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.

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.

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%.

Distribution of antibodies to influenza A virus in chickens in Japan.

A serological surveillance was carried out to detect antibody against influenza A virus in chicken sera. A total of 8,850 field samples were collected from 47 prefectures in Japan. Initially, all the sera were screened by agar gel immunodiffusion and those sera showing positive reaction were investigated for haemagglutination-inhibition (HI) and neuraminidase-inhibition antibodies against influenza viruses. Only 6 samples had antibodies; 4 sera had antibodies against human subtype H1N1 virus; with HI activity against strain A/PR/34; three sera had strong HI activity to strain A/Tottori/4/87, which by haemagglutination test is closely related to A/Yamagata/120/86. The remaining two chicken sera had antibodies against avian subtypes H10N4 and H3N6 viruses respectively.

Phenotypic and molecular characterization of V-factor (NAD)-independent haemophilus paragallinarum.

In South Africa from early 1989 onward, strains of Haemophilus paragallinarum not requiring nicotinamide adenine dinucleotide (NAD) have been isolated from commercial chickens suffering from infectious coryza. Fifteen of these field isolates were characterized by biochemical typing, serotyping, restriction endonuclease analysis (REA), and ribotyping. The chosen isolates represented diversity in geographic location, time of disease outbreak, and type of flock. All were typical of the species in biochemical properties, except that they were NAD-independent, and all were Page serovar A. REA was performed with three enzymes: HindIII, HpaII, and SspI. All isolates gave identical REA profiles with all three enzymes. Ribotyping was performed using a probe that consisted of the plasmid pUC19 into which the 16S rRNA operon of H. paragallinarum had been inserted. All 15 isolates gave the same ribotyping profile using each of the three enzymes. As a group, the NAD-independent strains gave REA profiles and ribotypes that were very different from a range of classic South African strains isolated before 1989. Our results strongly suggest that the NAD-independent isolates are clonal in nature.

Measurement of antibody titer to fowl pox virus by enzyme-linked immunosorbent assay.

The usefulness of the measurement of antibody titer to fowl pox virus (FPV) by enzyme-linked immunosorbent assay (ELISA) was evaluated in SPF chickens with or without inoculation with FPV. The optimum concentration of purified antigen was 10 micrograms/ml of protein. The absorbance at 492 nm was less than 0.10 in the chickens negative to FPV from 1 to 63 days old. By contrast, a higher titer was detected in SPF chickens with various FPVs inoculated into the wing web than in non-inoculated chickens. Moreover, there was no cross response to chicken sera immunized with Haemophilus paragallinarum, Marek's disease virus, Newcastle disease virus or infectious bronchitis virus. The titers increased after vaccination were not increased after subsequent challenge with virulent FPV. These findings suggested the usefulness of the measurement of the antibody response to FPV vaccine by ELISA.

Expression of the Newcastle disease virus (NDV) fusion glycoprotein and vaccination against NDV challenge with a recombinant baculovirus.

The hybrid baculovirus constructed from Autographa california nuclear polyhedrosis virus (NPV) and Bombyx mori (silkworm) NPV was used for expression of fusion glycoprotein (F) of Newcastle disease virus (NDV) strain D26. The gene encoding F protein was introduced into the improved baculovirus expression vector derived from the host-range-expanded baculovirus. In Spodoptera frugiperda (fall armyworm) cells infected with a recombinant baculovirus, HyF121, the expressed F protein was properly located onto the cell surface. After silkworm pupae were infected with HyF121, a subunit vaccine against NDV was prepared from the HyF121-infected pupae. Chickens inoculated with the subunit vaccine were protected against virulent NDV challenge.

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.

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.

Characterization of isolates of Haemophilus paragallinarum from Argentina.

The biochemical and serological properties of Haemophilus paragallinarum isolates recovered from 11 recent outbreaks of infectious coryza in layer hens and one case of swollen-head syndrome in broilers in Argentina are described. Twenty-four isolates had the typical biochemical properties of H. paragallinarum. All isolates were serotyped according to the Page scheme. Ten of the isolates were serovar A, 11 were serovar B, one was serovar C, and two isolates could not be serotyped. The isolates were also examined using a panel of monoclonal antibodies (MAbs) for Page serovars A (one MAb available) and C (three MAbs available). The serovar B isolates all failed to react with any MAb. The serovar C isolate reacted with all three serovar C MAbs but not with the serovar A MAb. Only six of the 10 serovar A isolates reacted with the serovar A MAb. These results indicate that H. paragallinarum isolates from Argentina are antigenically distinct from those examined in other countries, and it is suggested that coryza vaccines intended for use in Argentina may be more effective if based on local strains.

