Title of Invention

"A PROCESS FOR PREPARATION OF A REAGENT FOR DETECTING HAEMORRHAGIC SEPTICAEMIA IN CATTLE AND BUFFALOES"

Abstract

This invention relates to a two minutes process for preparation of a reagent for detecting Haemorrhagic septicaemia in cattle and buffaloes, wherein causative organism is are detected comprising, inoculating staphylococcus aureus Cowna-1 strain on blood agar plates followed by incubating in Brain Heart infusion (BHI) broth for 16-20 hours at 37°C, layering broth on BHI agar plates and incubating for 48 hours at 37°C, scrapping the growth from plates followed by centrifuging the cells at 5000 rpm for 12-15 minutes at room temperature, washing the cells using Phosphate Buffer Saline (PBS) at 5000 rpm for 12-15 minutes, preparing 10% suspension in PBS and adding 2% formalin and keeping it overnight followed by keeping in hot water bath at 80°C for 5 minutes and then immediately immersing in ice, washing the cell with PBS at 5000 rpm for 12-15 minutes and preparing 10% suspension of cells in PBS, adding crystal violet dye in quantity 1% v/v and keeping if for 5 minutes followed by at least three washings with PBS to remove excess dye and then preparing 10% suspension in PBS.

Full Text

FIELD OF INVENTION This invention relates to a process for preparation of a reagent for detecting Haemorrhagic septicaemia in cattle and buffaloes. The reagent enables rapid testing of the disease under the field conditions. PRIOR ART Haemorrhagic septicaemia is one of the most serious bacterial diseases of cattle and buffaloes, particularly in India, where it is mainly responsible for the highest number of reported deaths of the cattle and buffaloes. The disease is caused by Pasteurella multocida. The disease occurs in acute, sub-acute and chronic forms. The sub-acute form of the disease is characterised by pyrexia (l05-l07F), edematous swelling in the sub-mandibular region, which may extend to the neck and dewlap, dyspnoea and death. Septicaemic pasteurellosis flares up as an outbreak during the environmental stress (high temperature and humidity or very low temperature). On stress, these organisms, which normally reside in tonsillar arid nasopharyngeal mucosae of animals, asume pathogenic role and cause clinical disease. The incubation period is 2-5 days, The clinical symptoms in acute form, which is more common these days, include high temperature, profuse salivation, lacrimation and nasal discharge, conjunctivitis, sharp drop in milk yield, dyspnoea and death. The drop in milk yield, causes economic loss to the cattle/buffaloe owners. The disease is transmitted to other healthy animals through contact with infected animals, its fomites or through droplets. Saliva also contains large member of organisms, which can contaminate fodder/water etc Compared to cattle, buffaloes have been found 3 times more susceptible to the disease. Young animals, particularly upto 2 years of age, have been found to be highly susceptible, since these may not have been vaccinated and most of the time, the outbreaks are initiated from such animals. In cases where the animals are treated with suitable antibiotic (after determining antibiotic sensitivity) at early stage, the animals can be saved. Otherwise, case fatality rate is very high, (59%) especially in buffaloes. If the animal is lactating, there is complete cessation of milk yield for remainder of lactation period, even if the animal survives. So, the disease causes huge economic losses in the form of mortality, loss of milk production, cost of treatment (which is around Rs 1000/- per animal) and such economic losses are estimated to be around Rs58million in one state of India, namely Haryana state, which is the home of world famous Murrah buffalo. Therefore quick diagnosis and immediate treatment of affected animals with half-yearly regular vaccination is necessary to avoid economic losses to the farmers. Conventional reagents and methods known in the art for diagnosing of the disease have several drawbacks. A drawback of these methods is that these methods take 2-3 days for confirmatory diagnosis, which is too long a period in epidemic control. Another drawback of the above methods is that these methods require laboratory facilities and expertise necessitating skilled personnel for carrying our diagnostic test. According to U.S patent no.6,410,021 a reagent known in the art for serotyping of Pasteurella of pig origin, which is prepared by the process involving the use of hyperimmune rabbit anti-sera complexed to Staphylococcus aureus whole cells. A drawback of the above reagent is that it cannot be used for the diagnosis of Haemorrhagic septicaemia. Another drawback of the above reagent is that it cannot detect Pasteurella multocida. Still another drawback of the above reagent is that it cannot diagnose pasteurellosis of sheep and goats. OBJECTS OF PRESENT INVENTION An object of the present invention is to provide a process for preparation of a reagent for diagnosis of Haemorrhagic septicaemia disease in cattle and buffaloes. Another object of the present invention is to provide a process for preparation of a reagent, which enables confirmatory rapid field testing of the disease in just 2 minutes (as compared to 2-3 