Rodents Used for the Propagation of Hookworms and Haemonchus contortus

Abstract

Helminthiasis due to hookworm infestations in humans and Haemonchus contortus in sheep cause untold levels of disease and economic losses worldwide. Drug resistance is an ever-growing problem with pathogenic helminths. Thus, there is a critical need for new treatment strategies for hookworms and H. contortus that depends on animal models. Because hookworms and H. contortus are obligate parasites, they can only be maintained using live animals. This review describes use of the Syrian golden hamster to propagate hookworms and Mongolian gerbil to propagate H. contortus.

Introduction

Soil-transmitted helminth infestations in humans are a global public health problem of staggering proportions, impacting approximately 25% of the world’s population.²² Of the major soil-transmitted helminths (roundworms, whipworms, hookworms), the predominant species of hookworms that cause intestinal disease, that is, Necator americanus, Ancylostoma duodenale, and the zoonotic Ancylostoma ceylanicum, were estimated in 2006 to infest 576 to 740 million people worldwide.⁷ Risk models place 2.6 to 5.1 billion people worldwide at risk for developing hookworm infestations.^16,31 Although estimating the economic and health impact of hookworm infestation is challenging, it is likely that lost productivity and health care costs worldwide are tens to hundreds of billion dollars annually.³ The helminth parasite Haemonchus contortus, also known as the ‘barber pole worm’, is one of, if not the most economically important and pathogenic parasites of small ruminants.^2,6 H. contortus is found worldwide and significantly impacts humans through food and income losses due to reduced animal productivity, fertility, and increased mortality in small ruminants. Drug resistance and difficulty controlling H. contortus and hookworms are worldwide problems.^15,23,41 Thus, there is a critical need for animal models and the development of novel treatment strategies for H. contortus and hookworms.^32,35 Hookworms and H. contortus have important common biologic features. Both hookworms and H. contortus have a complete life cycle and are obligate parasites, and they require a mammalian host to complete their life cycle.^18,22,40 This has important implications for research; that is, because the parasites cannot be cultured in vitro, they must be passed in an animal host. The life cycle for hookworms is shown in Figure 1, and H. contortus has a similar life cycle. Hookworms and H. contortus infest hosts after eggs hatch in the environment and larval development reaches the infective third stage.^26,41 H. contortus infests a host after the infective third-stage larvae (L3) enter the host through ingestion compared with skin penetration by hookworms. Experimental animal infestation with hookworms and H. contortus generally requires administration of infective L3 organisms. The factors that determine which animals are permissive or nonpermissive hosts for hookworms and H. contortus are poorly understood. Host immune responses and undefined parasite or host factors appear to be critical determinants for establishing infestation in a particular host.

Figure 1.

Hookworm life cycle. 1) Patent adult worms pass eggs, which are shed in stool. 2) Eggs hatch in 1 to 2 d, if conditions are favorable, to free-living L2 rhabditiform larvae. 3) After 5 to 10 d the L2 develop into the infective L3, which can live for 3 to 4 wk. 4) When in direct contact with a human, the L3 penetrate the skin and are carried through the bloodstream to the lungs. After penetrating alveoli, the L3 ascend the bronchial tree to the pharynx and are swallowed. 5) When L3 reach the jejunum they attach to the intestinal wall and develop into adults. Source: Centers for Disease Control and Prevention. The figure is in the public domain and developed by the Centers for Disease Control and Preventions’s Division of Parasitic Diseases and Malaria. Reference to specific commercial products, manufacturers, companies, or trademarks does not constitute endorsement or recommendation by the U.S. Government, Department of Health and Human Services, or Centers for Disease Control and Prevention (https://www.cdc.gov/dpdx/hookworm/index.html).

Syrian golden hamsters (Mesocricetus auratus) have been preferable to mice and other rodents for hookworm studies and propagation since they are a fully permissive species, produce patent infections, and can recapitulate the course of human disease given an appropriate infective dose.^27,34,37 The use of mice as a model has languished likely since hookworm infestations cannot be established or maintained unless mice are immunosuppressed.^1,24 This suggests that the immune response to hookworm inoculation in mice is a significant factor in the establishment of stable infestation. Despite the availability of immunocompromised mouse strains, it was not until 2024 that it was demonstrated that patent A. ceylanicum infestation could be established in NSG mice (NOD.Cg-Prkdc^scid Il2rg^tm1Wjl/SzJ).²⁴ The time course of worm development and patency in NSG mice is similar to that in hamsters and provides additional evidence that immune responses to A. ceylanicum determines host permissivity in mice. The observation that infestations with Ancylostoma caninum cannot be established in NSG mice suggests that for this organism host immune responses are not the only factor that determines host permissivity.²⁴ Whether NSG or other immunocompromised mice will eventually replace hamsters as a model remains to be determined.

Rodent models to study H. contortus have been explored in rabbits, guinea pigs, mice, and Mongolian gerbils (Meriones unguiculatus).^12,19,34,37 The gerbil is the only species in which H. contortus will develop into patent adults, and the model originally described has become the predominant paradigm for propagating and studying haemonchosis.^12–14 Short duration (7 d) H. contortus infestations have been demonstrated in mice treated with immunosuppressive drugs or cimetidine.¹ To date there are no studies reported that examine immune responses to H. contortus in mice or whether patent infestations with H. contortus can be established in immunocompromised mouse strains.

The purpose of this review is to discuss the use of hamsters and gerbils for the propagation of hookworms and H. contortus, respectively, in these rodents.

