Seroprevalence and associated risk factors of mycoplasmosis in layer chickens at southern region of Bangladesh

Mycoplasma gallisepticum (MG) is the most important pathogenic Mycoplasma spp. causing avian mycoplasmosis and brought about huge economic losses to poultry industry in Bangladesh. The present study was undertaken to know the seroprevalence of MG in layer birds in three different geographical areas of southern Barishal division, Bangladesh. Total 310 sera samples were collected from wing vein of 30 farms for this study. Sera samples were tested with Rapid Serum Agglutination (RSA) for MG using commercial Antigen Kit (manufactured by Lillidale Diagnostic) to detect the presence of antibodies against MG. The overall seroprevalence of MG by RSA was 36.13%. Seroprevalence of MG infection was dominant in winter season (45.54%) and significantly highest occurrence was recorded in age groups from 20-40 weeks of layer chickens (51.79%). Serological investigation in three different upazila of Barishal division showed the highest infection rate (45.26%) in medium scale flocks (1000-3000) in comparison to (21.43%) small (<1000) flocks. The seroprevalence of MG was highest in Swarupkathi (44.38%) than in Barishal Sadar (26%) and Banaripara upazila (28%). Biosecurity and managemental failure is the overall risk factor in all types of farm due to lack of proper knowledge among farmer. This study reveals the current scenario of mycoplasmosis in layer birds of three different areas of Barishal division. Asian J. Med. Biol. Res. 2021, 7 (3), 292-297

Investigation on Seroprevalence of Mycoplasma Gallisepticum Infection in Commercial Layer Farms in Rajshahi District Using Rapid Serum Plate Agglutination Test

Mycoplasma gallisepticum (MG) infection is very common in birds that cause respiratory infections in chickens, turkeys, and other avian species. It has brought about a considerable amount of financial losses to the poultry sector in Bangladesh. We conducted a study on the Seroprevalence of MG infection in two different geographical areas of Bangladesh under Rajshahi district, namely Paba and Bagmaraupazila. 800 sera samples were collected and tested with Rapid Serum Plate Agglutination Test (RSA) to identify the MG antibodies using commercial Mycoplasma gallisepticum antigen. The gross Seroprevalence of MG infection was 59.25% in the study area. The maximum rate (68.80%) of infection was found in the winter season, followed by the summer season (49.36%). The result further revealed that the condition was higher (69.01%) in larger-sized flocks than in small (53.63%). We noticed that younger birds having 10-20 weeks of age are more prone to be affected with avian mycoplamosis with an infection rate of 72% compared to their adult counterpart of 71+ weeks with 52% morbidity. Our study revealed that Mycoplasma gallisepticum infection is prevalent in Paba and Bagmaraupazila in Rajshahi. The farms should take strict bio-security measures to mitigate this infection in the mentioned areas. Proper medications for the affected birds and timely prophylactic measures for the healthy ones could be practical and preventive strategies against avian mycoplasmosis. Amid limitations, we conducted our experiments, and thus further research is warranted to substantially assess and validates our observations.

Isolation and molecular detection of avian mycoplasmosis in selected areas of Mymensingh district in Bangladesh

Avian mycoplasmosis caused by several species of Mycoplasma including Mycoplasma gallisepticum, M synoviae, M. meleagridis and M. iowae. Among these Mycoplasma gallisepticum is the most important poultry pathogen in Bangladesh. For effective control of Mycoplasmosis, proper early diagnosis is the corner stone. The research work was designed, a total of 20 samples, lung exudates, swabs from trachea and air sacs were collected from dead birds of different poultry farms in Mymensingh district during October-December, 2007. Samples were collected in 10% buffered formalin for histopathological study. Swabs were collected in mycoplasma broth supplemented with supplement-G. Additionally Kanamycin solution was added to prevent the growth of gram–Ve bacteria and then the organisms were transferred into mycoplasma agar for isolation. Histopathological studies were conducted using routine procedure in Hematoxylin and Eosin stain. Isolated Mycoplasma were subjected to DNA extraction, Nested PCR was done using a commercial PCR kit. The histopathological study revealed the presence of mycoplasmal related tissue changes, such as severe congestion and infiltration of mononuclear cells in different organs. The extracted DNA accumulated at the upper position of DNA ladder as band without any smear formation. The DNA from avian mycoplasmas was amplified and gave amplified product of 975 bp by outer primer and 395 bp by inner primer which was much smaller than the expected size. In this study, preliminary results from field samples suggest that culture using mycoplasma agar and broth supplement with Supplement-G and Kanamycin solution could be useful for the isolation of pathogenic avian mycoplasmas. Asian J. Med. Biol. Res. 2021, 7 (2), 182-190

