Immunogenicity of Haemophilus paragallinarum serovar B strains.

Immunogenicity of three Haemophilus paragallinarum serovar B strains was investigated in cross-protection tests using monovalent vaccines prepared from the B strains, as well as one strain each of serovars A and C. A bivalent vaccine composed of the serovar A and C strains also was used. In the studies with the monovalent vaccines, the immunogenicity of serovar B strains was different from that of serovar A and C strains, although only partial serovar B-specific protection with the three strains was observed. Chickens vaccinated with the bivalent vaccine protected against challenge with one serovar B strain, as well as serovar A and C strains, but not against the other two serovar B strains.     

An indirect enzyme-linked immunosorbent assay for detection of antibodies to Actinobacillus pleuropneumoniae serovar 7 in pig serum.

Lipopolysaccharide (LPS) antigen was purified from Actinobacillus pleuropneumoniae serovar 7 by phenol-water extraction and fractionated on a, S-100 Sephacryl column. High molecular weight fractions of LPS purified from the S-100 column were pooled and used as antigen in an indirect serovar 7 ELISA. The ELISA was evaluated with sera from pigs experimentally infected with 11 different A. pleuropneumoniae serovars of biotype 1. Estimation of sensitivity and specificity of the A. pleuropneumoniae serovar 7 ELISA was performed using pig sera from herds naturally infected with A. pleuropneumoniae serovar 7 as well as sera from herds free of infection with A. pleuropneumoniae serovar 7. When compared to the complement fixation test (CFT) as a reference test, the ELISA showed much higher sensitivity and statistically equivalent specificity.     

Serological characterisation of Actinobacillus pleuropneumoniae isolated from pigs in 1993 to 1996

OBJECTIVES: To clarify the serological identity of the prototype strain of a group of Actinobacillus pleuropneumoniae isolates that could not be serotyped in previous studies and to establish the serovar of 378 isolates of A pleuropneumoniae obtained from pigs in Australia over the period 1993 to 1996. DESIGN: After initial validation, QGD and IHA tests were used to characterise the prototype isolate (HS143) selected to represent the cross-reacting isolates that were found in a previous study. Next, 378 recent field isolates of A pleuropneumoniae were characterised using the existing gel diffusion serotyping technique and/or the IHA or QGD tests. RESULTS: The indirect haemagglutination test was shown to be capable of correctly recognising the reference strain for all serovars except serovar 11. While the quantitative gel diffusion test was not as effective as indirect haemagglutination, it could recognise serovar 11. When the two tests were used to examine the prototype strain (HS143) of the cross-reactive isolates, the results indicated that HS143 is serologically distinct from all 12 of the recognised serovars of A pleuropneumoniae. However, as HS143 was subsequently identified as serovar 12 by one of the leading international reference laboratories, the antiserum to isolate HS143 was used as the serovar 12 antiserum. A total of 346 of the 378 A pleuropneumoniae field isolates examined could be confidently serotyped with almost 90% of the isolates being either serovar 1 (104 isolates); serovar 7 (83 isolates) or serovar 12 (142 isolates). A range of other serovars and some cross-reactive isolates made up the remainder of the isolates. CONCLUSION: The serovar 12 antiserum produced against the international reference strain (1096) does not recognise Australian serovar 12 isolates. The antiserum raised against isolate HS143 does recognise the Australian serovar 12 isolates. The dominant serovars of A pleuropneumoniae infecting Australian pigs are (in decreasing order) serovars 12, 1 and 7.     

Serological characterisation of Haemophilus parasuis isolates from Australian pigs

A total of 31 isolates of Haemophilus parasuis obtained from Australian pigs were serotyped by the Kielstein-Rapp-Gabrielson scheme. The isolates were assigned to serovar 1 (1 isolate), serovar 2 (1 isolate), serovar 4 (4 isolates), serovar 5 (7 isolates), serovar 9 (2 isolates), serovar 10/7 (4 isolates), serovar 12 (1 isolate) and serovar 13 (6 isolates). The remaining 5 isolates could not be assigned to a serovar. Two different serovars (5 and 13) were detected in one herd. The only 2 isolates obtained from clinically normal pigs (from the same herd) were serovar 9. The common serovars were isolated from pigs with pneumonia as well as from pigs with conditions of the Glässer's disease type. The serological heterogeneity amongst Australian isolates of H parasuis has important implications for the use of vaccines to control Glässer's disease.     

