A Novel 16S rRNA PCR-Restriction Fragment Length Polymorphism Assay to Accurately Distinguish Zoonotic Capnocytophaga canimorsus and C. cynodegmi.

The zoonotic bacteria Capnocytophaga canimorsus and C. cynodegmi, the predominant Capnocytophaga species in the canine oral biota, can cause human local wound infections or lethal sepsis, usually transmitted through dog bites. Molecular surveying of these Capnocytophaga species using conventional 16S rRNA-based PCR is not always accurate due to their high genetic homogeneity. In this study, we isolated Capnocytophaga spp. from the canine oral cavity and identified them using 16S rRNA and phylogenetic analysis. A novel 16S rRNA PCR-restriction fragment length polymorphism (RFLP) method was designed based on our isolates and validated using published C. canimorsus and C. cynodegmi 16S rRNA sequences. The results showed that 51% of dogs carried Capnocytophaga spp. Among these, C. cynodegmi (47/98, 48%) was the predominant isolated species along with one strain of C. canimorsus (1/98, 1%). Alignment analysis of 16S rRNA sequences revealed specific site nucleotide diversity in 23% (11/47) of the C. cynodegmi isolates, which were misidentified as C. canimorsus using previously reported species-specific PCR. Four RFLP types could be classified from all the isolated Capnocytophaga strains. The proposed method demonstrates superior resolution in distinguishing C. cynodegmi (with site-specific polymorphism) from C. canimorsus and especially in distinguishing C. canimorsus from other Capnocytophaga species. After in silico validation, this method was revealed to have an overall detection accuracy of 84%; notably, accuracy reached 100% in C. canimorsus strains isolated from human patients. Overall, the proposed method is a useful molecular tool for the epidemiological study of Capnocytophaga in small animals and for the rapid diagnosis of human C. canimorsus infections. IMPORTANCE With the increased number of small animal breeding populations, zoonotic infections associated with small animals need to be taken more seriously. Capnocytophaga canimorsus and C. cynodegmi are part of common biota in the mouths of small animals and can cause human infections through bites or scratches. In this study, C. cynodegmi with site-specific 16S rRNA sequence polymorphisms was erroneously identified as C. canimorsus during the investigation of canine Capnocytophaga by conventional PCR. Consequently, the prevalence of C. canimorsus is incorrectly overestimated in epidemiological studies in small animals. We designed a new 16S rRNA PCR-RFLP method to accurately distinguish zoonotic C. canimorsus from C. cynodegmi. After validation against published Capnocytophaga strains, this novel molecular method had high accuracy and could detect 100% of C. canimorsus-strain infections in humans. This novel method can be used for epidemiological studies and the diagnosis of human Capnocytophaga infection following exposure to small animals.

Molecular Characterization and Pathogenicity of the Novel Recombinant Muscovy Duck Parvovirus Isolated from Geese.

Goose parvovirus (GPV) and Muscovy duck parvovirus (MDPV) are the main agents associated with waterfowl parvovirus infections that caused great economic losses in the waterfowl industry. In 2020, a recombinant waterfowl parvovirus, 20-0910G, was isolated in a goose flock in Taiwan that experienced high morbidity and mortality. The whole genome of 20-0910G was sequenced to investigate the genomic characteristics of this isolate. Recombination analysis revealed that, like Chinese rMDPVs, 20-0910G had a classical MDPV genomic backbone and underwent two recombination events with classical GPVs at the P9 promoter and partial VP3 gene regions. Phylogenetic analysis of the genomic sequence found that this goose-origin parvovirus was highly similar to the circulating recombinant MDPVs (rMDPVs) isolated from duck flocks in China. The results of experimental challenge tests showed that 20-0910G caused 100% mortality in goose embryos and in 1-day-old goslings by 11 and 12 days post-inoculation, respectively. Taken together, the results indicated that this goose-origin rMDPV was closely related to the duck-origin rMDPVs and was highly pathogenic to young geese.

Preparation of Chicken Anemia Virus (CAV) Virus-Like Particles and Chicken Interleukin-12 for Vaccine Development Using a Baculovirus Expression System.

