The development and application of a blocking ELISA kit for the diagnosis of infectious coryza.

A blocking enzyme-linked immunosorbent assay (B-ELISA) kit for the diagnosis of infectious coryza was developed in this study. The kit was based on a recently described blocking ELISA that uses monoclonal antibodies to achieve specificity for antibodies to either Haemophilus paragallinarum serovar A or serovar C. The results showed that the B-ELISA kit detected 96 and 90%, respectively, of chickens vaccinated or challenged with H. paragallinarum serovar A. When used on chickens vaccinated or challenged with H. paragallinarum serovar C, the kit detected 77 and 40%, respectively, as positive. The majority of sera from vaccinated chickens were still positive on the serovars A and C ELISAs 4 months after vaccination. Based on pen trial data, the serovar A B-ELISA kit had a sensitivity of 95% and a specificity of 100%. The serovar C B-ELISA kit had a sensitivity of 73% and a specificity of 100%. A range of field sera was examined with the kit, generating results that correlated with the known vaccination/disease history of the flocks examined. As freeze drying the monoclonal antibodies and the conjugate had some effect on optimal working concentration, the kit used liquid solutions of these two reagents. The kit could be stored for 7 days at 37 degrees C, 10 months at 4 degrees C and more than 1 yearat -20 degrees C. Our results suggest that the kit would be a useful aid in the diagnosis of infectious coryza in China and other countries where H. paragallinarum serovars A and C are the predominant or sole serovars.

Production and evaluation of a panel of monoclonal antibodies against Haemophilus paragallinarum.

We report on the production and characterisation of monoclonal antibodies (MAbs) against Haemophilus paragallinarum, the causative agent of infectious coryza. A bank of 8 MAbs were produced by traditional techniques - four against the reference strain for Page serovar A (0083) and four against the reference strain for Page serovar C (Modesto). Seven of the eight MAbs were shown to be IgG(1) with one being nontypable. None of the MAbs had HI activity and none gave any detectable reaction when examined by Western blotting. None of the MAbs gave a positive reaction in the indirect ELISA with any of the eight type strains of Pasteurella species or sub-species. None of our 8 MAbs gave serovar specific reactions when used in an indirect ELISA format. There was a trend for the serovar A MAbs to give a higher titre with serovar A isolates/strains and a similar trend for the serovar C MAbs to give higher titres with the serovar C isolates/strains.

Enhancement of hemagglutinating activity of Haemophilus gallinarum by trypsin.

It is shown that hemagglutination (HA) activity of Haemophilus gallinarum is enhanced by trypsin. HA activation was more effective at 37 C than at room temperature.

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.

Serologic response to Haemophilus gallinarum in artifically infected and vaccinated chickens.

Serologic responses to Haemophilus gallinarum (HG) were compared in chickens artifically inoculated with living HG and vaccinated with HG bacterin, using tube agglutination (AGG), hemagglutination-inhibition (HI), and agar-gel precipitation (AGP) tests. Infected and vaccinated chickens differed markedly, as did infection routes early after treatment. HI response was delayed and lower in chickens infected intranasally than in vaccinated chickens. Both AGG and AGP responses peaked sooner than HI. AGP response was earlier in infected chickens, but later in vaccinated chickens. A maximum of three lines were observed. All which appeared for vaccinated chickens were similar to those for inoculated ones. A residual line remaining for very long periods for inoculated and vaccinated chickens appeared near the side of well of the HG antigen. The duration of symptoms following HG infection had no correlation with HI, AGG, and AGP titers. From these results, the HI test seems not to be the only serological diagnosis of HG infection in the field.

Biological activities of crude polysaccharide extracted from two different immunotype strains of Hemophilus gallinarum in chickens.

Biological activities in chickens of crude polysaccharide extracted from two different immunotype strains of Hemophilus gallinarum (HG), strain 221 and S1, were studied to clarify a type-specific protecting antigen and a toxin. Crude polysaccharide materials extracted from strains 221 and S1 were not only protective but toxic to chickens. They also contained at least two heat-labile antigens. When the polysaccharide materials were subjected to gel filtration on Sepharose 6B by tracing at 280 and 490 nm, the protective and toxic activities could be fractionated as peak-1 and -2 polysaccharides, respectively. The fraction of peak-1 polysaccharides from strains 221 and S1 was eluted at void volume. Cross-protection was not found between strains 221 and S1 in the fraction of peak-1 polysaccharide. Hemagglutination-inhibition (HI) antibody to type 1 hemagglutinin of HG (trypsin-sensitive hemagglutinin) was detected in chickens immunized with this fraction from strain 221 but not with that from strain S1. Type specificity between both strains was also found in this fraction by agar-gel precipitation (AGP) test. On the other hand, the toxic fraction of peak-2 polysaccharide, which caused hydropericardium in chickens, had a lower molecular weight than did that of peak-1 polysaccharide. It did not give HI antibody in chickens. Common antigenicity between strains 221 and S1 was found in the peak-2 polysaccharide by AGP test.

