A culture medium and culture method for haemonchus contortus fourth instar larvae

Abstract

This invention discloses a culture medium for fourth-stage larvae of *Haemaphysema contortus*, comprising a basal culture medium and auxiliary components. The auxiliary components consist of pyruvate, non-essential amino acids, L-glutamine, antibiotics, fetal bovine serum, centrifuged blood, and L-glutathione. This invention also discloses a method for culturing fourth-stage larvae of *Haemaphysema contortus*, comprising the steps of developing third-stage larvae into fourth-stage larvae, and then adding them to the culture medium for culturing until the final stage of the fourth stage. This invention enables the in vitro culture of fourth-stage larvae of *Haemaphysema contortus*, which is beneficial for promoting the development of related drugs and vaccines for this parasite and has significant implications for the control of the parasite.

Description

Technical Field

[0001] This invention relates to a culture medium and culture method for fourth-stage larvae of Haemaphysalis contortus, belonging to the field of biology. Background Technology

[0002] Haemonchus contortus is a blood-sucking parasitic nematode that infests the abomasum of ruminants such as cattle and sheep. It is widespread globally, and its life cycle includes a free-living stage and a parasitic stage. Stage IV (L4) is the initial stage of the parasitic life cycle. During this stage, the living environment undergoes a significant change, and the nematode migrates to the base of the abomasal mucosa of the host to begin feeding on blood. Towards the end of stage IV, the increased blood intake causes anemia and emaciation in the host animal, and in severe cases, even death, resulting in significant economic losses to the livestock industry. Control of this parasite primarily relies on chemical drugs, but long-term irrational drug abuse has led to serious drug resistance problems worldwide.

[0003] *Haemaphysalis contortus* is a model organism for drug development in the treatment of blood-sucking parasitic nematodes. Establishing an in vitro culture platform during the parasitic phase can significantly reduce experimental costs in drug screening. Furthermore, natural proteins extracted from the intestines of *Haemaphysalis contortus*, such as H-11 and H-gal-GP, have been proven to possess very high immunoprotective efficacy. However, the high cost of obtaining live animals has prevented the large-scale production of commercial vaccines. Therefore, establishing an in vitro culture platform for *Haemaphysalis contortus*, especially a platform for the parasitic phase, is of great significance for parasite control and has significant application value in drug and vaccine research and development.

[0004] However, the development of in vitro culture platforms for Haemonchus contortus has been slow. Currently, in vitro culture systems for the free-living stage (eggs to third-stage larvae) have been established, including fecal microbiota culture and culture medium culture methods, which are mature. However, in vitro culture of the parasitic stage has been slow to develop and is currently mainly stuck at the early stage of the fourth stage. To date, there is no commercially available culture system for fourth-stage larvae of Haemonchus contortus.

[0005] References:

[0006] 1. Arsenopoulos KV, Fthenakis GC, Katsarou EI, Papadopoulos E. Haemonchosis: A challenging parasitic infection of sheep and goats. Animals (Basel), 2021, 11(2): 3632. Nikolaou S, Gasser RB. Prospects for exploring molecular developmental processes in Haemonchus contortus. Int J Parasitol, 2006, 36: 859 - 868

[0007] 3. Stoll NR. In vitro conditions favoring ecdysis at the end of the first parasitic stage of Haemonchus contortus (Nematoda). Growth, 1940, 4: 383 - 406

[0008] 4. Stringfellow F. Cultivation of Haemonchus contortus (Nematoda: Trichostrongylidae)

[0009] from infective larvae to the adult male and the egg - laying female. The Journal of Parasitology, 1986, 339 - 345

[0010] 5. Niciura SCM, Minho AP, McIntyre J, Benavides MV, Okino CH, Esteves SN, Chagas ACS, Amarante AFTD. In vitro culture of parasitic stages of Haemonchus contortus.

