HISTORY OF AVIAN INFLUENZA (BIRD FLU) IN PAKISTAN

Investment in poultry sector in Pakistan is about one billion dollars. Every family in rural areas and every fifth family in urban areas is associated directly or indirectly with poultry production activities in one way or the other (Sadiq., 2000). Poultry industry in Pakistan and all over the world is a major contributor of animal proteins substitute. Pakistan poultry industry is facing various managemental problems along with infectious diseases including avian influenza avian influenza (Alexander., 2000). Economics losses from avian influenza have varied depending on strain of virus, species of bird infected, number of farms involved, control methods used, and speed of implementation of control or eradication strategies. Direct losses in HAPI outbreak have included disposal costs, high mortality and morbidity losses, quarantine and surveillance costs and indemnities paid for elimination of marketing birds (Swayne., 2003).

In Pakistan outbreak of AI was first recorded in October 1994. The disease affected broiler breeder in Mansehra, Abbotabad, Rawalpindi and adjoining areas, killing approximately one million birds. The causative agent was confirmed as avian influenza a virus H7N3 (Naeem and Hussain.,1995).

In 1996, outbreak of AI broiler breeder and commercial layer was suspected in various areas of the Punjab. This outbreak did not cause considerable loss but was responsible for low production and immunosuppression. The causative agent was isolated and characterized as avian influenza virus H9N2. Keeping in view the virulence of this virus for poultry, this was also included in locally prepared vaccine. The AI vaccines containing locally isolated H7N3 have been extensively used since 1996 (Muhammad et al., 1997).

However, due to poor bio security and congested poultry colonies, the problem of AI started reappearing in Karachi in 1999 mainly in broilers. The disease was controlled by vaccination and strict bio security measures. However, later on due to poor bio security and no usage of vaccine the avian influenza was endemic in Karachi by the end of 1999 (Naeem et al.,1999).

In 2001, outbreak of a respiratory syndrome in broiler and layer, in Karachi and Abbotabad was recorded. The morbidity upto 100 percent and mortality was upto 50 percent. The HA agent was confirmed as avian influenza (H9 subtype) by using HI with AIV-H9 specific antis era (Muhammad et al., 2001). In 2003, the occurrence of avian influenza virus (AIV) infection in broiler, layer and broiler breeder flocks were reported in |Southern Pakistan. Data from this survey showed high levels of AIV antibodies, indicating unrecognized AIV infection occurring in these flocks. Based on this information, a second investigation was undertaken in selected broiler breeder, broiler and layer flocks. In this investigation, nine H9N2 AIV isolates were recovered. Chicks with the previous history of respiratory tract infection and some without overt clinical respiratory signs, had seroconverted to H9N2 (Naeem et al., 2003).

COLLECTION & TRANSPORTATION OF SPECIMENS

Here are few guidelines for those attached with veterinary practice specially poultry. Sticking to these guidelines can enhance their skill and performance.

Collection of blood samples

1. Restrain the bird properly.
2. Expose the wing vein, clean it with antiseptic.
3. Prick needle in the vein properly.
4. Smoothly take the blood in syringe.
5. Pull the plunger back to create a free space in the syringe.
6. Make a slant and keep the syringe undisturbed for 30-45 minutes so that blood can be clotted and serum be separated.

Collection of serum sample

1. When the blood clots in the syringe and the serum must be separated.
2. Take serum in the aliquot, taking care of that no shreds of blood clot should be in the serum.
3. Tightly close the lid of aliquot.
4. Precautionary pack and label the samples.

Collection of tracheal swab

1. Restrain the bird properly.
2. Open beak of the bird.
3. Open the sterilized swab and take a deep sample by inserting swab in the pharynx.
4. Close the swab tube, properly label and pack it.

Collection of cloacal swab

1. Restrain the bird properly.
2. Locate cloaca of the bird.
3. Open the sterilized swab and take a deep sample by inserting swab in the cloaca.
4. Close the swab tube, properly label and pack it.

