Phagocytic uptake and killing of virulent and avirulent strains of Pasteurella multocida of capsular serotype A by chicken macrophages

The susceptibility of two highly virulent (VP161 and VP138) and two less virulent (VP17 and VP21) strains of Pasteurella multocida to phagocytic uptake and killing by chicken macrophages was compared using in vitro phagocytosis and bactericidal assays. When compared with VP17 and VP21, particularly after they were preopsonised with specific immune serum, VP161 and VP138 were more resistant to phagocytosis by chicken macrophages. The uptake of these bacteria increased following the removal of the bacterial capsules with hyaluronidase. All strains preopsonised with specific immune serum were killed to some extent by chicken macrophages. However, the percentages of killing for VP17 and VP21 were higher than those of VP161 and VP138. When the capsules of VP161 and VP138 were removed, the susceptibility of the bacteria to bactericidal activity of chicken macrophages increased. It can be concluded that the virulent strains of P. multocida were more resistant to phagocytosis and phagocytic killing by chicken macrophages compared with the less virulent strains. The hyaluronic acid capsule was considered to be important in the resistance, but might not be the only factor contributing to the resistance since the less virulent strains of P. multocida also possess capsules.     

Survival of avian strains of Pasteurella multocida in chicken serum

The ability of bacteria to survive in serum is considered a likely virulence determinant in diseases where the infective bacteria become septicaemic. Optimal conditions were established to test the survival of Pasteurella multocida in chicken serum. Serum was used at 90%, the inoculum was 10(3)-10(4)cfu in phosphate buffered saline pH 7.4. Survival was measured after incubation for 2-4 h; if survival was <50% the strain was considered serum susceptible. Susceptible strains were either killed or their growth was inhibited. Some resistant strains not only survived but grew rapidly in unheated serum. Thirty-five strains, all originally isolated from clinical fowl cholera, were tested; eight were susceptible, of which three were killed and five inhibited, and the remainder (27) were resistant. Ten serum-resistant P. multocida serogroup A strains were grown in hyaluronidase to remove the capsule and survival in chicken serum was re-tested. Three strains became susceptible, while seven strains remained resistant. Three serum susceptible strains were then tested in the presence of cytidine monophosphate-N-acetylneuraminic acid (CMP-NANA). This substance is present in the human serum, and is known to mask the effect of complement on Neisseria gonorrhoeae rendering susceptible strains resistant. Two of the three serum susceptible strains became resistant in the presence of CMP-NANA. Serum susceptibility/resistance was more complex than that of Escherichia coli, and the role of resistance to avian complement in the pathogenesis of fowl cholera remains to be determined.     

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.     

Haemolytic and cytotoxic activities of the Tween 80-extracted putative haemolysin of Pasteurella multocida B:2

The objective of this study was to investigate the haemolytic and cytotoxic activity of Pasteurella multocida B:2 strains, originally from cases of haemorrhagic septicaemia in cattle. All six P. multocida B:2 strains were non-haemolytic on sheep blood agar (SBA) and horse blood agar (HBA) when grown aerobically and on SBA anaerobically but they were haemolytic on HBA when grown anaerobically. No haemolytic activity against horse red blood cells was detected in culture supernates from aerobically or anaerobically grown cultures and only very weak haemolytic activity was obtained in supernates or pellet fractions from sonicated cells. However, after repeated extraction of sonicated cells with Tween 80, haemolytic activity was found in various cell fractions, both Tween-soluble and -insoluble. The Tween-extracted putative haemolysin and other bacterial fractions were also cytotoxic for mouse macrophage-like J774.2 cells. Further characterisation of the putative haemolysin revealed it to be a heat-labile, non-pore-forming protein of molecular weight >10 kDa whose activity was completely destroyed by trypsin and greatly reduced with protease and proteinase K treatment. Congo red also reduced the haemolytic activity. Non-denaturing gel-electrophoresis and RBC agar overlay revealed clear haemolytic zones but suggested that Tween was bound to some component of the P. multocida B:2 fractions and was responsible, to some extent, for the haemolytic activity observed. However, the effect of heat and other reagents on the Tween-extracted fractions and the lack of haemolytic activity in different Tween-extracted cell fractions of organisms other than P. multocida suggested that some proteinaceous component of the organism could indeed act as a haemolysin. This putative haemolysin may be one of the virulence attributes of P. multocida, but its characterisation and role in pathogenesis require further study.     

Expression of iron-regulated outer membrane proteins by porcine strains of Pasteurella multocida.