Cloning and DNA sequence of a 29 kilodalton polypeptide gene of Mycoplasma gallisepticum as a possible protective antigen.

A lambda gt11 clone, designated M1 and having a 0.8 kilobase (kb) insert, was selected by screening a Mycoplasma gallisepticum (M.g.) genomic DNA library with antisera against M.g. cells and their membrane proteins. The sequence of a 1.7 kb EcoRI fragment of genomic DNA covering the entire M1 insert revealed a long open reading frame, TM-1, that encoded a polypeptide with a deduced molecular weight of 29 kDa. An antiserum raised in chicken against the TM-1 polypeptide, which was produced by recombinant Escherichia coli cells and purified by column chromatography, inhibited growth of M.g. cells in vitro. Moreover, chickens immunized with this polypeptide were partially protected from a challenge with virulent M.g. This polypeptide may serve as the basis for a vaccine against M.g. infection.

[The use of a metallic stent in 32 patients with benign prostatic hypertrophy. Preliminary results and 3 months of follow-up].

A prospective noncontrolled study of the safety and potential efficacy of the metallic stent was performed on 32 patients with benign prostatic hypertrophy. Mean age was 76.6 years (range, 56-98 years), and mean prostatic volume was 24.2 cm3. The patients were selected on the basis of a quantitative symptom score (QSS), uroflowmetry measurements, and residual urine volume (RU). Nineteen patients had urinary retention and remaining 13 patients had moderate symptoms and signs of prostatism. Placing the stent was successfully done in 31 patients (97%). It took 15 minutes to place the stent using transabdominal and/or endorectal sonography. After 3 months, 27 patients (87%) showed improved QSS. In patients with dysuria, maximum flow rate (MFR) and RU before treatment were 6.9 +/- 1.7 ml/sec and 112.3 +/- 61.8 ml, respectively. After treatment, they improved to 12.3 +/- 2.7 ml/sec and 12.7 +/- 6.7 ml, respectively. On the other hand, all patients who had urinary retention were able to urinate just after treatment, and MFR and RU were 12.9 +/- 3.6 ml/sec and 24.4 +/- 43.3 ml, respectively. Evaluation on the basis of improvement in MFR and reduction in RU showed that the stent was effective in 71% of total patients (22 out of 31 patients), 94% of the patients with urinary retention (17 out of 18 patients). The overall clinical efficacy of this stent was 68% (21 patients). There were no major complications such as urge incontinence and urinary tract infection during follow-up. Although proximal migration of the stent was observed in 6 patients, the stent could be taken out and replaced in 4 patients. From the above results, we conclude that the metallic stent is useful for the treatment of prostatism and urinary retention.

Clinical effects of long-term use of neutralized dialysate for continuous ambulatory peritoneal dialysis.

The long-term effects of neutralized dialysate used in continuous ambulatory peritoneal dialysis (CAPD) were evaluated in 8 well-controlled patients. Twelve milliliters of 8.4% sodium bicarbonate was added to Dianeal PD-1 immediately before every administration. The final pH was 6.8 and the concentration of sodium bicarbonate was 6 mmol/l. The final sodium level was 138 mEq/l. This dialysate was used for 5 months. For 2 months before and 3 months after this period, Dianeal PD-2 was used as the dialysate for comparison. Blood bicarbonate levels significantly improved during the use of the neutralized dialysate. Blood sodium, chloride and magnesium levels and the effluent volume significantly increased. Sodium balance improved during the period when neutralized dialysate was used. Total leukocyte counts in the effluent decreased, and leukocyte viability increased. Abdominal distention, abdominal pain during instillation, nausea and headache improved. No side effects, including peritonitis, occurred during the trial of neutralized dialysate. The results suggest that this dialysate was less irritating to the peritoneal membrane than the control dialysate and that the therapeutic effects were satisfactory.