An evaluation of inactivated infectious coryza vaccines containing a double-emulsion adjuvant system.

The efficacy of experimental inactivated infectious coryza vaccines produced by a commercial vaccine manufacturer was evaluated. The vaccines, containing as the adjuvant phase either a double-emulsion mineral oil system or aluminum-hydroxide gel, were administered to 6-week-old chickens as a single dose. Some vaccines were a monovalent product containing a Page serovar C Haemophilus paragallinarum strain, and others were a bivalent product containing both Page serovar A and serovar C strains. After 3 weeks, all chickens were challenged by infraorbital sinus inoculation of virulent H. paragallinarum, either Page serovar C (strain HP31) or Page serovar A (strain HP14). The monovalent serovar C double-emulsion-based vaccines gave significant protection against a serovar C challenge, with the level of protection varying from 60% to 100%. The monovalent serovar C aluminum-hydroxide-gel vaccine also gave significant protection (94%) against a serovar C challenge. The bivalent double-emulsion vaccine gave significant protection against challenge from both serovars (100% for serovar C and 83% for serovar A). Although no major adverse reactions were detected, some chickens receiving both the double-emulsion vaccines and the aluminum-hydroxide vaccine developed relatively minor granulomatous reactions at the site of injection.     

The presence of nicotinamide adenine dinucleotide-independent Haemophilus paragallinarum in México

Two isolates of Haemophilus paragallinarum were obtained from a layer chicken in Mexico. The isolates were confirmed as H. paragallinarum by polymerase chain reaction and conventional biochemical identification. The isolates were nicotinamide adenine dinucleotide (NAD) independent-growing on blood agar without the need of a nurse colony as well as on a complex medium that lacked both NAD and chicken serum. Both isolates were pathogenic, causing the typical clinical signs of infectious coryza in susceptible chickens. One isolate was Page serovar B/Kume serovar B-1 and the other isolate was Page serovar C/Kume serovar C-2. The isolates were associated with a field outbreak that involved an egg drop of 20% over a 3-wk period and a doubling of weekly mortality (from 0.1% to 0.2%). This is the first report of NAD-independent H. paragallinarum outside South Africa and is the first time that NAD-independent H. paragallinarum of serovar B has been reported.     

A monoclonal antibody-blocking enzyme-linked immunosorbent assay for the detection of serovar-specific antibodies to Haemophilus paragallinarum

Two monoclonal antibody-blocking enzyme-linked immunosorbent assays (B-ELISAs) were developed to detect serovar-specific antibodies to Haemophilus paragallinarum. One assay detected antibodies against serovar A and the other antibodies against serovar C. The assays were evaluated with sera derived from disease-free chickens as well as chickens experimentally immunized and/or challenged with H. paragallinarum strains 0083 (serovar A), Modesto (serovar C), or HP31 (serovar C). When tested with 440 negative sera (170 from a specific-pathogen-free and 30 from each of nine commercial layer flocks), both tests gave only a single false-positive reaction. The use of the B-ELISAs with the experimentally produced sera showed the assays to be serovar specific. With the exception of one serum, the serovar A B-ELISA detected antibodies only in the chickens vaccinated with 0083. Similarly, with the exception of one serum, the serovar C B-ELISA detected antibodies only in those chickens vaccinated with Modesto or those chickens challenged with HP31. Overall, the serovar A B-ELISA had a specificity of 99.7% and a sensitivity of 78.7%, whereas the serovar C B-ELISA had a specificity of 99.8% and a sensitivity of 64.7%.     

