Mycoplasma gallisepticum and Mycoplasma synoviae in broiler breeders

Mycoplasma agents are difficult to isolate; this, in many cases, makes diagnosis and proper characterization impossible (Ricci, 2005). In Venezuela, avian mycoplasmosis is generally diagnosed by means of serological methods and, eventually, by PCR, performed in foreign laboratories (Godoy, 1999). Isolation and genomic characterization in flocks of poultry can be used to modify vaccination plans, make them more assertive or to consider the use of antibiotic therapy with more efficient control, as the mycoplasmas are often associated with other diseases. This is why the use of advanced techniques such as PCR and partial nucleotide sequencing are a tool of great importance in avian mycoplasmosis, achieving more accurate diagnoses that allow for more accurate differentiation of vaccination field strains, which leads to a better understanding of the pathogen affecting avian populations (Raviv & Kleven, 2008). The objective of our work was to isolate and molecularly characterize strains of Mycoplasma gallisepticum and/or Mycoplasma synoviae, isolated from broiler breeders and commercial layer poultry in Venezuela.

The study was conducted on 28 farms: 12 farms of Broiler Breeders with 696 samples and 16 Commercial Layer farms with 734 samples, for a total of 1430 samples. Sampling was performed intentionally, with a non-probabilistic approach in areas of high poultry production in the country. The study was conducted in two stages.  Field Stage: Taking blood samples, tracheal swabs, using FTA^® sample cards. Laboratory Stage: Detection of specific antibodies against M.g and M·s using the PRP and ELISA techniques was performed. The isolation of M.g and M.s was performed using standard culture techniques (Anonymous, 1994), using tracheal swabs taken from living birds on farms and in 2 cases of autopsy. Molecular characterization of the M.g and M·s was performed using PCR (Moscoso et al., 2004) at the INIA-Venezuela and partial sequencing of nucleotides (ABI Prism DNA sequencing kit and ABI Prism 3100 genetic analyzer (Applied Biosystems, Foster City CA,) in the Department of Molecular Genetics Instrumentation of the University of Georgia, USA ). DNA extracted from Frey broths positive at isolation was analyzed by means of PCR, using Invitrogen^® primers: MG-14F 5´ GAG CTA ATC TGT AAA GTT GGT C-3´; MG-13R 5´ GCT TCC TTG CGG TTA GCA AC-3´ of 186 pb and MSLF 5´ GAG AAG CAA AAT AGT GAT ATC A-3´ and MSLR 5´ CAG TCG TCT CCG AAG TTA ACA A-3´of 214 pb, corresponding to the matrix protein, as screening. For the purpose of differential diagnosis between the vaccine strain and the field strain Invitrogen^® primers were used: MG-c2-F 5´ CGC AAT TTG GTC CTA ATC CCC AAC A-3´ and MG-c2-R 5´TAA ACC CAC CTC CAG CTT TAT TTC C-3´ of 237-303 bp; MS vhla-F 5´GAT GCG TAA AAT AAA AGG AT-3´, MS vhla-R 5´GCT TCT GTT GTA GTT GCT TC-3´ of 316-395 bp. The analysis of the nucleotide sequence was performed in the Avian Diagnostic Laboratory, in Georgia (USA), using the Lasergene program (DNASTAR, Inc. Madison, WI., and the nucleotide-nucleotide Blast database NCBI - National Center for Biotechnology Information-).

PRP of 696 samples in Broiler Breeders was obtained for direct M.g and dilutions ranges between 62.07-65. 66% and for M.s was maintained at 33.19%. In Commercial Layers, of 734 samples for direct M.g and dilutions between 60.49-68.39% and for M.s 32.43-43.19 %. 90% of farms of Broiler Breeders used vaccines against M.g (strain F), whereas Commercial Layer farms, where its use is more restricted, did not use it. For M.s, none of the farms reported use of vaccines, and it was noted that the Commercial Layer farms showed a higher percentage of positive tests. However, it has been suggested that poultry lots may show non-specific reactions or false positives, due to recent applications of oily vaccines or the use of biological materials produced in live tissue against other infectious agents (Kleven, 1994;) (Contreras, 2000). Commercial Layers farms showed a higher percentage of positive test results, which is considered as evidence of recent infections, possibly because this type of operation includes poultry of different ages with poor biosecurity conditions. The analysis of overall prevalence in Broiler Breeders and Commercial Layers reveals co-infections of both microorganisms. However, in many cases this co-infection goes unnoticed because of the increased importance commonly given to M.g (Godoy, 1999). For ELISA in the Broiler Breeders' 537 samples: M.g 54.38 % and M.s 55.87% of positive test results. Seroprevalence in Broiler Breeders was slightly higher for M.s, although we know that these poultry were vaccinated at 90 % against M.g. Commercial Layers in 588 samples showed 73.64% positive test results for M.g and 64.12 % positive test results for M.s. In this case, seroprevalence of M.g was higher; most of these lots were not given vaccination against M.g or M.s. The IgG in the case of avian mycoplasmas occurs between 1 and 6 weeks after vaccination or infection by field strains of M.g or M.s (Ross, 2009). For vaccinated Broiler Breeders, it is inferred that the serological response is due to the use of vaccine strains, which produce an immune response strong and detectable for a period longer than 12 weeks (Contreras, 2000, Ricci 2005). Contrary to this case, positives that where reported in Commercial Layers in which no M.g vaccine use was reported, are possibly due to chronic and circulating infection among batches. For M.s both populations in studies received no vaccinations, so that the obtained results correspond to seroconversion by exposure to field strains present in the examined operations.

