Capsule thickness and amounts of a 39 kDa capsular protein of avian Pasteurella multocida type A strains correlate with their pathogenicity for chickens

Capsule thickness of avian Pasteurella multocida type A strains was determined by transmission electron microscopy after labeling with polycationic ferritin and compared with their pathogenicity for chickens. The capsule thickness of P. multocida strains Pm-18 and X-73 was 81.4 and 50.1 nm on average, respectively. These strains were highly virulent for chicken, whereas the less virulent strains Pm-1 and Pm-3 had a thin and irregular capsule, 21.0 and 29.8 nm on average, respectively. However, the thickest capsule was observed in strain P-1059, 101.2 nm on average, and the strain revealed moderate virulence. The noncapsulated variant P-1059B, which was derived from strain P-1059, revealed low virulence. The six P. multocida strains were examined with regard to protein content on the capsule of organisms. Amounts of total proteins of crude capsular extract (CCE) from capsulated strains were approximately twice those of the noncapsulated strains. The amount of an antigenic 39 kDa protein in the CCE were found to correlate with the capsule thickness, since heavily capsulated strains exhibited the greatest amount, whereas noncapsulated strains including noncapsulated and low virulent variant P-1059B possessed little 39 kDa protein. The results demonstrated that the capsule thickness and the quantity of a 39 kDa capsular protein of avian P. multocida type A strains correlated with their pathogenicity for chickens.     

A 39 kDa protein mediates adhesion of avian Pasteurella multocida to chicken embryo fibroblast cells

To clarify the role of avian Pasteurella multocida capsule in pathogenesis, adhesion of capsulated strains P-1059, X-73 and Pm-18, and noncapsulated strains P-1059B, Pm-1 and Pm-3 to chicken embryo fibroblast (CEF) cells was compared. Number of adherent organisms of the capsulated strains to CEF cells were approximately three times as much as noncapsulated strains indicating that adhesive properties were enhanced by the presence of bacterial capsule. Pretreatments of the bacterial cells with heat, trypsin, or with antiserum caused a marked decrease in adhesion of capsulated strain P-1059 and its noncapsulated variant P-1059B. However, depolymerization of capsular hyaluronic acid with high dose of hyaluronidase enhanced adhesion of these strains. Combined treatments of the bacterial cells with both hyaluronidase and trypsin significantly (P < 0.05) inhibited the adherence of strain P-1059 as compared to the treatment only with trypsin, but strain P-1059B was not affected. SDS-PAGE profiles of crude capsular extract (CCE) prepared from capsulated strain P-1059 and its noncapsulated variant P-1059B grown on dextrose starch agar (DSA) plates by heating at 56 degrees C in a 2.5% NaCl solution demonstrated eight protein bands of 28, 34, 36, 39, 52, 56, 63 and 93 kDa. The 28, 34 and 36 kDa proteins were commonly major for both strains, and the 39 kDa protein was major only for strain P-1059 but poor in strain P-1059B. Outer membrane protein (OMP) profiles were identical with a major protein at 34 kDa and four minor proteins between the two strains. The adhesion of strain P-1059 and strain P-1059B to CEF cells was inhibited significantly (P < 0.01) by treatment with rabbit antisera against P-1059, P-1059B, CCE or 39 kDa protein of strain P-1059 as compared to the treatment with either PBS or with normal rabbit serum. These results indicated that an antigenic 39 kDa protein in the capsule may be responsible for adhesion of avian P. multocida type A strains to CEF cells as a virulence factor.     