Seroprevalence of leptospirosis in domesticated Asian elephants (Elephas maximus) in north and west Thailand in 2004

Serum samples from Asian elephants (Elephas maximus) in the Kanchanaburi, Chiang Mai and Lampang provinces of Thailand were tested using the microscopic agglutination test against 22 serovars of Leptospira interrogans. A titre of more than 1:100 was used as evidence of infection. In northern Thailand, the seroprevalence was 58 per cent and the prevalent serovars were Leptospira interrogans serovar Sejroe, Leptospira interrogans serovar Tarassovi, Leptospira interrogans serovar Ranarum and Leptospira interrogans serovar Shermani. In western Thailand, the seroprevalence was 57 per cent and the prevalent serovars were L Tarassovi, L Sejroe, L Ranarum, Leptospira interrogans serovar Bataviae and L Shermani. These results were similar to studies in domestic livestock and stray dogs in the Bangkok district. Among the elephants from Kanchanaburi there were significant associations between seropositivity and between the camp and between the prevalent serovars and the camp.     

Effect of vaccination with a pentavalent leptospiral vaccine containing Leptospira interrogans serovar hardjo type hardjo-bovis on type hardjo-bovis infection of cattle

Effectiveness of 2 pentavalent leptospiral vaccines containing Leptospira interrogans serovar hardjo was evaluated for protection of steers from infection with serovar hardjo type hardjo-bovis. The hardjo component of 1 vaccine was prepared from serovar hardjo type hardjoprajitno. The hardjo component of the other vaccine was prepared from serovar hardjo type hardjo-bovis. Two steers were vaccinated once and 4 steers were vaccinated twice with the pentavalent vaccine containing type hardjoprajitno. Four steers were vaccinated once and 4 steers were vaccinated twice with the pentavalent vaccine containing type hardjo-bovis. Four steers were maintained as non-vaccinated controls. Steers given vaccine containing type hardjo-bovis developed higher mean serum microscopic agglutination titers against serovar hardjo than steers given vaccine containing hardjoprajitno. Six months after the first vaccination, all steers were challenge-exposed on 3 occasions by conjunctival instillation of 10(7) serovar hardjo type hardjo-bovis organisms, and on 1 occasion by conjunctival instillation of urine from a steer shedding hardjo-bovis. All control and all vaccinated steers became infected and shed serovar hardjo type hardjo-bovis in the urine. Lesions were detected in kidneys of 3 of 4 nonvaccinated control steers, 5 of 6 steers given hardjoprajitno vaccine, and 6 of 8 steers given hardjo-bovis vaccine. Leptospires were detected in kidneys of 4 of 4 control steers and 13 of 14 vaccinated steers.     

Cytometric and cytochemical study of peritoneal and alveolar macrophages from Yersinia pseudotuberculosis infected ground squirrels (Citellus citellus).

The enzymic activity (succinate dehydrogenase, acid and alkaline phosphatase) of alveolar and peritoneal macrophages as well as leucocytic reaction of ground squirrels infected with Yersinia pseudotuberculosis (I and III serovar) have been investigated in dynamics from the 1st up to the 30th day. The animals infected with III serovar survived only to the 7th day, while those infected with I serovar survived up to the 30th day after inoculation. A massive influx of leucocytes having peak values (100-fold increase) on the 3rd day after infection has been found in the peritoneal cavity of the animals infected with I serovar. Moderate leucocytosis in the blood, and insignificant fluctuations in alveolar macrophage number have been established too. An earlier and higher activation of succinate dehydrogenase in alveolar and peritoneal macrophages from animals infected with III serovar in comparison with those infected with I serovar was observed. No differences in alkaline phosphatase activity of alveolar and peritoneal macrophages have been found between the animals infected with I and III serovar. A correlation has been found between the number of leucocytes and changes in the enzymatic activity of the macrophages. A metabolic transformation was demonstrated typical for different macrophages (peritoneal and alveolar), in the course of this experimental intraperitoneal infection. Obviously, more virulent serovar III of Y. pseudotuberculosis fails to attract leucocytes to the peritoneal cavity sufficiently quickly, so it overcome the local protective mechanisms with consequent systemic cytochemical changes. On the other hand the virulent serovar I attracts leucocytes to the peritoneum and is presumably destroyed by them.     

The isolation and identification of Leptospira interrogans serovar bratislava from a pig in Germany.