Chicken infectious anemia (CIA) is a poultry disease that causes huge economic losses in the poultry industry worldwide. Commercially available CIA vaccines are derived from wild-type chicken anemia viruses (CAVs) by serial passage in cells or chicken embryos. However, these vaccinal viruses are not completely attenuated; therefore, they can be transmitted vertically and horizontally, and may induce clinical symptoms in young birds. In this study, we sought to eliminate these issues by developing a subunit vaccine exploiting the CAV structural proteins, engineering recombinant baculovirus-infected Spodoptera frugiperda (Sf9) cells that contained both the viral protein 1 (VP1) and VP2 of CAV. Moreover, we produced single-chain chicken interleukin-12 (chIL-12) in the same system, to serve as an adjuvant. The recombinant VP1 was recognized by chicken anti-CAV polyclonal antibodies in Western blotting and immunofluorescence assays, and the bioactivity of the recombinant chIL-12 was confirmed by stimulating interferon-γ (IFN-γ) secretion in chicken splenocytes. Furthermore, the ability of the recombinant VP1 to generate self-assembling virus-like particles (VLPs) was confirmed by transmission electron microscopy. Specific pathogen-free (SPF) chickens inoculated with VLPs and co-administered the recombinant chIL-12 induced high CAV-specific antibodies and cell-mediated immunity. Taken together, the VLPs produced by the baculovirus expression system have the potential to be a safe and effective CIA vaccine. Finally, we demonstrated the utility of recombinant chIL-12 as an adjuvant for poultry vaccine development.

Prevalence and Genotyping of Chlamydia psittaci from Domestic Waterfowl, Companion Birds, and Wild Birds in Taiwan.

Chlamydia psittaci, the causative agent of avian chlamydiosis, an important zoonotic disease, infects a wide range of birds. Infected birds, whether symptomatic or asymptomatic, intermittently shed the agent through respiratory and intestinal routes. Therefore, it is essential to investigate the epizootiology of C. psittaci in poultry, pet birds, and wild birds. In this study, cloacal or fecal swabs collected from domestic waterfowl, psittacine birds, Columbidae, and wild birds were used to determine the prevalence of C. psittaci in Taiwan between 2014 and 2017. The C. psittaci infection rate was as high as 34.2% among domestic waterfowl farms. The waterfowl isolates clustered into two groups based on ompA phylogeny: one group (G1-like) clustered with the Polish G1 strains; the other group (waterfowl-TW) clustered near, but independently from, the classical ABE genotype cluster. Separately, 3.1% of parrot samples tested positive for C. psittaci belonging to genotype A. C. psittaci isolates of genotype B were detected in 10.1% of racing pigeons and other Columbidae. Wild bird samples from a wildlife refuge had a 2.2% prevalence rate; among these, two atypical C. psittaci genotypes were detected in samples from a Malayan night heron (Gorsachius melanolophus) and a Taiwan barbet (Megalaima nuchalis). Taken together, our results revealed that the risk of C. psittaci transmission from domestic waterfowl and Columbidae birds to humans could be underestimated, given the high prevalence rates in these birds. Furthermore, the free-range rearing system of waterfowl in Taiwan may promote C. psittaci transmission between poultry and wild birds. Pet birds and racing pigeons, which are in close contact with people, are also possible sources for cross-species transmission. Further studies are necessary to elucidate the virulence, biological and genetic characteristics, and modes of transmission of Taiwanese C. psittaci isolates to facilitate the prevention and control of C. psittaci infection.

Sequence diversity and associated pathogenicity of the hemagglutinin cleavage site of H5N2 avian influenza viruses isolated from chickens in Taiwan during 2013-2015.