[0011] Rev Bras Parasitol Vet. 2023 Jan 13; 32(1): e010122. Summary of the Invention

[0012] The purpose of this invention is to provide a culture medium and method for fourth-stage larvae of Haemonchus contortus, which can culture Haemonchus contortus to the end of the fourth stage and improve the survival rate and development rate of the larvae.

[0013] The above objective is achieved through the following technical solution:

[0014] A culture medium for fourth-stage larvae of *Haemaphysalis contortus* includes a basal culture medium and auxiliary components. The types of the auxiliary components and their proportions in the basal culture medium are as follows:

[0015]

[0016] Preferably, the basal culture medium consists of NCTC-109 and LB in a volume ratio of 1:2.

[0017] Preferably, the types of auxiliary components and their content in the basal culture medium are as follows:

[0018]

[0019] Preferably, the centrifuged blood is prepared as follows: fresh defibrinated sheep blood is repeatedly frozen and thawed, then centrifuged at high speed, the supernatant is collected, and stored at 4°C.

[0020] The culture medium provided by this invention can culture Haemaphysema contortus from the early stage of stage IV to the late stage of stage IV with a survival rate of over 90%.

[0021] The present invention further provides a method for culturing fourth-stage larvae of Haemaphysalis contortus, comprising the following steps: developing third-stage molting larvae into fourth-stage larvae, and then adding them to the culture medium for culturing until the end of the fourth stage.

[0022] Preferably, the cultivation conditions are: temperature 39°C, CO2 content 20%.

[0023] The beneficial effects of this invention are:

[0024] This invention enables the in vitro culture of late-stage fourth-stage larvae of Haemonchus contortus, which is beneficial for promoting the development of related drugs and vaccines for this parasite and has significant implications for the control of the parasite. The fourth-stage larvae cultured in this invention have high survival and development rates, and also have advantages such as short culture cycle, simple operation, readily available culture medium materials, and low production cost. Attached Figure Description

[0025] Figure 1 This study compares the effects of three culture media—M-199, NCTC-109, and LB—on the fourth stage of larvae.

[0026] Figure 2 This study compares the effects of three different proportions of culture media on the culture of larvae in the fourth stage.

[0027] Figure 3 Morphological observation of the reproductive system of the late fourth-stage larvae of Haemaphysalis contortus. Detailed Implementation

[0028] The technical solution of the present invention will be described in detail below with reference to specific embodiments and examples. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods. Unless otherwise specified, the materials and reagents used in the following embodiments are commercially available.

[0029] Material:

[0030] (1) Laboratory animals and insect strains

[0031] Healthy goats around 3 months old were purchased from the Hubei Academy of Agricultural Sciences, and the Hacon-5 strain of Haemonchus contortus was preserved in our laboratory.

[0032] (2) Main materials and reagents

[0033] NCTC-109 medium (Thermo Fisher, USA), M-199 medium (Sigma, USA), defibrinated sheep blood (Dening Biotechnology, China), fetal bovine serum (Gibco, USA), penicillin-streptomycin-amphoteric B solution (Sigma, USA), L-glutathione (Sigma, USA).

[0034] (3) Obtaining infective third-stage (iL3) larvae

[0035] Healthy goats aged 3-6 months were purchased from the Hubei Academy of Agricultural Sciences and artificially dewormed with ivermectin. Fourteen days after deworming, feces were examined for parasite eggs for three consecutive days. After confirming the absence of eggs, 7000 iL3 goats were artificially infected. Feces were examined for eggs again after 28 days. Once egg production stabilized, feces were collected and cultured in a 26℃, humidified incubator for 7 days until the eggs developed into iL3 goats. After 7 days of culture, iL3 goats were collected from the feces using the Bellman method, the specific steps of which are as follows:

[0036] ① Place two layers of filter paper on the gauze and wrap the feces inside.

[0037] ② Place the wrapped feces into a Bellman funnel and add dechlorinated water at a temperature below 40°C until the feces are submerged.