29 isolations of mycoplasma were obtained in 28 analyzed protocols, noting a strong correlation to the results obtained in the PRP to recent and active infections. Typical colonies were observed under an indirect light, low magnification microscope (40 X). Some modifications, such as the addition of 2000 IU penicillin were made to the Frey broths in samples taken in the field, in order to limit the growth of contaminants, improving the observation of colonies. With the PCR using primers MG-14F / MG-13R / MSLF y MSLR, 19 strains of M.g and 10 strains of M.s. were identified. Out of 12 Broiler Breeder farms analyzed, 11 turned out positive to M.g. (91,67%) and 3 to M.s. (25%). While out of the 16 Commercial Layers farms analyzed, 8 tested positive for M.g. (50%) and 7 for 7 M.s. (43.75%) . Using primers mgc2 and MS-vlha in Broiler Breeders 11 positive testst were identified for M.g, and 3 for M.s. In Commercial Layers 4 positive tests were identified for M.g. and 7 for M.s. The sequential analysis of the mgc2 gene in Broiler Breeders determined that 9 samples showed a 100% identity percentage with: Strain F, strain E4-08 (Egypt), strain K5152AC01 and strain K4781ATK99; one sample with 99.5 % identity with strain F, strain E4-08 (Egypt), strain K5152AC01 and strain K4781ATK99; one sample with 97.9 % identity with strain F, strain E4-08 (Egypt), strain K5152AC01 and strain K4781ATK99, when compared to the strains in the GenBank data base. The product of the amplification of gene M.s could not be sequenced with the method used. In the vaccinated Broiler Breeders the vaccine strain competes with the field strains, so that both strains may be present. A strain with a similarity of 97.9% with strain F, which could be considered as a field strain was obtained in this study. Strains of M.s showed no sequencing results.  Harada et al. (2009), considers that the analysis of this gene can not always determine the molecular differences between field strains and vaccine strains. Form the sequential analysis of the gene mgc2 in Commercial Layers 4 sequences were obtained, which were analyzed, noting that two samples showed a 100% identity with strain F, strain Rab E4-08 (Egypt), strain K4781ATK99, strain K5058ETK01 and with strain K5152AC01; one sample with a 99% identity with strain F and 98.9% identity with strain Rab E4 - 08 (Egypt) strain K4781ATK99, strain K5058ETK01 and strain K5152AC01< one sample with 93.7% identity with strain F, strain Rab E4-08 (Egypt), strain K4781ATK99, strain K5058ETK01 and with strain K5152AC01 of the GenBank database. Sequencing of the other 4 isolated items for M.g. was not possible. For M.s with M.s the sequencing of 7 samples was obtained, 3 of which showed a percentage of 100% identity with: strain EsPKUAF08 (Pakistan), strain B95-04-K4412 (Spain); 90.4% with strain H and strain 94011 (Australia); one sample with 99.6% identity with strain EsPKUAF08 (Pakistan), strain B95-04-K4412 (Spain); 90% with strain H and strain 94011 (Australia); one sample with 98.8% identity with strain EsPKUAF08 (Pakistan), strain B95-04-K4412 (Spain); 89.2% with strain H and strain 94011 (Australia); two samples with 90.4% identity with strain EsPKUAF08 (Pakistan), strain B95-04 - K4412 (Spain); 99.2% with strain H, strain 94011 (Australia) and with the uniclone strain V8 MSPB. The results confirm that there is a wide variety of strains of mycoplasmas in these populations of Venezuelan poultry, which poses a major problem in this type of farming. Non-sequenced strains of M.s in Broiler Breeders and of M.g in Commercial Layers, may be due to strains that differ from those commonly identified using the mgc2 and M.s vlha primers. These genes minimize the possibility of cross reactions in the diagnosis of mycoplasmas by PCR (Takahashi-Omoe et al., 2004), and have been applied in molecular characterization studies of M.g and M.s in chicken (Evans & Leigh, 2008). Our study found that in 100% of the isolated cases collated a result of genomic identification using the PCR technique was obtained, either for M.g or for M.s, or for both at the same time, thus confirming that the technique is effective for the diagnosis of Mycoplasma strains and that a combination of both pathogens within a same avian population can occur. (Godoy, 1999, Ricci, 2005). For sequencing, mgc2 primers and M.s vlha primers achieve differentiation among strains of M.s and M.g and differentiation between vaccine strains of M.g (Raviv & Kleven, 2008), or the identification of vaccine strains of M.s using the vhla gene (Harada et al., 2009). Nevertheless, other primer designs exist which also have been used for differentiation of vaccine strains of Mycoplasma gallisepticum ts-11 and strain 685 (Evans & Leigh, 2008).

The serologic, isolation and M.g and/or M.s genomic identification results indicate that there is a considerable challenge in the field in the Venezuelan poultry farms examined, showing the presence of live strains (vaccine and/or field strains) in the avian populations.

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