Development and evaluation of a novel subunit vaccine for Mycoplasma gallisepticum / Desenvolvimento e avaliação de uma nova vacina para Mycoplasma gallisepticum

Adhesion proteins from Mycoplasma gallisepticum (MG) encoded by cytadhesion genes mgc1 and mgc2 were cloned into plasmid vectors and transformed into E. coli. Seventeen groups of specific-pathogen free (SPF), birds at four weeks of age were used to inoculate these two proteins (MGC1 and MGC2) mixed into an oil emulsion creating a novel MG vaccine. Six different protein concentrations (50, 100, 200, 400, 800, and 1000µg/bird) were tested with two equal concentration doses at four and seven weeks of age. In addition, many control groups were needed such as bacterin, membrane, no vaccine or challenge, oil emulsion alone, and no vaccine but challenged. Three weeks following the second vaccination, 50% of the birds in each treatment group were challenged with MG strain S6. The remaining birds were left as contacts to verify protection against horizontal transmission. All birds were bled before vaccinations, challenge and euthanasia. Birds were negative for MG at the first vaccination, as shown by serum plate agglutination test. At necropsy, tissue samples (trachea, lungs, and air sacs) were collected for histopathological examination. Swabs from trachea were used for PCR analysis. ELISA results showed a strong immune response to both protein preparations and almost the same response level for different doses tested, proving the immunogenic features of MGC1 and MGC2. However, humoral responses failed to prevent MG infection and disease when challenged as demonstrated by PCR and histopathology. MGC1 contact birds showed some degree of infection by PCR analysis. In addition, histopathological and ELISA results suggest that contact birds did not have enough time to develop lesions and to mount an immune response.

Os genes mgc1 e mgc2, codificadores de duas proteínas de adesão (MGC1 e MGC2) da bactéria Mycoplasma gallisepticum, foram clonados em E. coli. Dezessete grupos de aves livres de patógenos específicos (SPF), com quatro semanas de idade, foram inoculados com uma emulsão oleosa contendo as proteínas MGC1 e MGC2 purificadas. Seis concentrações (50, 100, 200, 400, 800, e 1000µg/ave) foram testadas com duas doses idênticas, às quatro e sete semanas de vida, respectivamente. Além disso, grupos controles foram avaliados com uma vacina comercial contra micoplasmose aviária, membrana de MG, grupo sem vacina/sem desafio, grupo vacina oleosa de MGC1 sem desafio, grupo com vacina oleosa de MGC2 sem desafio, grupo desafiado mas sem vacina. Três semanas após a segunda e a última vacinação, 50% dos animais dos grupos tratamentos foram desafiados com a cepa S6 de MG. O restante dos animais foi deixado como contato para averiguar proteção contra a transmissão horizontal da doença. Amostras de sangue de todas as aves foram coletadas antes das vacinações, do desafio e da eutanásia. As aves eram negativas para MG às quatro semanas de vida, conforme visto na aglutinação em placa. Na necropsia, tecidos (traqueia, pulmão e sacos aéreos) foram coletados para exame histopatológico. Suabes da traqueia foram utilizados para a PCR. Os resultados do ELISA demonstraram forte resposta imune contra as duas proteínas testadas e resposta similar independentemente do número de doses, provando a sua capacidade imunogênica. Porém, esta resposta humoral gerada foi incapaz de prevenir a infecção e a doença após desafio, conforme demonstrado pelos exames PCR e histopatológico. Aves-contato, inoculadas com MGC1, demonstraram estar infectadas nas análises de PCR. Além disso, os resultados do histopatológico e ELISA sugerem que os animais-contato não tiveram tempo suficiente para demonstrar lesões ou resposta imune.

Characterization of MGC2, a Mycoplasma gallisepticum cytadhesin with homology to the Mycoplasma pneumoniae 30-kilodalton protein P30 and Mycoplasma genitalium P32.

A second cytadhesin-like protein, MGC2, was identified in the avian respiratory pathogen Mycoplasma gallisepticum. The 912-nucleotide mgc2 gene encodes a 32.6-kDa protein with 40.9 and 31.4% identity with the M. pneumoniae P30 and M. genitalium P32 cytadhesins, respectively. Functional studies with reverse transcription-PCR, immunoblotting, double-sided immunogold labeling, and attachment inhibition assays demonstrated homology to the human mycoplasmal P30 and P32 cytadhesins. These findings suggest that there is a family of cytadhesin genes conserved among pathogenic mycoplasmas infecting widely divergent hosts.

Cloning and characterization of a putative cytadhesin gene (mgc1) from Mycoplasma gallisepticum.

