Rapid and specific detection of Mycoplasma gallisepticum and Mycoplasma synoviae infection in poultry using single and duplex PCR assays.

Avian mycoplasmosis, mainly caused by Mycoplasma gallisepticum (MG) and Mycoplasma synoviae (MS), is an economically important disease of poultry industry. The present study was aimed to develop duplex PCR as a rapid, specific and economical method for accurate detection of MG and MS in poultry and its comparison with single (monoplex) MG/MS PCR. During present investigation, a total of 146 poultry flocks having clinical history of respiratory disease were screened. Pooled tissue samples (trachea, lungs and air sacs) from 4-5 birds of each flock were collected during necropsy at disease investigation laboratories, Hisar, Haryana, India. The single and duplex PCR assays were standardized using primers of intergenic spacer region (IGSR; 16S-23S rRNA) for MG and hemagglutinin vlhA gene for MS, with expected amplicon size of 812 bp and 1200 bp products, respectively. In single PCR, 6.85%, 2.74% and 2.74% tissue samples were found positive for MG, MS and both MG and MS, respectively. However, duplex PCR showed, 7.53%, 2.74% and 1.37% positivity for MG, MS and both MG and MS, respectively. Taking the results of monoplex PCR as a gold standard, sensitivity and specificity of the developed duplex PCR was found to be 94.44% and 100%, respectively. Moreover, Cohen's kappa statistic (k = 0.97) measured a 'perfect' agreement between monoplex and duplex PCR assays. The positive and negative predictive values of duplex PCR was found to be 1.0 and 0.9922, respectively at 95% confidence interval (CI), as compared to monoplex PCR. The simultaneous use of two genes in a duplex PCR was more rapid and economical than two separate single PCR reactions.

Decrease of Mycoplasma gallisepticum seroprevalence and introduction of new genotypes in Dutch commercial poultry during the years 2001-2018.

Almost two decades ago, in addition to a compulsory M. gallisepticum (Mg) monitoring programme of breeding stock based on European Union regulations, the Dutch poultry industry added national regulations to further reduce the Mg prevalence in Dutch commercial poultry. Currently, all commercial chicken and turkey flocks except broilers are monitored for Mg. All breeding flocks on a farm where one or more flocks tested Mg positive are culled. Mg positive layer pullets are channelled and layer pullets placed on Mg positive multi-age farms are vaccinated. The monitoring data obtained were analysed covering a period of 17 years. Moreover, 31 Dutch Mg isolates from the same period were analysed by multilocus sequence typing (MLST) and compared to available PubMLST data. The results show that in breeding stock the seroprevalence decreased from 1.6% to 0.0%, in commercial layers from 6.3% to 1.9%, and in meat turkeys from 17.6% to 2.4%. The MLST results showed the presence of closely related and identical sequence types (STs) within the different Dutch poultry types. Similar STs were found in Northern and Southern Europe only. The results show a fast decline in the Mg prevalence since 2001, although in layers the Mg prevalence has stabilized and suggests backyard poultry might pose a risk for commercial poultry. The need for Mg control across poultry sectors and in trade was confirmed by the similarity in STs found in different types of poultry and regions. These results from the Dutch poultry industry can be extrapolated to Mg control in general.

Preparation of ELISA and Lateral Flow Kits for rapid Diagnosis of Mycoplasma gallisepticum in Poultry.

Avian mycoplasmas were mainly the cause of poultry industry economic losses; reduced meat and egg production and increases the antibiotic treatment cost. Mycoplasma gallisepticum (MG) infection is designated as infectious sinusitis of turkeys and chronic respiratory disease of chickens (gasping, depression, semi closed eyes, infraorbital sinuses edema and decrease in egg production). This study aimed to prepare, evaluate and Compare in-house ELISA kits and lateral flow assay (LFA) from a local strain of MG with commercial ELISA kits and PCR consequently. A total of 54 samples (27 tracheal swabs, 10 trachea and 17 lung) and 50 serum samples collected from birds suffering from chronic respiratory disease were tested by prepared in-house ELISA, commercial ELISA kits, PCR and LFA; a high correlation coefficient between in-house ELISA using whole antigen or sonicated antigen and commercial kit was recorded. Lateral Flow assay (LFA) performance indicate a low sensitivity (77.5%) but maintain a high specificity (92%) compared to PCR. The in-house ELISA kits and LFA prepared could be used as a fast diagnostic technique for detection of MG in Egypt. According to the available knowledge the prepared LFA for diagnosis of MG infection in chickens was developed for the first time in Egypt.

