CA1113394A - Anti-hemocytic serum and a method for the preparation thereof - Google Patents

Abstract

ABSTRACT OF THE DISCLOSURE
An anti-hemocytic serum for suppressing the immunolo-gic mechanism of insects which is inactivated blood plasma of immunized animals produced by the immunization of animals with a hemocyte suspension separated from cells of the bodies of insects: the titre of hemocytoagglutinins of the anti-hemocytic serum being 1:256 to 1:2048. A method for the preparation of the anti-hemocytic serum comprises in removing the alimentary tract and the hemolymph from insects, preserving the remaining cells of the bodies of insects and homogenizing them to form a hemocyte suspension subjecting the suspension to centrifugation, washing off the preservative from the suspension before immunization and immunizing animals 4 times at intervals of 5 days by injecting a 50% hemocyte suspension in an amount of 0.07 ml per 100 g of the weight of the animal, isolating the blood plasma of animals after immunization and inactivating the plasma.

Description

.3~ :

The present invention relates to a new anti-hemocytic ~rum for suppressing the imrnunologic mechanism of insects and a method for the preparation thereof. The new serum finds ~ ~;
application in pest control of such insects as the cabbage white butterfly, the bee moth, and the Colorado potato bettle. ~
Various biological preparations are ~no~,in for ' ~, .
protecting plants from pests, for example preparationssupplied under tlle trade ~ari~ Agritlol, Bact~n an~ ~isopore 28~. However, pests soon acqulre i~nunity to these ~reparations, and with pro--~ 10 longed application they do not produce a positive effect.
According to the present invention there is provided an anit-hemocytic serum for suppressing the immunologic mechanism of insects which is inactivated plasma of the blood of immunized animals obtained by the immunization of the animals with a , . . .
suspension of hemocytes isolated from the cells of the bodies of ;~
the insects; the titre of hemocytoaggulutinis of the anti-hemocytic serum being 1:256 to 1:2048.
Unlike conventional biological means of pest control the serum suppresses the-i~nunological reactivity of insect pests by retard~ing the-synthesis of antibodies. The advantages of the serum include the destruotion of insects by suppressing their immunity, the weakening of resistance of insects to bacteria, and the harmlessness of the serum of the environment.
The serum can be used for controlling any species of insect pests, and for every species of insects a serum is used which has been prepared on the basis of cells of the body of ~;
an insect of the respective species.
According to the invention, the method for the prepara-tion of the anti-hemoctyic serum for suppressing the immunolo-gic mechanism of insects comprises removing the alimentary tractfrom the insects and the hemolymph, preserving the remaining cells of the bodies of insects with a preservative and homo-., ,. . .,.,, , .~

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genizing them to form a suspension of hemoctyes, washing off thepreservative from the suspension. before lmmunization, and immunizing animals four-times at intervals of five day~ by .~
injecting a 50~ suspension of hemoctyes in an amount of 0.07 ~`
:~. ml per 100 g of the weight of an animal, removing the whole blood ~ :
of animals after immunization, isolating the plasma of the blood ~:. and inactivating the blood.
The suspension of hemocytes is preferably preserved by adding a mixture of the following components, in percent . 10 by weight: -sodium citrate . 3.5 glucose 2.0 chloramphenicol 0.015 distilled water up to 100, and then a solution of 0.24 M of saccharose, 0.004 M of ethylene diamine tetra-acetic acid, 0.01 M of sodium phosphate per 1000 . ml of distilled water having a pH of 7.0, and glycerin at a ~:
weight ratio of 1:2:1 respectively, is added to the resultant mixture which is kept at a temperature of~ -20C to -2C.
~, - The:method for the preparation.of the anti-hemoctylc ;

serum is effected in the following manner. -Pest insects may be of different species such as Galleria mollonella L, and Pieris brassicae L. The alimentary :
tract of insects is removed to prevent the action of enzymes ~ ;
and the hemolymph is also removed. The remaining cells of the bodies of insects are preserved in a..suitable preservative and homogenized to form a suspension of hemoctyes. The following ;~
composition is used as a preservative ensuring the prevention of :
agglutination, melinization and oxidation, in percent by weight:

30 sodium citrate 3-5 ~ ~
glucose 2.0 . ~;
chloramphenicol 0.015 :
distilled water up to 10

- 2 - :
: ~ ., . ~.

