CA3057970C

CA3057970C - Diagnostic agent for cancer containing nematode mutant, and method for assessing tactic behavior using said mutant

Info

Publication number: CA3057970C
Application number: CA3057970A
Authority: CA
Inventors: Takaaki HIROTSU
Original assignee: Hirotsu Bio Science Inc
Current assignee: Hirotsu Bio Science Inc
Priority date: 2017-03-31
Filing date: 2018-03-30
Publication date: 2022-05-03
Anticipated expiration: 2038-03-30
Also published as: JPWO2018181869A1; AU2018246554B2; WO2018181869A1; CA3057970A1; AU2018246554A1; JP6778449B2

Abstract

Provided are a diagnostic agent for cancer containing the nematode mutant AB1 strain, and a method for assessing migratory behavior using said mutant.

Description

Description Title of Invention: DIAGNOSTIC AGENT FOR CANCER CONTAINING
NEMATODE MUTANT, AND METHOD FOR ASSESSING TACTIC BEHAVIOR USING
SAID MUTANT
Technical Field [0001]
The present invention provides a diagnostic agent for cancer that contains a nematode mutant and a method for assessing tactic behavior using the nematode mutant.
Background Art

[0002]
A nematode exhibits attractive behavior toward cancer patient's urine, and exhibits avoidance behavior toward healthy subject's urine. Accordingly, it is possible to determine whether a subject from whom urine is derived is a cancer patient or a healthy subject based on tactic behavior of the nematode (Patent Literature 1). More specifically, according to the description in Patent Literature 1, a medium-scale study involving 242 specimens was able to detect cancer patients with a sensitivity of 100% and a specificity of 95%.
Citation List Patent Literature

[0003]
Patent Literature 1: WO 2015/088039 Summary of Invention

[0004]
The present inventors have been wOrking on the development of a technique for identifying a healthy subject who gets a false-positive result in the above described assessment based on the tactic behavior. The present inventors found that a nematode registered as an AB1 strain at Caenorhabditis Genetics Center (CGC) at College of Biological Sciences at the University of Minnesota in the United States was a nematode mutant and usually exhibited avoidance behavior in a cancer patient and a healthy subject but exhibited attractive behavior in some healthy subjects. Furthermore, the AB1 strain exhibited attractive behavior toward the urine of a healthy subject who gets a false-positive result on a test using a wild strain. The present invention is an invention based on these findings.

[0005]
Specifically, according to the present invention, the following embodiments are provided.
(1) A composition including a nematode, for use in assessing whether a subject suffers from cancer or not based on the tactic behavior of the nematode toward urine of the subject, wherein the nematode is an AB1 strain.
(2) The composition including a nematode, for use in assessing whether a subject suffers from cancer or not based on tactic behavior of the nematode toward urine of the subject, according to the above described embodiment (1), wherein the nematodes are a mixture of a wild-type strain and the AB1 strain.

(3) The composition according to the above described embodiment (1), wherein the composition is used for urine toward which the wild strain exhibited attractive behavior.
(4) The composition according to the above described embodiment (3), wherein the composition is a composition for use in detecting a false-positive subject.
(5) The composition according to the above described embodiment (2), wherein at least one of the wild-type strain and the AB1 strain is labeled.

(6) A method for determining whether a subject suffers from cancer or not, including observing tactic behavior of an AB1 strain toward urine of the subject, and determining that the subject is or may be a healthy subject when the AB1 strain exhibited attractive behavior toward urine derived from the subject.

(7) A method for determining whether a subject suffers from cancer or not, including observing tactic behavior of a wild strain and an AB1 strain of nematodes toward urine of the subject, and determining that the subject does not or may not suffer from cancer when the urine derived from the subject induced attractive behavior of both the wild strain and the AB1 strain.

(8) The method according to the above described embodiment (7), wherein the method further includes determining that the subject does not or may not suffer from cancer when the urine derived from the subject induced approach behavior of both the wild strain and the AB1 strain.

(9) The method according to the above described embodiment (8), wherein the method further includes culturing a mixture of the wild strain and the AB1 strain of nematodes.