The sequence at the hemagglutinin (HA) cleavage site (CS) plays a key role in determining the pathogenicity of avian influenza viruses. Three types of HA CS sequences, QREKR/GL, QRKKR/GL and QRRKR/GL, were previously reported in Taiwanese H5N2 viruses that were isolated from chickens from 2003 to 2013. However, no HA CS sequence was reported for viruses isolated after 2013. This article presents the HA CS sequences and pathogenicity of H5N2 viruses that were isolated from chickens in Taiwan during 2013-2015. Two novel HA CS sequences, QKEKR/GL and KREKREKR/GL, were found in the viruses isolated in 2013 and 2014, and pathogenicity tests showed that the viruses with these novel HA CS sequences are low and high pathogenic viruses, respectively. In contrast, the HA CS sequence QREKR/GL was found in all viruses that were isolated in 2015, and all of these viruses were low pathogenic viruses. After 10 passages in embryonated chicken eggs, a virus strain that was isolated in 2003 evolved into a viral quasispecies that contained at least four distinct types of HA CS sequences. These results highlight the potential of Taiwanese H5N2 viruses to change their pathogenicity and HA CS sequences via mutations. Furthermore, viruses with the HA CS sequence QREKR/GL were more prevalent than others in 2015. These findings are useful for understanding the mechanism of sequence changes at the HA CS and for refining H5N2 virus control measures in Taiwan.

Loss of the Capsule Increases the Adherence Activity but Decreases the Virulence of Avibacterium paragallinarum.

Avibacterium paragallinarum is the causative agent of infectious coryza, an important respiratory disease of chickens. The capsule is an important virulence determinant of many pathogenic bacteria, but the function of the capsule in Av. paragallinarum is not well defined. In this study, acapsular mutants of Av. paragallinarum were constructed by inactivation of the hctA gene using the TargeTron gene knockout system. The acapsular mutants were found to have greater hemagglutination activity than did the wild-type strain. Further, acapsular mutants exhibited an increased ability to adhere to DF-1 cells and to form biofilms on abiotic surfaces. Virulence assays showed that acapsular mutants were less virulent than the wild-type strain. Taken together, these results indicated that loss of capsule increases hemagglutination and adhesion activities but decreases the virulence of Av. paragallinarum. These results could be valuable to further elucidate the function of the capsule and the mechanism of pathogenicity of Av. paragallinarum.

The haemagglutinin of Avibacterium paragallinarum is a trimeric autotransporter adhesin that confers haemagglutination, cell adherence and biofilm formation activities.

The haemagglutinin (HA) protein plays a key role in the immunogenicity and pathogenicity of Avibacterium paragallinarum. A 210-kDa protein (HMTp210) was previously reported to be the HA of Av. paragallinarum, but the biological function of HMTp210 is not well defined. In this study, mutant strains that lacked HMTp210 were constructed using the TargeTron(®) gene knockout system. Haemagglutination and haemagglutination-inhibition (HI) assays showed that the HMTp210-deficient mutants exhibited no HA activity and failed to elicit HI antibodies in immunized chickens. Additionally, HMTp210-deficient mutants exhibited reduced ability to adhere to HeLa cells and to form biofilms on abiotic surfaces. Virulence assays showed that HMTp210-deficient mutants are less virulent than their isogenic wild-type strains. HMTp210 bears significant similarity to proteins of the trimeric autotransporter adhesin (TAA) family, and recombinant HMTp210 expressed in E. coli formed a trimeric structure. Taken together, these results indicated that HMTp210 is a trimeric autotransporter adhesin that confers haemagglutination, cell adherence and biofilm formation activities. These results should prove valuable to further elucidate the biological function of HA and the mechanism of pathogenicity of Av. paragallinarum.

Anti-influenza virus activity of the ethanolic extract from Peperomia sui.

ETHNOPHARMACOLOGICAL RELEVANCE: Peperomia sui Lin and Lu (Peperomia sui), a well-known Taiwanese folk medicine, has a broad range of biological effects, especially in treatment of upper respiratory tract diseases. However, no previous study has explored the activity of Peperomia sui against influenza virus infections. This study was carried out to evaluate the anti-influenza virus activity and the potential virucidal effect of the ethanolic extract of Peperomia sui (PSE). METHODS: The anti-H6N1 avian influenza viral activity of PSE against the influenza virus A/Chicken/TW/0518/2011 (H6N1) in chicken fibroblast DF-1 cells was evaluated by cell viability assay, hemagglutination assay, neuraminidase activity assay, indirect immunofluorescence assay and quantitative RT-PCR assay. RESULTS: PSE significantly increased the viability of cells that were infected by the H6N1 virus. PSE also suppressed the synthesis of viral nucleoprotein (NP), and inhibited the growth of the virus in DF-1 cells. Further, PSE inhibited the neuraminidase activity of H6N1 virus. CONCLUSIONS: The findings of this study provide important information for the exploitation and utilization of Peperomia sui in treatment of influenza infection.