To keep the hemoctye suspension at a temperature of ,~--~0C to -2C and to preserve the integrity of cells a solution of 0.24 M of saccharose, 0.004 M of ethylene diamine tetra-acetic acid, 0.01 M of sodium phosphate per 1000 ml of distilled water being a pH of 7.0, and glycerin in a ratio of 1:2:1 respectively, is added to the resultant solution. Immunization ' ~ of animals is carried out with the produced hemocyte suspension.
- For this purpose the hemocyte suspension is washed beforehand ~ -` with a physiological solution to remove the preservative.
~` 10 Immunization of animals is then carried out four times at inter~
vals of 5 days by injecting a 50% hemocyte suspension in an amount of 0.07 ml per 100 g of the weight of the animal. After `-immuni~ation the whole blood of animals is removed, the plasma `
of the blood is separated and inactivated by keeping it at 56C
for 30 minutes. The resultant anti-hemocytic serum has a titre of hemocytoagglutinins of 1:256 to 1:2048.
The anti-hemocytic serum wàs tested on different `
species of insercts. It was applied by spraying. The test results showed a high activity of the serum. The death rate of insects of the first generation was from 35 to 75% of the second genera~
tion from 85 to 100%, depending on the dose of the serum applied to insects. The serum may also be used in com~ination with entomopathogenic bacteria. ; `~
The present invention will be further illustrated by way of the following Examples in which methods for the preparation of the anti-hemocytic serum and methods for testing its activity are described.
Example 1 2000 Caterpillars of Galleria mellonella L in their dèvelopment period (nistars V and VI) are disinfected with !
ethyl alcohol and left for 5 to 6 hours for cleaning the alimentary tract. The live caterpillars are placed in a preservative

- 3 -consisting of the following components, in grams; sodium citrate 3.5; glucose 2.0; cloramphenicol O.OlS; disti]led water up to 100.
The preservative is used in an amount of 1 ml per 10 caterpillars.
Then one segment is removed from the front portion and one segment from the rear por~ion of the bodies of caterpillars. The alimentary tract is removed to prevent enzymatic action. The residual bodies of the caterpillars are ground and placed to-gether with said perservative in a magnetic mixer for 7 to 10 minutes for the complete separation of hemolymph. The resultant homogenate is filtered and~centrifuged 6 to 7 times for 15 minutes at 1000 r.p.m. The centrifugate is left for 30 minutes at room temperature and the upper fat-like layer is removed with a cottonwool wad. The plasma of the hemolymph is poured o~f and a solution consisting of 0.24 M of saccharose, 0.004 M of `~
ethylene diamine tetra-acetic acid, 0.01 M of sodium phosphate per 1000 ml of distilled water having a pH of 7.0, and glycerin ~ ~-in a weight ratio of 1 2:1 respectively, is poured onto the cells.
The resultant hemocyte suspension is kept until immunization of animals at a temperature of -20C ~ 20C. Prior to immunization the hemocyte suspension is washed with a physiological solution to remove the preservative. Immunization of 12 300 g white rats is carried out 4 times at intervals of 5 days. Each time 0.2 ml of a 50% hemocyte solution is injected. The first immunizatlon is done subcutaneously, injecting 0.2 ml of a 50%
hemocyte solution into each rat. The subsequent immuniæations are done intramuscularly and each time 0.2 ml of a S0~ hemocyte solution is injected in mixture wïth 16,666 units of penicillin to prevent possible infection.
Blood samples are taken from rats prior to the first immunization to determine the presence of natural antibodies in them against hemocytes of caterpillars. Seven days after the last immunization the rats are dehematized. The separated blood

- 4 -is placed in glass cy]inders and left at room temperature for 24 hours. The settled plasma of the blood ls poured off and inactivated at 56C for 30 minutes. The anti-hemocytic serum is produced in an amount of 15 ml. The titre of hemocytoagglu~
tinins is 1:2048.
The antl-hemocytic serum was tested on caterpillars of Galleria mellonella L in their development period (II-IV and .
V-VI nistars). Caterpillars were sprayed with the anti-hemocytic serum in doses of 0.25 to 2.5 microliters for each caterpillar. `~
The effect of the anti-hemocytic serum on the immunologic ;~
reactivity of the insects organisms was checked every 6 hours~
At the same time the anti-hemocytic serum was tested in com-bination with entomopathogenic bacteria Bac. Thuringiensis cereus var. galleriae. The anti-hemocytic serum was used in doses of from 0.25 to 2.5 microliters for each caterpillar. Bac. ~ ~
thuringiensis cereus var. galleriae was used as a 0.1% water sus- ~ `' pension in treating caterpillars of the first generation and as a 0.01% water suspension in treating caterpillars of the second generation.
, ~
A control experiment (without treating caterpillars) and treatment of caterpillars with heterogenous proteins and entomopathogenic bacteria Bac. thuringiensis cereus var. galleriae were carried out to compare the activity.
The effect on the viability of caterpillars was checked ~;
every 6 hours from the beginning of the experiments.
The maximum intensity of destruction of caterpillars ~, Galleria mellonella L was achieved in the period from 12 to 18 hours from the beginning of the experiment.
The test results are given in Table~ 1 and 2.