(10) The method according to the above described embodiment (9), wherein at least one of the wild-type strain and the AB1 strain is labeled.
Brief Description of Drawings [0006]
[Figure 1] Figure 1 is a plan view of a petri dish 1 used for assessing the behavior of a nematode as described in Example 1.
[Figure 2] Figure 2 is a graph showing taxis indexes of wild strains and AB1 strains for urine of patients with various cancers.
Detailed Description of the Invention [0007]
As used herein, "subject" means a mammal such as a primate (for example, a monkey, a chimpanzee, a gorilla, a bonobo, an orangutan, a marmoset, and a human). For example, "subject"
means a human. Herein, the subject is preferably a human.
Herein, "subject" is used to include a healthy subject, a subject suspected to suffer from cancer, and a subject suffering from cancer.
[0008]
As used herein, "cancer" means a malignant tumor. Cancers may be broadly classified into three groups: a hematopoietic tumor, an epithelial cancer (carcinoma), and a nonepithelial sarcoma (sarcoma). Examples of the hematopoietic tumor include leukemia, malignant lymphoma, and myeloma. Examples of the epithelial cancer include lung cancer, breast cancer, gastric cancer, large bowel cancer, uterine cancer, ovarian cancer, head and neck cancer, and tongue cancer. Examples of the sarcoma include osteosarcoma, chondrosarcoma, rhabdomyosarcoma, leiomyosarcoma, fibrosarcoma, liposarcoma, and angiosarcoma. As used herein, "cancer" includes early cancers such as a stage 0 early cancer and a stage 1 early cancer.
[0009]
Herein, "a composition for use in diagnosing cancer" may be simply referred to as "diagnostic agent for cancer."
[0010]
As used herein, "nematode" means Caenorhabditis elegans.
Nematode strains isolated from various environments have been registered at and published by Caenorhabditis Genetics Center (CGC) at College of Biological Sciences at the University of Minnesota in the United States, and can be distributed thereby.
Accordingly, those of skill in the art can obtain most of known strains from CGC. A hermaphrodite may be preferably used because the hermaphrodite can propagate by self-fertilization.

[0011]
As used herein, "tactic behavior" means attractive behavior or avoidance behavior, attractive behavior means behavior by which a physical distance from a certain substance is reduced and avoidance behavior means behavior by which a physical distance from a certain substance is increased. A substance that induces attractive behavior is referred to as an attractant and a substance that induces avoidance behavior is referred to as a repellant. It is a characteristic of a nematode that the nematode, based on olfaction, approaches the attractant and avoids the repellant. Behavior in which an organism approaches the attractant is referred to as attractive behavior (may be referred to herein as "positive") and behavior in which an organism avoids the repellant is referred to as avoidance behavior (may be referred to herein as "negative"). Furthermore, the attractive behavior and the avoidance behavior are collectively referred to as tactic behavior.

[0012]
As used herein, "wild strain" means a wild strain of a nematode and examples of the wild strain include an N2 Bristol strain. The wild strain as used herein can be a strain that exhibits attractive behavior toward the urine obtained from a subject with cancer and exhibits avoidance behavior toward the urine obtained from a subject without cancer.

[0013]
As used herein, "AB1 strain" refers to a nematode strain registered at CGC as AB1 (registration number 5459) (see WBG 10 (2) 140-141 and WBG 8 (2) 52). The AB1 strain is believed to be a nematode mutant separated from nature.

[0014]
The nematode exhibits attractive behavior toward cancer patient's urine, and exhibits avoidance behavior toward healthy subject's urine, as the present inventors already described in W02015/088039. Accordingly, it is possible to determine whether a subject is a cancer patient or a healthy subject based on tactic behavior of the nematode toward the urine obtained from the subject. However, more detailed examination of tactic behavior of the nematode has revealed that a certain percentage of healthy subjects may exhibit attractive behavior falsely (hereinafter also referred to as "false-positive"). The present inventors tested various nematode strains to detect a false-negative. Consequently, the present inventors succeeded in isolating a strain that exhibits attractive behavior toward a false-positive (AB1 strain).

[0015]
Method for Analyzing Urine Based on an Assessment System for Tactic Behavior of Nematode A method for analyzing urine based on an assessment system for tactic behavior of a nematode can be performed by placing a test sample (e.g., urine) obtained from a subject and a nematode at a certain distance apart and observing whether the nematode exhibits attractive behavior or avoidance behavior toward the test sample. Then, when the wild strain exhibited attractive behavior, it is possible to determine that the subject suffers from or may suffer from cancer. In W02015/088039, a medium-scale study involving 242 specimens was performed in this way and successfully detected cancer patients with a sensitivity of 100%
and a specificity of 95%.