Pathogenesis of fowl cholera: influence of encapsulation on the fate of Pasteurella multocida after intravenous inoculation into turkeys

The role of the capsule in the pathogenesis of fowl cholera was studied in turkeys. Avian Pasteurella multocida P-1059 was used in an encapsulated form, an enzymatically decapsulated form, and a mutant form lacking capsule-productivity (strain T-325). These forms were inoculated intravenously into normal or immune turkeys, and the numbers of viable bacteria in the blood, liver, and spleen were enumerated over a 120-minute period. Both normal and immune birds rapidly removed all three forms of organisms from the blood-stream at similar rates and trapped in the liver and spleen. In the liver of normal birds, the non-encapsulated mutant T-325 was readily inactivated, but the encapsulated P-1059 strain was not. When the decapsulated form of P-1059 was used, the bacterial counts in the liver temporarily decreased at 60 minutes PI. In immune birds, all three forms of organisms were equally inactivated in the liver. In the spleen, however, the bacterial numbers did not change throughout 120 minutes PI with all three forms of organisms in both normal and immune turkeys. The results indicated that the blood-borne P. multocida were readily trapped by reticuloendothelial phagocytes. The trapping process was not affected by encapsulation of the organism or by the immune status of turkey. Both factors, however, appeared to greatly influence the subsequent killing of P. multocida by hepatic, but not splenic, phagocytes.     

Characterization of a 39kDa capsular protein of avian Pasteurella multocida using monoclonal antibodies

The role of a 39kDa protein of avian Pasteurella multocida in pathogenesis of fowl cholera was investigated using monoclonal antibodies (Mabs). Mabs were prepared by immunization of BALB/c mice with a crude capsular extract (CCE) of P. multocida strain P-1059 (serovar A:3). Totally eight hybridomas producing Mab were obtained. Immunoblot analysis of the hybridomas revealed that all the Mabs recognized a 39kDa protein of CCE. Treatment of CCE antigen with proteinase K or periodic acid indicated that the epitope recognized was proteinaceous. The Mabs reacted with a major 39kDa protein of CCE from encapsulated strains but not with any protein of non-capsulated strains indicating that a direct correlation between encapsulation and the 39kDa protein. Immunoelectron microscopy on strain P-1059 and the non-capsulated derivative P-1059B (serovar -:3) reacting with the Mabs and gold-labeled anti-mouse IgG indicated that the protein is associated with the capsule. The Mabs significantly inhibited the adherence of encapsulated P. multocida strains to chicken embryo fibroblast cells, but only slightly that of non-capsulated strains. Mice passively immunized with the Mabs were protected from lethal challenge with virulent strains P-1059 and X-73 (serovar A:1). Thus the capsular 39kDa protein was determined to be an adherence factor and a cross-protective antigen of avian P. multocida type A strains.     

Treatment of experimentally induced Pasteurella multocida infections in broilers and turkeys: comparative studies of different oral treatment regimens

Experimental fowl cholera was induced in 60 healthy 10-week-old broiler chickens and 8-week-old turkeys by intramuscular inoculation with approximately 80 colony-forming units (cfu) of Pasteurella multocida X-73 strain and with approximately 70 cfu of P. multocida P-1059 strain, respectively. This method of infection proved to be useful for evaluating the efficacy of anti-microbial medication, by measuring mortality, weight gain, pathological responses and frequency of re-isolation of P. multocida. The efficacies of two different dosing methods, continuous and pulse dosing, were compared. Using the continuous-dosing method, norfloxacin was administered to drinking water at 100 mg/l for 5 days in chickens. Efficacies were slightly improved compared with pulse dosing at 15 mg/kg bodyweight for the same length of time. The opposite was observed in turkeys, to the degree of control of mortality and maintenance of weight gain.     

