Understanding worldwide declines in reptiles due to factors such as habitat loss and emerging infectious disease has become an increasingly important focus in conservation biology. Here, I use novel approaches from the field of landscape genetics to combine spatial genetic data with landscape data at both regional and local spatial scales to explore natural and anthropogenic landscape features that shape population structure and gene flow in a federally threatened reptile, Gopherus polyphemus. I also utilize approaches from the field of spatial epidemiology to examine the extent to which environmental variables can be used to predict the seroprevalence of an associated pathogen Mycoplasma agassizzi in gopher tortoise populations. Using mitochondrial data, I find evidence of a historical barrier to gene flow that appears to coincide with the Apalachicola River. I also discover low genetic diversity and evidence of population bottlenecks in the western portion of the range. My evaluation at the regional scale shows that dispersal is limited by geographic distance, areas of low elevation and major roads ways. A finescale study reveals no evidence of spatial genetic structure within a 14 x 35 km area. However, soil type is significantly correlated with pairwise genetic distances between individuals, suggesting that this variable influences fine-scale population structure in the gopher tortoise. In addition to soil, high density canopy cover is an important factor impeding gene flow at the local level for females, while land cover type explains some of the genetic variance between males. Finally, temperature and precipitation appear to be important predictors of the seroprevalence of the pathogen Mycoplasma agassizii in gopher tortoises. The probability of an individual testing seropositive for exposure to this disease increased with high temperature and low precipitation values. The methods presented in this dissertation evaluate novel approaches for assessing the influence of environmental variables on population structure, dispersal and disease occurrence and could be applied in future studies of other threatened and endangered taxa.

<p><a>The hemotrophic mycoplasma<i>, Mycoplasma ovis</i>, is found in sheep and goats throughout the world. This pathogenic bacterium is capable of causing an acute, life-threatening infection as well as chronic or subclinical infections in these animals. The purposes of the present studies were to develop <i>M. ovis</i>-specific assays for detection of this hemoplasma, and to better understand infection dynamics within pregnant ewes and lambs. </a>The first study describes the development and validation of a SYBR^® Green quantitative PCR (qPCR) assay, which was subsequently used to determine the prevalence of <i>M. ovis</i> infection within a population of goats and to evaluate risk factors for infection. This highly sensitive and specific assay consistently detected as few as 10 copies of plasmid/reaction. Convenience-based sampling of 362 goats from 61 farms located in Indiana revealed a prevalence of infection of 18% (95% confidence interval (CI), 14% to 22%). Bacterial loads of <i>M. ovis</i> ranged from 1.05 x 10³ to 1.85 x 10⁵copies/mL of blood with a mean of 1.31 x 10⁴copies/mL of blood. The only risk factor associated with hemoplasma infection was the production use of the goat; dairy goats had a 3.3 fold increase compared with the prevalence in goats used for meat. This study not only demonstrates that <i>M. ovis</i> infection is common in goats in Indiana, but shows the variability of bacterial loads that can be found in chronically-infected animals. While sub-clinically infected goats may have a bacteremia, levels are characteristically less than 2.0 x 10⁵copies/mL.</p><p> The second project utilized a combination of cross-sectional and longitudinal studies to estimate the prevalence of <i>M. ovis</i> infection from a cohort of naturally-infected pregnant ewes, assess changes in their bacterial loads, and determine the incidence of <i>M. ovis</i> in lambs pre- and post-weaning. The prevalence of <i>M. ovis</i> infection in ewes was not found to be significantly different during pregnancy, and before and after weaning of the lambs, with prevalence estimates of 45% (95% CI, 23.1 – 68.5), 36% (95% CI, 17.9 – 57.4), and 44%, (95% CI, 24.4 – 65.1), respectively. Bacterial loads of the ewes from the cross-sectional study ranged from 10⁴to 10⁹copies/mL of blood, with the median bacterial load at 10⁵ copies/mL of blood. While higher bacterial loads are typical of an acute infection, none of the ewes in this study had overt clinical signs. The data suggest that <i>M. ovis</i> loads may be higher in pregnant sheep, particularly in ewes half-way through pregnancy. Most of the <i>M. ovis</i> infections in the study lambs were detected post-weaning which suggests that transplacental or transmammary infection of <i>M. ovis</i> are unlikely routes.