days taken by conventional methods). Such rapid diagnosis enables quick treatment and control of outbreak of the disease. Still another object of the present invention is to provide a process for preparation of a reagent, which enables on-the-spot diagnosis in the field at the door steps of farmers/cattle or buffaloes owners and does not require any laboratory facilities or any instrumentation or any laboratory equipment. Further object of the present invention is to provide a process for preparation of a reagent, which provides an easy method of diagnosis of disease, which can be carried out even by unskilled personnel. Yet further object of the present invention is to provide a process for preparation of a reagent, which provides a cost-effective method of diagnosis of the disease. Still further object of the present invention is to provide a process for preparation of a reagent, which can detect not only causative organism but also its antigens thereby making it superior to culture isolation. Even further object of the present invention is to provide a process for preparation of a reagent, which can be carried out even during postmortem, in dead deteriorated samples, which enable implementation of suitable measures to prevent spread of the disease to the cattle/ buffaloes. Yet further object of the present invention is to propose a process for preparation of a reagent, which is stable with a shelf life of more than 1 year. Still further object of the present invention is to propose a process for preparation of a reagent, which requires only one drop of the blood sample form the animal suspected to be infected with the disease. Even further object of the present invention is to provide a process for preparation of a reagent, which can be effectively used for testing Pasteurella multocida in sheep and goats. STATEMENT OF INVENTION According to this invention there is provided a process for preparation of a reagent for detecting Haemorrhagic septicaemia in cattle and buffaloes consisting the steps of:- (a) inoculating staphylococcus aureus Cowan-I strain on blood agar plates followed by incubating in Brain Heart Infusion (BHI) broth for 16-20 hours at 37°C; (b) layering the broth on BHI agar plates and incubating for 48 hours at 37°C, scrapping the growth from plates followed by centrifuging the cells at 5000 rpm for 12-15 minutes at room temperature; (c) washing the cells at least three times, using Phosphate Buffer Saline (PBS) at 5000 rpm for 12-15 minutes; (d) preparing 10% suspension in PBS and adding 2% formalin and keeping it overnight followed by keeping in hot water bath at 80°C for 5 minutes and then immediately immersing in ice; (e) washing the cells at least five times with PBS at 5000 rpm for 12-15 minutes and preparing 10% suspension of cells in PBS; (f) adding crystal voilet dye in quantity 1% v/v and keeping if for 5 minutes followed by at least three washings with PBS to remove excess dye and then preparing 10% suspension in PBS; (g) raising hyper immune serum in calves as per standard protocol, obtaining Pasteurella multocida bovine; (h) adding Pasteurella multocida bovine serum thus obtained by step(g) to the suspension obtained by step(f), taking said bovine serum and the said suspension in the volume ratio of 1:9, incubating for 2-3 hours at 37°C with intermittent shaking, followed by centrifugation at 2000 rpm for 12-15 minutes, discarding the supernatant and dissolving the pellet in PBS to prepare 10% suspension thereby obtaining the desired co-agglutination reagent of the present invention. DE8CRIPTION OF FIGURES Fig 1:- Flow chart shows the process for preparation of a diagnostic reagent for testing of Haemorrhagic septicaemia in cattle and buffaloes. DESCRIPTION OF INVENTION According to this invention, the reagent of the present invention is prepared by the process as under:- a) Preparation of Staphylococcus aureus suspension in PBS Staphylococcus aureus Cowan-I strain is inoculated on blood agar plates and from there transferred to Brain Heart Infusion (BHI) broth. BHI broth is incubated for around 16-20 hours at 37°C. Layering of broth is done on BHI agar plates which are incubated for 48 hours at 37°C. The growth is scrapped from plates using a bent glass rod. The cells are centrifuged at 5000 rpm for 12-15 minutes at room temperature. The harvested cells are washed at least three times using "Phosphate Buffer Saline (PBS) at 5000 rpm for 12-15 minutes. Pellet is weighed and a 10% suspension is made in PBS and then 2% formalin is added and kept overnight to inactivate the cells. The cells are then kept in hot water at 80°C for 5 minutes in water bath and cooled immediately by immersing in ice. The cells are then washed at least five times with PBS at 5000 rpm for 12-15 minutes. A 10% suspension of cells is made in PBS and 1% crystal voilet is added. It is kept for 5 minutes. Three washings are given with PBS to remove excess dye. Finally 10% suspension is made in PBS (b) Hyper Immunisation Hyper immune serum is raised in calves as per standard protocol. (c) Preparation of Co-Agglutination Reagent Co-Agglutination Reagent is prepared by conjugation of protein A on Staphylococcus aureus with hyper immune serum, obtained by step(b) . This