A test in vero cell monolayers for toxin production by strains of Pasteurella multocida isolated from pigs suspected of having atrophic rhinitis

Pennings, A.M.M.A.; Storm, P. K.

doi:10.1016/0378-1135(84)90071-3

Cited by 32 publications

(29 citation statements)

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“…The suspensions were treated by sonication g for 75 sec, centrifuged (10 min, 5,000 rpm), and filtered through a sterile 0.22-m-pore membrane filter. 18,19 The toxins were kept frozen at Ϫ20 C until use.…”

Section: Methodsmentioning

confidence: 99%

“…2,13,17,26 Isolates are then tested for the production of DNT. To differentiate toxigenic and nontoxigenic strains, both in vitro and in vivo diagnostic methods have been used, including enzyme-linked immunosorbent assay (ELISA), 4,7,8,16,23,29 polymerase chain reaction (PCR), 4,11,15,17 guinea pig skin test, 5,7 mouse lethality test, 7,15,21 and tissue cell culture in various cell lines, such as Vero cells 19 and embryonic bovine lung (EBL) cells. 9,25,28 The objective of the present investigation was to compare the techniques of PCR, ELISA and cell culture to evaluate the occurrence of DNT-producing strains of P. multocida and to validate the cell culture test using fetal lung feline (FLF) cells.…”

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confidence: 99%

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Evaluation of Techniques for the Detection of ToxigenicPasteurella MultocidaStrains from Pigs

1998

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Abstract.Recently acquired field isolates and archived isolates from our collection of Pasteurella multocida were analyzed for production of dermonecrotic toxin. Detection of the toxin was carried out using a fetal lung feline (FLF) cell line and a commercial enzyme-linked immunosorbent assay (ELISA) kit. The dermonecrotic toxin gene (ToxA) was also detected using a polymerase chain reaction (PCR) technique. Results from the 3 methods were compared. Field isolates (group 1) came from a commercial herd that had clinical signs of atrophic rhinitis. Fifty-six (17.9%) strains were isolated from 312 nasal swabs. Thirty-five of these strains belonged to serotype A and the rest (21/56), although probably serotype D, were not characterized further. All of these strains were toxin negative based on both the ELISA and FLF cell culture results. Five isolates gave faint bands in the PCR reaction, and the rest (51/56) were PCR negative. PCR and ELISA were also performed from the initial swab cultures (mixed cultures); 7 samples gave faint PCR bands, but ELISA results were all negative. Archived strains (group 2) had been isolated from clinical cases of atrophic rhinitis and from cases of pulmonary pasteurellosis. A total of 76 strains were analyzed; 46 were serotype A, and the rest (30) were serotype D. ELISA and FLF cell culture tests were negative for all serotype A strains; however, 3 strains showed faint bands in the PCR reaction. Fourteen serotype D strains showed positive results in both the ELISA and the FLF cell culture tests. PCR from these samples also gave positive results showing a strong band in the gel. However, 4 strains that were ELISA and FLF cell culture negative showed a faint band in the PCR reaction. The 3 methods gave similar results in the detection of the P. multocida dermonecrotic toxin. However, complete agreement among the tests was achieved only when strong PCR bands were considered positive. This is the first report that demostrates the use of FLF cell line for the detection of toxigenic P. multocida.Pasteurella multocida is a common commensal and pathogen of the respiratory tract of animals. In swine, toxigenic strains (both capsular types A and D) are most often associated with atrophic rhinitis (AR), whereas type A strains alone are commonly associated with pneumonia, pleuritis, or abscessation. 31 AR is a major respiratory disease in pigs that causes severe economic losses to the pig industry. Bordetella bronchiseptica and P. multocida strains that produce dermonecrotic toxin (DNT) are recognized as etiologic agents of this disease. These P. multocida strains synthesize a 145-kD toxin encoded by the chromosomal ToxA gene. The ToxA protein is an essential virulence factor for progressive AR; nontoxigenic P. multocida isolates do not cause this disease and are considered commensal flora of the upper respiratory tract of pigs. Toxigenic and nontoxigenic P. multocida isolates do not differ in diagnostic biochemical reactions or morphology. 15 Therefore, a more rapid, accurate detection assay is n...

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Section: Methodsmentioning

confidence: 99%

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confidence: 99%

Evaluation of Techniques for the Detection of ToxigenicPasteurella MultocidaStrains from Pigs

1998

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“…This, together with the 'phage type' motives of the terminal sequences, makes the horizontal gene transfer probable. PMT production can be detected by the biological activity (Pennings and Storm, 1984) or immunological characteristics of PMT (Magyar and Rimler, 1991) or by identification of the toxA gene (Kamps et al, 1990). Nagai et al (1994) used a PCR technique combined with hybridisation for demonstrating the toxA gene.…”

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Phenotypic and genotypic characterisation of Pasteurella multocida strains isolated from pigs in Hungary

