Serological and Molecular Diagnosis of Mycoplasma gallisepticum and Mycoplasma synoviae in Poultry FarmsABSTRACT
The objective of this research was to evaluate the efficacy of serological and molecular tests in the detection of Mycoplasma gallisepticum (MG) and Mycoplasma synoviae (MS) in laying and broiler flocks. For this analysis, 344 blood serum samples, 220 tracheal swab samples, and 66 trachea samples were collected. The serum samples were subjected to a rapid agglutination (RSA) test, a hemagglutination inhibition (HI) assay, and an enzyme-linked immunosorbent assay (ELISA). Polymerase Chain Reaction (PCR), nPCR, and vaccine strain PCR were performed on the trachea and tracheal swab samples. RSA was conducted at 1:10 dilution, and resulted in 14.8% (51/344) of samples positive for MG and 28.5% (98/344) for MS. Regarding the results of the HI test, 8.7% (30/344) and 20.3% (70/344) were positive for MG and MS, respectively. Based on the ELISA, 45.6% (177/344) of the samples showed seropositivity for MG, and 57.3% (189/344) for MS. Out of all tracheal swab and trachea samples subjected to PCR, 25.9% (57/220) and 42.8% (31/66) were positive for MG, while 10.0% (22/220) and 18.2% (12/66) were positive for MS, respectively. As determined by the vaccine strain PCR, 56.1% (32/57) of the tracheal swab samples that were positive for MG originated from the MG-F strain, whereas, of the positive trachea samples, 40.6% (13/32) were positive for that vaccine strain. The importance of using different serological and molecular tools in the diagnosis of MG and MS is clear, considering the great variation of results between the techniques, in addition to the possibility that DNA of the agent detected in flocks not having presented seroconversion.
Keywords:
Broiler; commercial egg layers; Serology; PCR; Mycoplasma sp
INTRODUCTION
Mycoplasma gallisepticum (MG) is the etiological agent of chronic respiratory diseases (CRD) in chickens. These cause decreased egg production, reduced growth and feed conversion efficiency, decreased meat quality, and increased mortality (Fergunson-Noel & Williams, 2015). CRD can cause sneezing, coughing, nasal discharge, respiratory rales, and airsacculitis (Umar et al., 2010).
Infection by Mycoplasma synoviae (MS) in poultry may manifest due to immunosuppression, resulting in an increase in the mortality rate, a drop in egg laying and quality, poor hatchability, and a reduction in feed efficiency. Control programs for both MG and MS are expensive, and mismanagement may lead to a synergistic effect when associated with other diseases (Nascimento et al., 2005).
Diagnostic methodologies based on serological tests have been established to ensure flock vigilance against pathogens. Rapid serum agglutination (RSA) is the most common screening test to detect Mycoplasma sp. within flocks. When the serum reacts with the target antigen at dilutions of 1:1, 1:5, and, finally, 1:10, the result is considered positive and confirmed using ELISA or hemagglutination inhibition (HI) tests, as well as isolation and molecular biology techniques (Brasil, 1994, 1999).
Polymerase chain reaction (PCR) standardization allows for the detection of microorganisms without cultivating them (Madigan, 2004). This approach is especially useful for agents that are difficult to cultivate or have a long cultivation period, such as Mycoplasma spp., which has high sensitivity and specificity (Machado et al., 2012). This technique can also be used to obtain accurate results regarding the presence of subclinical infections (Machado et al., 2012), mixed infections with various mycoplasma species, infections caused by secondary bacterial contamination, and antibiotic growth inhibition, antibodies, and other inhibiting factors (Kempf et al., 1994).
Considering the importance of a reliable diagnosis of MG and MS, the objective of this research was to evaluate the efficiency of serological and molecular tests in the detection of MG and MS in laying and broiler flocks.
MATERIALS AND METHODS
Location and sampling
Samples were collected from 22 flocks from six egg layer and three broiler farms located in the state of Pernambuco, Brazil. Information regarding flock age, the presence of respiratory symptoms, MG vaccination, and the number of samples is given in Table 1. For convenience, non-probability sampling was used in this study (Sampaio, 1998).
