Genesize Polymorphism and Pathogenicity in Embryonated Eggs of Mycoplasma Gallisepticum Isolated From Commercial Chickens

Chronic respiratory disease (CRD) is caused by Mycoplasma gallisepticum (MG). Infected birds show respiratory and reproductive problems which lead to severe economic losses in poultry industry. There are only few published data on avian mycoplasmosis in Malaysia, thus, this study was carried out to determine the strain variability and pathogenicity of MG isolates, towards understanding the control of the infection. A total of 4605 samples were collected from chickens and their progeny from various commercial farms using sterile cotton swabs for culturing onto mycoplasma agar. Twenty three (23) MG isolates were obtained from suspected MG infected commercial chickens. Although MG could be isolated from various sites of the host, in this study, choanal and tracheal sites proved to be the best sites in live birds. On the other hand, trachea and airsac samples were the best sites for the detection of MG for chick embryos or chicks. Size variations among polymerase chain reaction products divergence of the MG-specific gene were the basis for strain differentiation. The local isolates exhibited gene size polymorphism in pvpA gene, 16S-23S rRNA intergenic spacer region gene, ermA gene and pMGA or vlhA gene with the presence of insertion or deletion observed in PCR products. However, the gapA gene, LP gene, F-strain-specific DNA fragment gene, CrmB gene, CrmC gene, p47 gene, HMW3-like protein gene and pneumoniae-like protein A gene sequences were constant in size. The embryonated eggs were each inoculated with "pleuropneumonia like organism" (PPLO) broth containing MG strains, via yolk sac. Mycoplasma gal/isepticum embryos, broth inoculated and uninoculated control embryonated eggs were examined at necropsy days 7, 10, 13 and 15 post-inoculation. The pathogenicity of the isolates in chicken embryonated eggs showed variations among each other. The MG isolates and strains that showed a wide variation in genes were examined for virulence in ovo. In this study, the presence of caseous airsac lesion correlated with virulence ofMG and presence of high maternal antibody titer. MG were isolated only in embryos that did not develop any caseous airsac lesions. MG inoculated embryos were polymerase chain reaction (PCR) positive regardless of the absence or presence of caseous airsac lesion, suggesting that caseous airsac lesion maybe the result of formation of antigen-antibody complex. Caseous airsacs were found to be one of the prominent lesions associated with MG infection. For certain highly pathogenic strains, there was clear relationship between the caseous airsac lesion and the presence of maternal antibody titer and embryo mortality. Less pathogenic strains that grow well usually caused embryo mortality during later stages of incubation and there was no strict correlation between caseous airsac lesion and the presence or absence of maternal antibody and embryo mortality. Based on the presence of the gene size polymorphism in pvpA gene and pMGA or vlhA gene; MGS6 (reference strain), 144 and 1-18 strains of MG showed a similar pattern of pathogenicity in that they are highly pathogenic, whereas, H21 8T, H21 lIT, H24 5C and H26 9C have similar pattern of molecular characterization and pathogenicity with ts-l1 (vaccine strain), characterized by their less pathogenicity in embryos. MGS6, 144 and 1-18 strains caused early embryonic death compared to ts-11, H21 8T, H2 1 lIT, H24 5C and H26 9C strains that caused embryo mortality during later stages of incubation. At this point, the postulation is that, when maternal antibody of MG is high and MG challenge is present, caseous airsac may occur. This would be due to maternal antibody in the eggs which may bind to MG that served as antigen to form antigen-antibody complexes. The immune complexes may help to release cytokines and attract more macrophages and other inflammatory cells, which help to increase the severity of the air sac lesion. When the MG strain with the gene size polymorphism in pvpA gene and pMGA or vlhA gene that has similar pattern with MGS6, it correlates with the formation of caseous air sac, as well as the increase in severity of the caseous airsac. This study showed that the combination of the gene size polymorphism in pvpA gene and pMGA or vlhA gene can be used as pathogenic markers for MG in determination of its pathogenicity towards chick embryos. Based on characterization and pathogenicity, MG field strains H2 1 8T, H21 HT, H24 5C and H26 9C showed similar pattern of molecular and pathogenicity characteristic with ts-l1 and therefore are potential candidates for live MG vaccine

Detection and characterization of chicken anemia virus from commercial broiler breeder chickens

