Molecular characterization of Mycoplasma gallisepticum isolates from turkeys

Mycoplasma gallisepticum was isolated from several turkey flocks at different locations in the United States that were clinically affected with respiratory disease. Five of these isolates from four series of outbreaks had patterns similar to the 6/85 vaccine strain of M. gallisepticum by random amplified polymorphic DNA (RAPD) analysis using three different primer sets, whereas with a fourth primer set (OPA13 and OPA14), only two of the isolates were similar to 6/85. Results obtained by sequencing portions of the pvpA, gapA, and mgc2 genes and an uncharacterized surface lipoprotein gene indicated that the field isolates had DNA sequences that ranged from 97.6% to 100%, similar to the 6/85 results. In some of the outbreaks there was an indirect association with the presence of commercial layers in the area that had been vaccinated with this vaccine strain, but there was no known close association with vaccinated birds in any of the outbreaks. Turkeys were challenged with two of the field isolates and with 6/85 vaccine strain. Turkeys challenged with the field isolates developed respiratory disease with airsacculitis and a typical M. gallisepticum antibody response, whereas birds challenged with 6/85 developed no respiratory signs or lesions and developed only a weak antibody response. Although these isolates were very similar to the 6/85 vaccine strain, it was not possible to prove that they originated from the vaccine strain-it is possible that they could be naturally occurring field isolates.     

Fingerprinting of Mycoplasma gallisepticum strains isolated from multiple-age layers vaccinated with live F strain

Mycoplasma gallisepticum (MG) isolates were obtained from three multiple-age commercial layer farms on which live F strain vaccine had been administered to each replacement flock for at least 2 years. All such isolates had restriction endonuclease DNA and sodium dodecyl sulfate-polyacrylamide gel electrophoresis protein patterns characteristic of F strain. These cultures also hybridized in dot blot assays with both the MG strain-specific and species-specific DNA probes. In contrast, the original MG isolate that came from one of the farms before vaccination began clearly was not F strain. These results suggest that continuous use of live F strain vaccine in each replacement pullet flock on multiple-age commercial layer sites will result in displacement of the original field strain of MG with the vaccine strain.     

Floor pen study to evaluate the serological response of broiler breeders after vaccination with ts-11 strain Mycoplasma gallisepticum vaccine

To determine the Mycoplasma gallisepticum (MG) rapid serum plate agglutination (RSPA) test response of broiler breeders after ts-11 strain vaccination, 55 Cobb pullets derived from a nonvaccinated, MG-negative, commercial, broiler breeder grandparent flock were monitored from 8 to 20 wk of age (over a 12-wk trial period). To evaluate the effect of lateral spread of the ts-11 vaccine strain on RSPA test results from commingled and adjacently penned birds, treatment groups included (A) birds vaccinated with ts-11strain MG at 8 wk of age, (B) commingled nonvaccinates in the same pen as the vaccinated birds, (C) nonvaccinates in a second pen separated from the first pen by a distance of 2 m, and (D) birds vaccinated with ts-11 strain MG at 8 wk of age and kept in a separate room. Rapid serum plate agglutination tests were performed once a week for 6 wk and then every 2 wk for 6 more wk, postvaccination. A polymerase chain reaction (PCR) assay specific fbr ts-11 strain MG was used to confirm vaccination, and a second PCR specific for non-ts-11 strain MG was used to confirm the absence of field infection. Seroconversion was first detected by the RSPA test 2 wk postvaccination and attained maximum positive rates of 58% at 12 wk postvaccination in treatment A and 60% at 8 wk postvaccination in treatment D. Seroconversion rates in nonvaccinated, commingled pullets was 10% at 5 wk and 30% at 12 wk after the vaccination of pen mates. The ts-11-specific PCR detected the vaccine strain in 80%-100% of the vaccinated birds 2 wk after vaccination. One of 15 nonvaccinated birds penned 2 m from vaccinated birds yielded ts-11 by PCR assay 12 wk after vaccination, which indicates that the spread of ts-11 over short distances may be possible in situations in which there is a common caretaker. PCR on tracheal swabs taken 12 wk postvaccination detected ts-l1 in 50% and 60% of the vaccinated birds in treatments A and D, respectively; in 30% of the commingled nonvaccinates; and in 6.6% of the separately penned nonvaccinates. In contrast, choanal swabs collected from vaccinated birds at 12 wk were 21% and 40% PCR positive for ts-11 strain MG, while those from nonvaccinates were negative. All samples were PCR negative for field strain MG. The pattern of seroconversion as measured by RSPA test in small groups of broiler breeders was different from that previously reported for leghorns. Lateral spread of the ts-11 strain to commingled pen mates occurred rapidly, causing RSPA seroconversion patterns that mimicked those of the vaccinated pen mates.     