A 150-kDa cytadhesin-like protein from Mycoplasma gallisepticum has been identified. A previously described 583-bp fragment (J.E. Dohms, L.L. Hnatow, P. Whetzel, R. Morgan and C.L. Keeler, Jr., Avian Dis. 37:380-388, 1993) was used to probe a genomic library of M. gallisepticum DNA. An 8.0-kb SacI fragment was identified, cloned, and partially sequenced. Analysis of the resulting 3,750-bp sequence revealed the presence of a 3,366-nucleotide open reading frame, mgc1. The 1,122-amino-acid protein encoded by this open reading frame, MGC1, has characteristics of a class I membrane protein and has homology with the MgPa cytadhesin of Mycoplasma genitalium (26.3%) and the P1 cytadhesin of Mycoplasma pneumoniae (28.7%). A portion of MGC1 was expressed as a glutathione S-transferase fusion protein and used to produce antiserum in rabbits. The antiserum recognizes a 150-kDa protein from M. gallisepticum. The protein is sensitive to trypsin, confirming that it is surface exposed. Primer extension analysis indicates that the mgc1 RNA starts within an upstream open reading frame, suggesting complex control of its expression. This is the first description of a functional gene from M. gallisepticum showing homology to cytadhesin genes from human mycoplasmas.

OSHA recordkeeping guidelines for occupational injuries and illnesses.

The U.S. Department of Labor's Bureau of Labor Statistics administers the occupational injury and illness recordkeeping regulations established by the Occupational Safety and Health Act of 1970. Medical facilities must comply with the law's requirements. This article provides both an overview of the regulations and information on how to obtain the required instructions and forms. While some states administer their own occupational safety and health programs, they must adopt standards and enforce requirements that are at least as effective as federal requirements.

Presence of lesions without virus replication in the thymus of chickens exposed to infectious bursal disease virus.

Specific-pathogen-free (SPF) chickens were exposed to the IM and VA isolates of virulent infectious bursal disease virus (IBDV). Both viruses induced rapidly progressing lymphoid cell depletion in the bursa. The bursal lesions persisted through the observation period of 16 days. The virus-exposed birds also had histologic lesions in the thymus. Thymic lesions peaked at 3-4 days postinoculation (PI) and then subsided. Immunofluorescence (IF) and antigen-capture enzyme-linked immunosorbent assay (ELISA) detected abundant viral antigen in the bursa, but not in the thymus, of chickens during the first week after infection with IM-IBDV or VA-IBDV. This result indicated that the presence of histologic lesions in the thymus was not associated with active infection and replication of the virus in thymic cells. Inoculation of homogenates of bursal and thymic tissues from virus-exposed chickens into embryonated chicken eggs revealed the presence of infectious virus from both tissues. We speculated that the virus recovered from thymus may have been contributed by virus-infected cells that were circulating through the thymus at the time when this tissue was homogenized.

Identification of the putative cytadhesin gene of Mycoplasma gallisepticum and its use as a DNA probe.

A portion of the putative Mycoplasma gallisepticum (MG) cytadhesin gene was identified and used as a diagnostic DNA probe. Degenerate oligonucleotide primers corresponding to conserved regions of the cytadhesin proteins from two human mycoplasmas, M. pneumoniae and M. genitalium, were synthesized for use in the polymerase chain reaction (PCR) on genomic MG DNA. A 583-base-pair MG DNA fragment was amplified and subsequently cloned and sequenced. The MG DNA fragment is predicted to encode a 193-amino-acid peptide. This peptide demonstrates significant homology to the expected portions of the two human mycoplasmal cytadhesin proteins. Used as a probe to study the distribution of this fragment in pathogenic and nonpathogenic avian mycoplasmas, the PCR product hybridized to genomic DNA from all seven MG strains tested. However, it failed to hybridize to M. synoviae, M. meleagridis, M. iowae, or M. gallinarum DNA.

OSHA safety requirements for hazardous chemicals in the workplace.

This article outlines the Occupational Safety and Health Administration (OSHA) requirements set forth by the Hazard Communication Standard, which has been in effect for the healthcare industry since 1987. Administrators who have not taken concrete steps to address employee health and safety issues relating to hazardous chemicals are encouraged to do so to avoid the potential of large fines for cited violations. While some states administer their own occupational safety and health programs, they must adopt standards and enforce requirements that are at least as effective as federal requirements.

OSHA's bloodborne pathogens standard: new job-safety requirements for healthcare.

This article outlines the OSHA requirements set forth by the new Occupational Exposure to Bloodborne Pathogens Standard. While some states administer their own occupational safety and health programs, they must adopt standards and enforce requirements that are at least as effective as federal requirements.

Mechanisms of immunosuppression.

