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WO1997029120A1 - Isolement de la sarcocystine - Google Patents

Isolement de la sarcocystine Download PDF

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Publication number
WO1997029120A1
WO1997029120A1 PCT/AU1996/000445 AU9600445W WO9729120A1 WO 1997029120 A1 WO1997029120 A1 WO 1997029120A1 AU 9600445 W AU9600445 W AU 9600445W WO 9729120 A1 WO9729120 A1 WO 9729120A1
Authority
WO
WIPO (PCT)
Prior art keywords
toxin
sarcocystine
mammal
contaminated
sarcocystis
Prior art date
Application number
PCT/AU1996/000445
Other languages
English (en)
Inventor
Jose Luis Azumendi
Original Assignee
Sarco Research Corporation Limited
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from AU43334/96A external-priority patent/AU4333496A/en
Priority claimed from GB9603304A external-priority patent/GB2310212A/en
Application filed by Sarco Research Corporation Limited filed Critical Sarco Research Corporation Limited
Priority to AU63492/96A priority Critical patent/AU6349296A/en
Publication of WO1997029120A1 publication Critical patent/WO1997029120A1/fr

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/44—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from protozoa
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to a method for the isolation of the toxin sarcocystine which is produced by a parasite of the Sarcocystis genus of protozoa.
  • the present invention relates to a step by step procedural technique for isolating the toxin produced by a parasite of the Sarcocystis genus.
  • material preferably heart muscle, from an Intermediate Host (IH) with Sarcocystis cysts is used to contaminate a Final Host (FH).
  • FH Final Host
  • a percentage of its blood is extracted and by means of a centrifugation process the serum is separated, then dialysed, preferably by passing through a sterile Physiological Saline Solution (PSS), and it is finally held for storage at a specific temperature.
  • PSS sterile Physiological Saline Solution
  • One of the current known methods for isolating the toxin includes developing the toxin in macrocysts in sheep contaminated with the parasite. According to this method, the macrocysts which are formed in the oesophagus of the sheep can reach a visible size and contain relatively high quantities of toxin. Such a method entails the inconvenience that the animal must be sacrificed to obtain a few millilitres of pure toxin. By contrast the animal is kept alive and in good health according to the method of the present invention, so becoming a good toxin donor from which a substantial volume thereof may be extracted regularly for a long time.
  • cell cultures may be utilised as supports for in vitro cultures of Sarcocystis, which allows for a totally-controlled environment to be maintained.
  • the inconvenience arises from the fact that the parasite only begins to produce the toxin 30 or more days after inoculation and then production ends shortly thereafter. Maintenance of these cultures for a long time becomes expensive and they yield a supernatant compound as complex as the animal's blood serum. This latter problem can be overcome using a purification procedure of the present invention.
  • Protozoa of the Sarcocystis genus were reported for the first time in 1843 when a researcher by the name of Miescher found tubules in the skeletal muscle of a house mouse. Doctors Rommel and Heidorn in 1972 found that the protozoa's life cycle was heterogeneous, with the asexual phase in the prey, the intermediate host, and the sexual phase in the pillager, the final host. At present, 122 species of the protozoa have been identified and in 56 of these two hosts are known. From the zoonosis point of view, those developing the asexual phase in humans are interesting, the final hosts of which are unknown to date, and the two which develop the sexual phase in humans which are known.
  • the present invention provides a method for isolating protozoa sarcocystine, i.e., the toxin of the Sarcocystis protozoa.
  • This toxin may then be used in the production of toxides or vaccines and also in the production of specific antibodies against the toxin, as described and claimed in our co-pending British Patent Application No. 9603303.0.
  • a method of isolating a sarcocystine toxin from a mammal including the steps of:
  • a mammal be used as a FH, e.g. a carnivore such as a canine animal.
  • a FH a carnivore
  • the mammal is infected with Sarcocystis parasite by muscle, and especially cardiac muscle, containing Sarcocystis cysts.
  • the dialysis solution consists of about 9 gm per litre of sodium chloride, although any salt solution of equivalent osmotic pressure can also be used.
  • a mammal is used as an IH, wherein the IH is a herbivore, and wherein the IH is infected with sporozoites from the stools of the FH.
  • the contaminated mammal has an absolute lymphocyte count at least 20% higher than its pre-contamination count.
  • the invention also relates to the determination of the optimum time for bleeding the donor as explained herein below. This involves the use of the toxicogenic characteristics of sarcocystine.
  • a 98.4% specificity which means that examination of 100 patients positive to the toxin shows that 98 of them are detected by the test
  • 99.00% sensitivity which means that, upon examination of 100 patients negative to the toxin, 99 of them are detected by the test
  • the toxin can be titrated in biological tests with a specificity of 99.80% (which means that upon examining 100 patients positive to the toxin, 100 of them are detected by the test), and a sensitivity of 99.99% (which means that upon examining 100 patients negative to the toxin, 100 of them are detected by the test).
  • the present invention also relates to the desiccation of the toxin isolated by dialysis in order to stabilise it for storage purposes.
  • a FH eg, a canine animal
  • IH eg. a herbivore such as a bovine animal
  • the heart muscle does not contain any other aetiological agent.
  • the absolute lymphocytes count of the FH is an indication of when the next step in the procedure can occur.
  • lymphocyte count reaches a minimum of 20% above the pre-contamination count
  • biological titration of the toxin as described herein below, can be applied to obtain a minimum titre of 3 units.
  • blood may be obtained from the FH. This is likely to occur between 50 and 80 days after contamination.
  • the absolute lymphocyte count of the FH is an indicator of the toxin concentration due to the mitogenic capacity of sarcocystine.
  • Sarcocystine is highly specific with respect to lymphocytes B, a feature not found in other substances from the blood of a mammal.
  • the FH is bled to obtain the required blood which is collected in a siliconed 500 millilitre sterile Erlenmeyer flask without anticoagulant.
  • the serum is then separated at 800g for 20 minutes in sterile centrifuge tubes in order to eliminate the corpuscular blood components. The presence of a small degree of haemolysis is acceptable.
  • the serum is then dialysed against a sterile PSS composed of about 9 grams of sodium chloride in 100 cubic centimetres of distilled water for 36 to 48 hours, keeping the temperature between 18 and 25°C and shaking continuously.
  • PSS sterile PSS
  • the toxin is isolated in the PSS with a minimum purity of 80%. Due to the size of the toxin molecule which is being isolated, temperature and dialysis time, it is possible to eliminate all those molecules which may cause alterations in the toxin usage.
  • the dialysed solution is then dried at a temperature of 37°C in order to remove the water and obtain the toxin concentrate.
  • the dried toxin is then stored in sterile amber flasks and they are kept at room temperature in a dry place.
  • the remaining contaminants located in the desiccated portion of the dialysis are glucose and some materials, none of which affect the toxicogenic, physical and/or biochemical characteristics of sarcocystine.
  • the IH from which the strain is to be obtained must come from an area where the chronic sarcosporidiosis is frequent and with very severe symptoms of the chronic disease demonstrated by between 10,000 and 30,000 bradozoids per gram of wide dorsal muscle. It must also be verified through serological tests that the animal does not suffer from any other disease.
  • the procedure should be applied to larger size mammals (eg, equine and bovine animals) with slight variations.
  • a stool sample of previously contaminated FH which has developed the whole picture described above, is taken and the sporozoites contained in the sample are purified. This is done by dissolving the stool sample in over-saturated glucose solution at the proportion of 1 volume of stool per 3 volumes of over-saturated solution.
  • the uppermost part of the preparation is collected, diluted in PSS and centrifugated at 300g for 10 minutes. The supernatant is discarded and the bottom residue is re-suspended in PSS to perform the toxin count.
  • the LH is contaminated with the parasite so prepared at a dosage of 700 sporozoites per kilogram. After 90 days, the IH is found in the chronic phase of the disease with high toxin contents in its blood, which makes it useful for extraction of the toxin.
  • the same techniques as described above may apply to the FH, ie, lymphocyte count and/or biological titration of the toxin.
  • the IH may show some symptoms of the severe disease, which may be treated symptomatically but no medicines for killing the Sarcocystis are to be used.
  • mice Three groups of 5 to 10 20-gram mice are selected.
  • the toxin is diluted with PSS at two known concentrations, between 0.5 and 1 mg per ml.
  • the first group of mice are orally administered with 0.2 ml of the toxin at dilution 1
  • the second group of mice are oral y administered with 0.2 ml of the toxin at dilution 2
  • the third group is used as a control and receives 0.2 ml of PSS without the toxin.
  • Eighteen hours later blood is obtained from all mice to make a total count of lymphocytes. They are then sacrificed to perform a necropsy.
  • the mice that received the toxin must present haemoperitoneum among other haemorrhages, while the control animals do not present any haemorrhage at all. Furthermore, the average of the total counts of lymphocytes in the inoculated animals will be higher than the average of the total counts of lymphocytes of the control animals.
  • a Sarcocystis type toxin unit is the amount of this toxin needed to increase by 10% the average of the total lymphocyte count of the contaminated mice as compared to the non-contaminated mice.