Use of gonococcal agar medium for preparation of antigen of Haemophilus paragallinarum hemagglutinin.

Some solid media for antigen preparation of Haemophilus paragallinarum (HPG) hemagglutinin (HA) were studied. When HA activities of HPG strain 221 cultivated on chicken meat infusion (CMI) and gonococcal (GC) agar media were compared, the titer of the GC agar medium appeared faster and was higher than the titer on the CMI agar medium. For both agar media, the HA titer and growth increased with the concentration of the chicken serum added. In addition, HA activity on GC agar medium showed a higher titer than activity on other media, such as brain-heart infusion, Trypticase soy, blood, and Kato's agar media. When various HPG strains were inoculated onto GC agar medium, HA titers in strains 0083 and F were equal to that in strain 221. These results confirm that the GC agar medium is useful for preparation of HA antigen of HPG.

Serological response of chickens either vaccinated or artificially infected with Haemophilus paragallinarum.

The serological response of chickens either vaccinated or artificially infected with Haemophilus paragallinarum (Hpg) serovar A or C was investigated using both a specific hemagglutinin (HA) antigen and a common HA antigen. With Hpg serovar A, both vaccinated and artificially infected chickens produced hemagglutination-inhibition (HI) antibodies to Hpg serovar-specific and Hpg common HA antigens. Most chickens vaccinated with Hpg serovar C had detectable HI antibodies to both types of HA antigen by 3 weeks postvaccination, after which titers gradually declined. In contrast, most chickens artificially infected with serovar C produced HI antibodies to only the common HA antigen; very few of these chickens produced HI antibodies to the serovar-specific HA antigen.

Latex agglutination test for measurement of type-specific antibody to Haemophilus paragallinarum in chickens.

A latex agglutination test employing serotype-specific Haemophilus paragallinarum polysaccharides as the antigen has been developed. It can be used to measure the antigen-antibody reaction in chicken sera when spontaneous latex particle agglutinin is removed without loss of detectable antibody by kaolin pretreatment. The test was tried to detect the serological response in immunized chickens with two different serotype strains of H. paragallinarum; it could detect the type-specific antibody, and no crossreaction was observed. This latex-agglutinating antibody may be related mainly to immunoglobulin M in primary antisera in which the latex titer was reduced by 2-mercaptoethanol treatment. The latex agglutination test should be valuable as a routine diagnostic method because it is type-specific, simple, and economic and its sensitized latex suspension is stable for at least four months.

Production and properties of hemagglutinin of Haemophilus gallinarum.

Production of hemagglutinin (HA) of Haemophilus gallinarum was compared in some media, and its properties were studied. HA was produced in Kato's media, brain heart infusion (BHI) broth containing beta-diphosphopyridine nucleotide, and chicken meat infusion (CMI) broth. The HA in CMI broth different according to the concentration of the chicken serum; no HA titer was found in 0.5% or more chicken serum, but HA was activated by storage in a refrigerator. Cells of H. gallinarum cultured for a long time had markedly decreased HA titer. A weak HA was produced in blood and Kato's agars, but no titer appeared in CMI and BHI agars. HA of H. gallinarum was heat-labile and inactivated by formalin, ethanol, methanol, and ethyl acetate. On the other hand, HA was resistant to chloroform, acetone, and some enzymes. Moreover, the HA titer of cell cultured in CMI broth was enhanced by hyaluronidase. H. gallinarum in Kato's and BHI broths were pleomorphic rods with or without a capsule, but were capsulated ovoid cells in CMI broth, according to electron microscopy.

Three substrains with different hemagglutination patterns isolated from a Japanese lentogenic strain of Newcastle disease virus.

Three substrains were cloned from chick kidney cell monolayers infected with the Ishii strain of lentogenic Newcastle disease virus isolated in Japan. These substrains, which differed in plaque morphology, were distinguished as having no +24R+ and -24R- patterns in the hemagglutination (HA)-elution test.

Slide-agglutination test of Haemophilus gallinarum antigen treated by trypsin to inhibit spontaneous agglutination.

Spontaneous agglutination of Haemophilus gallinarum (HG) cells cultured in Kato's broth was inhibited by trypsin treatment. The treated cell had the same antigenicity as the common antigen prepared from chicken meat infusion broth. Trypsinized antigen could be used in the slide-agglutination test to detect HG antibody in chicken serum.

Isolation and characterization of a Haemophilus paragallinarum mutant that lacks a hemagglutinating antigen.