Immunosuppresive disease is a major economic concern in domestic poultry production. Although many immunosuppressive agents have been described, mechanisms of how infectious and noninfectious agents compromise the immune system are poorly understood in avian species. Two categories, generalized and antigen-specific immunosuppression have been described in mammals. Generalized immunosuppression produces overall reduced responsiveness and increased susceptibility to a wide variety of infectious and neoplastic diseases. The best characterized immunosuppressive mechanisms are described in HIV-1 infections that lead to acquired immune deficiency syndrome (AIDS) in humans. In contrast, the antigen-specific suppression observed in human leprosy illustrates how an infecting agent selectively suppresses host responses against itself favoring bacterial spread. Both diseases have well-defined clinical staging classifications that correlate with specific immunological defects. An approach to studying immunosuppressive mechanisms in the avian suggests the need for relating pathogenesis with tests of immune responsiveness using a series of increasingly more specific immunological assays to pinpoint defects.

Stress--mechanisms of immunosuppression.

Stress, a term commonly used to describe varied phenomena, should be restricted to describe an adaptive response by an animal to threats to homeostasis. The threats to homeostasis are called stressors. Stressors include a variety of physical, psychological, chemical, or infectious causes that are modified by intrinsic and extrinsic factors. Examples of modifiers include stressor severity, duration, novelty, host genetics and immune status. What may be a stressor to an animal in one situation, when modified, may not be a stressor in another situation. Mechanisms of stress once thought to involve a single pathway described by Seyle as the General Adaptation Syndrome, have been rejected. Four pathways, some incompletely defined, have been implicated in modulation of the immune system. They include autonomic nervous system, the hypothalamic adrenal axis, extra-adrenal pathways involving neuropeptides and neurotransmitters and neuroimmunological mediators. The mechanisms of stress-induced immunosuppression resistance are poorly defined in domestic fowl and will require careful experimentation linking defined stressors with altered physiological responses that affect specific immune function and result in increased disease susceptibility.

Protection afforded infectious bronchitis virus-vaccinated sentinel chickens raised in a commercial environment.

The protective efficacy of three infectious bronchitis virus (IBV) vaccines for sentinel chickens raised with commercial Delmarva broiler chickens was evaluated during winter 1987. Specific-pathogen-free leghorn sentinel chickens were vaccinated with Massachusetts (Mass) alone, Mass and JMK, or Mass and Arkansas (Ark) combination live vaccines, or they remained unvaccinated. Four weeks post-vaccination, sentinels were placed on broiler farms at weekly intervals for 3 weeks corresponding to weeks 4, 5, and 6 of the broiler growing cycle. Vaccine efficacy was evaluated based on IBV reisolation attempts from tracheal swabbings following a 1-week field exposure period. Sentinel chickens vaccinated with Mass and Ark combination vaccine were best protected against IBV field challenge. Only four IBV isolations were made out of a 3-week total of 36 attempts, for an 11% isolation rate. IBV vaccines containing either Mass alone or Mass and JMK offered much lower levels of protection.

Comparative pathogenesis of serotype 1 and variant serotype 1 isolates of infectious bursal disease virus and their effect on humoral and cellular immune competence of specific-pathogen-free chickens.

Specific-pathogen-free chickens inoculated with isolate VA (variant A) or isolate IM of infectious bursal disease virus (IBDV) were examined for mitogenic response to T-cell mitogens, primary and secondary antibody response to sheep erythrocytes and Brucella abortus, and gross and histologic lesions in thymus and bursa. Both isolates induced comparable depression in the mitogenic and antibody response, and both caused extensive gross and histologic lesions in the bursa of Fabricius. However, bursal necrosis induced by the IM isolate was accompanied by an inflammatory response, whereas the inflammatory component was lacking in the lesion induced by the VA isolate. Furthermore, the IM isolate induced extensive lesions in the thymus, but the VA isolate did not.

Plasma cell quantitation in the gland of Harder during infectious bursal disease virus infection of 3-week-old broiler chickens.

Histopathologic changes in the gland of Harder (GH) and bursa of Fabricius (BF) were studied during and after infection of 3-week-old broiler chickens with a pathogenic strain of infectious bursal disease virus (IBDV). Plasma cell (PC) necrosis in the GH was seen from 5 to 14 days postinoculation (PI), BF follicular necrosis was observed from 1 to 7 days PI. PC numbers within the GH, counted for 28 days after inoculation, declined and were reduced (P less than 0.01) by 51% at 7 days after inoculation, which coincided with PC necrosis and heterophil infiltration. After 14 days PI, however, PC numbers were equal to those in uninfected controls. Since the GH is a major antibody-producing site in the paraocular area, the reduction in PC number at 7 days PI might indicate compromise of local immunity in the paraocular region and upper respiratory tract associated with IBD.

