Serotyping of Haemophilus paragallinarum isolates from Mexico by the Kume hemagglutinin scheme

A total of 42 isolates of Haemophilus paragallinarum from Mexico were serotyped by the Kume hemagglutinin scheme. Serovars A-1, A-2, B-1, and C-2 were recognized among 11 (26.2%), 7 (16.6%), 4 (9.5%), and 14 (33.3%) isolates, respectively. A further six isolates (14.3%) showed hemagglutinating activity but could not be classified into any serovar. Commercial vaccines containing Kume serovars A-1, A-2, B-1, and C-2 may provide better protection than those bi- or trivalent infectious coryza vaccines currently used in Mexico.     

Haemophilus paragallinarum haemagglutinin: role in adhesion, serotyping and pathogenicity

It is suggested that Haemophilus paragallinarum requires at least three haemagglutinins for adhesion during infection. This paper reports the partial purification and characterization of the HA-L haemagglutinin from H. paragallinarum strain 46-C3, a heat sensitive, trypsin sensitive haemagglutinin that has been shown to be the serovar specific haemagglutinin in this organism. Using the pl and molecular mass obtained, it was shown that this protein shares similarities with other types of adhesins found in Gram-negative bacteria. The haemagglutination assay conditions were optimized at pH 7.5 at 37 degrees C. It was also shown that activity is enhanced by the addition of Ca2+ and Mn2+ ions.     

Evaluation of two monoclonal antibodies for serotyping Haemophilus paragallinarum

Two monoclonal antibodies (MAbs) were evaluated for their ability to serotype 108 isolates of Haemophilus paragallinarum. One MAb (E5C12D10) was raised against a Page serovar A strain and the other (F2E6) against a Page serovar C strain. In both dot blot and hemagglutination-inhibition tests, MAb E5C12D10 recognized the type strains of Page serovar A and Kume serovars A-1, A-2, A-3, and A-4. MAb F2E6 recognized the type strains of Page serovar C and Kume serovars C-1, C-2, and C-3. Neither antibody recognized the type strains of Page serovar B or Kume serovars B-1 and C-4. When evaluated with 97 field isolates in a dot blot test, the MAbs serotyped 81 isolates, which was better than agglutinin typing by the Page scheme (69 isolates serotyped). The field isolates that did not react with the MAbs were either Page serovar B/Kume serovar B-1 (three isolates), Page serovar C/Kume serovar C-4 (12 isolates), or nontypable by either the Page or Kume scheme (one isolate).     

Antimicrobial drug resistance and the occurrence of plasmids in Haemophilus paragallinarum

A broth microdilution method was used to examine the sensitivity of 75 Haemophilus paragallinarum isolates to six antimicrobial drugs (ampicillin, erythromycin, neomycin, penicillin, streptomycin, and tetracycline). A total of 55 of the isolates (73%) were sensitive to all six drugs. The remaining 20 isolates were resistant to streptomycin, with one of these isolates also being resistant to tetracycline and another also resistant to neomycin. No isolate showing antimicrobial drug resistance belonged to agglutinin serovar C, despite this being the single largest serovar (27 out of 75) in the study. No plasmids were detected among the 75 isolates, despite the use of five different plasmid-screening techniques.     

Pathogenicity of Australian isolates of Haemophilus paragallinarum and Haemophilus avium in chickens

Twenty-seven Australian avian Haemophilus isolates were tested for their ability to cause infectious coryza in specific pathogen-free chickens. All 15 isolates, identified as H. paragallinarum, produced infectious coryza, whereas all 12 H. avium isolates were nonpathogenic, but spread to in-contact chickens.     

Serological classification of Australian isolates of Haemophilus paragallinarum

Thirty-nine Australian isolates of Haemophilus paragallinarum were compared serologically with 3 reference serotype strains of H. paragallinarum using a plate agglutination test. Twenty-eight of the isolates were serotype C, 5 were serotype A, while the remaining 6 isolates could not be assigned to a serotype.     