Development of mismatch amplification mutation assay for the rapid differentiation of Mycoplasma gallisepticum K vaccine strain from field isolates.

Mycoplasma gallisepticum causes respiratory diseases and reproduction disorders in turkeys and chickens. The infection has considerable economic impact due to reduced meat and egg production. Because elimination programmes are not feasible in a large number of poultry farms, vaccination remains the only effective measure of disease control. Differentiating vaccine strains from field isolates is necessary in the control of vaccination programmes and diagnostics. The aim of this study was to develop a polymerase chain reaction based mismatch amplification mutation assay (MAMA) for the discrimination of K vaccine strain (K 5831, Vaxxinova Japan K.K.). After determining the whole genome sequence of the K strain, primers were designed to detect seven different vaccine-specific single nucleotide polymorphisms. After evaluating preliminary results, the MAMA-K-fruA test detecting a single guanine-adenine substitution within the fruA gene (G88A) was found to be the most applicable assay to distinguish the K vaccine strain from field isolates. The detected K strain-specific single nucleotide polymorphism showed genetic stability after serial passage in vitro, but this stability test should still be evaluated in vivo as well, investigating a large number of K strain re-isolates. The MAMA-K-fruA assay was tested on a total of 280 culture and field samples. The designed assay had 102 and 103 template copy number/µl sensitivity in melt-curve analysis based and agarose-gel based assays, respectively, and showed no cross reaction with other avian Mycoplasma species. The new MAMA provides a time- and cost-effective molecular tool for the control of vaccination programmes and for diagnostics.

Fluorescent microspheres as a positive indicator in an intratracheal infection model.

Animal models testing the ability of vaccines and therapeutic agents to prevent pathology from induced respiratory infection are an important means of testing and validating the vaccines and therapeutic agents. However, the lack of induced pathology in test subjects could be either indicative of protection or a problem with the animal model system. This work describes the improvement of a chicken model system of intratracheal infection using fluorescent microspheres as a positive indicator of infection. It was shown that fluorescent microspheres and Mycoplasma gallisepticum bacteria both dispersed to the same areas of the chicken respiratory system and that the microspheres remained detectable in the chicken lung tissue for at least 7 days following infection. The microspheres used are detectable using a black light, allowing for visualization during necropsy. Using the updated model system, three live M. gallisepticum vaccines were tested both for their ability to elicit a humoral immune response following vaccination, and for their ability to protect from air sac lesion pathology at two different time points following vaccination. Results showed the protective effects of the different M. gallisepticum vaccines prevented the induction of pathology, consistent with previous results. The presence of the fluorescent microspheres provided a positive method of identifying the properly infected chickens and a means of differentiating failed experimental infections so that those samples could be removed, resulting in improved consistency in infection results.

Investigation of Mycoplasma spp. in birds of the Rio de Janeiro Zoo by isolation and PCR / Investigação de Mycoplasma spp. em aves do Zoológico do Rio de Janeiro por isolamento e PCR