<p>Abstract</p> <p>Background</p> <p>Chicken anemia virus (CAV) is the causative agent of chicken infectious anemia (CIA). Study on the type of CAV isolates present and their genetic diversity, transmission to their progeny and level of protection afforded in the breeder farms is lacking in Malaysia. Hence, the present study was aimed to detect CAV from commercial broiler breeder farms and characterize CAV positive samples based on sequence and phylogenetic analysis of partial VP1 gene.</p> <p>Results</p> <p>A total of 12 CAV isolates from different commercial broiler breeder farms were isolated and characterized. Detection of CAV positive embryos by the PCR assay in the range of 40 to 100% for different farms indicated high level of occurrence of vertical transmission of viral DNA to the progeny. CAV antigen was detected in the thymus and in the bone marrow but not in spleen, liver, duodenum, ovary and oviduct by indirect immunoperoxidase staining. The 12 CAV isolates were characterized based on partial sequences of VP1 gene. Six isolates (MF1A, MF3C, M3B5, NF4A, P12B and P24A) were found to have maximum homology with previously characterized Malaysian isolate SMSC-1, four isolates (M1B1, NF3A, PYT4 and PPW4) with isolate BL-5 and the remaining two (NF1D and NF2C) have maximum homology both with isolates 3-1 and BL-5. Meanwhile, seven of the isolates with amino acid profile of 75-I, 97-L, 139-Q and 144-Q were clustered together in cluster I together with other isolates from different geographical places. The remaining five isolates with amino acid profile of 75-V, 97-M, 139-K and 144-E were grouped under cluster II. All the CAV isolates demonstrated omega values (K_a/K_s) of less than one (the values ranging from 0.07 to 0.5) suggesting the occurrence of purifying (negative) selection in all the studied isolates.</p> <p>Conclusion</p> <p>The present study showed that CAV is widespread in the studied commercial broiler breeder farms. The result also indicated the occurrence of genetic variability in local CAV isolates that can be divided at least into two groups based on characteristic amino acid substitutions at positions 75, 97, 139 and 144 of the VP1 protein.</p

Molecular identification of two genetic markers that distinguish between pathogenic and nonpathogenic strains of Mycoplasma gallisepticum.

A total of 571 Mycoplasma gallisepticum (MG) field isolates originated from progenies and commercial poultry farms in Malaysia and 7 reference and vaccine strains were characterized by amplification of selected gene target specific sequences to MG pMGA and pvpA genes using conventional PCR of sequence specific primers. A total of 281 MG positive field isolates out of 571 MG samples were detected with the primer targeted pMGA gene and a total of 188 MG positive field isolates out of 571 MG samples were detected with the primer targeted pvpA gene. Similar and identical banding pattern among MG isolates obtained from progenies samples however, there was a variable on the banding pattern among MG isolates obtained from commercial chickens using the agarose gel electrophoresis. The sequencing analysis results of MG based on selected genes targeted specific sequences were obtained. The genetic diversity of the pMGA and pvpA genes of MG field isolates detected in progenies and commercial chickens were investigated. The gene size variation patterns of the pMGA and pvpA genes among MG field isolates shared identical variations with the pathogenic reference and vaccine strains that is an insertion bp fragments by using the pMGA gene primer set and a deletion bp fragments by using the pvpA gene primer set. However, the gene size variation patterns are quite different from the variation pattern of the less pathogenic vaccine strain that can’t be transmitted vertically. The polymorphism pattern of the primer for pMGA gene might be considered as a pathogenic vertical marker and the polymorphisms patterns of the two primers sets for both pMGA and pvpA genes might be useful for determining the two genetic potential pathogenic marker for MG infection that can differentiate between the highly and the less pathogenic MG isolates

Polymerase chain reaction-based discrimination of viable from non-viable <i>Mycoplasma gallisepticum</i>

The present study was based on the reverse transcription polymerase chain reaction (RT-PCR) of the 16S ribosomal nucleic acid (rRNA) of Mycoplasma for detection of viable Mycoplasma gallisepticum. To determine the stability of M. gallisepticum 16S rRNA in vitro, three inactivation methods were used and the suspensions were stored at different temperatures. The 16S rRNA of M. gallisepticum was detected up to approximately 20–25 h at 37 °C, 22–25 h at 16 °C, and 23–27 h at 4 °C. The test, therefore, could detect viable or recently dead M. gallisepticum (< 20 h). The RT-PCR method was applied during an in vivo study of drug efficacy under experimental conditions, where commercial broiler-breeder eggs were inoculated with M. gallisepticum into the yolk. Hatched chicks that had been inoculated in ovo were treated with Macrolide 1. The method was then applied in a flock of day 0 chicks with naturally acquired vertical transmission of M. gallisepticum, treated with Macrolide 2. Swabs of the respiratory tract were obtained for PCR and RT-PCR evaluations to determine the viability of M. gallisepticum. This study proved that the combination of both PCR and RT-PCR enables detection and differentiation of viable from non-viable M. gallisepticum