Histopathological lesion of Arthritis in Mycoplasma synoviae naturally infected breeder chicken in Egypt

Avian mycoplasmosis consider one of the main poultry industry problems all over the world. The present short communication records the pathological lesions in Mycoplasma synoviae (MS) positive samples. Our study was applied to joint tissue samples collected from chicken suspected of mycoplasma infections during a previous study. Joint tissue samples positive MS infection; from broiler breeder flocks aging 46-57 weeks with clinical arthritis were fixed in 10% neutral buffered formalin. Histopathological examined sections from MS positive joint samples showed detachment of the synovial sheet, moderate to severe tendonitis with moderate intensive inflammatory cells infiltration. Infiltration in/and around the blood vessels with lymphocytes of the synovial sheet. Few cases showed mild inflammation in tendon sheet characterized by inflammatory cells infiltration. In was concluded from the study that te detection of histopathological changes can be of value in diagnosis of MS natural infection in chicken with clinical arthritis but not sufficient alone. Although, histopathological examination is an auxiliary diagnostic tool besides isolation and molecular identification techniques.

OCCURRENCE, MOLECULAR IDENTIFICATION AND ANTIBIOTIC RESISTANCE PROFILING OF Mycoplasma gallisepticum AND Mycoplasma synoviae FROM CHRONIC RESPIRATORY DISEASE CASES IN POULTRY BIRDS AND FARM ENVIRONMENT