Characterization of a ts-1-like Mycoplasma galisepticum isolate from commercial broiler chickens

Several commercial broiler flocks in northeastern Georgia that were the progeny of the same parent flock (Flock 40) were diagnosed as Mycoplasma gallisepticum (MG) positive by serology, culture, and PCR. Flock 40 had been vaccinated with ts-11 live MG vaccine. Several isolates were obtained from the MG-positive broiler flocks, and these isolates were indistinguishable from the ts-11 vaccine strain by the molecular strain differentiation methods used. A pathogenicity study was performed to compare the virulence of one of the isolates, K6216D, to the ts-11 vaccine strain. K6216D elicited a significantly stronger antibody response and significantly increased colonization of the tracheas and air sacs. K6216D also elicited significantly greater air sac and tracheal lesions than the ts-11 vaccine strain at 10 and 21 days postinoculation (P < or = 0.05). This is the first report of a field case of the apparent reversion to virulence and vertical transmission of the ts-11 vaccine.     

Use of mgc2-polymerase chain reaction-restriction fragment length polymorphism for rapid differentiation between field isolates and vaccine strains of Mycoplasma gallisepticum in Israel

Increasing use of Mycoplasma gallisepticum (MG) live vaccines has led to a need for a rapid test for differentiation of MG field strains from the live vaccine strains ts-11 and 6/85. We examined the differentiating potential of diagnostic polymerase chain reaction (PCR) primers targeted to the gene mgc2, encoding a cytadherence-related surface protein uniquely present in MG. The mgc2-PCR diagnostic primers are specific for MG in tests of all avian mycoplasmas or bacteria present in the chicken trachea and are sensitive enough to readily detect MG in tracheal swabs from field outbreaks. Differentiation of vaccine strain ts-11 was based on identification of restriction enzyme sites in the 300-base-pair (bp) mgc2-PCR amplicon present in ts-11 and missing in MG isolates from field outbreaks in Israel. Restriction sites for the enzymes HaeII and SfaN1 were identified in the amplified region in strain ts-11 and were not found in 28 field isolates of MG, comprising a representative cross section of all the MG isolates from the period 1997-2003. In practice, differential diagnosis of MG is achieved within 1 day of submission of tracheal swab samples by mgc2-PCR amplification and restriction of the amplicon with HaeII, giving a 270-bp fragment for ts-11 or no restriction for other MG strains tested. Application of the mgc2-PCR-restriction fragment length polymorphism (mgc2-PCR-RFLP) assay enabled differential diagnosis of both components of a mixture of ts-11 and non-ts-11 DNA, detecting the field strain in the presence of a large excess of ts-11. The test was successfully applied in vivo for monitoring vaccinates in a ts-11 vaccine trial. In principle, the test may also be used to identify the 6/85 vaccine strain, which yields a 237-bp product, readily differentiated from the approximately 300-bp PCR product of all other strains tested. Further testing of field isolates will be necessary to determine the applicability of this test in the United States and other countries.     