For the study, serum (n = 344), tracheal swab (n = 220), and trachea (n = 66) samples were collected from randomly selected birds. Serum samples were obtained by drawing approximately 3 mL of blood from the brachial vein. The tracheal swab sample was obtained by inserting the swab into the bird’s trachea, rotating it, and then removing it.
Approximately one to six birds, depending on the farm, were euthanized to collect trachea samples, which were stored in PBS with 10% glycerol. All biological samples were kept in refrigerated isothermal boxes and transported to the laboratory for serological and molecular analyses. The samples were processed at the Laboratory of Infectious-Contagious Diseases of Domestic Animals (LDIC) at the Department of Veterinary Medicine (DMV) of the Universidade Federal Rural de Pernambuco (UFRPE). The research was approved by the Ethics Committee for Animal Use of UFRPE, under license number 093/2017.
Serological tests
Serological tests for MG followed the techniques recommended by the National Poultry Health Plan (PNSA; Brasil, 2001). The RSA commercial antigen test (INATA®) was employed to detect MG and MS. Equal parts of antigen and serum (1:1) were added and homogenized, and after 3 minutes, the presence or absence of agglutination (antigen-antibody complex) was verified. Positive sera were diluted to 1:5 and 1:10 with 0.85% NaCl solution. Samples that exhibited agglutination at 1:10 dilution were considered positive (Brasil, 1994).
Commercial antigens (INATA®) were used to perform the HI test, and the protocol followed the manufacturer’s instructions. After 90 min, the samples that presented a “tear effect” (red blood cell button drainage) in the 1:80 titration were considered positive. The FlockCheckIdexx® antibody detection kit was used for the ELISA for MG and MS, following the manufacturer’s guidelines. Absorbance was measured at 650 nm, and the ELISA threshold to differentiate reactive and nonreactive samples, the geometric mean titers (GMT), and other statistical variables such as the coefficient of variation (CV), the standard deviation (SD), and the maximum and minimum values, were calculated using the xCheck® software version 3.3 of Idexx®.
Molecular tests
Firstly, DNA extraction was performed from trachea and tracheal swab samples with the commercial Wizard® Genomic DNA Purification kit (Promega®), following the manufacturer’s protocol. After DNA extraction, PCR was performed using specific primers (Table 2). The conventional PCR reactions consisted of 2.75 µL of ultrapure water (Milli-Q), 6.25 µL of GoTaq® Green Master Mix (Promega®), 0.5 µL of each primer (20 µM), and 2.5 µL of extracted DNA, yielding a final volume of 12.5 µL. American Type Culture Collection strains from MG (ATCC 19610) were used as positive controls
To generate a thermal profile of the reactions, the following methodology recommended by Buim et al. (2009) was used: the samples were preheated at 94°C for 5 min, followed by 35 cycles of denaturation at 94°C for 1 min, annealing at 55°C for 1 min, and extension at 72°C for 2 min, with a final extension step at 72°C for 10 min, and cooling at 4°C for 5 min. Subsequently, 6 µL of the amplified DNA product was transferred with 0.5 µL of Blue Green Loading Dye (LGC Biotecnologia) to 1.5% agarose gel, subjected to electrophoresis, visualized under ultraviolet light, and photo-documented. The 100-bp DNA ladder (LGC Biotecnologia) molecular weight marker was used.
To perform the nPCR, the previous reaction amplicons were used as DNA. As previously described, the reaction was standardized to a final volume of 12.5 µL per microtube, with a primer concentration of 10 µM. The methodology recommended by Buim et al. (2009) was followed to obtain the thermal profile of the reactions. Furthermore, 6 μL of the reaction was transferred with Blue Green Loading Dye (LGC Biotecnologia) to 1.5% agarose gel, subjected to electrophoresis, visualized under ultraviolet light, and photo-documented.
After MG DNA detection using conventional PCR and nPCR, positive samples were subjected to additional PCR to differentiate field strains from the vaccine strain F. As previously described, this was achieved by standardizing the reaction to a final volume of 12.5 µL per microtube, with a primer concentration of 30 µM. The extracted vaccine strain (Conn-F) was used as a positive control, and ultra-pure water as a negative control. Electrophoresis was conducted as previously described.