Identification and characterization of an RTX toxin-like gene and its operon from Avibacterium paragallinarum.

Avibacterium paragallinarum is the causative agent of infectious coryza, an important respiratory disease of chickens. Whole-genome sequencing analysis showed that A. paragallinarum strain H18 contains an RTX toxin-like operon with strong similarity to the RTX operons of other members of the Pasteurellaceae. Four genes, designated avxIC, avxIA, avxIB, and avxID, were found in this operon. The avxIA gene encodes the structural RTX toxin-like protein, which has a predicted molecular mass of about 250 kDa. The AvxIA protein contains a peptidase S8 domain and a proprotein convertase P-domain, neither of which has been found in other RTX toxins. Recombinant AvxIA proteins expressed in Escherichia coli showed neither hemolytic nor cytotoxic activity. Polymerase chain reaction and sequencing analysis revealed that the avxIA gene was present in all strains and field isolates of A. paragallinarum examined in this study. Sera collected from chickens exposed to A. paragallinarum exhibited strong reactivity to the AvxIA protein, which suggests that AvxIA is immunogenic. This is the first report of the identification of an RTX toxin-like operon from A. paragallinarum. The gene products of this operon may be related to disease pathogenesis and potentially represent a useful vaccine target of A. paragallinarum.

Polymerase chain reaction-restriction fragment length polymorphism analysis of the genes involved in the biosynthesis of the lipopolysaccharide of Pasteurella multocida.

The lipopolysaccharide, also known as the somatic antigen or O-antigen, is an important virulence factor of Pasteurella multocida. In the current study, the genes involved in the biosynthesis of the outer core region of the lipopolysaccharide, which were obtained from somatic type reference strains and field strains of P. multocida, were subjected to polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis. The PCR-RFLP analysis classified 11 out of the 16 serotypes into 5 PCR-RFLP types (I-V). Types I and V contain strains belong to serotypes 1 and 13, respectively. The rest of the PCR-RFLP types contain strains belong to certain groups of serotypes. Typing of 38 field strains from poultry using PCR-RFLP analysis and the gel diffusion precipitation test showed consistent results. These results indicate that the PCR-RFLP analysis can be a useful tool for rapid somatic typing of some strains of P. multocida.

Different immune responses to three different vaccines following H6N1 low pathogenic avian influenza virus challenge in Taiwanese local chicken breeds.

BACKGROUND: H6N1 low pathogenic avian influenza virus (LPAIV) are frequently isolated in Taiwan and lead to significant economic losses, either directly or indirectly through association with other infectious diseases. This study investigates immune responses to three different vaccines following a H6N1 challenge in different local breeds. METHODS: Experimental animals were sampled from six local chicken breeds maintained at the National Chung-Hsing University, namely Hsin-Yi, Ju-Chi, Hua-Tung (Taiwan), Quemoy (Quemoy Island), Shek-Ki (China), Nagoya (Japan) and a specific pathogen free (SPF) White Leghorn line. A total number of 338 chickens have been distributed between a control and a challenge group, H6N1 challenge was performed at 7 weeks of age; vaccination against Newcastle Disease (ND), Infectious Bursal Disease (IBD) and Infectious Bronchitis (IB) was performed at 11 weeks. The anti-H6N1 LPAIV antibody titers were measured by ELISA at days 0, 7, 14 and 21 after challenge, and the anti-ND, anti-IBD and anti-IB antibody titers were measured by inhibition of hemagglutination test and ELISA at days 0, 14, 28 after vaccination. RESULTS: There was no effect of the H6N1 LPAIV challenge at 7 weeks of age on the subsequent responses to ND and IBD vaccine at 11 weeks of age, but, surprisingly, the H6N1 LPAIV challenge significantly affected antibody levels to IB vaccine in some breeds, since IB0 and IB14 antibody titers were lower in the challenge groups. However, there was no significant difference in IB28 antibody titers among the experimental groups. CONCLUSIONS: Local breeds have different immune response to H6N1 LPAIV challenge and subsequent vaccines. Differences dealt mainly with kinetics of response and with peak values. Quemoy exhibited higher antibody levels to H6N1, ND and IBD. The negative effect of the H6N1 LPAIV challenge on IB vaccine response may be related to the fact that both viruses target the lung tissues, and the type of local immune response induced by LPAIV challenge may not be favourable for birds to make optimum IB-specific antibody response.