</p><p> In the third study, a subset of <i>M. ovis</i> genes for use in a multi-locus sequence typing assay (MLST) were evaluated. Next-generation sequencing was performed to generate data from pooled DNA amplicons in order to identify single nucleotide polymorphisms (SNPs) of <i>M. ovis </i>from five genes. Evaluation of the quality and depths of coverage for the reads and SNPs indicated that the pooled DNA amplicons produced reads and SNPs having high quality and sufficient depth. This pooling technique is a cost-effective alternative to whole-genome sequencing. While the MLST has good discriminatory power and may be used to identify genetically distant and divergent clusters of <i>M. ovis</i> from different geographical origins, within a herd the discrimination power is low, which may hamper its usefulness in transmission studies. </p><p> The fourth and final study was the development of a loop-mediated isothermal amplification (LAMP) assay targeting the dnaK gene of <i>M. ovis</i>, with comparison of the assay to conventional PCR (cPCR). The metal ion indicator hydroxynaphthol blue (HNB) was added prior to the reaction, which allowed for visual detection of LAMP-positive samples as indicated by a color change from violet to sky blue. <i>Mycoplasma ovis</i> was consistently detected in 45 minutes with the LAMP assay at a reaction temperature of 64°C, with more infected sheep being detected than by cPCR. Therefore, the LAMP assay is fast and reliable in the detection of <i>M. ovis</i>. The developed LAMP assay may have applications in diagnostics, surveillance and disease management as well as prevalence studies. However, a more robust molecular technique is necessary for <i>M. ovis</i> isolate or stain discrimination to investigate transmission or disease spread in an outbreak.</p><p> </p><p> In conclusion, three new molecular tools for the detection of <i>M. ovis</i> in goats and sheep were developed as results of these studies. We have shown that the qPCR assay is an efficient tool for detection and quantification of <i>M. ovis</i> loads in blood from both of these species. On the other hand, the value of the LAMP assay is for reliable detection of infection (not quantification), especially in resource-limited situations. The five-locus MLST protocol developed herein, a typing assay based on the polymorphism of five gene sequences, is a laborious technique requiring DNA extraction, PCR amplification, purification and sequencing of target loci. The value of this technique is not as a routine diagnostic, but rather it may be used to better understand the genetic diversity of <i>M. ovis</i> and investigate strain variations. Most importantly, the scheme is sufficiently robust to allow direct genotyping of <i>M. ovis</i> in total blood DNA extracts without culture isolation. The MLST approach may prove useful as a tool for future investigations of transmission and disease spread. These studies have also expanded our understanding of the infection dynamics of <i>M. ovis</i> in pregnant sheep and lambs. It is shown herein that despite the high prevalence and sometimes high bacterial loads in pregnant ewes, <i>M. ovis</i> does not appear to be transmitted to the lambs in utero or during the perinatal period. The lambs become infected mostly after weaning; this may suggest a protective effect during the pre-weaning period and/or subsequent exposure/infection from their environment. </p><br>

Orientador: Márcia Guimarães da Silva / Banca: Rodrigo Paupéro de Camargo Soares / Banca: Vera Lúcia Mores Rall / Não disponível / Abstract: Microbial invasion of the amniotic cavity has been described in term deliveries and its role on the immune modulation is of interest to the better understanding of the underlying labor processes. The aim of this study was to determine the prevalence of Mycoplasma hominis and Ureaplasma urealyticum in the amniotic fluid of term pregnancies and to evaluate its influence on cytokines production at the end of pregnancy. A cross sectional study was conducted with fifty five pregnant women out of labor with intact membranes and gestational age between 37 and 41 weeks seen at the Bom Jesus hospital in Ariquemes, Rondônia, between June 2009 and May 2010. Amniotic fluid samples and fragments of chorioamniotic membranes were collected at cesarean section. M. hominis and U. urealyticum detection was performed by PCR and Interleukin (IL)-1β, IL-6, IL-8, IL-10 and Tumor Necrosis Factor (TNF)- levels were determined by