Statistical analysis
The Kappa coefficient (Cohen, 1960) was employed in the agreement test, with the following conventional interpretation values: <0.20 = poor agreement; 0.21-0.40 = weak agreement; 0.41-0.60 = moderate agreement; 0, 61-0.80 = good agreement ;> 0.80 = very good agreement. Negative values were interpreted as equivalent to 0.0. The chi-square test was used to determine associations between biological materials (Landis & Koch, 1977).
RESULTS
The results of each serological test are described in Table 3 per flock. Based on the results of the RSA (1:1 dilution), 23.5% (81/344) of the samples were positive for MG, and 41.6% (143/344) for MS. At the 1:10 dilution, 14.8% (51/344) of the samples were positive for MG, and 28.5% (98/344) for MS. In the flocks with a history of clinical respiratory symptoms subjected to the 1:10 dilution RSA, 33.3% (40/120) of the samples were positive for MG, and 55% (66/120) for MS. In addition, in flocks without a clinical respiratory history, 5.8% (13/224) of the samples showed positive results for MG, and 14.2% (32/224) for MS.
According to the HI test, 8.7% (30/344) and 20.3% (70/344) of samples were positive for MG and MS, respectively. Specifically in vaccinated flocks, a serum prevalence of 15.54% (24/165) was detected for MG. The flocks with clinical respiratory histories exhibited positivity rates of 20.8% (25/120) for MG, and 35% (42/120) for MS. In flocks with no respiratory history, 2.2% (5/224) of the samples were positive for MG based on the HI, and 12.5% (28/224) for MS.
In the ELISA assay, 45.64% (177/344) of the samples showed seropositivity for MG and 57.3% (189/344) for MS. Of the total number of sampled vaccinated birds, 69.6% (115/165) presented seroreactivity for MG. In flocks with a history of clinical respiratory symptoms, 93.3% (112/120) of the collected samples were positive for MG, and 97.5% (117/120) for MS. In flocks with no respiratory history, 29% (65/224) of the samples were positive for MG according to the ELISA, and 32.1% (72/224) for MS. Table 4 provides data on the agreement among the different tests.
The tracheal swab and trachea samples were submitted to conventional PCR, and 10% (22/220) and 24.2% (16/66) were positive for MG, respectively. According to the nPCR, 25.45% (56/220) of the swab samples and 46.97% (31/66) of the trachea samples were positive for MG. Moreover, 10% (22/220) of the tracheal swab samples and 18.2% (12/66) of the trachea samples were positive for MS, all of which were from laying hens. The results for each flock are presented in Table 5.
In the flocks with clinical respiratory histories, 40% (24/60) and 68.7% (8/12) were positive for MG when testing the tracheal swab and trachea samples, respectively. In non-clinical lots, 20% (32/160) of swab samples and 50% (24/48) of trachea samples were positive for MG. During the MS detection procedure, 11.67% (7/60) of the tracheal swab samples and 58.33% (7/12) of the trachea samples from flocks with clinical respiratory symptoms were positive, whereas in flocks without a clinical history, 9.4% (15 / 160) of swab samples and 8.33% (5/60) of trachea samples were positive for MS.
Regarding the type of biological material used to perform the PCR, a greater efficiency for the detection of MG was observed in the trachea samples than in the tracheal swab samples (p=0.001475). When testing for MS, no significant difference was found between these biological materials (p=0.113).
According to the PCR performed to differentiate vaccine strains, 56.1% (32/87) of the MG positive samples originated from the MG-F strain. In the vaccinated flocks, 77.8% (28/36) of the tracheal swab samples and 66.7% (12/18) of the trachea samples were positive for the vaccine strain. In lots with a clinical respiratory history, 60% (15/25) of tracheal swab samples showed positive results for the MG-F strain, whereas 43.3% (13/30) of the trachea samples were positive for the vaccine strain. Positive samples from unvaccinated flocks originating from the MG-F strain amounted to 26.7% (4/15) and 7.1% (1/14) for the trachea and tracheal swab samples, respectively. The results per flock are presented in Table 6.