Recombinant proteins containing the hypervariable region of the haemagglutinin protect chickens against challenge with Avibacterium paragallinarum.

The haemagglutinin (HA) protein plays a key role in the immunogenicity and pathogenicity of Avibacterium paragallinarum, but the domain organization and antigenicity exhibited by different domains of this protein remain unknown. This study reports the presence of a hypervariable region in the HA proteins of strains of serovars A and C of A. paragallinarum. This hypervariable region is located approximately at residues 1100-1600 of the HA protein. The sequence identity found in this hypervariable region was only 18.1%, whereas those upstream and downstream of this region were 83.8 and 97.8%, respectively. Western blot analyses using antisera against the whole-cell antigens of A. paragallinarum showed that the hypervariable region was more antigenic than other regions of the HA protein. Moreover, the antigenicity of the hypervariable region was serovar-specific. Chickens immunized with recombinant proteins that contained the hypervariable region were protected (83-100% protection rate) against challenge infection with A. paragallinarum of the homologous serovar. These results suggest that recombinant proteins containing the hypervariable region may be useful antigens for use in the development of a vaccine against A. paragallinarum.

Baculovirus as an avian influenza vaccine vector: differential immune responses elicited by different vector forms.

Baculovirus is an enveloped virus that infects insects in nature and has emerged as a novel vaccine vector. We previously constructed a recombinant baculovirus displaying the hemagglutinin protein (HA) of avian influenza virus (AIV) on the viral envelope (Bac-HA64), and demonstrated the induction of humoral responses in immunized mice. To improve the vector design and explore how the vector forms influence the vaccine efficacy, we constructed two more baculoviruses Bac-CHA and Bac-CHA/HA64. Bac-CHA expressed HA after transducing the host cells while Bac-CHA/HA64 not only expressed HA but also displayed HA on the envelope. After administration into BALB/c mice, all three vectors elicited HA-specific humoral (IgG1, IgG2a and hemagglutination inhibition titers), mucosal (IgA titers) and cellular (interferon (IFN)-γ and IL-4 producing T cells and IFN-γ(+)/CD8(+) T cells) immune responses. Intriguingly, the magnitudes and types of responses hinged on the vaccine form and administration route. Via intranasal (i.n.) and subcutaneous (s.c.) inoculation, the HA-displaying vectors Bac-HA64 and Bac-CHA/HA64 triggered stronger humoral and mucosal responses than Bac-CHA, but upon intramuscular (i.m.) injection the HA-expressing vectors (Bac-CHA and Bac-CHA/2HA64) elicited more robust humoral and cellular responses than Bac-HA64. Via either administration route, the dual form vaccine Bac-CHA/HA64 gave rise to superior or at least comparable HA-specific immune responses than the other two vaccine forms, implicating the potential of Bac-CHA/HA64 as a vaccine candidate against AIV infection.

Genetic and antigenic characterization of foot-and-mouth disease viruses isolated in Taiwan between 1998 and 2009.