[0016]
More specifically, the method for analyzing urine based on an assessment system for tactic behavior of a nematode may include, for example, placing a test sample (e.g., urine) obtained from a subject on a petri dish (e.g., a petri dish containing a solid medium), placing a nematode at a certain distance apart from the test sample in the petri dish with the test sample placed thereon, allowing the nematode to move after the placement, and evaluating that the subject suffers from or may suffer from cancer when the nematode exhibited tactic behavior toward the test sample.

[0017]
The tactic behavior of the nematode can be assessed by using a difference or a ratio between the number of nematodes that approached the test sample and the number of nematodes that avoided the test sample. In the assessment based on the difference in the numbers of nematodes, when the difference is a positive value, it is possible to determine that the test sample induces attractive behavior in total and is derived from a cancer patient; and when the difference is a negative value, it is possible to determine that the test sample induces avoidance behavior in total and is derived from a healthy subject. The tactic behavior of the nematode can also be assessed for example by using a taxis index as an indicator, as described below.
[Formula 1]
(Taxis index) ¨ A¨ B
A+ B
wherein A is the number of nematodes that exhibited attractive behavior toward the test sample, and B is the number of nematodes that exhibited avoidance behavior toward the test sample.

[0018]
When the taxis index is a positive value, it is possible to determine that the test sample induces attractive behavior in total and is derived from a cancer patient; and when the taxis index is a negative value, it is possible to determine that the test sample induces avoidance behavior in total and is derived from a healthy subject.

[0019]
The nematode may be labeled by a genetic modification technique. Examples of the label include a fluorescent protein.
Nematodes that express a fluorescent protein emit fluorescence of a specific wavelength when irradiated with excitation light of a specific wavelength, and this allows for identification of the individual nematodes. Examples of the fluorescent protein include, but are not particularly limited to, green fluorescent protein (GFP), yellow fluorescent protein (YFP), blue fluorescent protein (CFP), and GCaMP.

[0020]
AB1 strain The AB1 strain of the nematode exhibits avoidance behavior toward the cancer patient and the healthy subject, unlike the wild strain. However, the AB1 strain exhibits attractive behavior toward some of the healthy subjects. The urines toward which the AB1 strain exhibited attractive behavior were urines derived from a subject diagnosed as a healthy subject for example by a medical doctor. Therefore, a subject who was positive in the assessment using the AB1 strain is likely to be a healthy subject.

Accordingly, in the present invention, the AB1 strain can be used in assessing whether a subject suffers from cancer or not based on tactic behavior of the AB1 strain toward urine of the subject. In this aspect, the present invention provides a composition including a nematode, for use in assessing whether a subject suffers from cancer or not based on tactic behavior of the nematode toward urine of the subject, wherein the nematode is an AB1 strain. In other words, the present invention provides a composition including an AB1 strain, for use in detecting a healthy subject in cancer diagnosis.

[0021]
The subject who was positive in the assessment using the AB1 strain was also positive in the assessment using a normal wild strain, and this subject was a false-positive subject. Thus, the subject who is positive in the assessment using the AB1 strain and is positive in the assessment using the normal wild strain is likely to be false-positive.
Accordingly, in the present invention, the AB1 strain can be used in assessing whether a subject who is positive in the assessment using the wild strain is false-positive or not. In this aspect, the present invention provides a composition including a nematode, for use in assessing whether a subject suffers from cancer or not based on tactic behavior of the nematode toward urine of the subject, wherein the nematodes are a mixture of a wild-type strain and an AB1 strain. The present invention also provides a composition including a nematode, for use in assessing whether a subject suffers from cancer or not based on tactic behavior of the nematode toward urine of the subject, wherein the nematode is an AB1 strain and the subject is a subject in which urine of the patient induced approach behavior in the assessment using a wild strain of the nematode.

[0022]
Method for Assessing Tactic Behavior toward Urine by Using AB1 Strain (1) In the present invention, tactic behavior toward urine can be assessed by using the AB1 strain, for example as described below.