Field efficacy of different vaccines against infectious bursal disease in broiler flocks

A field study was performed to determine the efficacy of three commercially available vaccines against infectious bursal disease (IBD) in commercial broilers raised in a high IBD virus (IBDV) risk area. Live attenuated intermediate and intermediate plus vaccines were used in four flocks. Birds were vaccinated orally at the estimated vaccination time. Three broiler flocks were vaccinated subcutaneously with a turkey herpesvirus (HVT)-IBD vector vaccine at one day old. Evaluation of the efficacy of different vaccines was focused on humoral immune response, bursa/body weight (B/Bw) ratio, molecular detection of IBDV in ileocaecal tonsils and bursa of Fabricius, and production parameters. The serological results showed that although the uptake of all three vaccine strains was confirmed in the lymphoid organs, no significant antibody response to vaccination was detected in flocks vaccinated with intermediate and intermediate plus vaccines. A significant increase in antibody titres detected in flocks vaccinated with the vector vaccine indicated its ability to induce an immune response in birds with a high level of maternally derived antibodies. Observations obtained in this field trial did not confirm the expected reduction of the B/Bw ratio in flocks vaccinated with less attenuated vaccines. No significant differences were observed between birds vaccinated with the vector vaccine and those immunised with the intermediate plus vaccine. Very virulent IBDV was confirmed in the flock vaccinated with the intermediate vaccine. The infection induced reduced B/Bw and moderate mortality but did not affect the production parameters. Field infection was not detected in broilers vaccinated with the intermediate plus vaccine and the vector vaccine.     

Differentiation of Mycoplasma gallisepticum strains using amplified fragment length polymorphism and other DNA-based typing methods

Amplified fragment length polymorphism (AFLP) was used to type 34 strains of Mycoplasma gallisepticum (MG) including vaccine strains ts-11, 6/85, and F. Using AFLP, a total of 10 groups, with 30 distinguishable AFLP typing profiles, were generated in the analysis. The AFLP method was able to identify and differentiate both MG field strains from recent outbreaks and those that were epidemiologically related. The AFLP procedure will provide assistance in identifying the sources of mycoplasma infections. Vaccine strains were also differentiated from other field strains, which will be useful in the evaluation of vaccination programs. The AFLP discrimination potential was compared to other molecular typing techniques such as gene-targeted typing by DNA sequence analysis of the MG cytadhesin-like protein encoding gene, mgc2, and random amplified polymorphic DNA assay on the same MG isolates. The three assays correlated well with one another, with AFLP analysis having a much higher discriminatory power and reproducibility.     

Field observations with Salmonella enteritidis bacterins

A study involving 11 commercial layer flocks was conducted to determine the efficacy of Salmonella enteritidis bacterins (autogenous or federally licensed). The criterion for evaluation of vaccine efficacy was the presence or absence of S. enteritidis in the environment, the organs of the bird (including ovary and oviduct), and eggs. Environmental, rodent, and organ specimens from dead birds as well as eggs were cultured throughout the life of the flock. All layers were obtained from pullet sources that were negative for S. enteritidis, as determined by organ and environmental cultures. Despite the use of S. enteritidis vaccination, 63.6% of the houses had S. enteritidis-positive environmental cultures and 100% of the flocks had S. enteritidis organ-culture-positive birds. The range of positive cultures for S. enteritidis in the environment in vaccinated flocks was between 0 and 45.5%. Birds in vaccinated flocks were organ-culture positive for S. enteritidis between 10% and 40% of the time. The unvaccinated portion of flocks in the same house and the unvaccinated flock in a complex had similar results compared with the vaccinated portion of the flocks.     

Mycoplasma gallisepticum vaccination: further studies on egg transmission and egg production

Leghorn hens vaccinated twice with an inactivated Mycoplasma gallisepticum (MG) bacterin before egg production and subsequently challenged with virulent MG were protected against transmission of MG through the egg. Unvaccinated control hens transmitted MG through the egg at a high rate. When unvaccinated hens were vaccinated with MG bacterin 2 weeks after challenge with MG, there was no significant decrease in egg transmission. Hens vaccinated twice before laying did not suffer as severe egg-production drops as unvaccinated hens did when challenged with virulent MG. In a natural MG outbreak in a leghorn breeder flock, 50 hens were separated from the remainder of the flock and vaccinated with MG bacterin approximately 3 weeks after initial exposure. The unvaccinated hens transmitted MG through the egg at a rate three times higher than the rate of transmission of the vaccinated hens.     