Brazil is one of the countries with the most abundant avifauna in the world. The confinement of birds associated with close contact with other animals and humans favor the spread of agents of respiratory diseases. Among them, mycoplasmas can cause asymptomatic or apparent disease that manifests in birds by coughing, sneezing, rales, conjunctivitis, ocular and nasal discharge. Several described mycoplasmas cause disease in birds, especially Mycoplasma gallisepticum(MG) andMycoplasma synoviae(MS). The diagnosis ofMycoplasmaspp. can be done by clinical observation and laboratory analysis. Molecular diagnosis by PCR was boosted by its speed, sensitivity, and low cost of agent isolation techniques that take up to 21 days to complete. This study aimed to verify the occurrence ofMycoplasmaspp. in birds of the Rio de Janeiro Zoo (Rio Zoo), by isolation and PCR. Of the total 635 birds from the Rio Zoo, 81 were studied for detection ofMycoplasmaspp., when taken for routine health assessment exams. These birds belonged to the following orders: Psittaciformes (45), Accipitriformes (18), Galliformes (7), Piciformes (5), Strigiformes (4), Falconiformes (1) and Cariamiformes (1), all individuals already identified by microchip or leg-ring. There was no isolation of mycoplasmas in any of the samples tested, whereas, in the PCR, 62.96% (51/81) were positive, with 1.96% (1/51) identified as MG and 19.61% (10/51) as MS, representing 1.23% (1/81) and 12.34% (10/81) of the total population studied. PCR was shown to be a more effective technique than isolation in the detection ofMycoplasmaspp. in birds. It was possible to detect mycoplasmas in birds from Riozoo with no clinical respiratory signs, with higher MS prevalence than MG. The positivities forMycoplasmaspp., MS, and MG were different among the orders studied, being the highest occurrence in birds of prey, followed by Galliformes and Piciformes. The presence of MG and MS in birds of Rio de Janeiro Zoo confirms the circulation of these agents and the need for further studies on the dissemination of mycoplasmas in zoos for the epidemiological analysis of these bacteria in these places.(AU)

O Brasil é um dos países com maior avifauna do mundo. O confinamento de aves associado ao contato próximo a outros animais e seres humanos favorece a disseminação de agentes etiológicos causadores de doenças respiratórias. Dentre eles, os micoplasmas podem causar doença assintomática ou aparente que se manifesta em aves por espirros, estertores, conjuntivite, corrimentos oculares e nasais. São diversos os micoplasmas descritos causadores de doença em aves, com destaque para Mycoplasma gallisepticum (MG) e Mycoplasma synoviae (MS). O diagnóstico de Mycoplasma spp. pode ser feito pela observação clínica e análises laboratoriais. O diagnóstico molecular pela Reação em Cadeia da Polimerase (PCR) ganhou impulso por sua rapidez, sensibilidade e baixo custo em relação às técnicas de isolamento do agente que levam até 21 dias para conclusão do gênero Mycoplasma. Objetivou-se verificar a ocorrência da infecção por Mycoplasma spp. em aves no Zoológico do Rio de Janeiro (Rio Zoo), por isolamento e PCR. Do plantel de 635 aves do Rio Zoo, foram estudadas 81 para detecção de Mycoplasma spp., quando contidas para exames rotineiros de avaliação da condição de saúde. Essas aves eram pertencentes às ordens Psittaciformes (45), Accipitriformes (18), Galliformes (7), Piciformes (5), Strigiformes (4), Falconiformes (1) e Cariamiformes (1), todas já identificadas por microchip ou por anilha. Não houve isolamento de micoplasmas em nenhuma das amostras testadas, enquanto na PCR, 62,96% (51/81) foram positivas, sendo 1,96% (1/51) identificadas como MG e 19,61% (10/51) como MS, representando 1,23% (1/81) e 12,34% (10/81) da população total estudada. A PCR demonstrou ser uma técnica mais efetiva que o isolamento na detecção de Mycoplasma spp. em aves. Foi possível detectar micoplasmas nas aves do Riozoo sem sinal clínico respiratório, tendo MS maior prevalência do que MG. As positividades para Mycoplasma spp., MG e MS foram diferentes entre as ordens de aves estudadas, sendo a maior ocorrência nas aves de rapina, seguida dos Galliformes e dos Piciformes. A presença de MG e MS nas aves do Rio de Janeiro Zoo confirma a circulação destes agentes e a necessidade de mais estudos sobre a disseminação de micoplasmas em zoológicos para análise epidemiológica dessas bactérias nesse local.(AU)

Molecular detection and characterization of Mycoplasma gallisepticum and Mycoplasma synoviae strains in backyard poultry in Italy.