[0023]
The AB1 strain exhibits approach behavior toward urine of some healthy subjects. Accordingly, the AB1 strain can be used in detecting the healthy subject's urine. The assessment method can be performed, for example as described below.
First, the tactic behavior of the AB1 strain toward urine of the subject (that is, urine obtained from the subject) is observed. The urine toward which the AB1 strain exhibited approach behavior is or may be urine obtained from a healthy subject. Accordingly, it is possible to determine (or assess, expect, speculate, or diagnose) that a subject is or may be a healthy subject when the AB1 strain exhibited approach behavior toward the urine derived from the subject. Thus, it is shown that a subject is or may be a healthy subject when the AB1 strain exhibited approach behavior toward the urine derived from the subject.

[0024]
Method for Assessing Tactic Behavior toward Urine by Using AB1 Strain (2) In the present invention, the AB1 strain shows positive in subjects who were false-positive in the assessment test of tactic behavior using other wild strains of nematodes; and shows negative in other subjects. Accordingly, the AB1 strain can be used in detecting a subject who is or may be false-positive from subjects who were positive in the assessment of tactic behavior using other wild strains of nematodes. The assessment method can be performed according to, for example, an assessment method (A) or an assessment method (B) as described below.

[0025]
Assessment Method (A) First, the tactic behavior of the wild strain toward urine of the subject (that is, urine obtained from the subject) is observed. The urine toward which the wild strain exhibited approach behavior (that is, "positive" urine) is likely to be urine obtained from a cancer patient. Then, the tactic behavior of the AB1 strain toward the above described positive urine is observed. The urine toward which the AB1 strain exhibited approach behavior is or may be urine obtained from a healthy subject. Accordingly, it is possible to determine (or assess, expect, speculate, or diagnose) that a subject is or may be false-positive when the AB1 strain exhibited approach behavior toward the urine derived from the subject. Thus, it is shown that a subject is or may be a healthy subject when the AB1 strain exhibited approach behavior toward the urine derived from the subject.

[0026]
Assessment Method (B) In the above described assessment method (A), the wild-type nematode (wild strain) and the AB1 strain were used separately in the tests. In the assessment method (B), the wild strain and the AB1 strain are mixed and used.
The wild strain and the AB1 strain can be cultured separately. When the wild strain and the AB1 strain are cultured separately, they can be mixed and used when the behavior thereof is assessed. The wild strain and the AB1 strain can be mixed and used, without particular limitations, at a quantitative ratio of 2:1 to 1:2, and preferably at a quantitative ratio of about 1:1.
The wild strain and the AB1 strain can also be cultured, without particular limitations, under an identical condition.
The wild strain and the AB1 strain can also be mixed together and cultured. For coculture, the wild strain and the AB1 strain can be mixed at the time of culture and cultured together. The wild strain and the AB1 strain can be mixed and cultured, without particular limitations, at a quantitative ratio of 2:1 to 1:2, and preferably at a quantitative ratio of about 1:1. The growth speed of the AB1 strain is almost the same as that of the wild strain, and thus, when the AB1 strain and the wild strain are mixed at a ratio of 1:1 at the beginning of culture, the quantitative ratio of nematodes obtained after culture may be about 1:1.
The AB1 strain can be marked by a label. For example, a visualizable label, for example a fluorescent label, and more preferably a fluorescent protein label can be used as a label, without particular limitations. A fluorescent protein can be stably expressed in a nematode by the genetic modification technique. In an aspect of the present invention, an AB1 strain that expresses a fluorescent protein is provided. Alternatively, the wild strain can be marked by a label. For example, a visualizable label, for example a fluorescent label, and more preferably a fluorescent protein label can be used as a label, without particular limitations. A fluorescent protein can be stably expressed in a nematode by the genetic modification technique. The label can be introduced into one or both of the wild strain and the AB1 strain. When both of the wild strain and the AB1 strain are labeled, the wild strain and the AB1 strain may be labeled distinguishably.
Then, urine of a subject and the nematode mixture thus obtained can be used to observe the tactic behavior.
Consequently, it is possible to determine (or assess, expect, speculate, or diagnose) that a subject does not or may not suffer from cancer when the urine derived from the subject induced approach behavior of both the wild strain and the AB1 strain. Thus, it is shown that a subject does not or may not suffer from cancer when the AB1 strain exhibited approach behavior toward the urine derived from the subject.
The above described assessment method (B) may further include determining (or assessing, expecting, speculating, or diagnosing) that a subject suffers from or is likely to suffer from cancer when the urine derived from the subject induced approach behavior of the wild strain and induced avoidance behavior of the AB1 strain. Furthermore, the above described assessment method (B) may further include determining (or assessing, expecting, speculating, or diagnosing) that a subject is or is likely to be a healthy subject when the urine derived from the subject induced avoidance behavior of both the wild strain and the AB1 strain. Thus, it is shown that a subject is or may be a healthy subject when the AB1 strain exhibited approach behavior toward the urine derived from the subject.