DISCUSSION
This study demonstrated a higher seroprevalence of Mycoplasma synoviae (MS) antibodies than MG antibodies, which is consistent with some previous studies (Mettifogo et al., 2015; Rajkumar et al., 2018), but contrasts with others (Ball et al., 2018; Rehman et al., 2018; Tomar et al., 2017).
Khalifa et al. (2013), stated that in the last decades, Mycoplasma sp. infections have been characterized by the growth of M. synoviae in comparison to M. gallisepticum, as well as the transition from articular tropism towards pronounced respiratory tropism. This increase in the prevalence of MS may be related to the intensification of the control measures against MG (Balen & Fiorentin, 1990). Unlike MG, once a broiler’s breeder flock is positive for MS, it can be treated with antibiotics and tested again after a period, when antibiotic residues are eliminated (Brasil, 2001). Considering that MS vaccinations are not performed at poultry farms in the state of Pernambuco, it can be concluded that all detected seroconversion was attributed to field strains.
For many decades, the rapid or serum plate agglutination (RPA) test and the hemagglutination (HI) test were the standard tests for the monitoring programs of MS and MG. However, the ELISA assay was the serological test that showed the highest sensitivity. This corroborates the findings of Ewing et al. (1998), who reported a high ELISA accuracy for detecting MG in populations of birds with low prevalence or low levels of antibodies, suggesting that ELISA is the most effective confirmatory test, due to the difficulty in standardizing HI and the lack of commercial kits, and may therefore be considered as an alternative to RSA as a screening test.
Difficulties regarding the interpretation of the HI and RPA tests have already been recognized in early monitoring programs (Fahey & Crawley, 1954a). The absence or low frequency of positive results for both agents in healthy birds may be related to the low sensitivity of the SAR test and the high specificity of the HI test in subclinical infections. SAR only detects IgM, which is formed a few days after infection and persists for only 70 to 80 days, making negative RSA results unreliable (Nascimento & Pereira, 2009). In a study conducted by Ewing et al. (1996b) in a commercial multiplier-breeder, both HI and RPA showed a low sensitivity compared to ELISA. Nascimento et al. (1994) observed that breeding hens tested negative for MG based on an RSA test (1:1 dilution) tested positive in other diagnostic tests. Regarding the HI test, it demonstrates a poor ability to detect antigenic variants that differ from the strain used as the hemagglutinin antigen (Kleven et al. 1988), while also having a low sensitivity to low titers of IgG (Ewing et al., 1996).
These are concerning findings, since RSA false negatives may be sources of contamination for uninfected populations, hampering sanitary control (Dos Santos et al., 2007). For this reason, solely relying on one test in monitoring programs for MG and MS is not recommended (OIE, 2018; Feberwee et al., 2020). The further confirmation of serological results may happen through a second antibody test, or the detection and identification of MG or MS via isolation or PCR (Luciano et al., 2011; OIE, 2018).
Molecular techniques offer a dependable and accurate method for identifying Mycoplasma spp. strains in poultry as a substitute for serological testing (World Organization for Animal Health [OIE], 2019). However, molecular techniques should be used alongside conventional serological surveys and standard culture procedures to detect Mycoplasma infections. Molecular techniques not only enable rapid, accurate laboratory diagnosis of Mycoplasma infections in birds, but also allow for field isolate characterization and validation or exclusion of serological test results (Kursa et al., 2016).
In the present study, nPCR was able to detect Mycoplasma gallisepticum DNA even in samples that were negative according to conventional PCR. This indicates that this technique is useful for cases of seronegative flocks. The higher efficiency is due to nPCR using two primer pairs in two successive PCR reactions, significantly improving the sensitivity and specificity of DNA amplification (Carr et al., 2010). However, nPCR is not traditionally used for the molecular diagnosis of Mycoplasma spp., and the use of conventional PCR alone may therefore lead to false negative flocks, jeopardizing control measures.