[0038] ③ After standing for 4 hours, collect the lower liquid in a 50mL centrifuge tube, let it stand for another 4 hours, discard the upper 45mL liquid, and collect the bottom 5mL liquid.

[0039] ④ Add 2 mL of BU solution to a glass test tube, and place filter paper on the top of the liquid surface. Add the liquid retained in the previous step to the glass test tube, let it stand for 4 hours, collect the bottom iL3, and store it in 10 times the amount of BU solution in a 16℃ saturated humidity incubator.

[0040] Other materials whose sources are not specified are all conventional materials in this field.

[0041] Example 1: Screening of L4 basal culture medium

[0042] 1. Obtaining the third-stage molted larvae of *Haemaphysalis contortus*:

[0043] (1) Add 0.15% NaClO to a 1.5mL centrifuge tube and desheath at 37℃ for 10min;

[0044] (2) After desheathing is completed, centrifuge at 1300g for 3 min at room temperature;

[0045] (3) After centrifugation, discard 1 mL of supernatant and add 1 mL of 0.85% NaCl solution containing three antibiotics;

[0046] (4) Centrifuge the larvae at 1300g for 3 minutes, and repeat step (3) five times;

[0047] (5) Discard 1 mL of supernatant and add 1 mL of M-199 medium containing three antibodies;

[0048] (6) Centrifuge the larvae at 1300g for 3 minutes and repeat step (5) three times.

[0049] 2. Culture of three stages of molting larvae

[0050] (1) Add 100 U / mL penicillin, 0.1 mg / mL streptomycin, 0.25 μg / mL amphotericin B, 1 mM pyruvate, 0.1 mM non-essential amino acids, 2 mM L-glutamine, and 20% fetal bovine serum (FBS) to M-199 medium.

[0051] (2) The treated molting third-stage larvae were placed in a 24-well plate at a density of 100 larvae / well, with 500 μL of culture medium per well. The plates were then cultured in a 39℃ 20% CO2 incubator for 7 days until they developed into fourth-stage larvae.

[0052] 3. Screening of basal culture media for four stages of larvae

[0053] Fourth-stage larvae were collected by centrifugation at 1300g for 3 minutes, added to different culture media and cultured under the same conditions; every two days, 200μL of the upper layer of culture media was aspirated and the medium was changed; body length and body width were measured every five days, for a total of 3 times, and the tail development rate was also measured at the last time.

[0054] This experiment compared five different basal culture media: M-199, NCTC-109, LB, NCTC-109:LB = 1:1, NCTC-109:LB = 1:2, and NCTC-109:LB = 2:1. Each culture medium was supplemented with 100 U / mL penicillin, 0.1 mg / mL streptomycin, 0.25 μg / mL amphotericin B, 1 mM pyruvate, 0.1 mM non-essential amino acids, 2 mM L-glutamine, and 20% FBS to culture fourth-stage larvae.

[0055] Figure 1 The study compared the effects of three culture media—M-199, NCTC-109, and LB—on the fourth-stage larvae. It was found that NCTC-109 medium was more effective in promoting tail development in the fourth-stage larvae, while LB medium was more effective in promoting body length growth. Therefore, NCTC-109 and LB media were combined and their ratio was further investigated.

[0056] Figure 2 The study compared the culture effects of three different ratios of NCTC-109:LB = 1:1, NCTC-109:LB = 1:2, and NCTC-109:LB = 2:1 on L4 larvae. It can be seen that when NCTC-109:LB = 1:2, the tail development rate, body length, and body width are all at a higher level. Therefore, NCTC-109:LB = 1:2 was determined as the optimal basal culture medium for fourth-stage larvae, and will be referred to as "L4 medium" from now on.