is followed by mixing of anti P. multocida bovine serumthus prepared with suspension obtained by step (a) in the ratio of 1:9 and then incubating for 2-3 hours at 37°C with intermittent shaking. Centrifugation is done at 2000 rpm for 15 minutes and supematent is discarded. Pellet is dissolved in PBS to prepare 10% desired suspension which is kept at 4°C till used. WORKING EXAMPLE The invention will now be illustrated with a working example, which is intended to illustrate the working of the invention and is not intended to be taken restrictively to imply any limitation on the scope of the present invention. Staphiilococcus aureus Cowan 1 strain was streaked on blood agar. Then it was incubated for 37°C When pure colony was obtained. The colony was streaked on Brain heart infusion broth and again incubated for 18 hours at 37°.Thus a liquid culture of bacteria was obtained. This broth was layered on BHl agar plates and incubated for 48 hours at 37°C.Now growth was harvested from these BHI plates using Phosphate buffer saline(PBS).Centrifugation of PBS was done on 5000 rpm for 15 minutes. A 10% suspension in PBS was prepared( 1 gram bacterial pellet was dissolved in 9 ml PBS). To this 0.2 ml formalin was added to make a 2% suspension. Then kept overnight to inactivate the bacteria. From here the suspension was transferred to 80°C warm water bath for 5 minutes. Then it was immediately immersed in crushed ice and kept for about 30 seconds. The cells were washed 5 times with PBS at 5000 rpm for 15 minutes and a 10% suspension was made in PBS.To it crystal violet dye was added to make 1% concentration by volume.Excess crystal violet was removed by washing with PBS and a 10% suspension was made. Then 9 ml of the above 10% suspension was taken and to it 1 ml of Anti Pasteurella multocida hyperimmune serum was added.This was incubated for 37°C for 2-3 hours..Centrifugation at 2000 rpm was done and pellet was again made to 10% suspension in PBS. The reagent was stored at 4°C till used. Haemorrhagic Septicaemia is commonly occurring disease in Haryana. Many outbreaks are reported during monsoons and winters. Field trials of the reagent were conducted in field epidemics of Haemorrhagic Septicaemia in Haryana. As many as 18 outbreaks of this disease could be diagnosed instantly in different villages and immediate treatment/ control measures were initiated -This was very helpful in timely control of the epidemic. This finding was further confirmed and corroborated by carrying out other time consuming conventional laboratory tests.The results from these tests matched with the results from the rapid diagnostic reagent in all the cases. This rapid Coagglutination test could also be performed on dead animals during their postmortem examination to find the cause of the disease. We could confirm 2 to 3 deaths from this disease in buffaloes. We also performed the test on tracheal fluid,lung swab culture etc. and could detect the bacteria in animals suffering fromHaemorrhagic Septicaemia. METHOD OF USE When the suspected animal is at the height of temperature, one drop of co-agglutination reagent is added to one drop of blood from the suspected animals on a glass slide. It is mixed with the toothpick and a visible co-agglutination reaction is recorded after 2 minutes which confirms the presence of the disease in the affected animal. It is to be understood that the process of the present invention is susceptible to modification, changes adaptations by those skilled in the art. Such variant embodiments are intended to be within the scope of present invention, which is further set forth under the following claims:- We Claim 1. A process for preparation of a reagent for detecting Haemorrhagic septicaemia in cattle and buffaloes, comprising:- a) inoculating staphylococcus aureus Cowna-1 strain on blood agar plates followed by incubating in Brain Heart infusion (BHI) broth for 16-20 hours at 37°C; b) layering broth on BHI agar plates and incubating for 48 hours at 37°C, scrapping the growth from plates followed by centrifuging the cells at 5000 rpm for 12-15 minutes at room temperature; c) washing the cells using Phosphate Buffer Saline (PBS) at 5000 rpm for 12-15 minutes; d) preparing 10% suspension in PBS and adding 2% formalin and keeping it overnight followed by keeping in hot water bath at 80°C for 5 minutes and then immediately immersing in ice; e) washing the cell with PBS at 5000 rpm for 12-15 minutes and preparing 10% suspension of cells in PBS; f) adding crystal violet dye in quantity 1% v/v and keeping if for 5 minutes followed by at least three washings with PBS to remove excess dye and then preparing 10% suspension in PBS; 2. A two minutes process for preparation of a reagent for detecting Haemorrhagic septicaemia as claimed in claim 1, wherein washing the cells in 1 using Phosphate butter Saline is done at least three times. 3. A two minutes process for preparation of a reagent for detecting Haemorrhagic septicaemia claimed in claim1, wherein washing the cells in 1e sing PBS is done at least 5 times. 4. A process for preparation of a reagent for detecting Haemorrhagic septicaemia in cattle and buffaloes as substantially herein described and illustrated with accompanying figure.