ELISA. Chorioamniotic membranes were submitted to histopatological analyses. Presence of M. hominis was detected in 36.4% of amniotic fluid samples and any of them was positive for U. urealyticum. Regarding cytokines levels, 63.6% and 90.9% of samples have not shown detectable concentrations of TNF- and IL-1β. The median concentration of IL-6 and IL-8 were 107.9 pg/mL (0-517.1) and 208.1 pg/mL (0-1897.4), respectively. Interleukin-1, IL-6, IL-8 and TNF- concentrations were not associated with the presence of M. hominis in amniotic fluid, regardless the gestational age. No sample had detectable IL-10 levels. The histopatological analyses have shown no chorioamnionitis in any of the membranes, only a discreet mononuclear infiltration in the decidua could be observed in 40.4% of the samples. Presence of M. hominis was detected in 36.4% of amniotic fluid samples and any of them was positive for U. urealyticum. Regarding cytokines levels, 63.6% and 90.9% of samples have not... (Complete abstact click electronic access below) / Mestre

Made available in DSpace on 2016-04-28T20:16:28Z (GMT). No. of bitstreams: 1 2009 - Sandra Batista dos Santos.pdf: 939793 bytes, checksum: fad008b3d49abcd0c16da05450ee089f (MD5) Previous issue date: 2009-02-16 / Conselho Nacional de Desenvolvimento Cient?fico e Tecnol?gico / Mycoplasma are the smallest and more fastidious prokaryotes known, being responsible by high economical losses in livestock production. Mycoplasmas has been found frequently in the ear canal of goats. In these cases, are very closely related with mites, Psoroptidae and Raillietidae, that carried and disseminate mycoplasmas among the flocks. In Brazil, studies related mycoplasmas in the ear canal of bovine inexist. Like this, in the Chapter I, were surveyed up data on prevalence of mycoplasmas in the ear canal of bovine and your association with mites Raillietia spp., and identification of the two Raillietia species that occur in bovine of southeast, State Rio de Janeiro. The prevalence of Mycoplasma spp. in the ear canal of bovine was considered high, 80% (48/60). In these animals high prevalence was verified Raillietia spp. 76.7% (46/60). The parasitism by mycoplasmas and mites was verified in 40 animals (74.1%), this association was highly significant (p<0.001). Of the females of identified Raillietia 52.3% (101/193) were R. auris and 47.7% (92/193) were R. flechtmanni. In this study, was proven that mycoplasmas and mites in the ear canal of the bovine are closely related and these habitat occur pottentially patogenic mycoplasmas for cattle herds. In the Chapter 2, Mycoplasma mycoides Cluster (GMM) was diagnosed by PCR-REA and indirect immunoperoxidase (IPI), both, carried out in mycoplasmas isolates of hte ear canal. In this study, 35 strains selected in agreement with their biochemistry and physiologic proprieties, were used. Under IPI the prevalence obtained for GMM was 20.0% (12/60) while by PCR-REA it was 41.7% (25/60). The IPI typing of these isolates resulted in 58.3% (7/12) for M.mycoides mycoides LC and 41.7% (5/12) for M. capricolum. PCR-REA for M. mycoides Cluster was confirmed by the amplicon size of 785bp, compatible with this group. After restriction analysis with AluI in all M. mycoides cluster strains and ear canal samples the fragments size obtained were compatible with this group, and neither fragment of 370bp that is compatible with MmmSC of bovine origen it was visualized. / Mycoplasmas s?o os menores e mais fastidiosos procariotos conhecidos, sendo respons?veis por altas perdas econ?micas relacionadas a produtividade em ruminantes. Micoplasmas tem sido encontrados com frequ?ncia em conduto auditivo de caprinos. Nestes casos, est?o estreitamente relacionados com ?caros Psoroptidae e Raillietidae, que carreiam e disseminam micoplasmas entre os rebanhos. No Brasil, estudos sobre Mycoplasma em conduto auditivo de bovinos inexistem. Assim, no Cap?tulo I, foram levantados dados sobre preval?ncia de micoplasmas presentes no conduto auditivo de bovinos e sua associa??o com ?caros Raillietia spp., al?m da identifica??o das duas esp?cies de Raillietia spp. parasitas de conduto auditivo de bovinos da regi?o sudeste do Estado do Rio de Janeiro. A preval?ncia de Mycoplasma spp. no conduto auditivo de bovinos foi considerada alta, 80% (48/60). A preval?ncia de Raillietia spp. foi de 76,7% (46/60). Em 40 (74,1%) animais verificou-se parasitismo por Raillietia spp. e Mycoplasma spp., esta associa??o foi altamente significativa (p<0,001). Das f?meas de Raillietia identificadas 52,3% (101/193) foram R. auris e 47,7% (92/193) foram R. flechtmanni. comprovou-se que micoplasmas e ?caros do conduto auditivo de bovinos est?o estreitamente relacionados e que neste