During a study on the improvement of selective media for the isolation of leptospires from clinical material, leptospires were isolated from the kidneys of a pig. Test material was cultured in EMJH-medium containing 0.4% rabbit serum and 3 inhibitory substances (Rifampicin 10 micrograms/ml, Amphotericin B 2 micrograms/ml, 5-Fluorouracil 100 micrograms/ml). The cultured leptospira-strain (Berlin 406) was identified to serogroup level using the cross agglutination method and it was further typed to serovar level by MAB's, factor analysis and restriction endonuclease analysis (REA). MAB typing of Berlin 406 gave an agglutination pattern profile typical of serovar bratislava. Factor serum identification clearly identified this strain as serovar bratislava. On REA examination, Berlin 406 gave a profile identical to that of the serovar bratislava type strain Jez Bratislava i.e. genotype B1. This is the first report of a confirmed isolate of Leptospira interrogans serovar bratislava from domestic animals in Germany. There exist a number of possibly important implications as this agent is being incriminated in the cause of clinical disease not only in pigs, but also in horses and dogs in other countries.     

Antibody response to genus- and serovar-specific leptospiral antigens in Leptospira-infected cows

In cows inoculated with Leptospira interrogans serovar pomona or hardjo, the 2-mercaptoethanol-sensitive microscopic agglutination test (MAT) antibody to the serovar appeared 3 to 8 days after inoculation and peaked at 10 to 20 days, whereas the 2-mercaptoethanol-resistant MAT antibody was predominant at 35 to 80 days. A persistent antibody response, probably associated with serovar-specific leptospiral antigens, was detected in the cows inoculated with serovar pomona, using a sonicated or an alkaline-extracted antigen derived from serovar pomona in the enzyme-linked immunosorbent assay (ELISA). In contrast, a short-lived antibody response to the same antigens was demonstrated in cows inoculated with serovar hardjo, probably more typical of the response to genus-specific leptospiral antigens. Antigens derived from L biflexa serovar patoc only detected the latter type of antibody response in cows inoculated with serovar pomona or hardjo. Correlative studies revealed that the antigens derived from serovar patoc seem to be genus specific and serologically closely related, but not identical. The antigens derived from serovar pomona were genus specific on the basis of the early antibody response to leptospiral inoculation in the cows, but serovar specific based on the subsequent more persistent response to leptospiral inoculation. These antigens were also serologically closely related, but not identical. Examination of sera from cows that aborted and were MAT-positive for serovar pomona or hardjo revealed a more serovar-specific antibody response, indicating that there may have been a less recent leptospiral antigenic stimulus, thus emphasizing the caution with which results of the ELISA and other serologic assays for the detection of bovine leptospirosis must be interpreted.     

The isolation of Leptospira interrogans serovar pomona and related serological findings associated with a mixed farming unit in the Transvaal

This is the first known isolation in the Republic of South Africa (RSA) of the serovar pomona from the organs of porcine foetuses as well as from the renal lymph nodes of slaughter pigs showing chronic nephritis. In addition, the serovar pomona was isolated from the kidneys of 87.5% of the slaughter pigs examined. The success of these isolations was attributed in part to the refining of 2 existing isolation techniques which complement each other. Using the microscopic agglutination test, serum samples taken from the same farming unit showed evidence of antibodies to the serovar pomona in 89 out of the 170 bovines (52%), 9 out of the 52 porcines (17%), 2 of the 2 canines (100%), 5 out of the 13 equines (38%) and 2 out of the 152 ovines (1%) that were tested. As far as is known, serological evidence of the serovar pomona in porcines, ovines, equines and canines has never previously been published in the RSA.     

Effect of vaccination with a bacterin containing Leptospira interrogans serovar bratislava on the breeding performance of swine herds

Swine herds suspected to be infected with Leptospira interrogans serovar bratislava were vaccinated with bacterins containing 5 or 6 leptospiral serovars in which serovar bratislava was the unique component. The principal diagnostic feature indicating an infection by this organism was demonstration of antibody against serovar bratislava in sera from stillborn pigs. For 1 breeding cycle after vaccination of herds on 3 farms, 255 of 266 (95.9%) sows and gilts given the 6-serovar bacterin farrowed. In contrast, 233 of 311 (74.9%) sows and gilts given the 5-serovar bacterin farrowed. These results, as evaluated by analysis of variance techniques, showed a significant improvement (P less than 0.01) in reproductive performance for groups vaccinated against serovar bratislava.     