Documents:

607-DEL-2004-Abstract-(09-03-2009).pdf

607-DEL-2004-Abstract-01-04-2008.pdf

607-del-2004-abstract.pdf

607-DEL-2004-Claims-(09-03-2009).pdf

607-DEL-2004-Claims-(13-03-2009).pdf

607-DEL-2004-Claims-(14-10-2008).pdf

607-del-2004-claims.pdf

607-DEL-2004-Clams-01-04-2008.pdf

607-del-2004-complete specification (granted).pdf

607-DEL-2004-Correspondence-Others-(09-03-2009).pdf

607-DEL-2004-Correspondence-Others-(14-10-2008).pdf

607-DEL-2004-Correspondence-Others-(20-02-2009).pdf

607-DEL-2004-Correspondence-Others-01-04-2008.pdf

607-del-2004-correspondence.pdf

607-DEL-2004-Description (Complete)-(09-03-2009).pdf

607-DEL-2004-Description (Complete)01-04-2008.pdf

607-del-2004-description.pdf

607-DEL-2004-Drawings-(09-03-2009).pdf

607-DEL-2004-Drawings-01-04-2008.pdf

607-del-2004-drawings.pdf

607-DEL-2004-Form-1-(09-03-2009).pdf

607-DEL-2004-Form-1-01-04-2008.pdf

607-del-2004-form-18.pdf

607-DEL-2004-Form-2-(09-03-2009).pdf

607-DEL-2004-Form-2-01-04-2008.pdf

607-DEL-2004-Form-3-(09-03-2009).pdf

607-del-2004-form1.pdf

607-del-2004-form2.pdf

607-del-2004-form26.pdf