s?tio de localiza??o est?o presentes Mycoplasmas com alto potencial patog?nico para bovinos. No Cap?tulo II, foi realizada tipifica??o molecular dos isolados de Mycoplasma pertencentes ao Grupo Mycoplasma mycoides (GMM) atrav?s da t?cnica de PCR-REA, sendo estes resultados comparados com os obtidos na t?cnica de imunoperoxidase indireta (IPI). A preval?ncia obtida para o GMM na IPI foi de 20,0% (12/60) enquanto na PCR-REA foi de 41,7% (25/60). Das esp?cies de Mycoplasma tipificadas pela IPI 58,3% (7/12) foram M. mycoides subsp. mycoides LC e 41,7% (5/12) foram M. capricolum. Na PCR-REA, GMM, foi confirmado pela visualiza??o de um amplicon de 785bp, compat?vel com este grupo. Na clivagem do produto da PCR com a enzima de restri??o AluI, os fragmentos obtidos foram compat?veis com cepas padr?o do GMM, e dos isolados de conduto auditivo de bovinos estudados, nenhum fragmento de 370pb compat?vel com MmmSC, biotipo bovino foi encontrado.

Amostras de sangue total e suabes de orofaringe e conjuntiva foram coletadas de 57 felídeos Neotropicais (1 Leopardus geoffroyi, 17 L. wiedii, 22 L. tigrinus, 14 L. pardalis e 3 Puma yagouaroundi) mantidos em cativeiro no Refúgio Bela Vista, Foz do Iguaçu. Dados clínicos, hemograma e histórico dos animais foram disponibilizados. Materiais clínicos obtidos a partir dos suabes de orofaringe e conjuntiva foram submetidos ao cultivo para Mycoplasma spp em meio específico. DNA do sangue e suabes foram extraídos por meio de um kit comercial e pelo método de fervura, respectivamente. DNA extraído de amostras de sangue foram submetidos à PCR para detecção de Mycoplasma haemofelis (Mhf), Candidatus M. haemominutum (CMhm), DNA proviral do vírus da leucemia felina (FeLV) e Bartonella spp. DNA extraído dos suabes foram submetidos à PCR para detecção de Mollicutes e M. felis. Foi realizada uma análise de associação entre alterações clínicas e a infecção por Bartonella spp e um estudo de fator de risco da infecção por esse microrganismo. Apenas 1 (1,75%) animal foi positivo a reação para Mhf e nenhum foi positivo a reação para CMhm. Dois (3,5%) animais foram positivos a reação para FeLV e 6 (10,52%) foram positivos para Bartonella spp. Não houve co-infecção entre os agentes pesquisados nas amostras de sangue. Foram obtidos 5 (8,77%) isolados de Mycoplasma spp da orofaringe e nenhum de conjuntiva. DNA de Mollicutes foi detectado em 53 (93%) e 27 (47,36%) amostras de orofaringe e conjuntiva, respectivamente. Nenhuma amostra apresentou resultado positivo na detecção de DNA alvo de M. felis. Não houve associação entre as alterações hematológicas (anemia, desidratação, leucocitose, leucopenia, histórico de anemia) e a infecção por Bartonella spp. Machos apresentam maior risco de adquirir bartonelose do que fêmeas. Este é o primeiro relato da presença de DNA proviral de FeLV em L. tigrinus e L. pardalis no sul do aís, de DNA de B. henselae em L. tigrinus, L. pardalis, L. geoffroyii e P. yagouaroundi, e de um estudo de fator de risco associado a infecção por Bartonella spp em felídeos neotropicais. / Total blood samples and oropharinx and conjunctival swabs were collected from 57 neotropical felids (1 Leopardus geoffroyi, 17 L. wiedii, 22 L. tigrinus, 14 L. pardalis and 3 Puma yagouaroundi) maintained in captivity at Refúgio Bela Vista, Foz do Iguaçu. Clinical data, hemogram and clinical history of these animals were available. Clinical materials obtained from oropharinx and conjunctiva were cultured in specific media for Mycoplasma spp isolation. DNA of blood and swabs were extracted using a commercially available kit and a boiling method, respectively. DNA samples from swabs were submitted to a PCR for the detection of Mollicutes and M. felis. DNA samples from blood were submitted to a PCR for detection of Mycoplasma haemofelis (Mhf), Candidatus M. haemominutum (CMhm), feline leukemia virus (FeLV) proviral DNA , and Bartonella spp. The association between hematological alterations and bartonella infection was evaluated and a risk factor analysis was performed. Only 1 (1.75%) animal was positive for Mhf reaction, whereas all animals were negative for CMhm detection. Two (3.5%) animals were positives for FeLV and 6 (10.52%) animals were positive for Bartonella spp, by PCR. Co-infections among these agents were not observed. Five (8.77%) mycoplasma isolates were obtained from oropharinx samples and none was obtained from conjunctival samples. Mollicutes DNA was detected in 53 (93%) and 27 (47.36%) samples from oropharinx and conjunctiva, respectively. All samples were negative for M. felis detection. Hematological alterations (anemia, dehydration, leukocytosis, leucopenia, history of anemia) were not associated to Bartonella spp infection. Males are more likely to be infected than females. This is the first report of FeLV proviral DNA in L. tigrinus and L. pardalis in Southern Brazil, of B. henselae DNA in L. trigrinus, L. pardalis, L. geoffroyi and P. yagouaroundi, and the first study of risk factors for Bartonella spp infection in neotropical felids.