Mycoplasma gallisepticum (MG) and Mycoplasma synoviae (MS) represent the most important avian Mycoplasma species in the poultry industry, causing considerable economic losses. In Italy, the presence of MG or MS has been investigated especially in commercial poultry farms. To our knowledge, no systematic investigations on MG or MS presence using highly specific diagnostic assays have been performed in backyard poultry. The aim of this study was to detect and molecularly characterize MG and MS strains in 11 backyard poultry flocks located in different regions of Italy. Tracheal swabs were collected and DNA was extracted. For MS, a PCR targeting a vlhA gene fragment was performed, and typing and subtyping was attempted. The presence of MG was investigated by a screening PCR, then MG typing by gene-targeted sequencing (GTS). All the amplicons were sequenced, then MG and MS dendrograms were constructed. All the flocks examined resulted Mycoplasma positive: 5 out of 11 (45.45%) were MG and MS positive, 3 (27.27%) were MG positive, and the remaining 3 (27.27%) were MS positive. The MS detections were assigned to types C, D, and F. All strains of type D belonged to subtype D1 and 2 unknown subtypes were identified. A MS sequence showed peculiar characteristics, which did not allow assignment to a known MS type or subtype. MG GTS analysis identified 6 MG strains belonging to 5 subclusters circulating in Italian backyards chicken flocks. The results of this study provide evidence of a risk for commercial poultry farms, especially in areas where backyard and commercial farms are close, suggesting the implementation of biosecurity measures.

Influences of swab types and storage temperatures on isolation and molecular detection of Mycoplasma gallisepticum and Mycoplasma synoviae.

Routine diagnosis of Mycoplasma gallisepticum (MG) and Mycoplasma synoviae (MS) is performed by collecting oropharyngeal swabs, followed by isolation and/or detection by molecular methods. The storage temperature, storage duration and the type of swab could be critical factors for successful isolation or molecular detection. The aim of this study was to compare the influence of different types of cotton-tipped swab stored at different temperatures, on the detection of MG and MS. To achieve this, combined use of traditional culture analysis (both agar and broth), with modern molecular detection methods was utilized. Performances of wooden and plastic shaft swabs, both without transport medium, were compared. Successful culture of M. gallisepticum was significantly more efficient from plastic swabs when compared to wooden, whereas no difference was seen for the re-isolation of M. synoviae. Storage at 4°C compared to room temperature also increased the efficiency of culture detection for both Mycoplasma species. When stored at room temperature, PCR detection limits of both MG and MS were significantly lower for wooden compared to plastic swabs. The qPCR data showed similar detection limits for both swab types when stored at both temperatures. The results suggest that swabs with a plastic shaft are preferred for MG and MS detection by both culture and PCR. While a lower storage temperature (4°C) is optimal for culture recovery, it seems that both temperatures investigated here are adequate for molecular detection and it is the swab type which carries a greater influence.

Complete Sequence-Based Genotyping of mgc2/pvpA Genes in Chicken-Derived Mycoplasma gallisepticum Isolates of Iran.

Mycoplasma gallisepticum (MG) is a major pathogen of the poultry industry throughout the world. MG causes chronic respiratory disease in chickens and infectious sinusitis in turkeys. Despite constant improvements in the biosecurity of the poultry industry in Iran, MG infection still occurs and causes significant economic issues. To evaluate genetic variability, 10 Iranian MG isolates along with 17 available sequences were characterized by gene-targeted sequencing (GTS) analysis of complete mgc2/pvpA genes. According to the findings, 21 different sequence types within the sample set of 27 strains were typed by this method. The discriminatory power of this typing assay was established to be 0.97. Although no insertions and deletions of nucleotides were observed in the mgc2 gene among the Iranian strains, different lengths of pvpA genes with 1086, 1095, and 1101 nucleotides were detected within direct repeats (DRs) 1 and 2. Generally, eight tetrapeptides Pro-Arg-Pro-Met/Gln/Asn were found in the DRs of PvpA. Analysis of the carboxyl ends of PvpA proteins exhibited various repeats of prolines. In the phylogenetic tree of partial and complete mgc2/pvpA genes, all Iranian MG isolates were clustered into two distinct groups. Because this typing assay could provide a higher discriminatory power than the previously reported GTS scheme of partial mgc2/ pvpA genes, these results can be considered a blueprint for future national control and diagnostic strategies. Furthermore, consistent surveillance with larger datasets will be needed to clarify the epidemiologic characteristics of MG outbreaks in different poultry hosts.