[0057] Example 2: In vitro culture methods and conditions for fourth-stage larvae of Haemonchus contortus

[0058] Preparation of centrifuged blood: Fresh defibrinated sheep blood was frozen at -20℃ and thawed at room temperature twice. Then it was dispensed into 1.5mL centrifuge tubes and centrifuged at 12000g, 4℃ for 10min. The supernatant of the centrifuged defibrinated sheep blood was collected and stored in a 4℃ freezer to obtain the centrifuged product of freeze-thawed defibrinated sheep blood (abbreviated as: centrifuged blood).

[0059] Fourth-stage larvae were collected by centrifugation at 1300g for 3 min and added to culture media with different formulations as follows. They were placed in 24-well plates at a density of 100 larvae / well, with 500 μL of culture medium per well. The plates were incubated at 39℃ in a 20% CO2 incubator. The upper 200 μL of culture medium was aspirated every two days for medium replacement. After 15 days of incubation, the survival rate and development rate were calculated.

[0060] The criteria for distinguishing between the early, middle, and late stages of the four larval stages are as follows: In the early stage, the reproductive primordia cells are 12, the tail is slender and curved towards the dorsal side, and the reproductive primordia of the female are farther from the excretory opening than those of the male; In the middle stage, the reproductive system of the larva is long and spindle-shaped, the tail of the female is slender and curved towards the dorsal side into a small hook shape, and the tail of the male is shorter and thicker; In the late stage, the reproductive system of the larva has developed into a tubular shape, and the various organs of the female reproductive system are clearly visible, especially the vulva under a microscope, and the mucus sac and copulatory umbilicus of the male tail are clearly visible.

[0061] Table 1 Culture medium formulation

[0062]

[0063]

[0064] Table 2 Survival and development rate of fourth-stage larvae

[0065]

[0066] The data in Table 2 are the means of three independent replicate experiments. Regarding survival rate, formulations 1 and 2 were significantly better than formulation 3. Regarding development rate, formulation 1 was significantly better than formulations 2 and 3. The results indicate that formulation 1 is more suitable for the growth and development of fourth-stage larvae of *Haemaphysalis contortus*.

[0067] Figure 3 For morphological observation of the reproductive system of late-stage fourth-stage larvae of *Haemaphysalis contortus* cultured in vitro, both male and female worms developed clear reproductive systems. The female worms ( Figure 3 The vulva, vagina, uterus, and ovaries of the male worm (left) can be clearly distinguished. Figure 3 (Right) The larvae develop a "small hook-shaped" tail, and the internal copulatory umbrella is clearly visible through the transparent cuticle. This invention enables the in vitro culture of late-stage fourth-stage larvae of *Haemaphysalis contortus*. The significant development of the reproductive system represents a marked advancement in the in vitro culture technology of *Haemaphysalis contortus*.

Claims

1. A culture medium for fourth-stage larvae of Haemonchus contortus, comprising a basal culture medium and auxiliary components, characterized in that: The types of auxiliary components and their content in the basal culture medium are as follows: The method for preparing the centrifuged blood is as follows: Fresh defibrinated sheep blood is repeatedly frozen and thawed, then centrifuged at high speed, the supernatant is collected, and stored at 4°C.

2. The culture medium for fourth-stage larvae of Haemaphyseudomonas contortus as described in claim 1, characterized in that: The basal culture medium consists of NCTC-109 and LB in a volume ratio of 1:

2.

3. The culture medium for fourth-stage larvae of Haemaphysema contortus as described in claim 1, characterized in that: The types of auxiliary components and their content in the basal culture medium are as follows:

4. The use of the culture medium according to any one of claims 1-3 in culturing fourth-stage larvae of Haemaphysalis contortus.

5. A method for culturing fourth-stage larvae of *Haemaphysalis contortus*, characterized in that, Includes the following steps: The molting third-stage larvae are developed into fourth-stage larvae and then cultured in the culture medium described in any one of claims 1-3.

6. The method for culturing fourth-stage larvae of *Haemaphysalis contortus* as described in claim 5, characterized in that: The cultivation conditions were: temperature 39°C and CO2 content 20%.

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