Isolation and identification of leptospires from raccoons (Procyon lotor) in Indiana, USA

The present study was conducted to determine whether raccoons ( Procyon lotor) carry leptospires in their kidneys and thus are capable of infecting livestock and other domesticated animals in landscapes where these species co-occur. Isolation of leptospiral organisms and identification of leptospiral species and serogroups from raccoon kidneys using culture in conjunction with PCR and sequencing was performed. Thirty-four raccoons were live-trapped from a single forest patch in a highly agricultural region of central Indiana, USA. A portion of kidney (2cm2) from each raccoon was homogenized and used for leptospiral culture. Leptospiral cultures were subjected to DNA extraction and various PCR procedures. Serum sample from each raccoon also was collected and antibody titers to leptospiral serovars were determined by microscopic agglutination test (MAT). Twenty-nine out of 34 raccoon kidney cultures (85.3%) were found to have characteristic thin slender and curved spiral-shaped leptospiral organisms with hooked ends by dark field microscopy (DFM). All leptospiral cultures (34/34), 2 including those negative for leptospires by DFM, were positive for Leptospira by various PCR procedures. The PCR with primers targeting the conservative region of LipL32 gene was the most sensitive PCR in detection of pathogenic leptospires. Twenty-two raccoon kidney cultures (64.7%) were positive for leptospirosis by PCR amplification with the primers flanking the variable region of LipL32 gene. The PCR amplicons were directly sequenced and the sequences were compared to those of the reference strains available in GenBank. Twelve kidney cultures had Leptospira interrogans, 8 had L.kirschneri and 2 had L. borgpettersenii. In addition, serogroup-specific primers designed from the O-antigen gene were used to determine leptospiral serogroups. They were predominantly Grippotyphosa serogroup. Anti-leptospire antibodies were detected in 16 out of 34 raccoons (47.1%) by MAT. There were titers of ≥ 1:80 in 16 raccoons (47.1%) and titers ≥ 1:400 in 3 raccoons (8.8%). The highest leptospiral serovar-specific seroreactivity among 34 raccoons was L.interrogans Bratislava (38.2%) and L.interrogans Grippotyphosa (32.4%), respectively. The results indicated that the raccoon kidneys carry leptospiral organisms that can be detected and identified by culture in conjunction with PCR and sequencing which the leptospires were identified predominantly L. interrogans species and of the Grippotyphosa serogroup. Given the high densities of raccoons that exist in urban and agricultural ecosystem, the results of this study suggest that raccoons likely are an important reservoir for the maintenance and transmission of leptospiral organisms between wildlife and livestock, domesticated animals, and humans

A method for detection and differentiation of Mycoplasma gallispeticum.

The present invention provides a method of simultaneously detecting and differentiating Mycoplasma gallisepticum strains that cause respiratory infections in avian species. The method includes providing a nucleic acid prepared from a suspected biological sample, amplifying the nucleic acid with amplification primers specific for target genomic regions of Mycoplasma gallisepticum, and detecting the amplification products. The invention also relates to a diagnostic kit for carrying out the inventive method

Detection of mycoplasma gallisepticum by real time PCR using gapA gene

Mycoplasma gallisepticum (MG) is responsible for chronic respiratory disease which leads to a lot of economic losses to the poultry industry. Therefore, it is very important to detect this organism early and efficiently. MG can be diagnosed by three ways: isolation identification, serological method and molecular detection method. In this study, a SYBR green real time PCR assay by using gapA gene was developed for the detection of MG. This assay was powerful both from the aspect of specificity and sensitivity as it amplified only MG at early cycle when run together with 20 other avian Mycoplasma species and it can detect a minimum of 26 pg/μl of DNA template

Major trends in the manifestations and treatment of rheumatoid arthritis in a multiethnic cohort in Singapore

10.1007/s00296-012-2602-2Rheumatology International3371693-1703RHIN