The outer membrane protein (OMP) profiles of two strains of capsular type A Pasteurella multocida isolated from the lungs of pigs with enzootic pneumonia were studied. Sarkosyl extracted OMPs from P. multocida grown under iron-restricted and iron-replete conditions were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analysis. Results showed that the iron-regulated outer membrane proteins (IROMPs) with molecular masses of 74 kDa, 94 kDa, 99 kDa and 109 kDa were expressed by strain A52, while 74 kDa, 82 kDa, 94 kDa and 99 kDa IROMPs were expressed by strain B80. Swine immune sera, obtained from pigs which were first immunized with a polyvalent P. multocida type A and type D bacterin and subsequently challenged with type A strain of P. multocida, contained antibodies against the IROMPs. These antibodies cross-reacted with the IROMPs expressed by avian strain P1059 of P. multocida. Convalescent-phase serum obtained from turkeys which survived fowl cholera, also cross-reacted with the IROMPs from porcine strains of P. multocida. These results suggested that IROMPs from porcine and avian strains of P. multocida may share common epitopes that were recognized by swine immune serum as well as turkey convalescent-phase serum.     

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.     

The inefficacy of polivalent Pasteurella multocida vaccines for sheep

Immunity assays on sheep sera using passive mouse protection tests showed that vaccines containing more than 4 strains of Pasteurella multocida did not give a good immunity. The immune response was not enhanced by the use of an oil adjuvant, and high concentrations of bacteria had only a partial positive effect. Attempts to extract selectively the protection-inducing antigen(s) from P. multocida by veronal, phenol or potassium thiocyanate extraction were unsuccessful. Furthermore, it was found that sheep antisera to the recognized type strains of P. multocida afforded only limited protection against a number of field strains. We concluded from this that successful immunization against ovine pasteurellosis will depend on either the identification of a strain of P. multocida that gives a wide spectrum of immunity or the discovery of a live mutant suitable for vaccine production and the definition of cultural conditions that promote the expression of a common immunizing antigen.     

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.     

Strain differences in the susceptibility and resistance of Pasteurella multocida to phagocytosis and killing by rabbit polymorphonuclear neutrophils

The interactions of 2 capsular serotype A and 4 serotype D strains of Pasteurella multocida with rabbit polymorphonuclear neutrophils (PMN) were compared in vitro, using a PMN phagocytic and bactericidal assay. Bacteria and rabbit PMN were incubated for 15 minutes. The suspensions were subjected to differential centrifugation and the percentage of phagocytosis (cell association) was determined from the number of viable noncell-associated bacteria. The cell pellets and the associated bacteria were resuspended and PMN bactericidal activity was calculated from the number of remaining viable cell-associated bacteria at 45 and 75 minutes after the start of the assay. Test bacteria were not opsonized or were opsonized with immune serum containing active complement. One type A strain was ingested and killed by PMN in the presence and absence of opsonins. The 5 remaining strains were resistant to PMN killing, but only the type A strain resisted phagocytosis. Resistance of the type A strain was attributed to the hyaluronic acid capsule, since pretreatment of the bacteria with hyaluronidase rendered opsonized bacteria susceptible to ingestion and killing. The pattern of resistance of the 4 type D strains was different from that of the resistant type A strain. Both opsonized and nonopsonized type D bacteria became cell associated, but none were killed by PMN. The mechanism of resistance of these 4 strains to PMN bactericidal activity is currently unknown.     

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.     

Turkey macrophage and heterophil bactericidal activity against Pasteurella multocida.

Bactericidal activity of turkey macrophages and heterophils was demonstrated in an in vitro colorimetric bactericidal assay. Two vaccine strains and one field isolate of Pasteurella multocida A:3,4 and a single isolate each of Escherichia coli and Staphylococcus aureus were compared for susceptibility to the bactericidal activity of turkey macrophages and heterophils. Only P. multocida A:3,4-strain M-9 (the least virulent strain) was susceptible to macrophage bactericidal activity in the absence of specific immune serum, whereas all three P. multocida A:3,4 organisms were killed when opsonized with specific immune serum. E. coli was susceptible to the bactericidal activity of macrophages, and S. aureus was resistant. All bacteria tested were highly sensitive to the bactericidal activity of intact turkey heterophils, regardless of the opsonin treatment. Electron microscopic findings suggested that heterophils may kill extracellular P. multocida. Only S. aureus and E. coli were killed by lysed heterophils.     