Collection of tissue sample

1. Open the bird on a clean surface.
2. Take the trachea, lungs, liver, spleen and properly pack the sample in a plastic sticking bag.
3. Properly label sample

Transportation of Samples

1. The entire sample including serum, swabs, and tissues should be transported at 4ºC.
2. The cooler can be used for transportation by adding ice.
3. Tissue sample should be freeze before transportation.
4. Tissue, serum, cloacal & tracheal swab can also be preserved at freezing condition.

Note: Swab sample can be taken and transported only after adding transport medium.
Normal saline with antibiotic (Penicillin, Streptomycin, and Gentamycin) can also be used as transport medium.

COMMERCIALIZATION OF FORMALINIZED LIVER-ORGAN-VACCINE AGAINST HYDROPERICARDIUM SYNDROME (HPS)

Poultry industry in Pakistan has been confronted with a number of problems. Infectious diseases are the real threat to the flourishing poultry industry in Pakistan. Among these Hydropericardium syndrome (HPS) is of utmost importance. It affects broiler chickens in particular with colossal economic loss. The syndrome that occurred as a unique malady in the history of the country was firstly observed in the broiler growing area of Angara Goth in Karachi in August 1987 and extended rapidly among broiler units in densely populated areas surrounding the major cities. The syndrome is typically seen in 3-5 weeks old broiler chickens with a mortality of 30 -60% and is characterized by accumulation of straw colored fluid in the pericardium, swollen discolored and friable liver and pale enlarged kidneys.

The principle method to control the disease is by vaccination. Killed vaccines are available commercially but lack standard operating procedures (SOP) and good manufacturing practices (GMP) resulting in low quality vaccine. Titer of the seed virus is usually low in the final product that leads to poor immune response. Presence of contaminant biological materials further exacerbates the disease problem. Therefore there is need to develop a vaccine that is sterility, safety, potency tested and free of extraneous agents.

The liver specimens from acute freshly dead HPS cases are collected and washed with distilled water. The organs are homogenized in a blender to make 20% suspension in 0.9% saline solution. The suspension is sonicated in batches at 50 watts using 119 mm probe for 3 minutes, then centrifuged at 5000 rpm for 10 minutes. The biological titer (LD50) of the supernatant is determined in 04 weeks old broiler chicks. Formaldehyde (37%) is added at 0.3% concentration. Penicillin and streptomycin are added at 10,000 IU and 10 mg per ml respectively. Preparation is left at 4C^° for 24 hours. Laboratory and field trials of the formalinized liver organ HPS vaccine has been conducted and produced encouraging results.

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.

DEVELOPMENT OF A CONJUGATE VACCINE AGAINST STAPHYLOCOCCUS AUREUS BOVINE MASTITIS USING CAPSULAR POLYSACCHARIDES.

Introduction:

Mastitis is the most significant cause of economic loss to the dairy industry, and bacteria are the most common cause of mastitis. Staphylococcus aureus is the most ubiquitous and results in the greatest economic loss (Foster, 1986). Staphylcoccus aureus mastitis occurs despite numerous host defense mechanisms. Of these defense mechanisms, neutrophil phagocytosis is the most effective. However, 10high concentration of milk neutrophils (9 ×⁵ neutrophils/ml) is required to prevent infection. Because this concentration far exceeds the number of neutrophils in the normal healthy gland, increasing the phagocytic efficiency of neutrophils via opsonins is the most effective way of enhancing mammary gland resistance to S. aureus infection. A major obstacle to producing a protective immune response to S. aureus is the development of capsular polysaccharides (CP). It has been estimated that 94% to 100% of S. aureus isolated from cows possess CP (Norcross and Opdebeeck. 1993). The CP block neutrophil recognition of antibodies bound to the highly antigenic cell wall components of S. aureus. Polysaccharides are low in immunogenicity and do not readily elicit an antibody response. In addition to being low in immunogenicity, polysaccharides are T-cell independent antigens and thus do not stimulate immunological memory. However, it has been shown that antibodies to CP enhance phagocytosis (Guidry et al. 1994). Progress has been made toward increasing the production of antibodies to CP and stimulation of immunological memory by conjugating CP to carrier proteins (Fattom et al, 1993).