Rate of false positive reactions in 11 M. gallisepticum and M. synoviae serological tests in samples obtained from SPF birds inoculated with heterologous mycoplasma species.

No recent information is available on the specificity of current M. synoviae (Ms) and M. gallisepticum (Mg) serological tests. In this study the performance of a currently available Mg and Ms Rapid Plate Agglutination (RPA) test, and three Mg, three Ms and three Mg/Ms combination ELISAs were evaluated on SPF sera that were obtained from days (D) 0-28 after M. gallinarum, M. imitans or M. gallinaceum inoculation, after sham inoculation and without inoculation. Tracheal swabs for mycoplasma culture were obtained before inoculation (D0), 7 and 28 days post inoculation (d.p.i.) in all groups except the sham inoculated group. The different mycoplasma species colonized well. In the early stage after inoculation (7-14 d.p.i.) with heterologous mycoplasma species, the specificity varied from 85% to 100% in the Mg RPA test and from 70% to 85% in the Ms RPA test. The specificity of both Mg and Ms RPA test was 100% in the sham inoculated samples and ruled out the effect of sham medium. In the late stage (21-28 d.p.i.) specificity was 100% for both RPA tests. The test specificity was 100% for seven ELISAs except for two combination ELISAs where a specificity of 95% was found in the late stage after inoculation. However, this was not significantly different from the specificity of all other tests in the late stage of these groups. These results show that it is not advisable to establish Mg and Ms seromonitoring programmes on the Mg and Ms RPA test alone as other mycoplasma species frequently occur in poultry.

Molecular detection and genetic characterization of Mycoplasma gallisepticum, Mycoplama synoviae, and infectious bronchitis virus in poultry in Myanmar.

BACKGROUND: In Southeast Asian countries, including Myanmar, poultry farming is a major industry. In order to manage and maintain stable productivity, it is important to establish policies for biosecurity. Infectious respiratory diseases are a major threat to poultry farming. Avian influenza and Newcastle disease have been reported in Myanmar, but no scientific information is available for other respiratory pathogens, such as mycoplasmas and infectious bronchitis virus (IBV). Identifying the genotypes and serotypes of IBVs is especially important to inform vaccination programs. In this study, we detected Mycoplasma gallisepticum (MG), M. synoviae (MS), and IBV in several poultry farms in Myanmar. RESULTS: Samples were collected from 20 farms in three major poultry farming areas in Myanmar, and MG, MS, and IBV were detected on two, four, and eight farms, respectively, by polymerase chain reaction. Phylogenetic analysis revealed that the observed MG and MS isolates were not identical to vaccine strains. Three different genotypes of IBV were detected, but none was an unknown variant. CONCLUSIONS: Mycoplasmas and IBV were detected on poultry farms in Myanmar. Periodic surveillance is required to establish the distribution of each pathogen, and to institute better vaccine protocols.

gga-miR-146c Activates TLR6/MyD88/NF-κB Pathway through Targeting MMP16 to Prevent Mycoplasma Gallisepticum (HS Strain) Infection in Chickens.

Mycoplasma gallisepticum (MG), a pathogen that infects chickens and some other birds, triggers chronic respiratory disease (CRD) in chickens, which is characterized by inflammation. The investigation of microbial pathogenesis would contribute to the deep understanding of infection control. Since microribonucleic acids (miRNAs) play a key role in this process, gga-mir-146c, an upregulated miRNA upon MG infection, was selected according to our previous RNA-sequencing data. In this paper, we predicted and validated that MMP16 is one of gga-miR-146c target genes. Results show that MMP16 is the target of gga-miR-146c and gga-miR-146c can downregulate MMP16 expression within limits. gga-miR-146c upregulation significantly increased the expression of TLR6, NF-κB p65, MyD88, and TNF-α, whereas the gga-miR-146c inhibitor led to an opposite result. gga-miR-146c upregulation effectively decreased apoptosis and stimulated DF-1 cells proliferation upon MG infection. On the contrary, gga-miR-146c inhibitor promoted apoptosis and repressed the proliferation. Collectively, our results suggest that gga-miR-146c upregulation upon MG infection represses MMP16 expression, activating TLR6/MyD88/NF-κB pathway, promoting cell proliferation by inhibiting cell apoptosis, and, finally, enhancing cell cycle progression to defend against host MG infection.

