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A multiplex reverse transcription-polymerase chain reaction (RT-PCR) was developed to amplify the S1 and S2 genes of vaccine and recent Taiwanese isolates of infectious bronchitis virus (IBV). DNA fragments of 228 and 400 base pairs in length were amplified among IBV isolates in multiplex PCR, suggesting that there were no apparent deletions or insertions in these regions. No PCR products were amplified from unrelated avian viruses and negative controls. The results suggested that multiplex PCR provided a specific and sensitive approach for identification of IBV isolates. Sequence analysis of the hypervariable region (HVR) of S1 gene exhibited high variations among Taiwanese IBV isolates. The TWI and TWII groups were about 84-98 and 94-99% identity within the groups. American strains were most divergent sharing only 60% homology with TWI and TWII Taiwanese strains. The Mass group varied 0-10% among each other and had over 70% homology with TWI and TWII Taiwanese strains. A phylogenetic tree based on the nucleotide sequences of the HVR of S1 gene revealed that Taiwanese IBV isolates had evolved into three groups (TWI, TWII, and Mass). This suggested that there were multiple groups of viruses cocirculating in Taiwan.

A strain of Pasteurella anatis (PA) was isolated from the sinus of an adult leghorn laying chicken with sinusitis, nasal discharge, drop in egg production, and low mortality, symptoms initially thought to indicate infectious coryza. The tiny, smooth, whitish colonies were identified as PA. To compare its pathogenicity with that of commercial broilers, nine groups, 10 birds per group, of 10-day-old broilers were individually inoculated with the strain of PA, Pasteurella multocida (PM), or Escherichia coli (EC) by intravenous, intraperitoneal, intramuscular, or subcutaneous inoculation. The PA was determined to cause the signs, lesions, and septicemic death, which are similar to the symptoms of PM or EC infection. At 1 wk postinfection (PI), the mortality rate was between that of PM and EC infection at 1 wk PI. Twenty antimicrobial-containing discs were evaluated, and the isolate was highly sensitive to cetiofer, amoxicillin, lincopectin, and furazolidone. Furthermore, it was moderately sensitive to tetracycline and enrofloxacin and only slightly sensitive to cephalothin, chloramphenicol, flumequine, nalidixic acid, neomycin, oxolinic acid, streptomycin, and trimethoprim. The PA infection was treated successfully with amoxicillin.

The very virulent infectious bursal disease virus (vv IBDV) RNA in the bursa of Fabricius and spleen from experimentally infected chickens or field samples was detected by in situ hybridisation (ISH) with subsequent reverse transcription (RT)-polymerase chain reaction (PCR) and sequence analysis. The VP 2 gene of vv IBDV was detected by ISH in infected chicken tissues with a cloned digoxigenin (DIG)-labelled cDNA probe. To verify ISH, RT - PCR was used to amplify two 643- and 500-base pair fragments on the VP 2 gene of IBDV in the bursa of Fabricius. With all isolates, two c DNA fragments of 643 and 500 bp long, respectively, were generated as expected and further confirmed the specificity of ISH. Analysis of the hypervariable region (HVR) of the VP 2 gene revealed that a serine-rich heptapeptide SWSASGS located at amino acids 326-332 was conserved in recent Taiwanese strains, and two amino acid substitutions were found in the classical Taiwanese strains at positions 330M and 331W. Three amino acids were unique to the vv strains at positions 222A, 256I and 294I, compared with classical and variant strains. Sequence and phylogenetic analysis showed that the recent Taiwanese strains were closely related, very similar to vv IBDV s from Europe, China, Japan, and Africa, and distantly related to the Taiwanese classical strains., (Copyright 2001 Harcourt Publishers Ltd.)