Objective:

To prepare S. aureus mastitis vaccine using capsular polysaccharides conjugated to carrier proteins and evaluate its efficacy in increasing antibody production to capsular polysaccharides, and stimulation of immunological memory.

Methodology:

S. aureus serotypes 5, 8 and 336 will be isolated from clinical and subclinical cases of mastitis. S. aureus serotypes 5, 8 and 336 capsular polysaccharides (CP) will be prepared by treartment with lysostaphin and multiple alcohol purification followed by ion exchange chromatography. S. aureus serotypes 5,8 and 336 CP will be conjugated to purified carrier protein recombinant Pseudomonas aeruginosa exotoxin A as described by Fattom et al, 1993. In this study, cows will be immunized with a vaccine consisting of the conjugated S. aureus CP serotypes 5,8 and 336 emulsified in adjuvant and conjugated CP in microspheres emulsified in adjuvant. The antibody response will be tested for specificity, isotype, enhancement of neutrophil phagocytosis of three S. aureus serotypes, and prevention of S. aureus adherence to bovine mammary epithelial cells.

References:

• Fattom, A., R. Schneerson, D. C. Watson, W. W. Karakawa, D. Fitzgerald, I. Pastan, X. R. Li, J. Shiloach, D. A. Bryla, and J. B. Robbins. 1993. Laboratory and clinical evaluation of conjugate vaccines composed of Staphylococcus aureus type-5 and type-8 capsular polysaccharides conjugated to Pseudomonas aeruginosa recombinant exoprotein-A. Infect. Immun. 61:1023–1032.
• Foster, T. J. 1986. A new genetic approach to defining the virulence determinants of Staphylococcus aureus strains that cause bovine mastitis. Ir. Vet. J. 40:110–115.
• Norcross, N. L., and J. P. Opdebeeck. 1993. Encapsulation of Staphylococcus aureus isolated from bovine milk. Vet. Microbiol. 8:397–404.
• Guidry, A. J., C. N. O’Brien, S. P. Oliver, H. H. Dowlen, and L. W. Douglass. 1994. Effect of whole Staphylococcus aureus and mode of immunization on bovine opsonizing antibodies to capsule. J. Dairy Sci. 77:2965–2974

Production of recombinant fowl pox vaccine expressing Haemagglutinin of Avian Influenza H5N1

Introduction:

Avian influenza (AI) is an infectious disease caused by Avian Influenza virus (AIV), which is divides in to 16 Heamagglutinin (HA) subtypes and 9 Neuraminidases (NA) subtypes on the bases of antigenicity of HA and NA surface glycoprotein. H5 and H7 may cause High Pathogenic Avian Influenza (HPAI). Out breaks of severe disease caused by influenza virus have been reported frequently in the recent years, leading to substantial economic losses. H5N1 strain can de transmitted directly from birds to human (1-2) H7 and H9 AIV have been identified from humans and other mammals. These facts have made clear the zoonotic importance of the Avian Influenza virus. Presently H5 and H7 (HPAI) remain active in the south Asia and other part of the world (3-4). From Pakistan H9N2 (5), H7N3 have been isolated a no of time and in 2006 H5N1 was reported. Now in 2007 there is a severe outbreak of H5N1 in Islamabad and different districts of Punjab.

Objectives:

To produce avian influenza H5 vaccine, recombinant with Fowl Pox virus (FPV). Currently, inactivated whole vaccines are used which are creating problems in Epidemiological studies and it is difficult to differentiate between vaccinal response and outbreak from serological studies (6).

