toxic activity was determined by use of the guinea pig skin test and mouse lethality test. Three pigs were vaccinated twice with the modified PMT, and the remaining 2 pigs served as nonvaccinated control animals. Vaccinated and control pigs were challenge exposed with wild-type PMT. Pigs were euthanatized and necropsied on day 14 after challenge exposure. Turbinate atrophy was examined macroscopically and assigned a score. Serum anti-PMT antibodies were determined by use of an ELISA. RESULTS: The genetically modified PMT was characterized by a total lack of toxic activity. Pigs vaccinated with the modified PMT became seropositive; in contrast, control pigs remained seronegative. Necropsy revealed that the 2 control pigs had moderate and severe turbinate atrophy, respectively, whereas the 3 vaccinated pigs did not have any lesions in the turbinates or abnormalities in other organs. CONCLUSIONS AND CLINICAL RELEVANCE: Modification by use of S1164A and C1165S leads to a complete loss of toxic effects of PMT without impairment of the ability to induce protective immunity in pigs. Analysis of these results suggests that genetically modified PMT may represent a good candidate for use in developing a vaccine against progressive atrophic rhinitis in pigs.