Diagnosis and Control of Mycoplasma Gallisepticum Infection in Poltry

Abstract

THE OBJECTIVES AND SUMMARY OF THIS RESEACH. The Mycoplasma gallisepticm (MG) is the etiologic agent of chronic respiratory disease (CRD) and is responsible for significant losses to the poultry industry. The organism colonized in the upper respiratory tract epically tracheal epithelium, thoracic and abdominal air sacs. The infection is vertically transmitted to the progeny via the contaminated YOLK sac laid by affected parent hens. When such a situation occurs in breeder flocks used to produce hatching eggs for broilers or commercial layer chicks, the business does not remain feasible due to high morbidity, reduced growth rate, poor feed conversation, reduced egg production, poor hatchability, condemnation and down grading of carcasses and increased medication cost. In developed countries the focus of MG control necessitated the implementation of control programs based on eradication of MG from primary breeder stocks under voluntary or essential regulations, except commercial lying multiple-age flocks reared for table egg production, that are difficult to make MG clean due to continuous input of replacement flocks in contaminated premises. Only stick bio security measures can be minimising this problem in commercial layers. In some situation use of vaccine has been advocated in replacement pullets before they are shifted to the contaminated laying units. In spite of overall success of these programs. Economic losses from this MG infection are still significant. In less developed countries including Pakistan these losses are either because of absence of regions control program for serological monitoring, or the non availability of basic laboratory structure for prompt and accurate diagnosis of the aetiology agent that would help in eradication, isolation, and treatment of this ailment before it can inflict economical problems in susceptible flocks. Until 1991 the laboratory in fracture for the isolation and identification of avian mycoplasmas was not established poultry development centre, Punjab, Rawalpindi. Therefore total morality losses due to CRD and its direct impact on breeding operation was not reported in the region. However, since 1994 and epically after limited but timely financial grants provided by the Pakistan sciences foundation, Islamabad, the work on this most important practical problem facing the industry in this region and in the country as a whole was possible. Approximately 20-25 thousands live or freshly dead carcases are submitted annually to the disease diagnostic and research section of PDC, Rawalpindi for disease diagnostic and advice, from poultry flocks reared in and around capital territory of Islamabad, including abbatabad and Mnashara districts of North West frontier provinces. The major part of the population reared in this region comprised breeder stocks, aimed at supplying commercial day-old broiler and layer chicks for meet and egg purpose. Due to economic significance and nature of the business in the region, the two major concerns of the growers regarding MG control sera; How to maintain breeding stocks MG free. How to check vertical transmission and lateral spread of MG in progeny flocks for better performance. Therefore the studies carried out under this project were more of an applied value rather than just having an academic nature and were designed to achieve following major objectives. To determine the serologic status of MG in breeder and commercial flocks in the region. Isolation and identification of MG from field flocks and its rate of prevalence in the region. Study of pathogenicicty of isolated filed strain and their protein profiles in comparison to standard strain such as (S5, R AND S). Development of diagnostic plate antigen using indigenous field MG strains and or commercially available plate antigens. To study the effect of antimycoplasmal drugs on experimentally and or naturally expose MG strain of reduced virulence. To evaluate the efficiency of MG vaccine in broiler chicks using F strain of MG or indigenous MG strain of reduced virulence. The results of this research have been reported in six large chapters in manuscript Formate. For submission to scientific journals for publications. The brief summaries of each research study are reported as under. A. summary of chapter -1 In this study the prevalence of Mycoplasma gallisepticm and M. Synoviae was estimated by testing approximately 15655 serum samples obtained from approximately 400 random poultry flocks with approximately population of 0.75 million during five years period between 1991 to 1995, as a long term serologic surveillance. The percentage of agglutination reactions were 35.6 