[0027]
The composition including the AB1 strain of the present invention can be used in the above described method for assessing tactic behavior toward urine by using the AB1 strain.

[0028]
The present invention provides a method for preventing and/or treating cancer, the method including administering an anticancer agent to a subject who was determined to suffer from or be likely to suffer from cancer by the above described method of the present invention. In this embodiment, the method for preventing and/or treating cancer may further include determining that a subject suffers from or is likely to suffer from cancer when the AB1 strain exhibited approach behavior toward the urine derived from the subject.

[0029]
The present invention is described below with reference to Examples.
Example

[0030]
Example 1. Study of Tactic Behavior Based on Olfaction Using a Plurality of Different Nematode Strains In this Example, a plurality of different strains isolated from various soils were used to assess tactic behavior thereof based on olfaction.

[0031]
The nematode strain was purchased from Caenorhabditis Genetics Center (CGC) and the tactic behavior of the nematode based on olfaction was assessed. Specifically, culture of nematodes was performed using a NGM plate described in Table 1 below.

[0032]
[Table 1]
Table 1. NGM plate NaC1 3 g/L
Bacto peptone 2.5 g/L
Agar 17 g/L
Cholesterol (5 mg/ml, in ethanol) 1 ml/L
KPO4 (pH 6.0) 25 ml/L
CaCl2 1 ml/L
MgSO4 1 ml/L

[0033]
Assessment of the tactic behavior of the nematode was performed by using an assay plate (a petri dish with a diameter of 5 cm or 9 cm) described in Table 2 below.

[0034]
[Table 2]
Table 2. Assay plate (a petri dish with a diameter of 5 cm or 9 cm) Bacto agar 20 g/L
KPO4 (1 M) 5 ml/L
CaC12 (1 M) 1 ml/L
MgSO4 (1 M) 1 ml/L

[0035]
Urine samples of patients with various cancers were used as an odor-producing substance. More specifically, each of the urine specimens of patients with breast cancer, gastric cancer, bile duct cancer, large bowel cancer, a cecum, prostate cancer, pancreas cancer, or lung cancer and healthy subjects (including false-positive healthy subjects) was diluted 10 times or 100 times and used.

[0036]
The nematodes were cultured individually for four days at 20 C. Figure 1 shows the plate used in the Example. The above described odor-producing substance was spread on an area 5 for placing the sample on the plate and the nematodes were placed on an area 3 for placing the nematode on the plate, as shown in Figure 1. Then, the behavior of the nematodes were observed at 23 C.

[0037]
30 minutes after placing the nematodes, nematodes that existed in a region 2 and a region 4 were counted and a taxis index was calculated by using the following formula.
Taxis Index = {N (2) - N (4)1/1N (2) + N (4)1 wherein N (2) is the number of nematodes that existed in the region 2 in Figure 1, and N (4)is the number of nematodes that existed in the region 4 in Figure 1.

[0038]
Consequently, the normal wild strain of the nematode exhibited an approaching and moving-toward behavior toward cancer patient's urine and exhibited an avoiding and moving-away behavior toward healthy subject's urine. However, the present inventors discovered a strain that exhibited avoidance behavior toward both of the cancer patient's urine and the healthy subject's urine, among the nematode strains. The strain was an AB1 strain. The AB1 strain is a strain that was isolated and registered at CGC in 1984, and is available from CGC.

[0039]
It was found from further assessment of the behavior of the AB1 strain that the AB1 strain exhibited approach behavior toward false-positive healthy subject's urine toward which other wild strains exhibited approach behavior by mistake.

[0040]
This result revealed that the AB1 strain was a mutant that behaved differently from the wild strains in terms of approach behavior based on olfaction.
It can be understood that this result also indicates that urine toward which the normal wild strain exhibits approach behavior and the AB1 strain exhibits avoidance behavior is positive, and urine toward which both the normal wild strain and the AB1 strain exhibit avoidance behavior is false-positive.