Expression of pili and capsule by the avian strain P-1059 of Pasteurella multocida

The avian strain P-1059 of Pasteurella multocida was grown on blood agar (BA), on dextrose-starch agar (DSA), or in Heddleston's hydrogen sulfide test broth. Cells were examined for the presence of pili using electron microscopy after staining with phosphotungstic acid, and they were examined for capsule after ruthenium red staining. Pili were found on the capsulated iridescent type, P-1059I, and on two non-capsulated variants, the blue, P-1059B, and the gray, P-1059G. Many cells grown on BA were heavily piliated. In contrast, fewer cells grown on DSA had pili, and piliation was only slight to moderate. The P-1059I, P-1059B, and P-1059G produced pellicles when grown on broth medium. Pili were found on the circumference of the cells grown on either agar or broth medium. Occasionally a pilus connecting two cells was seen on cells cultured in broth. Cultivation of the P-1059I on DSA containing the iron-chelating agent alpha,alpha'-bipyridyl produced a non-capsulated blue variant. The non-capsulated variant reverted to P-1059I when grown on BA but did not revert when grown on DSA.     

Attenuated live fowl cholera vaccine. I. Development of vaccine strain M3G of Pasteurella multocida

A live cholera vaccine was developed from a virulent avian septicemia strain of Pasteurella multocida serotype 1. The virulent parental strain was mutagenized with N-methyl-N'-nitro-N-nitroso guanidine. Mutants were selected that had either smaller colonies at 37 C or temperature sensitivity for growth at 41 C. Four small-colony mutants and 2 temperature-sensitive mutants were studied. All the mutants were avirulent for turkeys. Sixteen days after turkeys were vaccinated with each mutant, both the vaccinates and unvaccinated controls were challenge-exposed to virulent P. multocida of the homologous serotype and the heterologous serotype 3. Two of the small-colony mutant strains protected against both homologous and heterologous challenge. Suggested for a live cholera vaccine is P. multocida M3G, a small-colony-forming mutant, innocuous for both mice and turkeys and stable against reversion.     

Protectivity of an immunoaffinity-purified 39 kDa capsular protein of avian Pasteurella multocida in mice

A 39 kDa protein of avian Pasteurella multocida strain P-1059 (serovar A:3) was purified from a crude capsular extract by immunoaffinity chromatography by using a ligand of purified mouse monoclonal antibody to the 39 kDa capsular protein of the strain. Protective activity of the purified 39 kDa protein antigen was determined by inoculation of ddY mice twice with 25 or 125 mug of the protein and challenge-exposure with 10 or 50 LD(50) of strains P-1059 or X-73 (serovar A:1). The results showed that the antigen gave high protection (60 to 100%). These results indicated that the 39 kDa protein of avian P. multocida is a cross-protective antigen over serovars A:1 and A:3.     

Characterization of outer membrane protein-enriched extracts from Pasteurella multocida isolated from turkeys

Outer membrane protein (OMP)-enriched extracts of avian strains of Pasteurella multocida were examined by use of sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Culture medium did not have a significant effect on the OMP profiles of strains of P multocida examined; however, in vivo propagation had an appreciable effect on the OMP profile composition of the reference strain P-1059. Such bacteria, expressed several additional OMP in the 27-kD, 48-kD, 56-kD, 60-kD, 80-kD, and 94-kD molecular mass regions. These OMP were not detected in the electrophorogram of strain P-1059 grown in vitro. The OMP profiles of reference strains of the 16 serotypes of P multocida did not identify any serotype-specific protein markers. Field strains of serotype A:3 had variation in OMP profiles and did not express OMP that all were identical to that expressed by the reference strain P-1059. The live attenuated CU and M9 bacterial vaccine strains expressed strain-specific OMP markers of 48-kD and 45-kD molecular masses, respectively. These strain-specific OMP markers may be used to differentiate these strains from virulent field strains that are of the same serotype and isolated from turkeys that have succumbed to pasteurellosis as a result of vaccine-related reactions or breakdown in immunity.     

Serum resistance is correlated with encapsulation of avian strains of Pasteurella multocida

Encapsulated avian strains of Pasteurella multocida possessing an A-type capsule were shown to be resistant to the bactericidal action of turkey serum, whereas unencapsulated variants as well as other unencapsulated strains were not. Removal of the capsule from serum-resistant strain P1059-1 resulted in this strain becoming susceptible to the bactericidal effects of turkey serum. Since complement was consumed when encapsulated or unencapsulated strain P1059-1 was incubated in turkey serum, we conclude that the capsule acts to shield the outer membrane rather than prohibiting the generation of an effective membrane attack complex.     