[Transurethral balloon dilatation of the prostate: initial results, indication and complication].

Transurethral balloon dilatation therapy was performed on 40 patients with benign prostatic hypertrophy (BPH) under local anesthesia. During the procedure, urinary urgency occurred in 80% of the patients. After prostatic dilatation, macrohematuria was observed in almost all patients. The longest follow-up period after dilatation was now 22 months, and the average was 9.5 months. After treatment, residual urine volume decreased, and average flow rate and maximal flow rate improved from 5.7 and 10.4 ml/sec to 8.2 and 15.6 ml/sec, respectively. Overall clinical efficacy was 67.5%. Urethral dilatation therapy was thought to be an effective and non-invasive therapy for BPH.

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.

Evaluation of a panel of monoclonal antibodies in the subtyping of Haemophilus paragallinarum.

A panel of four monoclonal antibodies (MAbs) was evaluated, using a hemagglutination-inhibition test, for its ability to subtype 76 isolates of Haemophilus paragallinarum. The results of the MAb reactions were compared with the results of both the Page and Kume serotyping schemes (the serovars of the Page scheme correspond to the serogroups of the Kume scheme). One MAb (E5C12D10) was raised against a Page serovar A strain and the remaining MAbs (F2E6, D6D8D5, and B3E6F9) against a Page serovar C strain. Six different reaction patterns were found among the 76 isolates of H. paragallinarum. There was total correlation between the MAb reaction pattern and the Page scheme, and thus the Kume scheme, to the serogroup level. All 19 Page serovar A (= Kume serogroup A) strains reacted only with MAb E5C12D10, whereas all five Page serovar B (= Kume serogroup B) strains failed to react with any of the MAbs. All 52 remaining strains were Page serovar C (= Kume serogroup C), and all failed to react with MAb E5C12D10 but showed varying reaction patterns with the three other MAbs. Although the MAbs recognized four subdivisions within Kume serogroup C, these subdivisions differed from the four Kume C serovars. This panel of MAbs can be used to assign isolates of H. paragallinarum to either Page serovars or Kume serogroups. Although the subdivisions recognized by the MAbs within the Page serovar C strains do not correspond to the Kume serovars, they may be useful in epidemiological applications.

Antibody response to Newcastle disease virus (NDV) of recombinant fowlpox virus (FPV) expressing a hemagglutinin-neuraminidase of NDV into chickens in the presence of antibody to NDV or FPV.

Antibody response of recombinant fowlpox virus (FPV) was studied in chickens inoculated with the virus in the presence or absence of antibodies against Newcastle disease virus (NDV) or FPV. In the case of NDV, high hemagglutination-inhibition titers to NDV were obtained when the antibody was present. No immune response to NDV was observed in the chickens previously vaccinated with FPV.

Pathogenicity and serovar-specific hemagglutinating antigens of Haemophilus paragallinarum serovar B strains.

The pathogenicity and presence of serovar-specific hemagglutinating (HA) antigens of Haemophilus paragallinarum serovar B reference strains 0222 and Spross and field isolates 24268 and 24317 were investigated. Chickens challenged with all strains except strain 0222 showed clinical signs of infectious coryza. For all four strains, challenged chickens had intrasinus lesions and were colonized by the challenge organism. Before and after hyaluronidase treatment, strains 0222, 24268, and 24317 showed HA activity against formaldehyde-fixed chicken erythrocytes but not against fresh chicken erythrocytes. Strain Spross expressed HA activity only after treatment. In cross-hemagglutination-inhibition tests, the four serovar B strains cross-reacted with each other but not with serovar A and C strains. Cross-adsorption tests indicated that strain 24317 has a wider range of HA antigens than the other two strains. Our results indicated that H. paragallinarum serovar B strains are pathogenic for chickens and that they possess six different HA antigens, one of which is specific for serovar B strains.

Recombinant fowlpox viruses inducing protective immunity against Newcastle disease and fowlpox viruses.

The haemagglutinin-neuraminidase gene of Newcastle disease virus was inserted into a non-essential region of the fowlpox virus genome and expressed under control of the vaccinia virus 7.5 kDa polypeptide gene promoter. Immunization with the recombinant fowlpox virus elicited protective immunity in chickens against both virulent Newcastle disease and fowlpox virus infection.