Differentiation of Mycoplasma gallisepticum vaccine strains ts-11 and 6/85 from commonly used Mycoplasma gallisepticum challenge strains by PCR

Mycoplasma gallisepticum (MG) is an important avian pathogen causing significant economic losses within the poultry industry. In an effort to develop tools to aid in MG research and diagnostics, we have compared sequences of the attenuated MG vaccine strain ts-11 to those of commonly used pathogenic challenge strains in search of a simple means of differentiation. Via gapA sequence alignments and comparisons, we have identified and designed primers facilitating strain differentiation. When applied to conventional polymerase chain reaction (PCR) assay at low annealing temperature, the primer sets allow for the differentiation of MG attenuated vaccine strains ts-11 as well as the attenuated MG vaccine strain 6/85 from the commonly utilized MG challenge strains R(low), R, and S6. Conventional PCR differentiation is based on the visualization of sole products with the attenuated MG strains ts-11 and 6/85 and the lack of the corresponding products from MG strains R(low), R, and S6. When applied to MG strain F, product visualization varies with the applied primer set. The differentiation of MG strains ts-11 and 6/85 from the pathogenic challenge strains was also accomplished via real-time analyses, however, the primer sets were not able to differentiate MG strains ts-11 and 6/85 from selected MG field isolates.     

The efficacy of three commercial Mycoplasma gallisepticum vaccines in laying hens

The efficacy of three commercial Mycoplasma gallisepticum (MG) immunizing agents-a bacterin, a recombinant fowlpox-MG vaccine, and a live F-strain vaccine-was compared in specific-pathogen-free hens in egg production. Three groups of 25 chickens were vaccinated with one of the vaccines at 10 wk of age and 25 birds were not vaccinated. At 25 wk of age (and approximately 50% egg production), 20 birds from each of the three vaccinated groups and 15 nonvaccinated controls were challenged with virulent R-strain via aerosol; the birds were necropsied and evaluated at 10 days post-challenge. The MG bacterin and live F-strain vaccinations were both protective and resulted in significant differences in air sac lesions, tracheal lesions, and ovarian regression compared to the nonvaccinated controls and the recombinant fowlpox-MG vaccine (P < or = 0.05). The evaluation of ovarian regression is a useful method of testing the efficacy of MG vaccines in laying hens.     

Randomly amplified polymorphic DNA (RAPD) analysis of Mycoplasma gallisepticum isolates from turkeys from the central valley of California.

Randomly amplified polymorphic DNA (RAPD) analysis was used to investigate the molecular epidemiology of 26 Mycoplasma gallisepticum (MG) isolates obtained from turkeys located in the central valley of California. The MG isolates were recovered from 5 different companies and 13 ranches. Each company had unique MG strains. No evidence of spread of MG between companies was detected. RAPD analysis of MG isolates within a ranch during an outbreak revealed only a single strain involved in each outbreak. RAPD analysis identified an isolate from 1 ranch with a banding pattern identical to that of the 6/85 vaccine strain, which had been used on that particular ranch. Similar RAPD banding patterns of isolates from different ranches within the same company suggested horizontal spread of MG between ranches. The use of 2 primer sets in RAPD analysis was critical to prevent misinterpretation of relationships between different isolates.     

Genotypic diversity in field isolates of Babesia bovis from cattle with babesiosis after vaccination

Objective To determine whether particular genotypes of Babesia bovis were common to field isolates obtained from cattle properties in Queensland where the B bovis vaccine had apparently failed.
Design A comparative study of polymerase chain reaction genotypes in different populations of B bovis .
Procedure Two polymerase chain reaction assays were applied to analyse DNA extracts of B bovis vaccine (K, T and Dixie strains) and 27 field isolates from 24 properties where disease outbreaks had occurred despite the use of the vaccine. To evaluate the stability of the genotypes identified, 11 of the field isolates were inoculated into experimental cattle that had either been previously vaccinated with T strain or not vaccinated.
Results No particular genotype of B bovis was responsible for the problems observed in previously vaccinated herds. None of the isolates had genotypes identical to the vaccine strains used. No geographic trends among the genotypes were observed. Isolates that originated from the same property also had different genotypes. Blood passage of the 11 field isolates in either previously vaccinated or nonvaccinated cattle did not alter the original genotype.
Conclusion No particular genotypes identified by the Bv80 and BvVA1 polymerase chain reaction assays could be associated with vaccine failures.     