8株禽多杀性巴氏杆菌外膜蛋白H基因序列分析

为了解国内禽多杀性巴氏杆菌（Pasteurella multocida,Pm）流行株外膜蛋白 H（OmpH）基因的变异情况,参考GenBank中已发表的多杀性巴氏杆菌序列设计1对特异性引物,采用PCR方法对不同来源的8株禽Pm菌株和3个荚膜型参考株（A、B、D）的OmpH基因进行扩增、测序。结果显示,11个菌株的OmpH基因开放阅读框在1002~1071 bp 之间;SignalIP 4.0预测结果表明,信号肽均为N端20个氨基酸残基,成熟蛋白氨基酸残基数量在314~337 aa之间,推测的分子质量在33.76~37.04 ku之间。与GenBank中15个菌株OmpH基因序列比对结果发现,核苷酸同源性在84.9%~100.0%之间;氨基酸同源性在81.5%~100.0%之间;其中C48-1、1010、9003、890920、921012、XJ-e 6个国内禽Pm分离株OmpH序列同源性为100.0%。试验结果表明,国内禽Pm菌株OmpH基因非常保守。     

Association of complement sensitivity with virulence of Pasteurella multocida isolated from turkeys

Two strains of Pasteurella multocida, both derivatives of strain P1059, were compared for virulence for 14-week-old turkeys and sensitivity to turkey plasma. Strain P1059-1, a nalidixic-acid-resistant mutant of P1059 with an LD50 of approximately 10(3) colony-forming units (CFU), was more resistant to the bactericidal effects of fresh turkey plasma at 37 C than avirulent strain P1059-1A. P1059-1A, with an LD50 of approximately 10(8) CFU, is an acapsular variant of P1059-1 that spontaneously arose after prolonged passage on artificial medium. The bactericidal effect on P1059-1A was removed when turkey plasma was treated with heat or with zymosan, maneuvers that removed hemolytic complement activity from turkey plasma.     

Vaccination of turkeys with cell-free culture filtrate of Pasteurella multocida

Turkeys given cell-free culture filtrate (CCF) of Pasteurella multocida strain R44/6 orally, via air sacs, or subcutaneously mixed 1:1 with incomplete Freund's adjuvant (IFA) at 6 and 9.5 weeks of age were compared with negative controls given bacteriologic medium and positive controls vaccinated with a commercial bacterin. At 13 weeks of age, serum antibody titers to P. multocida were detectable only in turkeys given CCF in IFA (low titers) and positive control turkeys (high titers), at which time turkeys were challenged orally with either the homologous strain or strain P-1059. Protection against challenge with strain R44/6 was provided by the commercial bacterin, CCF in IFA, and CCF given via air sacs. When turkeys were challenged with strain P-1059, protection was superior in turkeys given CCF via air sacs, intermediate in turkeys given commercial bacterin or CCF in IFA, and absent in negative control turkeys and turkeys given CCF orally. These results indicate CCF is an effective immunogen when administered via the lower respiratory tract for protecting turkeys against pasteurellosis.     

Monoclonal antibodies against surface antigens of Pasteurella multocida strain P-1059