and 28.3 for MG and MS respectively. During the years, 1996, an exclusive effort was made to determine the serologic status of MG only, by testing approximately, 7630 serum samples obtained from 60 sampled broiler breeder flocks with approximate population of 0.34 million as a short term serologic surveillance. The estimated true prevalence rate of MG ranged between 21 to 30 percent. All these flocks were reared around northern designated yielded significantly variable agglutination reactions when sera from the same source were tested. The percentage of agglutination reactions yielded by com-1 , ranged between 10.2 to 16.8, when sera were tested with or without dilution respectively ; whereas, percentage of agglutination reactions ranged between 8.2 to 9.9 respectively. When the same sera were tested with or without dilution by COM-2. Both antigens also varied significantly while yielding negative SPA reactions. Approximately 752 sera remained negative by COM-1 and 1942 sera remained negative by COM-2. When SPA positive sera were not significantly different from SPA positive sera when tested after dilution. Both MG SPA antigens invariably yielded false positive agglutination reaction with sera from MG negative field flocks inoculated with oil-emulsion vaccines against infection coryza and infection bursal disease. These agglutination reactions were eliminated when sera were diluted 1:4 or 1:8 in phosphate buffer saline. Antibodies to MG were frequently seen in day old progeny originated from serologically or culturally positive breeder flocks. These antibodies were undetected 14 days after hatch. The finding obviously emphasise the need to sample chicks as early as possible after hatch if presence or absence of maternal antibodies in progeny is to be the oriented for assessing the status of breeder flocks for MG. B. summary of chapeter-2 This study reports the efforts made to isolate MG strains from filed flocks and their flocks and their accurate diagnosis. The cumulative percentage diagnosis of chronic respiratory disease (CRD) in all breeds and in all season of the years, 1995 ranged between 17-25 percentage. Airsacultis was the most diagnostic pathologic lesions seen at necropsy during early age ( 1-8 weeks ) in commercial broilers infected with MG though vertical or lateral transmission. In adult birds reared in high endemic areas, Airsacultis was accompanied with fibrinopurulents exudates in the abdominal cavity along with tracheilis. Similarly, during production, the pathological lesions in females comprised thickening of the air sacs, tracheitis, pericarditis, per hepatitis with cheesy yellowish plaques and coagulated yolk material along with peritonitis and cheesy cores in the oviduct. The ovarian follicles showed regression along with salpingitis. In adult breeder male’s rachitic, thickening of air sacs and swollen joints and infraobital sinuses were the major lesions recorded. In acute case, the tastes of male showed engorged blood vessels. Modified Frey’s medium supplemented with 15% horse serum and 0.5% yaest extract was used to make primary isolations of MG strains from filed poultry flocks reared in northern areas of the country. On the basis of cultural techniques and biochemical tests, initially, 137 isolates from 473 field specimen’s thoracic and abdominal air cases, ovaries, oviduct and tests. Were identified as of MG, giving an overall 28.9% rate of isolation. The maximum form of isolation was form tracheas and air sacs respectively. Isolation attempts made from sullen infraorbital sinuses joints and cloacae swabs were not successful. Approximately, 19 isolate were subjected to identify by growth inhabitation, agar diffusion precipitation and immunofluorscence antibody techniques for final conformation and further studies. Only 15 isolates yielded positive serology with all of these tests. All the three serological tests were highly compatible in making final confirmation of field MG isolation. C. Summary of chapter-3 This study reports the trials that were conducted to determine the pathogen city and protein profiles of standard and indigenous MYcoplasma gallisepticum strains. MG strain R and the two indigenous MG isolates PMG-35 and PMG-198, induced the highest rate of morality and Airsacultis in ova and in Vivo studies respectively. These MG strains yielded sillier signs and lesions and embryo mortality in ovo pathogenicicty trails using embrocating eggs from commercials or species pathogen free source. MG strains F and one indigenous MG isolates , failed to induced embryo morality and also induced mild or moderate signs and lesions in vivo pathogen city trails. However, all MG strain induced week or moderate antibody response by 7th day post