<p>Chlamydia and gonorrhea are major causes of sexually transmitted infections (STI) in adolescents worldwide. The infections are caused by <em>Chlamydia trachomatis</em> or <em>Neisseria gonorrhoeae, </em>bacteria with clinical manifestations such as urethritis, prostatitis and epididymitis among men, and urethritis, cervicitis and upper genital tract infection (i.e. pelvic inflammatory disease) among women. However, in many cases of genital tract infection, the etiology remains uncertain. In light of this, <em>Mycoplasma genitalium</em> was somewhat accidentally isolated in 1980 after prolonged incubation of urogenital specimens from men with non-gonococcal urethritis. Following the initial isolation in 1980, repeated attempts have been made to recover the extremely fastidious organism from clinical samples by culture techniques, but isolates have been rare and difficult to obtain. With the development of PCR methods in the early 1990s, detection of <em>M. genitalium</em> infection became more feasible.</p><p>The aim in paper <strong>I</strong> was to compare three different PCR assays (conventional and real-time 16S rRNA gene PCR as well as real-time <em>Mycoplasma genitalium</em> adhesin protein (MgPa) gene PCR) for detection of <em>M. genitalium</em>. The study also determined the prevalence of <em>M. genitalium</em>. Clinical specimens collected from STI attendees, 381 men and 298 women, were used to determine the prevalence of <em>M. genitalium</em> and 213 of these specimens were used in the PCR comparative study. The prevalence of <em>M. genitalium</em> infection in men and women was 27/381 (7.1 %) and 23/298 (7.7 %) respectively. In the PCR comparative study, <em>M. genitalium </em>DNA were detected in 61/76 (80.3 %) of true-positive specimen by conventional 16S rRNA gene PCR, in 52/76 (68.4 %) by real-time 16S rRNA gene PCR and in 74/76 (97.4 %) by real-time MgPa gene PCR. Hence, real-time MgPa gene PCR is well suited for clinical diagnosis of <em>M. genitalium</em> in urogenital specimens from men and women.</p><p>The aim in paper <strong>II</strong> was to determine whether a patients’ endocervical swab specimen can be transported in first void urine (FVU) as combined specimens in detection of <em>Mycoplasma genitalium </em>by real-time PCR. The study also compared two different DNA extraction methods (manual Chelex DNA extraction and automated BioRobot M48 DNA extraction) for observation of possible PCR inhibition. Clinical specimens collected from 329 women attending a STI clinic were used in the study. A total of 100 endocervical swab specimens transported in FVU was used in the PCR inhibition analysis. <em>M. genitalium</em> was detected in 25/329 (7.6 %) women. Endocervical swab specimens transported in FVU demonstrate higher sensitivity compared to both FVU alone and specimens transported in 2-SP medium detecting 24/25 (96 %), 22/25 (88 %) and 17/25 (68 %) of <em>M. genitalium</em> positive women, respectively. Automated BioRobot M48 DNA extraction was shown to be superior to manual Chelex extraction leaving no PCR inhibition and slightly higher DNA yield and/or better sensitivity. The results from these two studies are important knowledge in establishing the future diagnostic level of this STI in our county and also nationally.</p>

Mollicutes (mycoplasmas) are pathogenic in a wide range of mammals (including humans), reptiles, fish, arthropods, and plants. Of the medically important mollicutes, Mycoplasma gallisepticum is of particular relevance to avian agriculture and veterinary science, causing chronic respiratory disease in poultry and turkey. Using two-dimensional electrophoresis based quantitative expression proteomics, the current study investigated the molecular mechanisms behind the phenotypic variability between a M. gallisepticum vaccine strain (6/85) and a competitive, virulent field strain (K5234), two strains which were indistinguishable using commonly accepted genetic methods of identification. Twenty-nine proteins showed a significant variation in abundance (fold change > 1.5, p-value < 0.01). Among others, the levels of putative virulence determinants were increased in the virulent K5234, while the levels of several proteins involved with pyruvate metabolism were decreased. It is hoped that the data generated will further the understanding of M. gallisepticum virulence determinants and mechanisms of infection, and that this may contribute to the optimization of diagnostic methodologies and control strategies.