Avian mycoplasmosis is an important risk for commercial poultry production leading to enormous losses in terms of disease and productivity. The main causative agents are Mycoplasma gallisepticum and Mycoplasma synoviae. To study the variable degree of resistance to commonly prescribed and used antibiotics in mycoplasmosis, a total of 115 samples including tissue specimen and swabs were collected from chronic respiratory disease (CRD) cases of broiler and layer birds and their contaminated farm environment. The samples were directly passaged into the Brain Heart Infusion broth (supplemented with 10 % horse serum, NAD, cysteine, penicillin and thallium acetate). Positive samples were transferred to Brain Heart Infusion agar (Difco) for the isolation of Mycoplasma spp. while negative samples were declared after the third passage. Of the samples, 61.5% were found positive for Mycoplasma spp., which were recovered mostly after second passage. Out of total culture positive cases, Mycoplasma gallisepticum (MG) was identified in 62% cases and Mycoplasma synoviae (MS) in 38%, as confirmed through Polymerase Chain Reaction (PCR) using specific primers. The MG and MS isolates showed variable degrees of sensitivity against the commercially available drug of choice, tylosin. The highest Minimum Inhibitory Concentration (MIC) of enrofloxacin (112.38±4.34 µg/ml) was recorded against MG, followed by tetracyclin (91.58±4.66µl/ml), gentamicin (54.33±2.98 µg/ml), spiromicin (52.23±3.99 µg/ml) and tylosin (52.58±2.69 µg/ml). The highest MIC for enrofloxacin (168.24 ±3.82 µg/ml) was recorded against MS followed by tetracyclin (115.48±2.62 µg/ml), spiromicin (95.96 ±2.17 µg/ml), tylosin (84.84±2.56 µg/ml) and gentamicin (46.4±2.18 µg/ml). Multiplex PCR is a time tested tool for the molecular diagnosis and confirmation of Mycoplasma species.Key words: avian mycoplasmosis; chronic respiratory distress; minimum inhibitory concertation; multiplex polymerase chain reaction POJAVNOST, MOLEKULARNA IDENTIFIKACIJA IN UGOTAVLJANJE ODPORNOSTI NA ANTIBIOTIKE MIKOBAKTERIJ Mycoplasma gallisepticum in Mycoplasma synoviae IZOLIRANIH IZ KOKOŠI S KRONIČNIMI DIHALNIMI OBOLENJI IN IZ NJIHOVEGA BIVALNEGA OKOLJA Povzetek: Ptičja mikoplazmoza je resno težava v perutninski proizvodnji, ki vodi v velike izgube zaradi obolevanja perutnine in posledično povzroča ekonomske izgube. Glavni povzročitelji mikoplazmoz so Mycoplasma gallisepticum in Mycoplasma synoviae. Za preučevanje spremenljive stopnje odpornosti na običajno predpisane in uporabljene antibiotike pri mikoplazmozi je bilo odvzetih skupno 115 vzorcev, vključno z vzorci tkiva in brisom, pitovnih piščancev, nesnic s kroničnimi boleznimi dihal (CRD) in iz njihovega bivalnega okolja. Vzorci so bili preneseni v tekoče gojišče BHI (iz angl. Brain Heart Infusion), z dodatkom 10 % konjskega seruma, NAD, cisteina, penicilina in talijevega acetata. Pozitivne vzorce smo prenesli v agar BHI (Difco) za izolacijo Mycoplasma spp. Vzorci so bili določeni kot negativni po tretji pasaži. Med vzorci je bilo 61,5 % pozitivnih na prisotnost Mycoplasma spp., ki smo jih večinoma ugotovili po drugi pasaži. Od vseh pozitivnih primerov je bila ugotovljena Mycoplasma gallisepticum (MG) v 62 % primerov, Mycoplasma synoviae (MS) pa v 38 %, kar je bilo potrjeno z verižno reakcijo s polimerazo (PCR) z uporabo specifičnih primerjev. Izolati MG in MS so pokazali spremenljivo stopnjo občutljivosti na komercialno dostopno zdravilo tilozin. Minimalna zaviralna koncentracija (MIC) pri MG je bila najvišja pri enrofloksacinu (112,38 ± 4,34 µg/ml), sledili pa so tetraciklin (91,58 ± 4,66 µl/ ml), gentamicin (54,33 ± 2,98 µg/ml), spiromicin (52,23 ± 3,99 µg/ml) in tilozin (52,58 ± 2,69 µg/ml). Najvišjo MIC proti MS smo ravno takougotovili pri enrofloksacinu (168,24 ± 3,82 µg/ml), ki so mu sledili tetraciklin (115,48 ± 2,62 µg/ml), spiromicin (95,96 ± 2,17 µg/ml), tilozin (84,84 ± 2,56 µg/ml) in gentamicin (46,4 ± 2,18 µg/ml).Ključne besede: ptičja mikoplazmoza; kronična bolezen dihal; minimalna zaviralna koncentracija; mnogokranta PCR reakcija

Treasing on Tylvamyco® as a Novel Immunomodulatory Medication on Broiler Chickens

Present study aims at the evaluation of the efficacy of Tylvamyco® as a new macrolides generation in control of avian mycoplasmosis in broilers chickens with special attention to its immunomodulation effects. A total of 500 day old broiler Ross 308 chicks were equally subdivided into two treatments of 250 birds in each. The Tylvamyco® treated group, and the control non treated group were kept in a separate house. Blood samples and tracheal tissues collected at one day old and also each week till the end of the trials for isolation M. gallisepticum and also measuring the immune status of the experimental chicks. M.gallisepticum occurrence rate in broilers chickens was 12% which confirmed by PCR. The minimal inhibitory concentration values Tylvamyco® against recovered 12 M.gallisepticum isolates standard strain showed that the Tylvamyco® has MIC90 value of 0.008. In the Tylvamyco® treated group the immune status profiles record that there are marked increase in the immunological parameters by age as; HI test results for Mycoplasma ,NDV , AI, INF-γ conc. ,IL-6 conc. ,Phagocytic cell count ,Nitric oxide conc. and Lysozyme conc. at 1, 15, 30 day old respectively. The molecular analysis of CXCL8 gene as an indicator for inflammation reduction potency in In the Tylvamyco® treated group by using real-time PCR showed that the Cycle Threshold of CXCL8 gene reduced by age from 13.6 to 10.7 at 15 day old and 30 day old with fold change 0.57 and 1.4 respectively. Performance parameter in Tylvamyco® treated group as 3.22 kg/bird, with mean weight gain 2.33 kg/bird and feed conversion rate 1.4. The mortality rate was 5% with slight air saculitis, as post-mortum records, in conclusion our study proved that Tylvamyco® act as a potent immunomodulatory medication in broilers.