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Suppresslon of Immunologic Mechanism of the Effect of Bac. thuringiensis cereus ~;
Galleria mellonella Types of experiments _ Effectiveness of Destruction of Destruction of caterpillars in caterpillars less than 24 hours after 24 Nistar II _Nistar IV Nistar II Nistar IV
1. Check experiment -(without treating caterpillars) 0 0 0 2. Treatment with he-terogeneous pro- ~
teins 0 0 1 1 ~;
3. Treatment with en-tomopathogenic bac-teria Bac. thuringi-ensls cereus var.
g leriae 0 0 16 16 4. Treatment wi~h anti-hemocytic serum32 32 32 32

5. Treatment with anti- ;~
hemocytic serum in combination with Bac. !:
thuringiensis cereus~
var. galleriae 75 75 75 75 :: , .

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Comparing the data given in the Tables it can be seen that in treating caterpillars G mellonella L with the anti -hemocytic serum their death rate in the second generation reaches 100% whereas due to -the use of entomopathogenic bacteria in the second generation the efficiency of destruction of caterpillars diminishessince they acquire immunity against ~hese bacteria.
Example 2 Caterpillars of the cabbage white butterfly Pieris brassicae L are used. The process of preparation of the anti-hemocytic serum is identical with that of Example 1.
The tests of the anti-hemocytic serum produced are identical with those of Example 1.
Caterpillars of Pieris brassicae L were treated with the anti-hemocytic serum in doses of 0.25 microliters to 4.5 microliters as well as with the anti-hemocytic serum in com-bination with entomopathogenic bacteria Bac. thuringiensis cereus var. galleriae which were used as a 0.1~ water suspension in doses of 0.016 ml of each caterpillar~ The test results are given in Tables 3 and 4.

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Table 3 Type of preparation Dose of treatment for Effectiveness of ;~
each caterpillar, in destruction of microlitres caterpillars, . in %
1. Anti-hem~cy~ic serum 0.25 30 ditto 0.5 32 ~.
ditto 0.75 40 ditto 1.0 40 :
ditto 1.25 50 ;~
- ditto 1.50 58 ditto 1.75 70 ditto 2.5 98 : ditto 4.5 100 :
2. Control experi- ~:
ment (distilled water 0.25 0 :
3. Ditto 4.5 0 :.

, 33~
Table 4 No. Type of preparation Dose of txeatment Effectiveness for each of destruction -~
caterpillar of cater pillars in %
l. Anti-hemocytic 0.25~1~0.016 ml of a -. .
serum with 0.1% water suspension of ~ ~:
entomopathogenic bacteria 50 bacteria Bac. 0.5~1~0.016 ml of a :~:
: thuringiensis 0.1% water suspension ` ~:
cerëus~~var. of bacteria 67 qalleriae . ~.
1.0~1+0.016 ml of a ` :
- 0.1% water suspension . of bacteria 83 2.5~1+0.016 ml of a 0.1% water suspension ::
of bacteria lO0 4.5~1+0.016 ml of a 0.1% water suspension of bacteria ` lO0 ~ :
: 2. Cdntroll.
experiment (without :~
treatment) - ~`
3. Control ~
experiment ` `
` (distilled ~ .
: water) 2.5 ~l 0 :
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~ :20 ' , ' .

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-`~ 'I 12 -. ,, ,~

Claims (3)

THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:

1. An anti-hemocytic serum for suppressing the immuno-logic mechanism of insects which is inactivated plasma of the blood of immunized animals produced by immunization of animals with a hemocyte suspension separated from cells of the bodies of insects; the titre of hemocytoagglutinins of the anti-hemocytic serum is 1:256 to 1:2048.

2. A method for the preparation of anti-hemocytic serum for suppressing the immunoligic mechanism of insects which comprises re-moving the alimentary tract and the hemolymph from the insects preserving the residual cells of the bodies of insects with a preservative and homogenizing then to form a hemocyte suspension, subjecting said suspension to centrifugation, washing off the preservative from the suspension prior to immunization, and immunizing animals 4 times at invervals of 5 days, by injecting a 50% hemocyte suspension in an amount of 0.07 ml per 100 g of the weight of the animal, isolating the blood plasma of animals after immuniza-tion and inactivating said plasma.

3. A method as claimed in Claim 2, whereby the hemocyte suspension is preserved by adding a mixture consisting of the following components, in percent by weight:
sodium citrate 3.5 glucose 2.0 chloramphenicol 0.015 distilled water up to 100, then a solution consisting of 0.24 M of saccharose, 0.004 M of ethylene diamine tetra-acetic acid, 0.01 M of sodium phosphate for 1000 ml of distilled water, pH of 7.0, and glycerin in a ratio of 1:2:1 respectively, is added to the resultant mixture, and the resultant mixture is kept at -20°C ? 2°C.