Fifteen hemolytic gram-negative bacteria were isolated from the respiratory tracts of sick birds suffering from a long-lasting respiratory syndrome or from the bone marrow of dead birds distributed in the southern part of Taiwan. These were classified as Pseudomonas aeruginosa (10 isolates), Pseudomonas fluorescens (2 isolates), Pseudomonas stutzeri (1 isolate), Pasteurella haemolytica (1 isolate), and Proteus morganii (1 isolate). Each isolate was inoculated intraperitoneally into one group of ten 4-week-old male white leghorn chickens. Mortality and lesions were scored daily for 1 week. Three of the 10 isolates of Pseudomonas aeruginosa caused 100% mortality. Six other isolates of Pseudomonas aeruginosa and the one isolate of Proteus morganii caused 50% mortality. The remaining isolates induced less than 30% mortality. The sole nonpathogenic sample was one isolate of Pseudomonas fluorescens. When therapeutic levels of 22 antibiotics or sulfa drugs were evaluated for their inhibitory activity against the 15 isolates, the most effective were apramycin (15/15), gentamicin (15/15), spectinomycin (13/15), oxytetracycline (8/15), and sulfachloropyrazine (7/15). The least effective were ampicillin, cloxacillin, and tiamulin, which were not effective against any of the isolates. The 14 other drugs were of very low (> 4/15) effectiveness. Most of the isolates studied were virulent for chickens and very resistant to currently used drugs.

The egg transmissibility of strains F and R of Mycoplasma gallisepticum (MG) was studied in 53 pullets by isolating MG from the eggs and embryos. MG was isolated at rates of 3.19% and 1.59% from the eggs of hens exposed by aerosol to the R or F strain, respectively. However, MG was isolated at rates of 0.19% and 0.00% from the eggs of chickens exposed by eye drop to the R or F strain, respectively. A statistically significant reduction in egg production was observed during the first 4 weeks postexposure in the group exposed to the R strain of MG by aerosol. An unexpected finding was significantly lower fertility of eggs from all MG-infected groups.

The sensitivity and specificity of the microagglutination (MA), serum-plate-agglutination (SP), and hemagglutination-inhibition (HI) test for Mycoplasma gallisepticum (MG) were compared in groups of chickens infected with MG, M. synoviae, or Pasteurella multocida or inoculated with bacterins prepared from Staphylococcus aureus or Erysipelothrix rhusiopathiae. Of the three tests evaluated, the HI test had the highest specificity, but it was the least sensitive. Both the MA and SP tests were more sensitive than the HI test but lower in specificity. The MA test was less sensitive than the SP test in detecting antibodies against heterologous MG strains.

Strains F and R of Mycoplasma gallisepticum (MG) were compared for their pathogenicity in broad-breasted white turkey poults in terms of induction of airsacculitis, depression of growth rate, and induction of antibody production against MG. Eye-drop inoculation with the F or R strain produced only minimal lesions. However, aerosol exposure with either MG strain resulted in a significantly higher incidence and severity of air-sac lesions (P less than 0.001), reduction of growth rate (P less than 0.005), and hemagglutination-inhibition (HI) antibody titers against MG than did eye-drop inoculation. There were no significant differences between the two MG strains in effects on incidence of air-sac lesions or on growth rate.

Of 44 representative isolates of yeast isolated from the poultry upper digestive tract from 40 clinical thrush cases in Taiwan during 1985-87, 39 (89%) isolates were classified as Candida albicans, and 5 (11%) were classified as Torulopsis pintolopesii. Fifteen commercial antifungal drugs, incorporated individually in Sabouraud's dextrose agar by serial twofold dilutions, were tested for their inhibitory effect against the 44 isolates. The MIC50 of these drugs in increasing order was less than or equal to 2 ppm GV-11, 6 ppm gentian violet, less than 16 ppm amphotericin B, 16 ppm hyamine 1622, 25 ppm econazole, 35 ppm chlorohydroxyquinoline, 40 ppm nystatin, 64 ppm miconazole, 747 ppm malachite green, 1550 ppm benzoic acid, 1536 ppm copper sulfate, 3144 ppm Monoprop, 4951 ppm Mold Zap, greater than 16,384 ppm propionic acid, and greater than 16,384 ppm sodium propionate.

Five strains of Mycoplasma gallisepticum (MG) were tested for their cross-protection, virulence, and antigenic relationship in young leghorn chickens. Chickens vaccinated at 1 day of age by eye drop with the F or R strain of MG had good protection against aerosol challenge with the F, R, or S-6 strain at 4 weeks postvaccination. Chickens vaccinated at 1 day of age with the 503 or 730 strain had only partial protection against challenge with R, F, or S-6; the degree of cross-protection was related to the virulence of the vaccine strain used. In the hemagglutination-inhibition test, serum from chickens with single infection (vaccination and/or challenge with only one strain) with the F, S-6, or R strain reacted strongly with antigens prepared from S-6 and A5969 and reacted weakly with 730 and 503 antigens. Chickens with a single infection with the 503 or 730 strain reacted only with antigen prepared from the homologous strain. Serum from chickens with a two-strain mixed infection had a wider spectrum of HI activities. In the serum-plate-agglutination test, serum from chicken infected with the S-6, F, or R strain reacted with all antigens. Chickens infected with the 503 or 730 strain reacted more strongly with the homologous antigen. Antigen prepared from the S-6 strain of MG detected antibodies against the 503 and 730 strains more efficiently than did antigen prepared from the A5969 strain.