Development of Molecular Methods for Rapid Differentiation of Mycoplasma gallisepticum Vaccine Strains from Field Isolates.

Mycoplasma gallisepticum is among the most economically significant mycoplasmas causing production losses in poultry. Seven melt-curve and agarose gel-based mismatch amplification mutation assays (MAMAs) and one PCR are provided in the present study to distinguish the M. gallisepticum vaccine strains and field isolates based on mutations in the crmA, gapA, lpd, plpA, potC, glpK, and hlp2 genes. A total of 239 samples (M. gallisepticum vaccine and type strains, pure cultures, and clinical samples) originating from 16 countries and from at least eight avian species were submitted to the presented assays for validation or in blind tests. A comparison of the data from 126 samples (including sequences available at GenBank) examined by the developed assays and a recently developed multilocus sequence typing assay showed congruent typing results. The sensitivity of the melt-MAMA assays varied between 101 and 104M. gallisepticum template copies/reaction, while that of the agarose-MAMAs ranged from 103 to 105 template copies/reaction, and no cross-reactions occurred with other Mycoplasma species colonizing birds. The presented assays are also suitable for discriminating multiple strains in a single sample. The developed assays enable the differentiation of live vaccine strains by targeting two or three markers/vaccine strain; however, considering the high variability of the species, the combined use of all assays is recommended. The suggested combination provides a reliable tool for routine diagnostics due to the sensitivity and specificity of the assays, and they can be performed directly on clinical samples and in laboratories with basic PCR equipment.

Diagnosis of Avian Mycoplasmas: A Comparison between PCR and Culture Technique.

Mycoplasma gallisepticum and Mycoplasma synoviae are the causative agents of avian mycoplasmosis in commercial poultry. Among the available tools, polymerase chain reaction (PCR) and culture are confirmatory tools for the diagnosis of mycoplasmosis after the initial serological screening of suspected birds. Overall, 181 samples were analyzed, 152 (84%) and 103 (57%) of which were found positive by PCR and culture, respectively. Further, 54 (92%) broiler samples were found positive for general avian mycoplasma. Among the total positive samples, MS positivity was as high as 72 (47%) by PCR, while it was 45 (44%) by culture. MG positivity was 23% and 25% in PCR- and culture-positive samples. MG grows more easily compared to MS. The agreement value between the tests was 67%. Overall, flock wise prevalence was not much varied. The prevalence of mycoplasmosis was higher during winter. Our study confirmed that PCR is the most sensitive and reliable tool for the diagnosis of avian mycoplasmosis in field samples.

Co-circulation of genetically diverse population of vaccine related and unrelated respiratory mycoplasmas and viruses in UK poultry flocks with health or production problems.

Respiratory diseases continue to have a major impact on poultry health, welfare and productivity. However, little information is available on their current status in UK poultry flocks. We investigated the presence of four economically important respiratory pathogens in healthy or problematic flocks; infectious bronchitis virus (IBV), avian metapneumovirus (aMPV), Mycoplasma gallisepticum (Mg) and Mycoplasma synoviae (Ms). Samples from 131 UK poultry flocks were received during the 12 month study period. Oropharyngeal (OP) swabs were taken from eight birds per flock and accompanied with flock health information. The study included 118 chicken, 6 pheasant and 5 turkey flocks, and 1 quail and 1 partridge flock. Chicken flocks were of layers (n = 98), broilers (n = 15), breeders (n = 3) and undisclosed (n = 2). Flock ages ranged from 3 to 72 weeks old, and the average flock size was 17,633 birds. PCR detected 65 (49.6%), 59 (45%) and 8 (6.1%) flocks as positive for IBV, Mg/Ms and aMPV respectively. Analysis of the mgc2 gene of the Mg isolates revealed high similarities to Mg TS-11 and Mg 6/85. Further gene analysis found that the TS-11-like isolates were unrelated to the TS-11 vaccine. Multi-locus sequence typing (MLST) analysis identified the majority of positive Ms as ST21, along with ST2 (MS-H-like), ST6 and ST43. IBV S1 gene sequencing identified strains as 793B (66.7%), Arkansas (23.8%) and Massachusetts (9.5%). All aMPV positive samples belonged to subtype B. Findings indicate that over half of the flocks sampled were positive for at least one of the four vaccine or field strains of mycoplasmas or viruses.