Four monoclonal antibodies (MAbs) were developed against serotype 3:A, P-1059 strain of Pasteurella multocida. Enzyme-linked immunosorbent assays were used to screen those hybridomas producing antibodies to either a surface protective (2.5 S) or lipopolysaccharide (LPS) antigen. MAbs 6EE11, D7H10, E11E3, and C11H2 were positive against 2.5 S antigen, and two of them, E11E3 and C11H2, were positive for the LPS antigen. MAbs 6EE11 and D7H10 reacted with a major protein band of molecular weight of 35,500, whereas E11E3 and C11H2 recognized a band with a molecular weight of 12,500 of the 2.5 S antigen. Treatment of the 2.5 S antigen with periodic acid abolished epitopes reacting with E11E3 but not with 6EE11. MAb 6EE11 did not recognize any band in Western blot after proteinase K treatment of the 2.5 S antigen, whereas antibody activity of E11E3 did not change. MAb 6EE11 reacted with serotypes 3, 4, 9, 10, 11, 12, and with M-9 strains in the immunofluorescence test. MAb E11E3 was positive only with serotype 3 or 10 strains, excluding M-9 strain. Electron microscopic studies with P-1059 strain indicated that antigens binding to 6EE11 and/or E11E3 were present in the capsule.     

牛多杀性巴氏杆菌外膜蛋白H的表达与纯化

试验对多杀性巴氏杆菌外膜蛋白H(OmpH)基因进行克隆、鉴定,并在原核系统中表达。以多杀性巴氏杆菌(CVCC448)强毒株基因组为模板,扩增OmpH基因,连接T载体,经测序鉴定正确后与表达载体pET-28a连接构建重组表达质粒OmpH-pET28a,将此重组质粒转化入表达宿主E.coli BL21菌株内,抽提质粒,酶切鉴定正确后对转化菌株以IPTG进行诱导,表达产物通过镍离子亲和层析纯化,之后进行SDS-PAGE和Western blotting分析。结果显示,OmpH基因的编码区为978 bp,编码326 个氨基酸残基,融合蛋白分子质量约为37 ku。Western blotting检测结果显示,表达的重组蛋白OmpH可与鼠抗多杀性巴氏杆菌全菌体多抗血清反应得到清晰的目的条带,表明表达的重组蛋白具有良好的免疫原性。多杀性巴氏杆菌OmpH基因的成功表达,为进一步研究其免疫作用奠定了基础。     

The effect of hypovitaminosis A on the pathogenesis of Pasteurella multocida in turkeys

It has been proposed that Pasteurella multocida can invade the host tissues via the mucous membrane. Vitamin A (VitA) deficiency has been associated with mucous membrane damage, such as squamous metaplasia. The objective of this study was to determine the early stages in the pathogenesis of P. multocida in VitA-deficient turkeys and clinically healthy turkeys. Fifteen-week-old VitA-deficient and clinically healthy turkeys were inoculated with P. multocida P-1059, a virulent strain, and the portal of entry, invasion, and localization of P. multocida were studied by microbial examination of the trachea, liver, and lung and histologic examinations of internal organs. Higher mortality was found in VitA-deficient turkeys. Pasteurella multocida was first reisolated from the trachea, secondarily from the liver and blood, and finally from the lung in both groups. Invasion of P. multocida into tissues occurred between 3 hr and 24 hr postinoculation in both groups. Our findings suggest that altered membrane integrity in VitA-deficient birds did not appear to change the time course of the systemic spread of P. multocida infection in turkeys and that the increased mortality seen in the VitA-deficient turkeys may be associated with immune system impairment.     

Evaluation of Pasteurella multocida mutants of low virulence. I. Development and pathogenicity

Mutagenesis of the Clemson University (CU) vaccine strain of Pasteurella multocida with N-methyl-N-nitro-N-nitrosoguanidine resulted in temperature-sensitive mutants that grew at 37 C but not at 42 C. Seven such mutants were evaluated for immunogenicity in turkeys. From these seven, only two, PM#1 and PM#3, provided turkeys with a level of protection against challenge with a virulent serotype 3 P. multocida strain (P-1059) comparable to the protection provided by the CU strain. Intravenous (IV) inoculation of PM#1, PM#3, or CU was used to assess differences in virulence. PM#1 and PM#3 resulted in lower rates of mortality and lameness than the CU strain. Histopathological evaluation of spleens 24, 48, and 72 hours after IV inoculation demonstrated that the CU strain induced significantly more fibrinoid necrosis of the spleen than either PM#1 or PM#3.     