Methodology:

Avian Influenza virus will be isolated from field or RNA will be isolated from inactivated whole H5 subtype vaccine, cDNA encoding whole H5 genes of H5N1 subtype will be cloned and introduce into expression vector plasmid. The plasmid will be introduced in Fowl Pox virus attenuated strain; the recombinant Fowl pox virus will be propagated in Chicken Embryo Fibroblast (CEF) to produce vaccine. Expression of H5 gene in the virus will be detected by PCR using specific primers. Immunity produced in the experimental birds will be titrated by Haemagglutination Inhibition (HI) test and experimental inoculation of H5N1 virus to the birds.

References:

1. Subbarao K, Klimov A, Katz J, Regnery H, Lim W, Hall H, et al. Characterization of an avian influenza A (H5N1) virus isolated from a child with a fatal respiratory illness. Science 1998; 279:393–6.

2. Claas EC, de Jong JC, van Beek R, Rimmelzwaan GF, Osterhaus AD. Links Human influenza virus A/Hong Kong/156/97 (H5N1) infection. Vaccine 1998; 16:977–88.

3. Spackman E, Senne DA, Davison S, Suarez DL. Sequence analysis of recent H7 avian influenza viruses associated with three different outbreaks in commercial poultry in the United States. J Virol 2003; 77:13399–402.

4. Fouchier RAM, Schneeberger PM, Rozendaal FW, Broekman JM, Kemink SAG, Munster V, et al. Avian influenza A virus (H7N7) associated with human conjunctivitis and a fatal case of acute respiratory distress syndrome. PNAS 2004; 101:1356–61.

5. Naeem, K., A. Ullah, R. J. Manvell and D. J. Alexander, 1999. Avian influenza A subtype H9N2 in poultry in Pakistan. Vet. Rec., 145: 560.

6. Beard CW, Schnitzlein W, Mand Tripathy DN. Protection of chickens against highly pathogenic avian influenza virus (H5N2) by recombinant fowlpox viruses. Avian Diseases 1991; 35:356–9.

VIRULENCE CHARACTERIZATION AND GENOTYPIC ANALYSIS OF SALMONELLA TYPHIMURIUM ISOLATED FROM FOOD AND PROCESSING ENVIRONMENTS

Introduction:

Nontyphoid salmonellosis is a worldwide disease of humans and animals. Infections due to Salmonella spps. range from gastroenteritis to enteric fever. Salmonella Typhimurium may cause nontyphoid salmonellosis and is a very important food-borne pathogen (World Health Organization, 2005). The causative organisms can pass through the food chain from primary production to households or food-service establishments and institutions. Large number of Salmonella outbreaks from contaminated food such as sprouts, poultry, fish, meat, and eggs has been reported (National Advisory Committee on Microbiological Criteria for Foods, 1999; van Duynhoven et al., 2002; Dominguez et al., 2007). The recent-most reported outbreak was in 2006 and was connected with the consumption of tomatoes contaminated with S. Typhimurium (Center for Disease Control and Prevention, 2006). Diarrheal diseases are common in Pakistan due to the consumption of contaminated water and food. No systematic guideline exists for the surveillance of Salmonella infections in human and animal in Pakistan. Therefore, the magnitude of nontyphoidal Salmonella infection is not well documented.

Salmonellae cause disease by invading the intestinal epithelium after ingestion. Researchers have studied the internalization of salmonellae, such as S. typhi, S. typhimurium, and S. choleraesuis, in epithelial cells and found that they multiply within formed vacuoles (Gahring et al., 1990). In vitro systems using different cell lines have been used to study the interaction between salmonellae and eukaryotic cells. Galan and Curtiss (1989) first characterized the Salmonella invasion gene invA, the first gene in an operon which is thought to trigger the internalization of S. typhimurium in cultured epithelial cells. Mutations within this operon render salmonellae incapable of invading Madin Darby canine kidney (MDCK) cells in culture. Virulence plasmids are one of several Salmonella virulence determinants involved in survival and growth in host cells. Virulence plasmids are thought to not be involved in the initial interaction between salmonellae and the intestinal mucosa or required for invasion into deeper tissue. However, they enable the organism to persist in the reticuloendothelial cells of liver and spleen (Gulig, 1990).