Varga

Sellyei

Magyar

2007

Acta Veterinaria Hungarica

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A total of 146 Pasteurella multocida strains isolated from swine in Hungary in the last 20 years were examined. Biochemical characterisation and PCRbased techniques were used to determine species, subspecies, biovar, capsule type and presence of the toxA gene. Eighty-seven percent of the isolates belonged to P. multocida ssp. multocida, and 98% of these had biovar 3 or were trehalose-or lactose-fermenting or ornithine decarboxylase negative variants of that. Ten percent of the strains were P. multocida ssp. septica, and within this group 80% of the strains showed sorbitol-negative biovars (5, 6 and 7). The rest of the strains (20%) were lactose positive. Only 3% of the porcine isolates were P. multocida ssp. gallicida and 3 out of the 4 strains belonged to the dulcitol-fermenting biovar 8. Using a capsule-specific multiplex PCR, 60% of the strains belonged to capsule type D, 38% to capsule type A, and only 1 isolate had capsule type F. In contrast with data published in the literature, only 3% of capsule type D isolates carried the toxA gene, while this ratio was 41% for the type A strains. A remarkable regional distribution of toxA gene positive strains was observed. All but two isolates were found in swine herds located in the Transdanubian region, separated from other parts of Hungary by the river Danube.

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“…23 However, toxicity appears to be cell specific; comparable concentrations of PMT act as a mitogen for the murine 3T3 fibroblast line.13.26 In vitro observations revealed that nanomolar PMT levels were toxic for both embryonic bovine lung and fibroblastic rat osteosarcoma cells. Neither cytotoxic effects nor inhibition of proliferation were observed after treatment of the osteoblastic rat ROS 17/ 2.8 osteosarcoma cell line with similar PMT doses.…”

Section: Discussionmentioning

confidence: 99%

Effects of thePasteurella multocidaToxin on Osteoblastic Cells in vitro

Sterner-Kock

Lanske²,

Überschär

et al. 1995

Vet Pathol

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Abstract. Pasteurella multocida toxin induces localized osteolysis in the turbinate bones of swine. Osteolysis appears to be due to an increased level of osteoclastic bone resorption, although osteoblast activity may also be impaired. We studied the effects of purified toxin on the osteoblastic phenotype of the ROS 17/2.8 rat osteoblastic osteosarcoma cell line. Treatment of both embryonic bovine lung cells and a nonosteoblastic rat osteosarcoma cell line (ROS 25/1) with nanomolar doses of toxin produced marked cytotoxic actions. In the osteoblastic ROS 17\23 cells, this level of toxin reduced expression of an osteoblastic marker (alkaline phosphatase), was associated with matrix mineralization, but had no cytopathologic action. The osteoblastic cell population may be resistant to a direct cytotoxic effect but is nevertheless a target for toxin action.Key words: Alkaline phosphatase; osteoblast; Pasteurella multocida toxin; rhinitis atrophicans. In pigs, colonization of the upper respiratory tract by Bordetella bronchiseptica and toxigenic strains of Pasteurella multocida leads to progressive osteolysis of the nasal turbinate bones (infectious atrophic rhinitis).** A dermonecrotic toxin (PMT) isolated from P. multocida reproduces the full spectrum of pathologic changes seen in infected a n i r n a l~.~ Morphologic examination of the nasal bones of PMT-treated animals revealed a dramatic increase in multinucleate osteoclastic cells and local inactivation and reduction of the osteoblast p~p u l a t i o n .~ These divergent actions of the toxin lead to a net reduction in bone formation and increased bone resorption in the infected t i s~u e .~,~~ Recent evidence has suggested that there may be a direct action of PMT upon the osteoclast cell system.6 Our goal was to determine if there is also a similar direct action of PMT on the osteoblast population. To determine this, we used the rat osteosarcoma cell line ROS 17/2.8 as an in vitro model of the osteoblast. This clonally derived cell line retains a full osteoblastic phe-'Present address: Department of Veterinary Pathology, School of Veterinary Medicine, University of California, Davis, CA. notype and is able to produce mineralized matrix both in vitro and in v~v o .~* , *~ Materials and Methods Isolation and purification of PMTPMT was isolated and purified from a toxigenic type D P. muirocida strain (A 1 1 -2A) according to established p r o t o c~l s~~~~~ with some minor modifications. Bacteria were grown for 18 hours at 37 C in brain-heart infusion broth. Bacteria were harvested in chilled phosphate-buffered saline, centrifuged (30 minutes at 9,000 x g, 4 C), resuspended in distilled water (pH 8.0), and disrupted by sonication (4 minutes at 150 W). Sterile filtered (0.22-wm filter) extract was concentrated by ultrafiltration (Amicon Witten D membrane PM lo), dialyzed against 20 mM Tris/HCl (pH 7.5), and fractionated by DEAE-Sephacel C26-40 anion-exchange chromatography using a linear salt gradient from 0.25 to 0.48 M. Fractions containing cytotoxic activity in t...

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A test in vero cell monolayers for toxin production by strains of Pasteurella multocida isolated from pigs suspected of having atrophic rhinitis

Cited by 32 publications

References 2 publications

Evaluation of Techniques for the Detection of ToxigenicPasteurella MultocidaStrains from Pigs

Evaluation of Techniques for the Detection of ToxigenicPasteurella MultocidaStrains from Pigs

Phenotypic and genotypic characterisation of Pasteurella multocida strains isolated from pigs in Hungary

Effects of thePasteurella multocidaToxin on Osteoblastic Cells in vitro

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