Characterisation of isolates of Haemophilus paragallinarum from Indonesia

OBJECTIVE: To characterise 18 isolates of Haemophilus paragallinarum isolated from chickens in Indonesia. PROCEDURE: The isolates were identified to species level by traditional phenotypic methods. Six of the isolates were also identified by a species-specific polymerase chain reaction. Fourteen of the isolates were examined for resistance to a panel of seven antimicrobial agents using a disc diffusion method. All 18 isolates were serotyped according to the Page scheme using reference antisera in a haemagglutination inhibition test. RESULTS: Four of the 18 isolates were obtained from indigenous (kampung) chickens, with the remainder being from typical intensive poultry production systems. The 18 isolates were obtained from 11 outbreaks that showed the typical clinical signs of infectious coryza and 11 of the isolates were obtained from chickens that had been vaccinated with infectious coryza vaccines. All 18 isolates were confirmed as H paragallinarum by biochemical testing and six isolates were also identified as H paragallinarum by the polymerase chain reaction test. Eleven isolates were resistant to erythromycin and streptomycin, 10 to neomycin, eight to oxytetracycline, five isolates to doxycycline, three to sulphamethoxazoltrimethoprim but only one to ampicillin. Seven isolates were Page serovar A, four were Page serovar B and seven were Page serovar C. CONCLUSION: The presence of all three Page serovars (A, B and C) has been confirmed for the first time in Indonesian chickens. As the majority of the infectious coryza vaccines in use in Indonesia contain only serovar A and C, the presence of serovar B in chickens indicates that the protection by these bivalent vaccines would be reduced. The use of trivalent infectious coryza vaccines that contain serovars A, B and C is recommended for use in Indonesia.     

副猪嗜血杆菌分离株耐药性、血清型及RAPD基因型分析

为弄清某规模化养猪场感染和流行的副猪嗜血杆菌(HPS)菌株的耐药性、血清型及基因型,本试验自该猪场发生浆膜炎和关节炎病猪的不同组织样品中分离到6株HPS菌株,通过细菌培养特性、生化试验和PCR扩增细菌16S rRNA基因片段并测序对分离株进行鉴定;进而鉴定分离株对不同类别抗生素的耐药性;最后对分离株进行血清分型和RAPD基因分型并分析两者的相关性。结果显示,自病猪不同组织脏器中分离鉴定了6株HPS菌株,体外培养具有典型卫星生长现象;生化试验结果符合HPS反应特性;PCR均可扩增出821 bp的HPS 16S rRNA基因片段,且序列与HPS一致。药敏试验结果显示分离株对头孢菌素类、青霉素类、氨基糖苷类、大环内酯类和四环素类抗生素敏感,而对喹诺酮类、磺胺类、林可霉素类和氯霉素类抗生素产生耐药性。琼脂扩散试验结果显示HPS分离株均为血清5型;RAPD分析显示HPS分离株与15个血清型参考菌株可分为4个基因型。本试验结果为HPS的流行病学调查及防控具有一定的借鉴价值。     

Serological classification of Japanese isolates of Haemophilus paragallinarum using two serovar-specific monoclonal antibodies

A serological classification of 106 Japanese isolates of Haemophilus paragalinarum recovered from 1960 to 1984 was performed by dot-blotting and hemagglutination-inhibition (HI) tests using two serovar-specific monoclonal antibodies (MAbs), E5C12D10 and F2E6. By the dot-blotting test, 49 of the isolates were serovar A and 55 isolates were serovar C, and the two remaining isolates did not react with either MAb. These two nontypable strains had no hemagglutinating activity against chicken erythrocytes and were nonpathogenic to chickens. Although 49 serovar A isolates were serotyped by the HI test, only 23 of the 55 serovar C isolates could be serotyped. The remaining 32 isolates could not be serotyped because no or low hemagglutinating activity could be detected. Our results indicate that H. paragallinarum serovars A and C have both been present in Japan since 1960, with serovar A isolates being dominant before 1970 and serovar C isolates more prevalent than serovar A since 1970.     

Expression of hemagglutinin of Haemophilus paragallinarum serotype A in Escherichia coli.