Detection of Mycoplasma gallisepticum and Mycoplasma synoviae among Commercial Poultry in Khouzestan Province, Iran.

Mycoplasmas are important avian pathogens, which can cause both respiratory disease and synovitis in poultry that result in considerable economic losses to the poultry industry all over the world. The aim of this study was to determine the prevalence of Mycoplasma gallisepticum and Mycoplasma synoviae infections among commercial poultry flocks in Khouzestan province, Iran, using the polymerase chain reaction (PCR) technique. Totally, 290 tracheal swab samples were collected from 19 broiler flocks and 4 layer-breeder flocks, with or without respiratory signs, in different areas of Khouzestan province within six months. The PCR tests were applied for the specific amplification of 16S rRNA (185 bp) and vlhA (392 bp) genes. Out of 100 swab samples obtained from the layer-breeder flocks, 1 and 72 specimens were positive for M. gallisepticum and M. synoviae, respectively. In this regard, out of the 4 layer-breeder flocks, 1 (25%) and 4 (100%) flocks were positive for M. gallisepticum and M. synoviae, respectively. However, none of the studied broiler flocks were M. gallisepticum- or M. synoviae-positive. According to the results, the PCR technique could be concluded as a rapid method for the accurate identification of M. gallisepticum and M. synoviae infections in commercial poultry flocks. The results were indicative of the low prevalence of M. gallisepticum in the studied flocks in Khouzestan province. On the other hand, M. synoviae was widely distributed among layer-breeder flocks in this province.

Application of high-resolution melting-curve analysis on pvpA gene for detection and classification of Mycoplasma gallisepticum strains.

Mycoplasma gallisepticum (MG) is an avian species pathogen which causes heavy economic losses in the poultry industry. The purpose of this study was to determine genomic diversity of 14 MG field strains from chicken, Chuker partridge and peacock collected during 2009-2012 in Iran by polymerase chain reaction and partial sequencing of the pvpA gene. A High-Resolution Melting (HRM) technique was also developed and applied to differentiate between field and vaccine strains. Sequencing of the pvpA gene revealed a 51 nucleotide deletion, within DR-1 and DR-2, among MG strains from chicken and partridge whilst 63 nucleotides were deleted in MG strain from peacock. One nucleotide substitution was also observed among chicken MG strains. Phylogenetic analysis of the sequences clustered all of the Iranian MG strains into two clades or phylogeny groups; the strains from chicken and partridge in one group (group 1) and the strain from peacock into another group (group 4). HRM analysis has also produced comparable outcome to those of sequencing; four distinct melting curves which correspond to the three MG strains from chicken, Chukar partridge and peacock and ts-11 vaccine strain. Overall, findings of this study point towards a single source of infection for the chicken and partridge MG strains and likelihood of the strains being native and endemic in Iran. Peacock considered as an exotic species in Iran, hence the genetic distance for the pvpA gene. MG can be transmitted easily among different avian species and this distinct peacock strain may pose a threat to poultry industry. Our findings also show that molecular variation among pvpA gene of MG strains could be revealed using the relatively rapid and affordable HRM technique.

Detection of Mycoplasma gallisepticum in House Finches ( Haemorhous mexicanus) from Arizona.