A devastating outbreak of foot-and-mouth disease (FMD), caused by a porcinophilic serotype O virus, occurred in Taiwan in March 1997. This outbreak was brought under control by means of a stamping-out policy and vaccination. Although mandatory vaccination was conducted in Taiwan between 1997 and 2007, sporadic outbreaks of FMD occurred between 1998 and 2009; however, the viruses that caused these outbreaks remain uncharacterized. This article reports the genetic and antigenic characterization of FMD viruses isolated in Taiwan during this period. Sequence analysis of the VP1 coding region showed that the viruses isolated in Taiwan between 1998 and 2009 were most similar to viruses isolated in Taiwan in 1997 and to viruses isolated from Hong Kong and Vietnam in 1991-1996. The results of phylogenetic analysis suggested that the viruses isolated in Taiwan in 1998-2009 were derived from the viruses isolated in Taiwan in 1997. However, substantial mutations were found in the viruses isolated in 2009, and some of these changes may have resulted from vaccine pressure in the field. Serum neutralization tests confirmed that viruses isolated in 2009 showed a significant change in antigenicity. This is the first report of changes in the VP1 sequence and antigenicity of porcinophilic FMD viruses isolated from an area in which long-term mandatory vaccination against FMD was practiced.

Analysis of the biosynthesis genes and chemical components of the capsule of Avibacterium paragallinarum.

The aim of this study was to investigate biosynthesis genes and chemical components of the capsule of Avibacterium paragallinarum. The sequence of a 10-kb region containing the capsule biosynthetic locus of Av. paragallinarum was determined. Two reference strains, i.e., 221 (serovar A) and H18 (serovar C), together with four Taiwanese field strains (all serovar C) were sequenced. The results showed that there are two genotypes (I and II) of the capsule biosynthetic locus in Av. paragallinarum, and the capsule genotype is independent of the serovar. The capsule biosynthetic loci of genotypes I and II consisted of six and five genes, respectively. The genotype I genes encoded proteins that are most similar to proteins from Pasteurella multocida capsule types A and F while the genotype II genes encoded proteins most similar to proteins from P. multocida capsule type D and Escherichia coli K5. The results suggested that genotype I strains contain hyaluronan or chondroitin in the capsule wall while genotype II contain heparosan. Enzymatic digestion of the capsule materials extracted from Av. paragallinarum showed that genotype I strains contained chondroitin while genotype II strains contained heparosan in the capsule. This is the first report on the existence of different genotypes of capsule biosynthesis genes in Av. paragallinarum and the presence of chondroitin and heparosan as chemical components of the capsule of Av. paragallinarum.

Surveillance of avian and swine influenza in the swine population in Taiwan, 2004.

BACKGROUND AND PURPOSE: We conducted serological and virological surveillance of pig farms in Taiwan from areas epidemic for low pathogenic avian influenza virus (AIV), H5N2 subtype, in order to determine the prevalence of AIV and swine influenza virus (SIV) in 2004. METHODS: Pig sera from 9833 animals from 1974 farms in 9 counties were examined using agar gel precipitation (AGP) to screen for the presence of antibody against influenza A virus. AGP-positive sera were subjected to hemagglutination-inhibition test against H1, H3, H5 and H7 AIV subtypes and H1 and H3 SIV subtypes. Nasal swabs from 881 pigs were also examined for the presence of SIV by virus isolation in specific pathogen-free embryonated chicken eggs. Virus isolates were identified by reverse transcriptase-polymerase chain reaction followed by DNA sequencing of hemagglutinin and neuraminidase genes. RESULTS: The AGP test on sera revealed the presence of antibodies against influenza A virus in 62.6% of farms and in 37.7% of pig sera. SIV antibodies to subtype H1 and H3 were found in 10.8% and 65.8% of sera, respectively. There were two peaks of the serological prevalence of SIV in pigs: one between January and February, and the other in October. By contrast, hemagglutinin tests against H5 and H7 AIV subtypes were negative in all sera, while there was a very low positive rate against H1 and H3 AIV subtypes. One H1N2 and one H3N1 viral isolate were obtained from nasal swabs of pigs. Phylogenetic analysis of hemagglutinin and neuraminidase genes revealed both isolates were reassortants of both classical and recent SIVs. CONCLUSIONS: Different subtypes of SIV co-circulate among swine from different farms within the same county and may cause clinical outbreaks of the disease in Taiwan.

Development of blocking ELISA for detection of antibodies against avian influenza virus of the H7 subtype.