The genomic DNA of Haemophilus paragallinarum (Hpg) serotype A strain 221 was cloned into vector plasmid pBR322. The recombinant plasmids were introduced into Escherichia coli (E. coli) strain C600. Subsequently, a total of 277 transformants were obtained. One, designated strain no. 6, expressed hemagglutination activity against chicken erythrocytes. Strain no. 6 contained the recombinant plasmid pNV102, and DNA of about 2.57 kb was inserted into pNV102. When strain no. 6 was cured of pNV102, the strain lost hemagglutination activity. When the cured strain was retransformed with pNV102, hemagglutination activity was restored. E. coli strain no. 6 reacted with monoclonal antibody specific to the hemagglutinin of Hpg serotype A in a dot-blotting analysis. Chickens immunized with the inactivated strain no. 6 produced the hemagglutination inhibition (HI) antibody, and chickens possessing the HI antibody showed protection against challenge exposure by Hpg strain 221.     

Hemagglutinin of Haemophilus paragallinarum serotype 2 organisms: occurrence and immunologic properties of hemagglutinin

Hemagglutinating properties of Haemophilus paragallinarum serotype 2 and serotype C against freshly collected and glutaraldehyde (GA)- fixed chicken RBC were investigated. Different from serotype 1, the nontreated organisms of serotype 2 and serotype C lacked hemagglutinating activity. However, when the organisms were treated with potassium thiocyanate (KSCN) and/or sonication, activity occurred not only against GA-fixed chicken RBC, but also against GA-fixed RBC of various animal species. The maximum hemagglutination titer (1:64 to 1:256) was obtained against GA-fixed RBC with the KSCN-treated organisms that were also sonicated. The activity was inactivated by heating at 100 C or by treatments with formalin or trypsin, but not by treatments with hyaluronidase or neuraminidase. By using KSCN-treated and sonicated organisms and GA-fixed chicken RBC, a detection of hemagglutination-inhibition (HI) antibody was possible. The HI tests showed that serotype 2 hemagglutinin was immunologically distinct from serotype 1 and that the HI antibody correlated to protective activity against challenge exposure with serotype 2 and serotype C. Chicks having HI antibody greater than 1:5 were protected against challenge exposure with homologous type, but were not protected with serotype 1. Applicability of the HI test was also shown for evaluating protective potency in the serotype 2 vaccine, as well as in the serotype 1 vaccine.     

Hemagglutinating activity and immunological properties of Haemophilus paragallinarum field isolates in Japan

Four field isolates (S4, S10, S15, and S17) of Haemophilus paragallinarum were recovered from chickens affected with infectious coryza in widely separated regions of Japan. Their hemagglutinating (HA) activity and immunological properties were compared with those of strain 221 of serovar A/1 and strains Modesto and S1 of serovar C/2. When treated with potassium thiocyanate or hyaluronidase, all the isolates showed HA activity against formaldehyde-fixed chicken erythrocytes but not against fresh chicken erythrocytes. In the hemagglutination-inhibition (HI) test, the isolates cross-reacted with strains Modesto and S1 but not with strain 221. The immunological properties of these isolates, as determined by cross-protection tests, were similar to those of strain S1 and, to a lesser degree, strain Modesto, but not to strain 221. Our results indicated that the four field isolates belong to serovar C/2 and that the HI test is a suitable method for serotyping H. paragallinarum.     

Virulence of South African isolates of Haemophilus paragallinarum. Part 1: NAD-dependent field isolates

The virulence of four South African field isolates of NAD-dependent Haemophilus paragallinarum, representing the four serovars known to occur in that country, was investigated. During this study an alternative challenge model for infectious coryza was used, in which the infectivity as well the virulence of different isolates could be evaluated. The challenge model consisted of the direct challenge, via intrasinus injection of one chicken in a row of interconnected layer cages, containing 10 chickens, which are subsequently infected by natural routes. A scoring system of the clinical signs was established in which a score is given to the ability of the isolate to produce clinical signs in the challenge birds. The mean daily disease score for the flock can be calculated and plotted on a graph to give a graphic representation of the disease profile. A mean disease score, calculated over a 20-day examination period can be calculated. Isolates can then be compared to each other, either graphically or by a comparison of the mean disease scores. It has been demonstrated using this scoring system that the South African serogroup C isolates appear to be more virulent than the South African serogroup A or B isolates. It was further established that the serovar C-3 isolate appeared to be the most virulent.     