Detection and antibiotic resistance of Mycoplasma gallisepticum and Mycoplasma synoviae among chicken flocks in Egypt

Background and Aim: Mycoplasma gallisepticum (MG) and Mycoplasma synoviae (MS) are the most significant pathogens of avian mycoplasmosis. This study aimed to isolate and identify MG and MS from chickens and detect the various virulence genes in the isolates. Moreover, the efficacies of different antibiotics were tested to identify suitable treatment regimens. Materials and Methods: We isolated MG and MS from 487 chicken samples of different ages located in different Governorates in Egypt using conventional isolation methods. The isolates were characterized by polymerase chain reaction (PCR) and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then tested for antibiotic sensitivity by the minimum inhibitory concentration (MIC) method. Results: The prevalence of MG among the isolates was 9.85%, with the highest percentage isolated from air sacs, while the prevalence of MS among the isolates was 1.6%. Moreover, the highest levels of the prevalence of both MG and MS were during the winter and autumn sampling, while the lowest levels were in the summer and spring. Following the 16S rRNA-based detection of Mycoplasma isolates, 14 MG and 5 MS isolates were identified by different PCR-based detection methods for various virulence genes. Nine MG isolates contain the mgc2 gene, six MG isolates contain the gapA gene, and three MS isolates contain the vlhA gene. We validated a duplex PCR method for the simultaneous identification of MG and MS, based on 100% of the MG and MS isolates generating common bands at 55 and 17 kDa, respectively. The MIC method identified tiamulin and spiramycin as the antibiotics of choice for the treatment of MG and MS infections, respectively. Conclusion: For more precise diagnosis of Mycoplasma infections in chicken flocks, conventional isolation methods must be confirmed by PCR. SDS-PAGE analysis helps in epidemiological studies and vaccine preparation. The MIC method can be used to help develop therapies to control avian mycoplasmosis infections.

Identification of Strain-Specific Sequences That Distinguish a Mycoplasma gallisepticum Vaccine Strain from Field Isolates

ABSTRACTDespite attempts to control avian mycoplasmosis through management, vaccination, and surveillance,Mycoplasma gallisepticumcontinues to cause significant morbidity, mortality, and economic losses in poultry production. Live attenuated vaccines are commonly used in the poultry industry to control avian mycoplasmosis; unfortunately, some vaccines may revert to virulence and vaccine strains are generally difficult to distinguish from natural field isolates. In order to identify genome differences among vaccine revertants, vaccine strains, and field isolates, whole-genome sequencing of theM. gallisepticumvaccine strain ts-11 and several “ts-11-like” strains isolated from commercial flocks was performed using Illumina and 454 pyrosequencing and the sequenced genomes compared to theM. gallisepticumRlowreference genome. The collective contigs for each strain were annotated using the fully annotatedMycoplasmareference genome. The analysis revealed genetic differences amongvlhAalleles, as well as among genes annotated as coding for a cell wall surface anchor protein (mg0377) and a hypothetical protein gene,mg0359, unique toM. gallisepticumts-11 vaccine strain. PCR protocols were designed to target 5 sequences unique to theM. gallisepticumts-11 strain:vlhA3.04a,vlhA3.04b,vlhA3.05,mg0377, andmg0359. All ts-11 isolates were positive for the five gene alleles tested by PCR; however, 5 to 36% of field isolates were also positive for at least one of the alleles tested. A combination of PCR tests forvlhA3.04a,vlhA3.05, andmg0359was able to distinguish theM. gallisepticumts-11 vaccine strain from field isolates. This method will further supplement current approaches to quickly distinguishM. gallisepticumvaccine strains from field isolates.