鸡传染性贫血病毒VP1、VP2基因的表达及免疫原性研究

为探索鸡传染性贫血重组蛋白亚单位疫苗的可行性,将鸡传染性贫血病毒基因分别克隆到转移载体p Fast Bac HTA中,再将其分别转化DH10Bac感受态细胞得到相应的重组表达载体,转染昆虫细胞Sf21获得含有VP1、VP2基因的重组杆状病毒v Bac-VP1、v Bac-VP2,应用悬浮培养的Sf21细胞表达重组蛋白。SDS-PAGE及Western blotting结果证明,VP1、VP2基因在昆虫细胞中得到了表达,动物试验进一步证实,重组蛋白免疫SPF鸡可产生ELISA抗体,提示杆状病毒表达的VP1、VP2具有良好的免疫原性。     

云南江城蓝舌病病毒16型毒株分离鉴定及其L2基因序列分析

为了解近年来云南江城县蓝舌病病毒16型（Bluetongue virus type 16,BTV-16）毒株的流行情况及其L2基因与国外流行株的遗传进化关系,本研究将江城县送检的300份牛肝素钠抗凝血提取红细胞后静脉接种10日龄鸡胚,将收集的鸡胚肝脏捣碎离心,上清液接种于C6/36和BHK21细胞传代。针对出现细胞病变的样品,应用群特异性VP7片段引物进行RT-PCR检测,应用BTV-16 L2基因特异性引物对检测出的BTV核酸阳性样品进行RT-PCR扩增和测序,采用DNAStar和Mega 6.0软件对获得的L2基因编码区序列进行核苷酸、氨基酸同源性比对及遗传进化分析,同时利用BTV-16标准阳性血清对分离到的病毒进行中和试验鉴定。结果显示,江城县发现30个可致细胞病变的样品,其中17个样品经RT-PCR初步确认为BTV;经L2基因序列分析和中和试验鉴定,确定其中6株为BTV-16型毒株;核苷酸、氨基酸同源性比对分析结果显示,6个毒株核苷酸和氨基酸同源性分别在93.4%~98.0%和94.2%~99.1%之间;遗传进化分析发现,其中5株与2001-2008年日本及1982-2011年印度分离的BTV-16毒株亲缘关系较近;1株与1985-1990年日本分离的BTV-16毒株亲缘关系较近。本研究发现,云南江城县BTV-16毒株呈现新旧毒株交叉持续流行态势,但在自然进化中遗传变异不大,有一定的稳定性,本研究在分子水平阐明了云南江城县地方流行BTV-16 L2基因间的遗传和差异,为进一步开展BTV分子流行病学及检测研究提供科学依据。     

Virulence and toxigenicity of capsular serogroup D Pasteurella multocida strains isolated from avian hosts

Five capsular serogroup D strains of Pasteurella multocida isolated from avian hosts were examined for virulence and toxigenicity. Virulence was based on development of lethal infections or lesions following intramuscular exposure of turkey poults. The four strains isolated from turkeys varied from slightly to moderately virulent; the strain isolated from a chicken was avirulent. Poults exposed by intra-airsac inoculation with relatively few organisms of the more virulent of the strains had a high mortality rate; however, intranasal exposure of poults with this strain did not cause clinical disease or establish infections. All strains from turkeys were toxigenic, producing heat-labile toxins that killed poults when administered intraperitoneally and caused focal dermal lesions when administered intradermally. Using these criteria, the strain from a chicken was not toxigenic. The demonstration of virulence, particularly the high mortality in poults exposed via air sacs, indicates avian capsular serogroup D strains are a potential cause of fowl cholera.     

The virulence and protective efficacy for chickens of pasteurella multocida administered by different routes

The relative virulence for chickens of five strains of Pasteurella multocida was evaluated. Twenty groups, each of ten chickens, were inoculated with a standard dose of 10(5) of each of five strains by the intramuscular (I.m.), intravenous (I.v.), intratracheal (I.tr.) or conjunctival (Co) routes. The highest mortality occurred in the groups dosed I.m. and I.v., followed by I.tr. inoculation. The relative virulence of each strain did not change when inoculated by the different routes. The most virulent strain, VP161, caused 100% mortality by all except the Co route. The least virulent strain, VP17, caused a single mortality by the I.v. route, but gave a high level of protection to birds inoculated by both the I.m. and I.v. routes, when challenged by intramuscular injection with (VP161). There was no protection against I.m. challenge in the birds inoculated by the I.tr. or Co routes. Serum antibody levels measured by ELISA correlated with the level of protection against virulent challenge for groups inoculated I.m. or I.v., but not I.tr. Western blots of pooled sera from each group did not show any specific antigen recognition that might explain the observed differences in protection. Inoculation with strain VP17, (both I.m. and I.tr.) also gave a high level of protection to birds challenged with strain VP161 by intratracheal instillation.     