The DNA from MG was detected more frequently than that of MS. This is different from the results obtained in the study by Barros et al. (2014), in which 1/24 (4.17%) samples were positive for MG and 7/24 (29.17%) for MS in poultry farms in the state of Pernambuco. Buim et al. (2009) reported different results, with only 2% of the samples testing positive for MG, and 32.4% for MS. Such differences may be related to vaccinated flocks and the great number of PCR-positive samples.
Higher percentages of positive samples were observed, both for MG and for MS, in flocks exhibiting reduced egg production and/or respiratory symptoms such as dyspinea, coughing, sneezing, open beak, tearing, and tracheal crackles. These findings are consistent with those observed in similar studies conducted by Barros et al. (2014), who also detected a high frequency of MS-positive samples from commercial laying hens with clinical signs.
When comparing the results obtained from serology with those from PCR, Mycoplasma gallisepticum DNA was found in trachea and tracheal swab samples from unvaccinated broilers that did not show serum conversion in any of the serological tests. These results suggest a recent infection in which an immunological response has not yet occurred. According to Levisohn & Dykstra (1987), it is possible to detect the presence of MG in the trachea before the first instance of a serological response because it is the preferred site of infection for most strains, acting as a reservoir. Therefore, the use of PCR analysis permits pathogen detection before the immune response is induced, providing a notable advantage over serological tests (Moreno et al., 2009).
An alternative explanation is that MG may have been in its latency period, during which it is not recognized by the host immune system due to intracellular location (Biberstiein & Zee, 1990; Razin et al., 1998), thereby justifying the higher number of positive findings in trachea samples rather than tracheal swab samples. In such a case, Mycoplasma waits for an immunosuppression situation (infection by viruses or other bacteria) to start disseminating and potentially cause a clinical disease (Whitford et al., 1994; Chin et al., 2003; Kleven, 2003a).
The presence of flocks testing negative on the SAR tests, HI assays, and ELISA and subsequently yielding positive results based on PCR analysis is an important finding that may be useful for the development of disease control and management strategies at poultry farms.
In Brazil, the use of vaccines against MG is prohibited in breeding flocks. Contrarily, commercial laying hens are commonly vaccinated, and it is thus difficult to differentiate antibodies from field strains and from the vaccine. Therefore, a differential diagnosis of the MG-F strain is particularly important in the country, since live vaccines are the most used by poultry farmers, especially in layer hens (Mettifogo et al., 2015). The efficiency of this vaccine is in the humoral and cellular response, and as a mechanism for the competitive exclusion of field strains from the farm (Nascimento & Pereira, 2009).
The PCR test of the F strain showed the presence of field strains in vaccinated and non-vaccinated flocks, as well as the presence of the MG-F strain in vaccinated and non-vaccinated flocks, including broilers. Therefore, such results indicate the dissemination of vaccine strains in non-vaccinated flocks and wild strains in vaccinated flocks, thereby requiring genetic sequencing to isolate strains. According to Correzola (2012), positive unvaccinated birds indicate the environmental spread of live vaccine strains from vaccinated lots from the same farm or neighboring farms. Strain F is the oldest and most studied strain in the replacement of wild strains, and continues to be transmitted among chicken flocks, even after vaccination has been suspended. Therefore, it is necessary to monitor the pathogenicity of the vaccine (Nascimento & Pereira, 2009).
Mycoplasmas in poultry farms can be associated with deficient sanitary barriers, which is a crucial risk factor for the spread of the disease. This aspect is important from an epidemiological point of view, because mycoplasmas can be transmitted horizontally and vertically, which facilitates the spread of these bacteria between flocks from the same farm and among different farms, increasing the frequency of cases and economic losses (Buim et al., 2009).
The importance of evaluating different Mycoplasma sp. diagnostic tools is evident. The great variation of results gathered by the different techniques highlights the necessity to update the standardized diagnostic protocol established by the National Poultry Health Plan. It is also necessary to consider the possibility of detecting the DNA of the agent in flocks that did not present seroconversion. Furthermore, the PCR technique has a demonstrated ability to differentiate field strains from vaccine strains.