In an in vivo cross-protection test with Haemophilus paragallinarum strains of serovars B and C, we isolated and characterized a mutant strain, S1M, which lacked a hemagglutinating (HA) antigen when compared biologically and immunologically with isogenic strain S1. Unlike the isogenic strain S1, the mutant strain S1M lacked HA activity against formaldehyde-fixed chicken erythrocytes, even after hyaluronidase treatment, and it did not stimulate hemagglutination-inhibition antibody in chickens immunized with bacterial cells. Dot-blot testing and immunoelectron microscopy with monoclonal antibodies against serovar C-specific HA antigens showed that strain S1M did not react with these monoclonal antibodies. Furthermore, strain S1M was found to be both non-pathogenic and non-immunogenic. In contrast, the isogenic strain S1 reacted with these monoclonal antibodies and was pathogenic and immunogenic. These results suggest that the HA antigen of H. paragallinarum serovar C. strain plays an important role in pathogenicity and immunogenicity.

Determination of types 1 and 2 hemagglutinins in serotypes of Hemophilus paragallinarum.

Type 1 hemagglutinin (HA) of Hemophilus paragallinarum (HPG) was found only in Page's serotypes A and B and in Kato's serotypes I and III of HPG. Type 2 HA was found in all serotypes of HPG. Hemagglutination-inhibition test revealed no serological differences among the type 2 HA of Kato's serotypes.

Characterization and use of monoclonal antibodies to identify Haemophilus paragallinarum serovars.

Two serovar-specific monoclonal antibodies (MAbs) to Haemophilus paragallinarum serovars A/1 and C/2 strains, respectively, were developed and characterized by hemagglutination-inhibition (HI) and dot-blotting tests using representative H. paragallinarum serovars A/1, B, and C/2 strains. In both the HI and dot-blotting tests, one MAb (E5C12D10), raised against strain 221, serovar A/1, reacted only with serovar A/1 strains, while the other MAb (F2E6), raised against strain S1 of serovar C/2, reacted with only serovar C/2 strains examined. In both tests, the two MAbs did not react with two serovar B strains. These results indicated that the two MAbs recognize serovar-specific hemagglutinating (HA) antigens of H. paragallinarum serovars A/1 and C/2 strains, respectively, and that a dot-blotting test using these MAbs is a practical alternative to the HI test for serotyping H. paragallinarum. Strains 0222 and Spross of serovar B, which did not react with these two MAbs, were found to possess serovar-specific HA antigen in cross-HI tests.

Characterization of two new monoclonal antibodies against Haemophilus paragallinarum serovar C hemagglutinating antigen.

Two new monoclonal antibodies (MAbs), D6D8D5 and B3E6F9, both directed against Haemophilus paragallinarum serovar C hemagglutinating (HA) antigen, were produced, and characteristics of the MAbs were compared with those of the previously described MAb F2E6 in dot-blot and hemagglutination-inhibition (HI) tests using two representative H. paragallinarum strains each of serovars A, B, and C strains and 55 Japanese serovar C field isolates. MAb D6D8D5 and MAb F2E6 reacted with all serovar C strains and field isolates in the dot-blot test. However, MAb D6D8D5 showed various degrees of inhibition of the HA activity of field isolates. In the enzyme-linked immunosorbent assay-competition test, MAb D6D8D5 did not compete with MAb F2E6. MAb B3E6F9 reacted with strain S1, serovar C but not with strain Modesto, serovar C in both dot-blot and HI tests. Three out of 55 field isolates did not react with MAb B3E6F9. Neither MAb reacted with the serovar A and B strains.

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.

Growth kinetics of embryo- and organ-culture adapted Beaudette strain of infectious bronchitis virus in embryonated chicken eggs.

Three Beaudette strains of infectious bronchitis virus (IBV) [two chicken-embryo trachea-culture-adapted (E71CET10 and E71CEK10CET30) and one embryo-adapted (E72)] were propagated in 10-day-old embryonated chicken eggs (CE) to investigate growth and lethality patterns. The E72 and E71CEK10CET30 viruses were similar in growth pattern as to logarithmic phase and maximum virus production, but 95% of the CE mortality with E72 virus occurred between 20 and 32 hours whereas with E71CEK10CET30 60% mortality occurred between 36 and 84 hours. The yield of the E71CET10 virus was minimum, with little lethality.

Characterization of isolates of avian haemophili from Brazil.

The biochemical and serological properties of 29 isolates of avian haemophili obtained from chickens in Brazil are described. Twenty-seven of the isolates had the typical biochemical properties of Haemophilus paragallinarum. The two remaining isolates had the typical properties of Pasteurella avium, formerly known as Haemophilus avium. All of the H. paragallinarum isolates were serotyped according to the Page scheme using a hemagglutination-inhibition test. Fourteen of the isolates were serovar A, one was serovar B, 11 were serovar C, and one isolate could not be serotyped. The isolates were also examined using a panel of monoclonal antibodies for Page serovars A (one monoclonal antibody available) and C (three monoclonal antibodies available). As expected, the serovar B isolate failed to react with any monoclonal antibody, whereas the 11 serovar C isolates reacted with all three serovar C monoclonal antibodies but not with the serovar A monoclonal antibody. Only eight of the 14 serovar A isolates reacted with the serovar A monoclonal antibody.

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.