Evaluation of a Mycoplasma gallisepticum strain exhibiting reduced virulence for prevention and control of poultry mycoplasmosis.

Two experiments were conducted to evaluate the virulence and vaccination efficacy of a Mycoplasma gallisepticum (MG) isolate designated MG Intervet 6/85. Virulence of the strain was determined by evaluation of airsacculitis scores following aerosol exposure to the isolate before and after 10 sequential passes in either commercial broiler chickens or commercial turkeys. Two-week-old specific-pathogen-free chickens were vaccinated by aerosol exposure. The birds were challenged with the R' strain of MG at either 4 or 8 weeks post-vaccination. Efficacy was evaluated by airsacculitis scores determined 21 days after challenge. Ten repetitive back-passes of the isolate in chickens and turkeys did not substantially increase the virulence. Virulence for both chickens and turkeys was minimal, while protection elicited by aerosol vaccination in young chickens against virulent R' strain was significant (P less than or equal to 0.05) compared with unvaccinated controls.     

Detection of wild-type Aujeszky's disease virus by polymerase chain reaction in sheep vaccinated with a modified live vaccine strain

An outbreak of Aujeszky's disease occurred in a flock of sheep which had been housed together with pigs. After the death of five sheep with clinical signs of Aujeszky's disease, the remaining sheep were vaccinated with the Bartha vaccine strain, and the pigs were vaccinated with the 783 vaccine strain of Aujeszky's disease virus. Despite vaccination, however, more sheep died. Brain tissues from four sheep were collected for virus isolation and for immunobistological examinations. Only vaccine virus (gE-negative) was detected in the tissue. After DNA restriction enzyme analysis of the isolated virus, DNA of one or both of the vaccine strains was detected in all sheep. In one sheep field virus DNA was also detected. However, when the polymerase chain reaction was performed on samples prepared from paraffin-embedded tissues, DNA of field virus (gE-positive) was detected in all four sheep. It was probable that the sheep had not yet mounted a sufficient immune response to the vaccine virus, or were already infected with field virus at the time of vaccination. We concluded that the sheep died from field virus infection and not from vaccine virus infection and that only the polymerase chain reaction made it possible to specifically detect even very small amounts of field virus DNA among vaccine virus DNA in all investigated sheep.     

Evaluation of Mycoplasma gallisepticum K-strain as a live vaccine in chickens

We evaluated the pathogenicity of three live Mycoplasma gallisepticum (MG) vaccine candidates by infection via aerosol of 3-wk-old chickens with log phase broth cultures (trial 1). Two of the candidates (K3020 and K4649A) colonized only 10% and 20% of the chickens, respectively, unlike K2101 (K-strain), which was reisolated from all of the vaccinated chickens tested. K-strain inoculation did not result in significant air sac or tracheal lesions in chickens at 10 and 39 days postinfection (P < or = 0.05). The efficacy of K-strain as a live vaccine was evaluated in trial 2, by challenge of vaccinated chickens with virulent R-strain via aerosol at 6 wk postvaccination. K-strain vaccination resulted in significant protection from air sac and tracheal lesions (P < or = 0.05). The K-strain was further investigated to evaluate transmissibility (trial 3), colonization and persistence of infection following aerosol administration (trial 4), genetic and phenotypic stability following back passage through chickens (trial 5), and vertical transmission (trial 6). The K-strain had a low rate of horizontal transmission; it remained primarily in the respiratory system of inoculated birds and persisted in the upper respiratory tract for the duration of the trial 4 (5 mo). There was no increase in virulence of K-strain when it was back passaged five times through chickens, and no vertical transmission of K-strain was detected. K-strain showed great potential as a safe and effective live MG vaccine.     