ACKNOWLEDGMENTS
The authors would like to thank the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) and the Fundação de Amparo à Ciência e Tecnologia de Pernambuco (FACEPE) for granting the scholarship that made this research possible. They also thank the Laboratório Avícola Uberlândia (LAUDO) and the Idexx Brasil Laboratórios for supporting the research through the donation of HI and RSA antigens and ELISA kits.
REFERENCES
- Balen L, Fiorentin LO. Mycoplasma synoviae e seu impacto econômico sobre a avicultura. Anais da Conferência APINCO de Ciência e Tecnologia Avícolas; 1990; Campinas, São Paulo, Brasil: FACTA; 1990. p.135-40.
-
Ball C, Forrester A, Ganapathy K. Co-circulation of genetically diverse population of vaccine related and unrelated respiratory mycoplasmas and viruses in UK poultry flocks with health or production problems. Veterinary Microbiology 2018;225:132-8. https://doi.org/10.1016/j.vetmic.2018.09.009
» https://doi.org/10.1016/j.vetmic.2018.09.009 -
Barros MR, Nascimento ER, Silva JSA, et al. Occurrence of Mycoplasma synoviae on commercial poultry farms of Pernambuco, Brazil. Pesquisa Veterinária Brasileira 2014;34(10):953-6. https://doi.org/10.1590/S0100-736X2014001000005
» https://doi.org/10.1590/S0100-736X2014001000005 - Biberstein EL, Zee YC. Review of veterinary microbiology. Oxford: Blackwell Scientific Publications; 1990.
- Bradbury JM. Biosecurity and vaccination control of Mycoplasma infections. World Poultry 2007;23(1):35-7.
- Brasil. Ministério da Agricultura, Secretaria de Defesa Agropecuária, Departamento de Defesa Animal. Normas técnicas para o controle e a certificação de núcleos ou estabelecimento avícola livres de micoplasmoses aviárias. Diário Oficial da União: Seção 1, Brasília, DF, n.24, 1 jul 1999.
- Brasil. Ministério da Agricultura. Secretaria de Defesa Agropecuária. Departamento de Defesa Animal. Programa Nacional de Sanidade Avícola; 2001; Brasília, DF.
- Brasil. Ministério da Agricultura. Secretaria de Defesa Agropecuária. Portaria Ministerial 193, de 19 de setembro de 1994. Portaria Ministerial 193. Diário Oficial da República Federativa do Brasil, Poder Executivo, Brasília, DF.
-
Buim MR, Mettifogo E, Timenetsky J, et al. Epidemiological survey on Mycoplasma and Mycoplasma synoviae by multiplex PCR in commercial poultry. Pesquisa Veterinária Brasileira 2009;29(7):552-6. https://doi.org/10.1590/S0100-736X2009000700009
» https://doi.org/10.1590/S0100-736X2009000700009 -
Carr J, Williams DG, Hayden RT. Molecular detection of multiple respiratory viruses. Molecular Diagnostics 2010:289-300. https://doi.org/10.1016/B978-0-12-369428-7.00024-0
» https://doi.org/10.1016/B978-0-12-369428-7.00024-0 -
Chin RP, Yan Ghazikhanian G, Kempf I. Mycoplasma meleagridis Infection. Enciclopédia Biosfera 2003;8(15):1539. https://conhecer.org.br/ojs/index.php/biosfera/article/view/3721
» https://conhecer.org.br/ojs/index.php/biosfera/article/view/3721 -
Cohen J. A coefficient of agreement for nominal scales. Educational and Psychological Measurement 1960;20:37-46. https://doi.org/10.1177/001316446002000104
» https://doi.org/10.1177/001316446002000104 - Correzola LM, Buchala FG, Vitagliano SMM, et al. Serological response of commercial laying hens to Mycoplasma gallisepticum in poultry farms in São Paulo State. Ars Veterinaria 2012;28(1):41-47.