In 1994, an endemic poultry pathogen, Mycoplasma gallisepticum (MG), was identified as the causative agent of a novel disease in house finches ( Haemorhous mexicanus). After an initial outbreak in Maryland, MG spread rapidly throughout eastern North American populations of house finches. Subsequently, MG spread slowly through the northern interior of North America and then into the Pacific Northwest, finally reaching California in 2006. Until 2009, there were no reports of MG in the southwestern United States east of California. In August 2011, after reports of house finches displaying conjunctivitis characteristic of MG infection in Arizona, we trapped house finches at bird feeders in central Arizona (Tempe) and southern Arizona (Tucson and Green Valley) to assay for MG infection. Upon capture, we noted whether birds exhibited conjunctivitis, and we collected choanal swabs to test for the presence of MG DNA using PCR. We detected MG in finches captured from Green Valley (in ∼12% of birds captured), but not in finches from Tucson or Tempe. Based on resampling of house finches at these sites in July 2014, we suggest that central Arizona finches likely remain unexposed to MG. We also suggest that low urban connectivity between arid habitats of southern and central Arizona or a reduction in the prevalence of MG after its initial arrival in Arizona may be limiting the spread of MG from south to north in Arizona. In addition, the observed conjunctivitis-like signs in house finches that were negative for MG by PCR may be caused primarily by avian pox virus.

HOUSE FINCH ( HAEMORHOUS MEXICANUS)-ASSOCIATED MYCOPLASMA GALLISEPTICUM IDENTIFIED IN LESSER GOLDFINCH ( SPINUS PSALTRIA) AND WESTERN SCRUB JAY ( APHELOCOMA CALIFORNICA) USING STRAIN-SPECIFIC QUANTITATIVE PCR.

: In 1994 Mycoplasma gallisepticum was found to be the etiologic agent of House Finch ( Haemorhous mexicanus) conjunctivitis, a rapidly expanding epidemic caused by a genetically discrete, House Finch-associated strain of M. gallisepticum (HFMG). While most prominent in House Finches, HFMG has been reported in other members of the family Fringillidae, including American Goldfinches ( Spinus tristis), Purple Finches ( Haemorhous purpureus), Pine Grosbeaks ( Pinicola enucleator), and Evening Grosbeaks ( Coccothraustes vespertinus). Herein we report two new potential host species of HFMG strain, the Lesser Goldfinch ( Spinus psaltria), belonging to the Fringillidae family, and the Western (California) Scrub Jay ( Aphelocoma californica), belonging to the Corvidae family. The latter is one of only two reports of HFMG being found outside the Fringillidae family, and of these is the only one reported outside of captivity. Furthermore, non-HFMG M. gallisepticum was identified in an American Crow ( Corvus brachyrhynchos), indicating presence of additional strains in wild birds. Strain typing of M. gallisepticum isolates was done via HFMG-specific quantitative PCR analysis and validated using random amplified polymorphic DNA analysis. Our results suggested an expanded host range of HFMG strain, and further suggested that the host range of HFMG was not limited to members of the family Fringillidae.

Attenuated Phenotype of a Recent House Finch-Associated Mycoplasma gallisepticum Isolate in Domestic Poultry.

Mycoplasma gallisepticum, known primarily as a respiratory pathogen of domestic poultry, has emerged since 1994 as a significant pathogen of the house finch (Haemorhousmexicanus) causing severe conjunctivitis and mortality. House finch-associated M. gallisepticum (HFMG) spread rapidly and increased in virulence for the finch host in the eastern United States. In the current study, we assessed virulence in domestic poultry with two temporally distant, and yet geographically consistent, HFMG isolates which differ in virulence for house finches-Virginia 1994 (VA1994), the index isolate of the epidemic, and Virginia 2013 (VA2013), a recent isolate of increased house finch virulence. Here we report a significant difference between VA1994 and VA2013 in their levels of virulence for chickens; notably, this difference correlated inversely to the difference in their levels of virulence for house finches. VA1994, while moderately virulent in house finches, displayed significant virulence in the chicken respiratory tract. VA2013, while highly virulent in the house finch, was significantly attenuated in chickens relative to VA1994, displaying less-severe pathological lesions in, and reduced bacterial recovery from, the respiratory tract. Overall, these data indicate that a recent isolate of HFMG is greatly attenuated in the chicken host relative to the index isolate, notably demonstrating a virulence phenotype in chickens inversely related to that in the finch host.