The preferred method for the control of Mycoplasma gallisepticum is eradication. Unfortunately, the advent of multiple-age farms for poultry production has made eradication impractical after the organism has been introduced. Live vaccination with the F strain of M. gallisepticum during the rearing period has been used to prevent egg production losses. More recently, an inactivated oil emulsion bacterin has become available commercially. Vaccine and bacterin offer protection against egg production losses, respiratory signs and lesions, and egg transmission. Vaccination of broilers, however, is not yet feasible, Bacterin offers minimal protection against infection. The use of bacterins and vaccines on multiple-age farms is being studied as a potential eradication tool.

Progeny chicks of Mycoplasma gallisepticum (MG)-infected and MG-free white leghorns were used to evaluate the protective effects of maternal antibodies against aerosol challenge with the virulent R strain of MG at 3, 7, or 14 days of age. Interference by maternal antibodies with day-1 vaccination with the F strain of MG was also studied. In another trial, 8, 4, or 2 ml of high- or low-titered antiserum was given to 20-day-old commercial white leghorn chicks. Protection was measured in terms of air-sac lesions post-challenge with the R strain of MG as above. Maternal antibody in the chicks conferred very little protection against challenge and did not interfere with day-1 vaccination with the F strain of MG. Low-titered antibody gave no protection against growth of MG in the tracheas, but the high-titered MG antiserum did confer some protection against airsacculitis. This protection might be due to growth-inhibition antibody in the transferred serum.

Twenty-nine antibiotics or drugs were incorporated individually into mycoplasma agar to evaluate their inhibitory activity against avian mycoplasmas: 100 recent Taiwan isolates of 7 serotypes and 10 standard strains of 7 serotypes were tested. All of the standard strains were very sensitive to erythromycin, chlorotetracycline, doxycycline, minocycline, and tetracycline, but the local isolates were highly resistant to these antibiotics. The drugs or antibiotics that possessed an MIC90 of 50 micrograms/ml or less against the local isolates were tiamulin (less than 0.4 micrograms/ml), lincospectin (2.7), josamycin (2.7), lincomycin (3.0), spectinomycin (4.8), tylosin (6.0), kanamycin (6.0), chloramphenicol (6.0), gentamicin (7.5), apramycin (24.5), doxycycline (27.4), minocycline (29.0), spiramycin (30.0), colistin (44.3), leucomycin (45.0), and streptomycin (50.0). The MIC90 of the other antibiotics or drugs was greater than 50 micrograms/ml. None of the isolates or strains were sensitive to nalidixic acid, ronidazole, penicillin, ampicillin, cephalexin, carbadox, or four sulfa drugs at a concentration about 5 times the therapeutic level.

Five trials were conducted to evaluate the virulence and the vaccination efficacy of the F, R, S6, and A5969 strains of Mycoplasma gallisepticum (MG) at different in vitro passage levels. Vaccination was done by eye-drop or aerosol, and efficacy was evaluated in terms of air-sac lesions after aerosol challenge with the R strain of MG. Continuing medium passage of these strains of MG resulted in gradual attenuation. Aerosol vaccination with highly attenuated MG at 21 days of age was more effective in stimulating the development of immunity than was eye-drop vaccination at 7 days, but aerosol vaccination induced more air-sac lesions. Aerosol vaccination with high-passaged F or S6 strain provided good immunity against MG challenge. Back-passage in chickens of the F strain of over 300 medium passages did not substantially increase the virulence, but back-passage of the S6 strain at the 258th passage and F strain at the 11th and 252nd passage did result in increased virulence. Both low- and high-passaged F and S6 strains were virulent for turkey poults, but only the high-passage F strain did not significantly (P less than 0.05) affect body weight.