-
Ewing ML, Cookson KC, Phillips RA, et al. Experimental infection and transmissibility of Mycoplasma synoviae with delayed serologic response in chickens. Avian Diseases 1998;42(2):230-8. https://doi.org/10.2307/1592472
» https://doi.org/10.2307/1592472 -
Ewing ML, Kleven SH, Brown MB. Comparison of enzyme-linked immunosorbent assay and hemagglutination-inhibition for detection of antibody to mycoplasma gallisepticum in commercial broiler, fair and exhibition, and experimentally infected birds. Avian Diseases 1996;40(1):13-22. https://doi.org/10.2307/1592366
» https://doi.org/10.2307/1592366 -
Ferguson-Noel, N, Williams S. The efficacy of Mycoplasma gallisepticum K-strain live vaccine in broiler and layer chickens. Avian Pathology 2015;44(2):75-80. https://doi.org/10.1080/03079457.2015.1005054
» https://doi.org/10.1080/03079457.2015.1005054 - Kempf I, Gesbert F, Guittet M, et al. Evaluation of two commercial enzyme-linked immunosorbent assay kits for the detection of Mycoplasma gallisepticum antibodies, Avian Pathology 1994;23(2):329-38. https:// doi: 10.1080/03079459408419000
-
Khalifa KA, Abdelrahim ES, Badwi M, et al. Isolation and molecular characterisation of Mycoplasma gallisepticum and Mycoplasma synoviae in chickens in Sudan. Journal of Veterinary Medicine 2013;2013:1-4. https://doi: 10.1155/2013/208026
» https://doi: - Kleven SH, Morrow CJ, Whithear KG. Comparison of Mycoplasma gallisepticum strains by hemagglutination-inhibition and restriction endonuclease analysis. Avian Diseases 1988;32(4):731-41.
- Kursa O, Tomczyk G, Sawicka A, et al. Molecular methods used in the diagnosis of Mycoplasma synoviae infection in poultry. Medycyna Weterynaryjna 2016;72(1):18-21.
- Landis JR, Koch GG. The measurement of observer agreement for categorical data. Biometh 1977;33:159-74.
- Lauerman LH. Manual of nucleic acid amplification assays for diagnosis of animal diseases. Turlock: American Association of Veterinary Laboratory Diagnosticians; 1998.
- Levisohn S, Dykstra MJ. A quantitative study of single and mixed infection of the chicken trachea by Mycoplasma gallisepticum. Avian Diseases 1987;31:1-12.
-
Machado LS, Nascimento ER, Pereira VLA, et al. Mycoplasma gallisepticum como fator de risco no peso de lotes de frangos de corte com condenação por aerossaculite na Inspeção Sanitária Federal. Pesquisa Veterinária Brasileira 2012;32(7):645-8. https://doi.org/10.1590/S0100-736X2012000700010
» https://doi.org/10.1590/S0100-736X2012000700010 - Madigan MT, Martusko JMD, Parkes J. Microbiologia de brock. 10th ed. São Paulo: Artmed; 2004.
-
Mettifogo E, Buzinhani M, Buim MR, et al. Evaluation of a PCR multiplex for detection and differentiation of Mycoplasma synoviae, M. gallisepticum, and M. gallisepticum strain F-vaccine. Pesquisa Veterinária Brasileira 2015;35(1):13-8. https://doi.org/10.1590/S0100-736X2015000100004
» https://doi.org/10.1590/S0100-736X2015000100004 -
Mohammed HO, Carpenter TE, Yamamoto R. Economic impact of Mycoplasma gallisepticum and M. synoviae in commercial layer flocks. Avian Diseases 1987;31(3):477-82. https://doi.org/10.2307/1590727
» https://doi.org/10.2307/1590727 - Moreno AM. Técnicas moleculares de diagnóstico. In: Revolledo L, Ferreira AJP, organizadores. Patologia aviária. Barueri: Editora Manole; 2009. 510p. ISBN:978-8520420584.
- Nascimento ER, Pereira VLA. Micoplasmoses. Doenças das aves. Campinas: FACTA; 2005. p. 485-95.
- Nascimento ER, Pereira VLA. Micoplasmoses. Doenças das Aves. Campinas: FACTA; 2009. p. 485-500.