Comparison of Haemophilus paragallinarum isolates by restriction endonuclease analysis of chromosomal DNA

Chromosomal DNA from Haemophilus paragallinarum was examined by restriction endonuclease analysis (REA) using the enzymes BamHI, EcoRI, HindIII, or SmaI. The enzyme SmaI had no apparent effect upon the DNA from eight representative H. paragallinarum isolates. The remaining enzymes cut the H. paragallinarum DNA to varying degrees, with the most useful patterns for distinguishing isolates being given by HindIII. The REA patterns given by HindIII were stable under both in vitro and in vivo conditions. The use of the enzyme HindIII showed that eight Australian isolates of H. paragallinarum were genetically similar. In contrast, 14 isolates of H. paragallinarum from outside Australia were markedly different from each other and the Australian isolates. A plasmid of approximately 6 kilobase pairs in size was found in one isolate of H. paragallinarum.     

副鸡嗜血杆菌的分离鉴定及防治

河南省中牟县某养殖户饲养7000只海兰褐商品蛋鸡，在开产前按常规进行了鸡新城疫等疫苗的免疫，但未接种过鸡传染性鼻炎菌苗。2003年7月5日，即240日龄时，个别鸡开始零星发病，8d内蔓延至全群，有80％的鸡出现症状，并零星死亡。产蛋率下降至’70％，其技术人员对发病鸡群曾用链霉素、红霉素等药物内服，但效果不明显。病鸡精神萎顿、垂头缩颈、食欲明显降低。病鸡鼻流浆液性分泌物，有的鸡甩头、打喷嚏；眼结膜发炎、眼睑肿胀，有的流泪；一侧或两侧面部肿胀。     

Further characterization of Haemophilus paragallinarum and Haemophilus avium

A total of 60 isolates of Haemophilus spp. from chickens, including four reference strains of H. paragallinarum and one of H. avium, were examined for their physiological and biochemical properties. The isolates could be placed into two groups. One group was identified as H. paragallinarum and consisted of 43 isolates including the four reference strains of H. paragallinarum. The other group was identified as H. avium and consisted of 17 isolates including the reference strain of H. avium. H. avium can be differentiated from H. paragallinarum by its possession of the enzymes catalase and alpha-glucosidase, capacity to grow in air, production of acid from galactose, and by the fact that its growth is not improved by the addition of chicken serum. In addition, the majority of H. avium isolates, unlike H. paragallinarum, possess a yellow pigment and produce acid from trehalose.     

An evaluation of agglutination and coagglutination techniques for serotyping of Haemophilus pleuropneumoniae isolates

A comparative evaluation of rapid slide agglutination, tube agglutination, 2-mercaptoethanol tube agglutination, and coagglutination tests was made for serotyping isolates of Haemophilus pleuropneumoniae. The results indicated that a majority of the isolates could be serotyped by any of these tests. But, it was not uncommon to find isolates which were inagglutinable or poorly agglutinable in homologous sera. Heat treatment of whole-cell suspensions of such isolates was essential to unmask the serotype-specific antigenic determinants; however, in the process of heat treatment, cross-reactive common antigens of minor nature were also exposed. The antibodies involved in such cross-reactions were mainly of immunoglobulin M type, because the cross-reactivities were completely abolished in coagglutination and 2-mercaptoethanol agglutination tests. Thus, both these tests were satisfactory for serotyping inagglutinable mucoid strains. For serotyping strains which were either polyagglutinating or autoagglutinating, agglutination tests could not be used, but the coagglutination test proved to be satisfactory. The coagglutination test was serotype-specific, sensitive, simple, rapid, reproducible, and easier to read and interpret than rapid slide or tube agglutination tests. This test could be used to serotype mucoid, smooth, or rough isolates.