BACKGROUND AND PURPOSE: The conventional method used for subtyping of antibodies against avian influenza viruses is hemagglutination inhibition (HI) test. However, the HI test is laborious and requires preparation of antigen from viable viruses that might be hazardous. The aim of this study was to develop a blocking enzyme-linked immunosorbent assay (B-ELISA) for detection of antibody of avian influenza of the H7 subtype. The B-ELISA is fast and avoids the need to culture whole viruses. METHODS: The B-ELISA was based on the reaction between a monoclonal antibody and a recombinant hemagglutinin protein purified from Escherichia coli. The specificity of the B-ELISA was determined by testing H7-negative field sera and the sensitivity of the B-ELISA was determined by testing sera collected from experimentally immunized chickens. RESULTS: The specificity of the B-ELISA was found to be 97.7% when compared with the HI test. The sensitivity was found to vary with the HI titer of sera. A sensitivity of 100% was achieved when test sera had HI titers >or=2(7). The sensitivity dropped to 33% and 20% when test sera had HI titers of 2(6) and 2(5), respectively. Nearly all test sera with HI titers

Protective immunity conferred by recombinant Pasteurella multocida lipoprotein E (PlpE).

The genes encoding Pasteurella multocida lipoprotein E (PlpE) and lipoprotein B (PlpB) were cloned from P. multocida strain X-73 (serotype A:1) and expressed in Escherichia coli. The protective immunity conferred by recombinant PlpE (r-PlpE) and PlpB (r-PlpB) on mice and chickens was evaluated. The results showed that mice immunized with 10microg of purified r-PlpE were protected (80-100% survival rate) against challenge infection with 10 or 20 LD(50) of P. multocida strains X-73 (serotype A:1), P-1059 (serotype A:3) and P-1662 (serotype A:4). In contrast, mice immunized with r-PlpB were not protected. Chickens immunized with 100microg of purified r-PlpE were protected (63-100% survival rate) against lethal challenge infection with strains X-73 and P-1662, whereas those immunized with r-PlpB were not. Sequence analyses showed that PlpE from different strains of P. multocida exhibited 90.8-100% sequence identity to each other, suggesting that PlpE might serve as a cross-protective antigen. This is the first report of a recombinant P. multocida antigen that confers cross protection on animals.

Immunogenicity and haemagglutination of recombinant Avibacterium paragallinarum HagA.

Inactivated vaccines of Avibacterium paragallinarum provide protection and reduce the economic losses caused by infectious coryza. However, inactivated bacterins provide protection only against the Page serovars included in the vaccine. In this study, we investigated the immunological properties of a functional recombinant haemagglutinin protein (rHagA) derived from a Taiwan isolate strain A9 as the immunogen for vaccination. The rHagA subunit vaccine protected 71% of immunized chickens against 10(10) colony-forming units (CFU) of viable A9. Vaccinated chickens which showed no clinical signs of coryza developed haemagglutination inhibition (HI) titers of 1:10 or greater. Haemagglutination (HA) of serovars A and C was not affected by the presence of rHagA specific antiserum. The HA of rHagA could only be induced against formaldehyde-fixed chicken red blood cells (FA-RBCs). These results suggested that HagA is a moderate immunogen and might not be a major haemagglutinin in vivo. However, HagA might be involved in haemagglutination when treated serovar C aggregates fixed RBCs in vitro.

Cloning of the gene and characterization of the enzymatic properties of the monomeric alkaline phosphatase (PhoX) from Pasteurella multocida strain X-73.

We have identified a new phoX gene encoding the monomeric alkaline phosphatase from Pasteurella multocida X-73. This gene was not found in the published genome sequence of Pasteurella multocida pm70. Characterization of the recombinant PhoX of Pasteurella multocida X-73 showed that it is a monomeric enzyme, activated by Ca(2+) and possibly secreted by the Tat pathway. These features distinguish phosphatases of the PhoX family from those of the PhoA family. All proteins of the PhoX family were found to contain a conserved motif that shares significant sequence homology with the calcium-binding site of a phosphotriesterase known as diisopropylfluorophosphatase. Site-directed mutagenesis revealed that D527 of PhoX might be the ligand bound to the catalytic calcium. This is the first report on identification of homologous sequences between PhoX and the phosphotriesterase and on the potential calcium-binding site of PhoX.