- Nascimento ER, Yamamoto R, Damassa AJ, et al. PCR versus isolamento e sorologia no diagnóstico da infecção por Mycoplasma gallisepticum em galinhas e perus. Anais da Conferência APINCO de Ciência e Tecnologia Avícolas; 1994; Santos, São Paulo. Brasil: FACTA; 1994. p. 89-90.
- Nascimento ER, Yamamoto R, Herrick KR, et al. Polymerase chain reaction for detection of Mycoplasma gallisepticum. Avian Diseases 1991;35(1):62-9.
-
Rajkumar S, Reddy MR, Somvanshi R. Molecular prevalence and seroprevalence of Mycoplasma gallisepticum and M. synoviae in Indian poultry flocks. Journal of Animal Research 2018;8(1):15-19. https://doi.org/10.30954/2277-940X.2018.00150.03
» https://doi.org/10.30954/2277-940X.2018.00150.03 -
Razin S, Yogev D, Naot Y. Molecular biology and pathogenicity of mycoplasmas. Microbiology and Molecular Biology Reviews 1998;62(4):1094-156. https://doi.org/10.1128/mmbr.62.4.1094-1156.1998
» https://doi.org/10.1128/mmbr.62.4.1094-1156.1998 -
Rehman A, Shah A, Rahman S. Seroprevalance of Mycoplasma gallisepticum and mycoplasma synoviae in commercial broilers and backyard poultry in five districts of Khyber Pakhtunkhwa-Pakistan. Pakistan Veterinary Journal 2018;38(2):149-52. https://doi.org/10.29261/pakvetj/2018.047
» https://doi.org/10.29261/pakvetj/2018.047 - Sampaio IBM. Estatística aplicada à experimentação animal. Belo Horizonte: Fundação de Ensino e Pesquisa em Medicina Veterinária e Zootecnia; 1998; p.221.
- Santos BM dos, Marín-Gómez SY, De Paula ACB. Confiabilidade de um teste de triagem para Micoplasmose aviária. Veterinária e Zootecnia 2007;1(1):18-23.
-
Stipkovits L, Kempf I. Mycoplasmoses in poultry. Revue scientifique et technique (International Office of Epizootics) 1996;15(4):1495-525. https://doi.org/10.20506/rst.15.4.986
» https://doi.org/10.20506/rst.15.4.986 -
Tomar P, Singh Y, Mahajan N, Jindal N, Singh M. Molecular detection of Avian mycoplasmas in poultry affected with respiratory infections in Haryana (India). International Journal of Current Microbiology and Appled Sciences 2017;6:2155-62. https://doi.org/10.20546/ijcmas.2017.606.254
» https://doi.org/10.20546/ijcmas.2017.606.254 -
Umar S, Munir MT, Ur-Rehman Z, et al. Mycoplasmosis in poultry: update on diagnosis and preventive measures. Worlds Poultry Science Journal 2017;73(1):12-7. https://doi.org/10.1017/S0043933916000830
» https://doi.org/10.1017/S0043933916000830 - Whitford HW, Rosenbush RF, Lauerman LH. Mycoplasmosis in animals: laboratory diagnosis. Hoboken: Wiley-Blackwell; 1994. p.12-14.
- World Organization for Animal Health. Avian mycoplasmosis (Mycoplasma gallisepticum, M. synoviae). In: WOAH. Manual of diagnostic tests and vaccines for terrestrial animals. Paris: OIE; 2019. v.2, p.844-59.
-
Funding
Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) and Fundação de Amparo à Ciência e Tecnologia de Pernambuco (FACEPE).
-
Data availability statement
The data used to support the findings of this study are available from the corresponding author upon reasonable request.
-
Disclaimer/Publisher’s Note
The published papers’ statements, opinions, and data are those of the individual author(s) and contributor(s). The editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions, or products referred to in the content.
Data availability
The data used to support the findings of this study are available from the corresponding author upon reasonable request.
Publication Dates
-
Publication in this collection
25 Nov 2024 -
Date of issue
2024
History
-
Received
15 May 2024 -
Accepted
02 Aug 2024
