DEVELOPMENT OF INACTIVATED OIL EMULSION VACCINE AGAINST MYCOPLASMA GALLISEPTICUM USING LOCAL STRAINS

INTRODUCTION:

Mycoplasma gallisepticum (MG) belongs to the class Mollicutes, order Mycoplasmatales, family Mycoplasmataceae. M. synoviae (MS), M. meleagridis and M. iowae can also cause disease in poultry, but MG is considered to be the most important of the pathogenic mycoplasmas and the OIE has designated the disease caused by MG as notifiable. MG occurs world-wide and is particularly important in chickens and turkeys as a cause of respiratory disease, called chronic respiratory disease (CRD) and decreased production (Bradbury, 2001). It can also cause upper respiratory disease in game birds. The severity of the disease is greatly affected by the degree of secondary infection with viruses such as Newcastle disease and infectious bronchitis, and bacteria such as Escherichia coli. In poultry the infection is spread vertically through infected eggs and horizontally by close contact; the MG nucleic acid has been identified in environmental samples (Marois et al., 2002).

The clinical signs in infected poultry can vary from asymptomatic to obvious respiratory signs including coryza, conjunctivitis, coughing and sneezing. Nasal exudate, tracheal rales and breathing through the partially open beak may occur. Unilateral or bilateral sinusitis may also be a feature, particularly in turkeys and game birds and the infraorbital sinuses may become so swollen that the eyelids are closed. Conjunctivitis, with frothy ocular exudate is also a common feature in turkeys and game birds, and sometimes in chickens. In turkeys there is often soiling of the wing feathers as the result of attempts to remove exudate from the eyes. Infected finches may reveal ocular and nasal discharge and swollen eyelids in addition to the conjunctivitis.

The major economic losses due to CRD are reduced growth rate, poor feed conversion, reduced egg production in breeders and commercial layers (15-25 less eggs per hen housed in 60 weeks), poor hatchability and livability, condemnation and downgrading of carcasses and increased medication cost (Jordan, 1975). According to disease diagnostic summary published each year (1989-94) at Poultry Research Institute, Rawalpindi; the economic losses in terms of mortality only due to CRD ranged from 16 to 21% annually in poultry flocks reared around capital territory of Islamabad (Anonymus, 1994). All these factors together make MG infection as one of the costliest disease problem confronting the poultry industry today.

The preferred method of control is to maintain MG-free flocks. Both live and killed vaccines are used in chickens. Vaccination is considered in situations where field exposure is inevitable. The usual use is to prevent egg production losses in commercial layers and to reduce egg transmission in breeding stock or to aid MG eradication on multi age sites. It is important to vaccinate before field challenge.

OBJECTIVE:

To prepare and evaluate the efficacy of inactivated oil adjuvanted MG vaccine from locally isolated strains of Mycoplasma gallisepticum.


REVIEW OF LITERATURE:

Mycoplasma is unique in the sense that they are not truly bacteria and not truly viruses. Since they are the smallest living organisms, they are often referred as bacteria (Lott et al. 1978). MG infection historically has been called as chronic respiratory disease (CRD), air sacculitis, and air sac infection. The CRD has been reported from poultry populations of over 100 countries worlwide (FAO/WHO, 1984). According to a survey of the poultry industry in the USA, CRD was reported to cause losses of about 125 million dollars per annum
(Bickford, 1986).

Vardman et al. (1973) reported the effects of Mycoplasma infection on broiler performance. They found that Mycoplasma exposed groups had significantly higher rates of condemnation due to airsacculitis than Mycoplasma free chickens. Lott et al. (1978) noted approximately 18 % reduction in egg production in broiler breeders. In lyers, there was a loss of weight, reduced feed consumption. Egg production may decline and be maintained at a lowered level without evidence of clinical signs (Branton et al. 1985).

There are few reprts on the incidence of Avian Mycoplasmosis in Pakistan. Khalil (1984) recorded 16% incidence of Mycoplasma gallisepticum and 4% of Mycoplasma synovae in and around Lahore. He recorded most of the infections in broilers between 4 & 6 weeks of age and 18-22 weeks of age in layers. Rizvi et al. (1994-95) reported the incidence of CRD in layers as 15, 1.66, 0.33 and 0.33 % at ages of 1-10, 11-20, 21-40 and 41-60 weeks respectively in Lahore. Khan et al. (1995-96) recorded the incidence of MG infection 9.84% on flock basis in Sahiwal and Bahwalnagar districts while Sahoota and Dil
(1995-96 ) reported the incidence of Mycoplasmosis 10.37% in different commercial poultry farms in Rawalpindi/Islamabad areas.