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  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Biochemistry (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Zoology (AREA)
  • Biophysics (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Toxicology (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)

Abstract

La présente invention concerne un procédé d'isolement d'une toxine, la sarcocystine, provenant d'un mammifère. Cette opération comprend: (a) la dialyse de sérum sanguin d'un mammifère contaminé par le parasite de la sarcocystine, au moyen d'une solution aqueuse de sel, et (b) le séchage de la solution dialysée pour isoler la toxine sarcocystine purifiée. L'invention concerne aussi la toxine qu'est la sarcocystine quand elle a été isolée par ce procédé.
PCT/AU1996/000445 1996-02-05 1996-07-16 Isolement de la sarcocystine WO1997029120A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU63492/96A AU6349296A (en) 1996-02-05 1996-07-16 Sarcocystine isolation

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
AU43334/96A AU4333496A (en) 1996-02-05 1996-02-05 Procedures for isolation and titration of sarcocystine or parasite toxin of sarcocystis genus
AU43334/96 1996-02-05
GB9603304A GB2310212A (en) 1996-02-16 1996-02-16 Isolation of the sarcocystine toxin from the genus Sarcocystis
GB9603304.8 1996-02-16

Publications (1)

Publication Number Publication Date
WO1997029120A1 true WO1997029120A1 (fr) 1997-08-14

Family

ID=25626324

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/AU1996/000445 WO1997029120A1 (fr) 1996-02-05 1996-07-16 Isolement de la sarcocystine

Country Status (1)

Country Link
WO (1) WO1997029120A1 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7169398B2 (en) * 2000-04-25 2007-01-30 Wyeth Equine protozoal myeloencephalitis vaccine

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0600396A2 (fr) * 1992-12-04 1994-06-08 University Technologies International Inc. Vaccins à partir de protozoaires intestinaux

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0600396A2 (fr) * 1992-12-04 1994-06-08 University Technologies International Inc. Vaccins à partir de protozoaires intestinaux

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
JOURNAL OF THE EGYPTIAN MEDICAL ASSOCIATION, Volume 52, No. 11, 1989, EI AKKAD et al., "On Some Pharmacological and Toxicological Effects of a Protozoan Toxin 'Sarcocystin'". *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7169398B2 (en) * 2000-04-25 2007-01-30 Wyeth Equine protozoal myeloencephalitis vaccine

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