Present study is designed to determine the relationship between the presence of both the invA and spvC genes and the degree of invasiveness among salmonella isolates recovered from diverse food sources. A comparison of the strains recovered of different origins will clarify the nature of human salmonellosis. The data generated by genotypic analysis, antibiotic profile, plasmid profile and presence of virulence genes in S. Typhimurium isolates from foods can be useful in epidemiological investigation of Salmonellosis.

Objectives:

1. To compare the isolates for the presence of virulence genes invA and spvC.

2. To compare Salmonella isolates obtained from various sources for invasiveness in epithelial tissues.
3. To characterize isolates in terms of antibiotic resistance and genetic relatedness

Methodology:

Bacterial strains:

Salmonella will be isolated from commercially processed spent hens, broilers, and eggs and the egg production environment. Poultry associated samples including ovaries and oviduct tissues, cecum tissue sections, whole egg contents and the samples from egg production environment will be taken. Other food products such as fish, meat, beef, fruits, vegetables, sprouts will also be sampled. Salmonella samples will be cultured and identified by API system. Stereotyping of recovered isolates will be performed.

InvA and spvC Primers and probes:

Primers for Salmonella spvC and invA will be designed using published sequences (GenBank accession number M64295 and M90846).Probes will be prepared using PCR and labeled PCR products will be used as probes in DNA-DNA hybridization with colonies or plasmid DNA.

Plasmid DNA isolation:

Plasmid DNA will be isolated and purified using Mini-Prep kit.Plasmid DNA will be characterized by gel electrophoresis on a 1% agarose gel according to standard methods (Sambrook et al., 1989).

Hybridization:

Colonies will be grown on Brain heart infusion agar and then patched onto nitrocellulose membrane for the colony blots. The colonies will be lysed and the DNA will be denatured and neutralized (Sambrook et al., 1989). The DNA-DNA hybridization will be performed by prehybridization for 1 h at 68C in 5XSSC. Hybridization will be conducted at 68C overnight in buffer containing boiled digoxigenin-labelled probe. Enzyme immunoassay for the detection of the presence of digoxigenin labeled probe will be performed.

Invasin assay:

Invasin assay of Finlay and Falkow will be used with modifications. Monolayers will be disrupted by pipetting and titres of internalized bacteria will be determined on MacConkey’s agar. Inoculum level will be determined and percent invasion will be calculated by the formula: (CFU recovered after cell lysis/CFU of innoculum) x 100. Each invasion assay will be run in triplicate along with positive and negative controls.

Genome typing by PFGE:

Intact genomic DNA isolation and PFGE analysis will be carried out using protocol described by Gautum (1997). Salmonella Typhimurium isolates will be incubated at 37C in 25ml Luria broth. 1ml of bacterial cells will be harvested and washed three times using TE buffer and cell density will be adjusted. 100ml of the cell suspension will be incubated with proteinase K and lysozyme at 37C for 15 minutes. Restriction digestion will be carried out with 50U of Xbal at 37C. PFGE will be performed in 1% agarose gel in Tris Borate EDTA buffer. Strains differentiating in one band will be considered as different pulsed field profiles (PFP’S).

Antimicrobial Susceptibility Test:

Antimicrobial susceptibility test, constituting commonly used antimicrobial agents, was performed on Muller-Hinton Agar by disc diffusion method as described by National Committee for Clinical Laboratory Standards (National Committee for Clinical Laboratory Standards, 2002).

References:

Centers for Disease Control and Prevention (2006). Salmonellosis. Available online at http://www.cdc.gov/ncidod/dbmd/diseaseinfo/ salmonellosis_2006/outbreak_notice.htm. Accessed 10 April2008.