Effects of differences in virulence of different serovars of Haemophilus paragallinarum on perceived vaccine efficacy

The virulence of four South African field isolates of NAD-dependent Haemophilus paragallinarum and two field isolates of NAD-independent H. paragallinarum has previously been tested in unvaccinated chickens. In this study, the disease profiles caused by the NAD-dependent isolates of H. paragallinarum in vaccinated chickens were studied. It was shown that the clinical signs induced in the vaccinated chickens were substantially less severe than were those in unvaccinated chickens, as was expected. However, due to the high virulence of the serovar C-3 isolates, clinical signs in the vaccinated chickens challenged with this isolate were still detected. These were as severe as those occurring in unvaccinated chickens challenged with serovar B-1 isolates. Although the clinical signs induced in unvaccinated birds challenged with serovar A-1 were more severe than those occurring when vaccinated birds were challenged with serovar C-3, the overall disease profiles were similar. Substantial clinical signs were recorded in vaccinated birds challenged with serovar C-3. This could be interpreted as vaccination failure if the disease profile obtained in unvaccinated birds is not considered. It was found that a high level of protection was provided by this vaccine against challenge by serovar C-3. The high virulence of this serovar resulted in the development of clinical signs in vaccinated birds. These findings could possibly explain the large number of so-called vaccination failures that are reported in South Africa.     

Antibody responses to avian encephalomyelitis virus vaccines when administered by different routes

Antibody responses to a commercial avian encephalomyelitis virus (AEV) vaccine administered by different routes were measured by an enzyme-linked immunosorbent assay (ELISA). Responses to single doses of vaccine administered by the ocular route to 10% of a flock were comparable with those obtained when all birds received a single dose in the drinking water. However, ocular vaccination of 5% of the flock resulted in significantly lower responses than those obtained when 10% were vaccinated. Maternal antibody was shown by the ELISA to persist in chickens from vaccinated flocks for up to 21 days after hatching. Day-old chickens with serum absorbances of < 0.3 at 492 nm, as determined by the ELISA, were shown to be susceptible to intracerebral challenge with the neurotropic Van Roekel strain of AEV.     

A single dose of inactivated oil-emulsion bivalent H5N8/H5N1 vaccine protects chickens against the lethal challenge of both highly pathogenic avian influenza viruses

In this study, two highly pathogenic avian influenza (HPAI) H5N8 viruses were isolated from chicken and geese in 2018 and 2019 (Chicken/ME-2018 and Geese/Egypt/MG4/2019). The hemagglutinin and neuraminidase gene analyses revealed their close relatedness to the clade-2.3.4.4b H5N8 viruses isolated from Egypt and Eurasian countries. A monovalent inactivated oil-emulsion vaccine containing a reassortant virus with HA gene of the Chicken/ME-2018/H5N8 strain and a bivalent vaccine containing same reassortant virus plus a previously generated reassortant H5N1 strain (CK/Eg/RG-173CAL/17). The safety of both vaccines was evaluated in specific-pathogen-free (SPF) chickens. To evaluate the efficacy of the prepared vaccines, 2-week-old SPF chickens were vaccinated with 0.5 mL of a vaccine formula containing 10⁸/EID₅₀ /dose from each strain via the subcutaneous route. Vaccinated birds were challenged with either wild-type HPAI-H5N8 or H5N1 viruses separately at 3 weeks post-vaccine. Results revealed that both vaccines induced protective hemagglutination-inhibiting (HI) antibody titers as early as 2 weeks PV (≥5.0 log₂). Vaccinated birds were protected clinically against both subtypes (100 % protection). HPAI-H5N1 virus shedding was significantly reduced in birds that were vaccinated with the bivalent vaccine; meanwhile, HPAI-H5N8 virus shedding was completely neutralized in both tracheal and cloacal swabs after 3 days post-infection in birds that had been vaccinated with either vaccine. In conclusion, the developed bivalent vaccine proved to be efficient in protecting chickens clinically and reduced virus shedding via the respiratory and digestive tracts. The applicability of the multivalent avian influenza vaccines further supported their value to facilitate vaccination programs in endemic countries.