[0041]

Then, the AB1 strain was fluorescently labeled.
Specifically, green fluorescent protein (GFP) was expressed in the body wall muscle of an AB1 strain to generate the AB1 strain that emits fluorescence when irradiated with excitation light.
Specifically, a plasmid that contains a gene including a GFP
gene operably linked downstream to myo-3 promoter was injected into the gonad of the AB1 strain to obtain a transformant. This strain was irradiated with y ray to integrate the injected plasmid into the chromosome.

[0042]
Then, an equal number of imagoes of the labeled AB1 strain and imagoes of the wild strain were plated on an agar culture medium sprinkled with feeds, and a mixture including the labeled AB1 strain and the wild strain was obtained after 3 to 5 days.
After confirming that each strain accounts for about half, these strains were used for the assessment of tactic behavior. In the assessment of behavior, the nematodes were observed under irradiation of visible light and excitation light for GFP and each taxis index could be calculated. The result is shown in Figure 2.

[0043]
As is shown in Figure 2, the normal wild strain (N2 Bristol strain in Figure 1) has a positive taxis index for cancer patient's urine and exhibits approach behavior theretoward; and has a negative taxis index for healthy subject's urine and exhibits avoidance behavior theretoward. In contrast to this, it was revealed that the AB1 strain had a negative taxis index for both the cancer patient's urine and the healthy subject's urine and exhibited avoidance behavior theretoward. Furthermore, eight different wild strains other than the N2 Bristol strain were tested and all the strains exhibited tactic behavior similar to that of the N2 Bristol strain.

[0044]
As described above, use of the AB1 strain and the normal wild strain allows for reducing the percentage of false-positives, and improvement in accuracy of assessment can be expected.

Claims

CLAIMS:

1. Use of a nematode for assessing whether a subject suffers from cancer or not based on taxis behavior of the nematode toward urine of the subject, wherein the nematode is an AB1 strain, and wherein attractive behavior of the nematode toward the urine indicates that the subject does not suffer from cancer or is unlikely to suffer from cancer.

2. The use according to claim 1, wherein the AB1 strain of nematode and a wild-type strain of nematode are used, and wherein (i) attractive behavior of both the AB1 strain of nematode and the wild-type strain of nematode toward the urine of the subject indicates that the subject does not suffer from cancer or is unlikely to suffer from cancer;
(ii) avoidance behavior of the AB1 strain of nematode and attractive behavior of the wild-type strain of nematode toward the urine of the subject indicates that the subject suffers from cancer or likely suffers from cancer; and/or (iii) avoidance behavior of both the AB1 strain of nematode and the wild-type strain of nematode toward the urine of the subject indicates that the subject does not suffer from cancer or is unlikely to suffer from cancer.

3. The use according to claim 1, wherein the urine of the subject is urine toward which a wild-type strain of nematode exhibits attractive behavior.

4. The use according to claim 3, wherein a false-positive subject is detected.

5. The use according to claim 2, wherein at least one of the wild-type strain of nematode and the AB1 strain of nematode is labeled.

6. A method for determining whether a subject suffers from cancer or not, comprising observing taxis behavior of a wild-type strain of nematode and an AB1 strain of nematode toward urine of the subject, and determining that the subject does not suffer from cancer or is unlikely to suffer from cancer when both the wild-type strain of nematode and the AB1 strain of nematode exhibit attractive behavior toward the urine of the subject.

7. A method for determining whether a subject suffers from cancer or not, comprising observing taxis behavior of a wild-type strain of nematode and an AB1 strain of nematode toward urine of the subject, and determining that the subject does not suffer from cancer or is unlikely to suffer from cancer when the urine of the subject induced avoidance behavior of both the wild-type strain of nematode and the AB1 strain of nematode.

8. A method for determining whether a subject suffers from cancer or not, comprising observing taxis behavior of a wild-type strain of nematode and an AB1 strain of nematode toward urine of the subject, determining that the subject suffers from cancer or likely suffers from cancer when the urine of the subject induced attractive behavior of the wild-type strain of nematode and avoidance behavior of the AB1 strain of nematode.

9. The method according to any one of claims 6 to 8, wherein the method further comprises culturing a mixture of the wild-type strain of nematode and the AB1 strain of nematode.

10. The method according to claim 9, wherein at least one of the wild-type strain of nematode and the AB1 strain of nematode is labeled.