Contagious Bovine Pleuropneumonia (CBPP), caused by Mycoplasma mycoides mycoides small colony (MmmSC), is a devastating respiratory disease of cattle in Africa, Asia and the Middle East. Little investigation has been done on molecular disease pathogenesis and host response beyond soluble cytokine detection. This study developed and characterized models for three strains of MmmSC of varying severity. Strains used were Gladysdale, Ondangwa and Shawawa. Samples of bronchoalveolar lavage fluid, bronchial biopsy, nasal epithelial cells and blood were obtained prior to and at weekly time points post-infection. Microarray analysis of RNA extracted from samples revealed host cellular pathways and genes important in the pathogenesis of CBPP, including multiple immune system and inflammatory response pathways. A number of pathways whose influence on disease pathogenesis was not immediately clear were also activated, including pathways involved in amino acid synthesis, fat metabolism, and endocrine hormone responses. Microarray results were confirmed with real-time polymerase chain reaction (RT-PCR) of selected genes. Comparative RT-PCR analysis of selected genes between the three strains of MmmSC revealed genes possibly responsible for differential strain virulence, including interleukins 1B, 6, 8, and 18 and the gene nuclear factor of kappa light polypeptide gene enhancer in B cells inhibitor, alpha (NFKBIA). A similar analysis of selected genes between survivors and nonsurvivors of the virulent Gladysdale strain of MmmSC suggested genes involved in survival, including interleukin 8, calmodulin 2 (CALM2), and NFKBIA. Avenues of additional study were identified.

The oxidative damage to DNA, protein and lipid, may be accumulated and play a role in the process of human cell aging. Oxidative stress may be due to the aerobic respiration and ozone-induced radiation which result in reactive oxygen species known as free radicals. Therefore, the antioxidant and free radical scanvenger which may reduce the oxidative damage, are of great interest, both in academic research and in the business world. The present study aims to evaluate the antioxidative potential of a very unique vegetable-fruit extract (Tsan-Ron-Bau-Yuan). The extract consists of over forty domestic vegetables and fruits, without using chemical fertilizers and pesticides during their entire growth period, is produced through a sophisticated freeze-dry technology. The anti-hydroxyl radical antioxidative potential were evaluated using the following four methods: (1) the plasmid DNA (2) the protein (3) the cell line and (4) the red blood cell . The results clearly demonstrat that the aqueous fraction of this extract can remarkably reduce the oxidative damage as evidenced by the DNA and protein model. In addition, the susceptibility of human red cells to oxidative stress can also be alleviated to some extents based on the RBC deformability studies. The efficacy of protection of the oxidative damage mediated by .OH generated by the Fenton¡¦s reaction was in the order of : aquaeous extract >100% ethanol extract > ethanol/ethylacetatee extract > 50% ethanol extract. Conversely, no significant protection to the action of hydrogen peroxide was observed in the cell line. Lysozyme which is ubiquitous among various natural products has negligible contribution as an antioxidant as revealed in the RBC deformability tests. Swine enzootic pneumoniae ¡]SEP¡^, is a disease caused by Mycoplasma hyopneumoniae infection, and usually lead to considerable economic loss. Though extensive research during the past years, the molecular mechanism about the infective pathway of M. hyopneumoniae was still elusive. The membrane proteins of this microorganism were considered as critical adhesion molecules and therefore are potential candidates for vaccine development. Through immunoscreening, we had isolated five recombinant phage clones expressing 10 kDa, 32 kDa, 36 kDa, 42 kDa and 60 kDa antigen proteins from the lEMBL3 library of Mycoplasma hyopneumoniae. The clone carrying the P42 gene was subcloned and further characterized as a heat shock protein gene. In the present study, the heat-shock protein gene encoding a 42 kDa/ 65 kDa protein¡]P42/P65¡^was cloned into the mammalian expression vector pcDNA3 and obtained plasmid pcDNA42. The immune response induced by pcDNA42 was evaluated in mice. The IgG titer was obviously elevated during the first eight weeks with the IgG1 titer slightly higher than IgG2a. However, the IgM titer was not changed signifcantly. Studies on the macrophage activity and T cell cytotoxity were still undergoning.