Rabbit pasteurellosis: induced disease and vaccination

Pasteurellosis was induced in rabbits by conjunctival inoculation with 2 strains of Pasteurella multocida. The LD50 of strain P1062 (a bovine isolate) was 10(5.1) colony-forming units and that of strain P1059 (a turkey isolate) was 10(5.5) colony-forming units. Pasteurella-free rabbits were vaccinated IV or mucosally with boiled cells of P multocida or a cross-reactive uridine diphosphogalactose epimerase-deficient mutant of Escherichia coli J5. In rabbits challenge exposed with P multocida strain 1062 or 1059, homologous P multocida strain gave the best protection against fatal bacteremia. Partial protection was provided by J5; mucosal routes of vaccination (aerosol or conjunctival) gave better protection than did the IV route. Serum antibody titers were lower in rabbits vaccinated by mucosal routes than in those vaccinated IV. Cross-reactive IgG and IgM titers to P multocida were demonstrated when rabbits were vaccinated with J5. On the basis of bacteriologic examination of nasal secretions, rabbits that died were considered culture positive sooner than were those that survived. On the basis of bacteriologic examination of blood, rabbits that died were considered culture positive, and those that survived were considered culture negative. Seemingly, heat-stable antigens were protective, the cross-reactive E coli J5 mutant (with only core lipopolysaccharide) provided partial protection against pasteurellosis, and the mucosal route was somewhat useful for cross-protective immunization.     

禽多杀性巴氏杆菌C48-3株hexABC基因缺失株的构建及其生物学特性分析

为研究荚膜在禽多杀性巴氏杆菌(Pasteurella multocida)致病过程中的作用,本研究利用同源重组基因敲除技术构建禽P.multocida C48-3株荚膜多糖输出蛋白基因hexABC的缺失突变株,并以小鼠为动物模型检测荚膜缺陷对细菌毒力的影响。PCR、RT-PCR和DNA测序结果均表明253 bp的hexC基因下游序列、798 bp的hexB基因全序列和290 bp的hexA基因上游序列完全被四环素抗性基因替代,表明构建了基因敲除突变株C48-3ΔhexABC。电镜观察结果表明突变株荚膜合成能力缺失,对小鼠的致病性试验结果显示突变株毒力基本丧失。本研究获得的无荚膜突变株为进一步研究禽P.multocida的致病机理奠定基础。     

The effect of concurrent infections with Pasteurella multocida and Ascaridia galli on free range chickens

Pasteurella multocida and Ascaridia galli are observed with high prevalences in free range chickens in Denmark, but the impact is unknown. A study was carried out to examine the interaction between A. galli and P. multocida in chickens and the impact on production.Five groups, each with 20 18-week-old Lohmann Brown chickens were infected. Group 1 was orally infected with 1000+/-50 embryonated A. galli eggs. Group 2 received 10(4) cfu P. multocida intratracheally. Group 3 was infected with A. galli and subsequently with P. multocida. Group 4 was infected with P. multocida followed by A. galli. Group 5 was the control. The study ran for 11 weeks where clinical manifestations, weight gain and egg production were recorded. Excretion of P. multocida was determined on individual basis and blood smears were made for differential counts. At the end of the study pathological lesions and the number of adult worms, larvae and eggs in the faeces were recorded.The birds were more severely affected when infected with both pathogens compared to single infections with A. galli or P. multocida, respectively. A lower weight gain and egg production was observed with dual infections. A. galli infection followed by a secondary P. multocida infection resulted in more birds with pathological lesions and continued P. multocida excretion.In conclusion a negative interaction between A. galli and P. multocida was observed and it is postulated that free range chickens are at higher risk of being subjected to outbreaks of fowl cholera when they are infected with A. galli.