Isolation of Actinobacillus pleuropneumoniae serovar 15-like strain from a field case of porcine pleuropneumonia in Japan

An Actinobacillus pleuropneumoniae strain isolated from a field case of porcine pleuropneumonia in Japan, was closely related to a reference strain of serovar 15, which is a newly proposed serovar according to an analysis of field isolates originating from Australia. The isolate had biological and biochemical properties consistent with A. pleuropneumoniae biovar 1, and reacted strongly to a rabbit antiserum raised against a reference strain of serovar 15 in an agar gel precipitation test. The nucleotide sequence of a hyper variable region in the 16S RNA gene of the isolate was identical to that of the reference strain of serovar 15. The isolate possessed A. pleuropneumoniae-RTX toxin (Apx) II, III, and IV genes, consistent with serovar 15. Its virulence in mice was lower than that of ApxI-bearing strains but higher than that of other ApxIII-bearing strains. This is the first report describing the isolation of A. pleuropneumoniae serovar 15-like strain from a country or region other than Australia.     

An evaluation of the apxIVA based PCR-REA method for differentiation of Actinobacillus pleuropneumoniae

A restriction analysis of PCR (PCR-REA) amplified apxIVA gene has been suggested as an alternative method for serotyping of Actinobacillus pleuropneumoniae by Jaglic et al. [Jaglic, Z., Svastova, P., Rychlik, I., Nedbalcova, K., Kucerova, Z., Pavlik, I., Bartos, M., 2004. Differentiation of Actinobacillus pleuropneumoniae by PCR-REA based on sequence variability of the apxIVA gene and by ribotyping. Vet. Microbiol. 103, 63-69]. The current study investigated whether this alternative method could distinguish between the reference strains of serovars 13-15 and the value of the method when applied to 47 field isolates representing serovars 1-3, 5, 7-9, 12 and 15 as well as non-typable isolates. The reference strains of serovars 13 and 14 had the same sized product after the apxIVA PCR, while the product for serovar 15 was of different size compared to all the other serovar reference strains. The CfoI digest profiles of the reference serovars 13 and 14 strains were different from each other and from all other serovars. The HpaII digest profiles of these two serovars were very similar to each other, but both were distinctively different from the other serovar profiles. The CfoI digest profile of serovar 15 strain was very similar to the serovars 3 and 12 strains except for two faint extra bands for serovar 15. The HpaII digest profiles of serovars 12 and 15 reference strains were identical. The PCR-REA method correctly recognized the serovar of 21 of 43 field isolates. It was concluded that the method was a useful additional tool to support, but could not replace, conventional serotyping.     

Development and initial evaluation of an indirect enzyme-linked immunosorbent assay for the detection of Leptospira interrogans serovar hardjo antibodies in bovine sera.

Outer sheath antigen from Leptospira interrogans serovar hardjo type hardjoprajitno and acetic acid extracted antigens from serovar hardjo types hardjoprajitno and hardjobovis were evaluated in an immunoassay for ability to detect hyperimmune rabbit serum to serovar hardjo. The degree of cross-reactivity with hyperimmune rabbit sera to L. interrogans serovars pomona, copenhageni, grippotyphosa, canicola and sejroe, and Leptospira biflexa serovar patoc was also measured for each antigen. All of the antigens reacted with the antiserum to L. interrogans serovar hardjo. The outer sheath antigen however, also showed wide cross-reactivity with the antisera to all of the serovars of L. interrogans tested and with the antiserum to L. biflexa serovar patoc. The acetic acid extracted antigen from either type hardjoprajitno, or type hardjobovis, showed a high degree of specificity for serovar hardjo antiserum. The hardjobovis acetic acid extracted antigen was characterized by sodium dodecyl sulfate polyacrylamide gel electrophoresis and immunoblotting, and was incorporated into an indirect ELISA for detection of anti-serovar hardjo antibodies in bovine serum. This ELISA showed a relative specificity of 100% with 156 bovine sera which were negative at a dilution of 1:100 in the microscopic agglutination test (MAT) for L. interrogans serovars hardjo, pomona, sejroe, icterohaemorrhagiae, copenhageni, canicola, and grippotyphosa. The relative sensitivity of this assay with 192 bovine sera which had serovar hardjo MAT titres of > or = 100 was 95.3% (95% confidence limit = 2.99%). The degree of cross-reactivity with 289 bovine sera which had serovar pomona MAT titres of > or = 100 (with no detectable serovar hardjo MAT titres) was approximately 1.0%. This assay was: easily standardized, scored objectively, repeatable, semi-automated and used a non-hazardous antigen that can be routinely prepared in gram amounts.     