exposure as detected by commercial MG serum plated agglutination antigen test. From 14 day post exposure to the end of trial , all group yielded strong positive SPA and H1 titters with the exception of F strain expose group, which yield weak SPA reaction and low H1 response that was delayed until 4 week postexpoure , except MG-F exposed group which was delayed by 14 days of isolation 90% or above in groups expose to R, RMG-35 or RMG-198, whereas in groups expose to F strain the rate of isolation was only 60% and 75% respectively. The histopathological changes induced of mg ,r trachea and air sac were similar. The two indigenous MG strains PMG-161 and PMG0-163 were similar. The two indigenous MG strains PMG-161 and PMG-163 were distinguished only by few missing bands of low molecular weight. Summary of chapter-4 In this study sensitively and specificity of serum plate agglutination PMG-35, PMG-191 was compared with SPA antigens prepared with standard MG strain S6 or two commercially available MG SPA antigens designed C1 and C2. Sensitively was measured using one group of 10 chickens exposed to R strain of Mycoplasma gallisepticum by intraocular and intranasal inoculation. Specifically was measured in one group of 10 chickens each inoculated with oil-emulsion vaccine. They were coryza bacteria, infection brusal disease virus and reveres inactivated virus vaccines. All sera were tested at 1, 2,3,4,5 and 6 week post inoculation. All antigens had a perfect sensitively score. The three laboratory prepared antigens and one commercial antigens. Even though these indigenous Mycolpasma gallisepticum strains showed marked differences in pathogenicicty in a previous study but evidently, they did not differ significantly in sensitively and specificity when used as agglutination antigens. When known Mycolasma gallisepticum negative sera were tested commercial MG SPA yielded significantly huger number of false positive asgglution reactions. When the overall performance of the laboratory prepared antigen and a commercial C2 antigen had scores above. E. Summary of chapter 5 This study reported the therapeutic efficiency of antimucoplasma drugs namely enrofloxcin, myosin and spiramycin when evolutes in two different in-vivo trials. In trial-1, the locally isolated Mycoplasma gallisepticum strain PMG-198 was used to infect experimental chicks through intrapulmonary route. In trial-2 naturally infected day old chicks were obtained from MG infected breeder flocks. The measures of such as clinical signs, mortality, weight gains, air sac lesions, seroconversions and frequently of resolution were recorded in each medicated and non-medicated groups. Severe clinical signs characterised with sneezing, respiratory distress and recumbence with significantly high mortality rate were seen in un-medicated controls. In both trails enrofloxicin or tylsion did not vary significantly but it varied significantly from spirmacin medicine group in trail-2 or non-medicated control. Among non medicated control group or trial the chicks in trial-1 had better weight gain compared to the chicks in trial. The group medicated with enrofloxanie and tylsion yielded least number of birds positive by [plates asgglution test, gross air sacs lesions, and reduced frequently controlled. Non medicated group infected control were almost 100% positive by aero logy, air sacs lesions and MG reiosolation in bath trails. F. Summary of chapter -6 This study reports the experiments to evolutes the efficacy of Mycoplasma gallisepticum vaccine in broiler chicks conducting under controlled conditions. An indigenous field isolate of reduced virulence, designed PMG-161, and F strain of MG r were used as vaccine candidate and were employed as live culture via eye drop at 11 days of age. the birds were challenged at 15 days post- vaccination with R strain of MG or with an indigenous MG strain of high virulence designated PMG-35. The F strain vaccinated group did not show any respiratory sign up till point of challenge whereas, PMG-161 vaccinated group showed mild vaccine groups by 14 days post vaccination. When examined at 7 and 14 days postcghallenge the F strain vaccinated and challenged subgroup showed least respiratory signs without mortality. As such the indigenous MG did not prove to be a good vaccine candidate in the control of respiratory sign or airsaccilitis in vaccinates. Further work is needed to determined the capability of F strain and indigenous MG isolate of reduced virulence’s as vaccine candidate in preventing air sacs lesions and respiratory signs in commercial broilers or as a mean to displace and level of immunity to withstand field challenge in relation to route of vaccination are other areas that needed to be explored.