The molecular epidemiology of four outbreaks of porcine pasteurellosis

Biochemical profiles, restriction endonuclease analysis (REA) and ribotyping were used to investigate a total of 38 Pasteurella multocida isolates from four separate outbreaks of pasteurellosis in Australian piggeries. Six isolates were obtained from Outbreak 1, 16 from Outbreak 2 and eight each from outbreaks 3 and 4. Outbreaks 1 and 2 were cases of pneumonic pasteurellosis while outbreaks 3 and 4 involved systemic pasteurellosis. Biochemical characterisation established that a number of different types of P. multocida were present in outbreaks 1 and 3 while outbreaks 2 and 4 were associated with a single type of P. multocida. Outbreaks 1 and 3 yielded isolates of P. multocida that belonged to the subspecies multocida and gallicida, with the subspecies multocida isolates being identified as biovar 3 (6 in total) or 12 (1 in total) and the subspecies gallicida isolates (7 in total) being identified as biovar 8. All 24 isolates from outbreaks 2 and 4 belonged to the subspecies multocida and were all biovar 3. REA and ribotyping showed that, in outbreaks 1 and 3, there were three different types of P. multocida in each outbreak with no common strains between the outbreaks. The molecular methods showed that only a single strain of P. multocida was associated with outbreaks 2 and 4, although the outbreaks were associated with strains that differed in REA profiles but shared a ribotype profile. This study has shown that both, systemic and pneumonic pasteurellosis can be associated with either a single strain or multiple strains of P. multocida. The results also indicate that the molecular typing methods of REA and ribotyping are superior to biochemical characterisation for epidemiological investigation of porcine pasteurellosis.     

利用BIOLOG系统对不同种类细菌鉴定的研究

为完善细菌鉴定工作,本试验通过利用BIOLOG细菌自动微生物分析系统(简称BIOLOG系统)对13株大肠杆菌、15株巴氏杆菌和7株金黄色葡萄球菌进行了鉴定,并与传统方法[1]包括形态学观察、革兰氏染色、生化实验等的鉴定结果进行了比对.实验结果表明,与传统方法相比BIOLOG系统具有准确、快捷、简便的优点,同时也有一定的局限性,从而为BIOLOG系统的更好使用提供了实验依据.     

In vivo antigen expression by Pasteurella multocida.

Pasteurella multocida was purified from the blood of turkeys affected with acute fowl cholera, and membrane preparations from those bacteria were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and visualized on immunoblots. Antigens were detected in the membranes of these in vivo-propagated bacteria that were not detected in membrane preparations of the same P. multocida strain grown in vitro. The unique antigens were detected in the detergent-insoluble phase and were enriched to various degrees by different detergents.     

Functional activities of the Tsh protein from avian pathogenic Escherichia coli (APEC) strains

The temperature-sensitive hemagglutinin (Tsh) expressed by strains of avian pathogenic Escherichia (E.) coli (APEC) has both agglutinin and protease activities. Tsh is synthesized as a 140 kDa precursor protein, whose processing results in a 106 kDa passenger domain (Tsh_s) and a 33 kDa β-domain (Tsh_β). In this study, both recombinant Tsh (rTsh) and supernatants from APEC, which contain Tsh_s (106 kDa), caused proteolysis of chicken tracheal mucin. Both rTsh (140 kDa) and pellets from wild-type APEC, which contain Tsh_β (33 kDa), agglutinated chicken erythrocytes. On Western blots, the anti-rTsh antibody recognized the rTsh and 106 kDa proteins in recombinant E. coli BL21/pET 101-Tsh and in the supernatants from APEC grown at either 37℃ or 42℃. Anti-rTsh also recognized a 33 kDa protein in the pellets from APEC13 cultures grown in either Luria-Bertani agar, colonization factor antigen agar, or mucin agar at either 26℃, 37℃, or 42℃, and in the extracts of outer membrane proteins of APEC. The 106 kDa protein was more evident when the bacteria were grown at 37℃ in mucin agar, and it was not detected when the bacteria were grown at 26℃ in any of the culture media used in this study. Chicken anti-Tsh serum inhibited hemagglutinating and mucinolytic activities of strain APEC13 and recombinant E. coli BL21/pET101-Tsh. This work suggests that the mucinolytic activity of Tsh might be important for the colonization of the avian tracheal mucous environment by APEC.     

传染性法氏囊病病毒VP2基因原核表达及抗原性分析

从辽宁省某鸡场分离到一株传染性法氏囊病病毒(IBDV)。以该毒株基因组核酸为模板,应用RT-PCR扩增得到VP2基因,构建表达质粒pET28a-VP2,再将其转化至大肠埃希菌BL21(DE3)用IPTG进行诱导表达。表达产物经SDS-PAGE分析在48ku处出现特异性蛋白条带;经Western blot分析VP2蛋白可以与IBDV阳性血清发生特异性反应。用VP2蛋白制备的油乳剂疫苗免疫接种SPF鸡,2周后体内可以检测到特异性抗体。证明VP2蛋白具有重要的应用价值,为IBDV基因工程亚单位疫苗的研制奠定了基础。