Dominguez A., Torner N., Ruiz L., Martinez A., Bartolome R., Sulleiro E., Teixido A. and Plasencia A. (2007). Foodborne Salmonella-caused outbreaks in Catalonia (Spain), 1990 to 2003. Journal of Food Protection 70: 209–213.

Gautom R.K. (1997). Rapid pulsed-field gel electrophoresis protocol for typing of Escherichia coli O157:H7 and other gram negative organisms in 1 day. Journal of ClinicalMicrobiology 35: 2977–2980.

Gahring, L. C., F. Heffron, B. B. Finlay, and S. Falkow. 1990. Invasion and replication of Salmonella typhimurium in animal cells. Infect. Immun. 58: 443–448.

Galan, J. E., and R. Curtiss III. 1989. Cloning and molecular characterization of genes whose products allow S. typhimurium to penetrate tissue culture cells. Proc. Natl. Acad. Sci. USA 86:6383–6387.

Gulig, P. A. 1990. Virulence plasmids of S. typhimurium and other salmonellae. Microb. Pathog. 8:3–11.

National Advisory Committee on Microbiological Criteria for Foods. (1999). Microbiological safety evaluation and recommendation of sprouted seeds. International Journal of Food Microbiology 52: 123–153.

National Committee for Clinical Laboratory Standards (NCCLS) (2002). Performance standards for Antimicrobial Susceptibility Testing, NCCLS Document no. M100-S12. Villanova, PA: National Committee for Clinical Laboratory Standards.

Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecular cloning: a laboratory manual, 2nd ed., vol. 1. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.

van Duynhoven Y.T., Widdowson M.A., de Jager C.M., Fernandes T., Neppelenbroek S., van den B.W., Wannet W.J., van Kooij J.A., Rietveld H.J. and van Pelt W. (2002). Salmonella enterica serotype Enteritidis phage type 4b outbreak associated with bean sprouts. Emerging Infectious Disease8: 440–443.

World Health Organization. (2005). Drug resistant Salmonella. Available online at http://www.who.int/mediacentre/factsheets/ fs139/en. Accessed 10 April 2008.

Development and Optimization of Quantitative Real Time PCR for the Detection of Mycoplasma Synoviae Infections

Introduction: Mycoplasma synoviae (MS) is a major pathogen of chickens and turkeys causing upper respiratory infection and synovitis in chickens and turkeys. Additionally, MS infection in turkeys has been associated with increased condemnation at processing due to airsacculitis, as well as with breast blisters and green discoloration of liver, which results in slower processing and further economic loss. Because of the high value of individual breeder hens and because of their ability to infect progeny by vertical transmission, economic losses are potentially most severe when MS infections occur in breeder flocks. Therefore a reasonable strategy for control of MS is to establish MS-free breeder flocks. Diagnosis of MS infections is often carried out by serological procedures but non specific reactions or insensitivity of the serological detection of MS infection often limits the effectiveness of these procedures. Isolation of the organism is also used but may be unsuccessful due to bacterial contamination, mixed mycoplasma infections or other reasons. Successful isolation may require 1-3 weeks. Species specific PCR tests are used based on 16s rRNA and vlhA genes. The major advantages of Real-Time PCR over standard PCR assay are the one tube and one time handling with reading of the reaction during the assay and less time needed to perform the test.

Objective: To develop and optimize real time PCR assay for quantitative detection of Mycoplasma synoviae infections from clinical samples.

Methodology: M. synoviae strains/isolates from chicken and turkey will be procured for standardization of the real time PCR assay. Clinical samples (trachea, air sac, synovial fluid) from affected chicken/turkey will be collected. Nucleic acids will be extracted directly from different M. synoviae strains/isolates and clinical samples and will be subjected to real-time PCR. Primer and fluorescent tagged probe set used in this investigation will be developed and characterized. The amplification of DNA fragment from the suspect samples detected by monitoring the increase in fluorescence from the dye labeled MS-specific probe will be investigated