The effect of infectious bursal disease virus infection on local and systemic antibody responses following infection of 3-week-old broiler chickens.

Broiler chickens infected at 3 weeks of age with infectious bursal disease virus (IBDV) were given Brucella abortus (BA) or sheep red blood cell (SRBC) antigens before, during, and after the acute phase of the infection. Gland of Harder (GH) extracts and serum samples were used to assay local and systemic antibody titer to each antigen 7 days after antigen was administered. Antibody titers to both BA and SRBC antigens were lower (P less than 0.05) in GH extracts and serum of IBDV-infected broilers than uninfected controls. The responses to BA, a thymus-independent antigen, took longer to become depressed than the responses to SRBC, a thymus-dependent antigen. The depression of antibody titers following IBDV inoculation suggests compromise of both local and systemic immune function, a finding of importance to the broiler industry.

Infectious bursal disease virus infection in the quail-chicken hybrid.

Hybrids produced from crossing Cornell K-strain white leghorn chickens and Line II Japanese quails were studied for susceptibility to infection with infectious bursal disease virus (IBDV). Quail-chicken hybrids were infected successfully following inoculation with IBDV at 14, 21, or 52 days of age. In most cases, precipitating antibodies were detected in serum by 10 days postinoculation (PI). Although no clinical signs or gross lesions were evident in the bursa of Fabricius of hybrids, histologic changes in the bursa were detected upon microscopic examination using hematoxylin and eosin staining. Chickens were successfully infected also; they had gross and microscopic lesions in the bursa and produced precipitating antibodies. In addition, staining of bursal sections with low concentrations of peroxidase-conjugated concanavalin A revealed a rearrangement of a leukocyte cell type (probably macrophages) in infected chickens and hybrids. Japanese quails were refractory to infection; they showed no bursal changes and did not form precipitating antibodies.

The immunization of broiler chickens against type C botulism.

Broiler chickens were inoculated with different amounts of a Clostridium botulinum type C toxoid at 1 or 14 or both 1 and 14 days of age. Immunity was assessed following challenge with type C botulism toxin at 3, 6, and 8 weeks of age. Protection induced by toxoid injection was affected more by time and number of inoculations than by the amount of toxoid administered. Single toxoid injections at one day of age furnished poor protection, whereas groups injected at 14 days of age were well protected at 6 and 8 weeks of age but not at 3 weeks of age. Variable results were observed in groups inoculated at both 1 and 14 days of age: immunity was evident in some groups following 3-, 6-, and 8-week toxin challenges.

Cases of type C botulism in broiler chickens.

Twenty-seven cases of type C botulism were studied in integrated broiler operations on the Delmarva Peninsula. Single and repeated outbreaks in broiler flocks were observed. No botulinum toxin was found in litter and feed samples despite repeated testing. Clostridium botulinum type C was cultured from litter, feed, and tissues of morbid and clinically normal chickens sampled from farms in which flocks experienced botulism. Clostridium botulinum type C could not be cultured from tissues of clinically normal chickens taken from a farm without a flock history of botulism or from caged broilers reared in semi-isolation.

Susceptibility of broiler chickens to Clostridium botulinum type C toxin.

As broiler chickens aged during the first eight weeks of life, they decreased in susceptibility to the lethal effects of Clostridium botulinum type C toxin. Eight-week-old broilers were 2,000 times less susceptible to the lethal effects of the toxin than hatched chicks. Different broiler crosses showed similar susceptibility to the lethal effects of type C toxin at seven weeks of age, but 2.5 times more toxin was required to produce lethality by oral inoculation than was required by subcutaneous inoculation.

Plasma cell changes in the gland of Harder following infectious bursal disease virus infection of the chicken.

Tissue changes in the gland of Harder (GH) were studied following infection of day-old chicks with infectious bursal disease virus (IBDV). Broiler chicks that lacked maternal antibody to IBDV were inoculated with IBDV at 1 day of age and were maintained segregated from uninoculated controls. Plasma cell (PC) content of the GH was lowered among infected chickens from 1 to 7 weeks postinoculation. Since the GH in uninoculated control birds was not densely populated with PCs until 3 weeks of age, the PC depletion found in young chickens infected with IBDV became more clear with age. After the chickens were 3 weeks old, fewer (P less than .01) plasma cells were observed in the GH of IBDV-infected chicks than in uninoculated controls. Lymphoid follicles and heterophil populations in the GH did not appear to be affected by IBDV infection, nor could necrotic or degenerative changes be found in the acini or excretory ducts. The observation of fewer PCs suggests that a major lymphoid tissue of the upper respiratory tract is comprised by IBDV infection of day-old chicks.U