Mycoplasma hyopneumoniae is the etiologic agent causing chronic pneumonia of swine. The lung lesions of swine produce the slower growth rate and lower feed conversion ratio and finally cause economic loss. Although four genome projects of mycoplasma species had been completed, the genome-sequencing project of M. hyopneumoniae also closed to the finished stage. However, only a few genes and proteins of M. hyopneumoniae have been studied, the molecular pathogenic mechanism remains elusive. The research of molecular vaccine is still preliminary. In order to obtain more information about epitope structures as the basis to develop molecular vaccine against this pathogen, two phage-displayed random heptapeptides libraries were used to identify epitopes recognized by purified IgG of rabbit anti-M. hyopneumoniae hyperimmune serum in this study. Individual phage clones were isolated and verified the binding specificity to the purified IgG by Western blot analysis and competitive ELISA after three rounds of biopanning. The selected clones were further characterized by DNA sequencing analysis and deduced to amino acid sequences. There are six consensus sequences contained tri- to hepta-peptide existing among the selected phage clones by aligning the sequences of foreign amino acids displaying on pIII protein. The consensus sequences may be serving as crucial epitopes of M. hyopneumoniae. By searching the protein database of M. hyopneumoniae deposited in NCBI, some surface proteins were matched by the selected mimotopes. Like P97, the essential protein for attaching to cilia of swine, the deduced epitopes mainly located at a.a. from 365 to 382, 395 to 403 and 436 to 452, the R1 and R2 repeated sequences also matched by the mimotopes. To evaluate the potential of these mimotopes as effective vaccine, several phage clones were chosen to immunize mice by intraperitoneal and intranasal administration. There are specific antibody responses to these mimotopes existing in serum IgG, fecal extracts and bronchoalveolar lavage fluids IgA. The serum IgG subclass profiles analysis reveals that these are mainly existed in IgG1 subclass. Base on the results of IgG subclass profiles analysis in sera, the results suggest that the phage-derived vaccines mainly activate Th2 cellular immunity pathway with the strategy used in this study. The similar results were found in the inactivated vaccine. The Th2 activation will promote the elimination of extracellular microorganism. Western blotting analysis showed that each serum raised by the phage clones could recognize 2 to 5 mycoplasma proteins. With the results of growth inhibition assay, we found that the selected phage clones CS4 and 58 are better vaccine candidates and some proteins (97 kDa¡B56 kDa and 30 kDa) may play crucial roles in block the bacteria growth. The advantage was taken of the natural property of M13 phage to infect E. coli, which is initiated by the N terminal of pIII coat protein binding with the F pili of E. coli. Plaque reduction tests were proposed to demonstrate the humoral immunity responses induced by phage-derived vaccine containing the antibodies specifically against the foreign peptide displayed on pIII coat protein. The present results provide more epitope information of M. hyopneumoniae. The mice immunization results reveal that the phage-displayed mimotopes can be used as potential vaccine candidates. The strategy presented in this study can shorten the time course for vaccine development and provide an alternative pathway for searching vaccine candidates against M. hyopneumoniae.