Experimental infection of lactating goats with Leptospira interrogans serovars pomona and hardjo

Pathogenesis of Leptospira interrogans serovars pomona and hardjo was evaluated in 14 lactating goats. Although mild clinical signs of leptospiral infection characterized by pyrexia and reduction in milk yield appeared in some animals, a consistent clinical pattern was not observed in the inoculated animals. The pomona serovar was isolated from the kidney of 1 of the 4 goats inoculated with serovar pomona. The hardjo serovar (strain UI 750) was isolated in the rabbit serum-supplemented bovine albumin polysorbate-80 liquid medium only from the mammary gland of 1 of 4 goats at 13 days after inoculation with serovar hardjo. The positive culture was detected after an 8-month incubation period.     

An evaluation of the cross-protection afforded by inactivated infectious coryza vaccines

The cross-protection afforded by three inactivated infectious coryza vaccines was evaluated. Each vaccine contained one of the following strains of Haemophilus paragallinarum, HP31, HP60 and HP14. Strain HP31 belongs to Kume serovar C-2, strain HP60 belongs to Kume serovar C-4 while strain HP14 belongs to Kume serovar A-4. Four groups of twenty six-week-old specific-pathogen-free chickens were either given a single dose of an aluminium-hydroxide based vaccine (three groups) or left as unvaccinated controls. Three weeks after vaccination, all four groups were challenged with virulent H paragallinarum. One half of each group was challenged with the strain HP31 and the other half with strain HP60. The efficacy of the vaccines was assessed in terms of the prevention of the typical clinical signs and macroscopic lesions of infectious coryza as well as the prevention of any colonisation by the challenge organism. The serovar C-2 and serovar C-4 vaccines protected more than 50% of birds against either a serovar C-2 or C-4 challenge while the serovar A-4 vaccine protected less than 50% of birds.     

Development of a monoclonal antibody-based competitive enzyme-linked immunosorbent assay for the detection of Leptospira borgpetersenii serovar hardjo type hardjobovis antibodies in bovine sera.

A murine monoclonal antibody (designated M553) that binds to an epitope on whole cell antigens prepared from Leptospira borgpetersenii serovar hardjo type hardjobovis and Leptospira interrogans serovar hardjo type hardjoprajitno, was produced and incorporated into a competitive enzyme-linked immunosorbent assay for the detection of bovine antibodies to serovar hardjo. The epitope recognized by M553 was susceptible to periodate oxidation. The M553 antibody was characterized by western blot with hardjobovis whole cell antigen. This antibody does not cross-react with whole cell antigens prepared from 11 other pathogenic Leptospira serovars, or, Leptospira biflexa serovar patoc. The sensitivity estimate of the competitive ELISA was 100% with field sera (n = 165) with serovar hardjo microscopic agglutination test (MAT) titres of > or = 100. The specificity estimate was 100% with sera (n = 128) obtained from a specific pathogen free herd of cattle that were negative in the MAT at a dilution of 1:100 for serovars hardjo, pomona, sejroe, copenhageni, canicola, and grippotyphosa. The specificity estimate with field sera (n = 301) with serovar hardjo MAT titres of < 100, was 98% (95% confidence interval = +/- 1.58%). There was no cross-reactivity with field sera (n = 306) with serovar pomona titres > or = 100 and serovar hardjo titres < 100. The specificity estimate with the combined populations of sera with serovar hardjo MAT titres of < 100 (n = 735) was 99.18% (95% confidence interval = +/- 0.65%). There was a high level of agreement (kappa = 0.977) between the results of the competitive ELISA and those of the MAT.