Kleven et al. (1984) descibed that the preferred method for the control of Mycoplasma gallisepticum is eradication. The advent of multiple-age farms for poultry production has made eradication impractical after the organism has been introduced. Live vaccination with the F strain of M. gallisepticum during the rearing period has been used to prevent egg production losses. More recently, an inactivated oil emulsion bacterin has become available commercially. Vaccine and bacterin offer protection against egg production losses, respiratory signs and lesions, and egg transmission. Vaccination of broilers, however, is not yet feasible; Bacterin offers minimal protection against infection. The use of bacterins and vaccines on multiple-age farms is being studied as a potential eradication tool.

METERIALS & METHODS:

Sample collection:

Tissue samples (trachea, lungs, air sacs) and tracheal swabs will be taken from suspected birds and kept at 4°C for further isolation and identification.

Isolation & Identification of Mycoplasma gallisepticum:

The samples will be innoculated in liquid Frey’s medium and incubated at 37°C 3-4 days. All samples showing colour change will be adopted by transfering 3-4 times in broth medium afterwords the active culture will be transferred on to solid medium under humid environment at 37°C in a bacteriological incubator for 3-5 days. Preliminary identification will be done through morphological, and biochemical tests. Disk Growth Inhibition test will be performed using standard antiserum of Mycoplasma gallisepticum as described by Stanbridge and Hayflick, 1987.

Vaccine Preparation:

For preparation of vaccine, broth medium will be inoculated with rapidly growing inoculum, at a rate of approximately 5% (v/v) and incubated at 37°C. Hrvesting will be done after 24 hours of inoculation. The antigen will be concentrated by centrifugation. Concentration will be standardized on packed cell volume, which 1% (v/v) is packed cell in final product. Inactivation of organism will be done by treatment with 1% formaline. Bacterins will be made as water in-oil emulsion, 80% mineral oil, 20% aqeous, with emulsifying agent (Tween 80).

Validity of vaccine:

To determine the validity of vaccine, sterility, safety, potency and stability tests will be performed.

REFERENCES:

Anonymous, 1988-94. Annual Progress Report, 1988-94. Poultry Development Centre (PRI), Punjab, Pakistan.

Bradbury J.M. (2001). Avian mycoplasmas. In: Poultry Diseases, Fifth Edition, Jordan F., Pattison M., Alexander D. & Faragher T., eds. W.B Saunders, London, UK, 178-193.

Bickford, A. A. 1986. Diseases affecting reproducing/laying birds and reproducing performance. Poc. Aust. Vet. Assan. 92: 759-761.

Branton, S. L. and J. W. Deaton, 1985. Egg production, egg weight, egg shell strength and mortality in three strains of commercial layers vaccinated with F strain of Mycoplasma gallisepticum. Avian dis. 29: 832-834.

FAO/WHO/OIE. 1984. Animal health year book: Geneva/Rome/Paris.

Jordan, F. T. W., 1975. Avian Mycoplasma and pathogenicity. A review. Avian Pathol. 4.

Khan, M. S., M. Mumtaz and R. U. Rehman, 1995-96. Incidence of major diseases in broiler flocks maintained in Sahiwal & Bahawalnagar. Annual Progress Report, Poultry Develop. Centre (PRI), Pakistan.

Kleven, S.H., J. R. Glisson and M. Y. Lin, 1984. Bacterins and vaccines for the control of Mycoplasma gallisepticum. Isr. J. Med. Sci. 20(10):989-991.

Khalil, M., 1984. Studies on serotypes of Mycoplasma isolated from poultry. MSc. Thesis, C.V.S, Univ. Agri. Faisalabad, Pakistan.

Lott, B. D., J. H. Drott and T. H. Vardmann, 1978. Effect of M. Synovae on egg quality, and egg production of broiler breeders. Poultry science. 57: 309-311.

Marois C.F., Dufour-Gesbert F. & Kempf I. (2002). Polymerase chain reaction for detection of Mycoplasma gallisepticum in environmental samples. Avian Pathol., 31, 163-168.

Rizvi, A. H., A. Raza and I. Bhatti, 1994-95. Astudy on the seasonal prevalence of chronic respiratory disease in broiler and layer flocks maintained in and around Lahore. Annual Progress Report, Poultry Develop. Centre (PRI), Pakistan.

Sahoota, A. W. and S. Dil, 1995-96. Astudy on the prevalence of different poultry disease in Rawalpindi/Islamabad areas. Annual Progress Report, Poultry Develop. Centre (PRI), Pakistan.

Stanbridge, E. and L. Hayflick, 1987. Growth inhibition test for identification of Mycoplasma species utilizind dried antiserud-impregnated paper discs. J. Bacteriol. 93: 1392-1396.

Vardmann, B. D., F. N. Reece and J. W. Deaton, 1973. Effect of M. Synovae on broiler performance. Poultry science. 55: 1909-1912.