Hookworms: Animals and Husbandry

The Syrian golden hamster has been used as a rodent model for studies of hookworm pathology, immunology, and vaccination since the mid-1980s. Although several different hamster strains, most of which are no longer available, have been described in the literature, HsdHan^®:AURA appears to be the strain most commonly used for hookworm propagation and experiments. Husbandry for hookworm-infested hamsters does not require special caging, bedding, food, or water. The most recent edition of Biosafety in Microbiological and Biomedical Laboratories does not specify an ABSL level for working with hookworm-infected animals or provide recommendations for disposing of bedding from hookworm-infested animals.²⁸ The only recommendation made in Biosafety in Microbiological and Biomedical Laboratories is to wear personal protective equipment that would prevent skin contact when working with infective larvae. If worms are harvested from hamsters during the prepatent period (1 to 13 d postinfection), there is no risk to human health.^18,22 For studies using hamsters during the patent period, for example, to collect fecal samples for eggs or in vivo drug studies, the life cycle of the organism, cage conditions and change intervals, and use of personal protective equipment would still make it very difficult for staff to contract hookworm disease from handling infested animals or their bedding and feces.^18,22 Male hamsters 4 to 6 wk old are usually used since females are significantly less susceptible to hookworm infestations.^30,38 Neonatal hamsters (1 to 2 d old) can also be used, however the difficulty in maintaining breeding colonies and caring for neonates has led to the use of animals at or past the age at which they reach sexual maturity.^4,5

A. ceylanicum and A. duodenale: Propagation

Animals are usually inoculated orally with parasite L3 at 3 to 5 wk of age. When inoculated with up to 200 L3 PO (with euthanasia 14 d later), animals do not develop clinical signs.¹¹ Although possible to infest hamsters with A. ceylanicum percutaneously, this route has not been employed.¹⁰ Animals may exhibit mild clinical signs during egg harvesting procedures since fecal collection to harvest eggs is usually done at 18 to 35 d postinoculation.²⁹ Lower parasite doses with extended duration of infestation can result in signs related to anemia (weight loss, lethargy, decreased PCV).^11,17 After L3 administration, animals should be observed daily and weighed at least weekly. To obtain infective L3, eggs are purified from the feces of infested animals, cultured on activated charcoal for 7 d at 28 °C, and harvested using a Baermann technique.²⁵ Although not necessary to produce patent infestations, treating A. ceylanicum inoculated hamsters with dexamethasone will increase egg production and prolong the duration of egg shedding, thus reducing the number of animals needed to produce eggs. This procedure was adapted from studies on N. americanus.³³

N. americanus: Propagation

Propagation of N. americanus strains that are not host adapted requires immunosuppression of animals or use of neonates in order for the parasites to complete their life cycle.^25,33,36 Hamster-adapted strains of N. americanus exist or can be developed that do not require immunosuppressed or neonatal animals to maintain the life cycle of parasites. However, it may take up to 100 generations (26 y) to generate a host-adapted N. americanus strain that may also have questionable translatability to human parasite strains.^20,21 The infective L3 of N. americanus are obligate epidermal parasites. Thus, hamster infestation requires L3 larval inoculation by a cutaneous or percutaneous route.^8,9 Infestations become patent at around 36 to 43 d postinoculation, and fecal egg counts become maximal at 57 to 67 d postinoculation.²⁰ There are no extant studies documenting clinical signs in hamsters resulting from N. americanus infestation. Infestation using parasite strains that are highly host adapted can result in transitory anemia.³⁸

H. contortus: Animals and Propagation

Unfortunately, all current publications using gerbils for H. contortus studies do not specify the strain used and/or the source. The source is often described as a breeding facility within the investigator’s institution. It appears that the only current commercially available gerbil strain is Crl:MON(Tum). Because H. contortus is not a human pathogen, housing infested animals does not require any special husbandry conditions or safety measures for staff other than wearing personal protective equipment usually used in rodent facilities. Both male and female gerbils can be infested with H. contortus; however, animals must be immunosuppressed, which is usually accomplished with glucocorticoids, to maintain infestations at 10 to 14 d postinoculation.¹⁴ Without immunosuppression L3 can develop into adult worms; however, the adults may not be patent and are rapidly expulsed after 14 d. Even with immunosuppression some strains of H. contortus may only develop to the L4 stage.³⁹ Animals inoculated with up to 2000 L3 and euthanized by day 14 postinoculation do not show clinical signs (R. Aroin, personal communication, February 2024). Because patent infestations apparently cannot be established in gerbils, L3 are isolated from eggs passed in H. contortus–infested sheep feces.^14,39 There are no extant publications describing clinical signs in gerbils after H. contortus infestations, and virtually all published studies conclude at 14 d postinoculation.

Conclusions

Research to develop new treatments and preventatives for hookworms and H. contortus is critically important for worldwide human and animal health. Despite this fact, there is a dearth of publications on the development of biologic or small-molecule treatments for these parasites. The fact that hookworms and H. contortus have particular requirements necessary for their propagation, most prominent being the need of a live animal host to maintain parasite stocks, certainly contributes to the lack of novel treatment development. In addition, host–parasite interactions that govern establishment of patent infestations remain poorly elucidated, which further hinders the development of new animal models. Because a wide variety of immunocompromised mouse strains are available, there are certainly sufficient resources that could be employed to provide better insight into host–parasite interactions. This would in turn provide better understanding of what constitutes permissive hosts and possibly lead to the availability of less resource-intense animals to propagate and study helminth parasites. Because the loss of parasite strains can be disastrous for researchers, further advances in cryopreservation can provide a margin of safety in preserving valuable parasite strains.²⁵ However, for the foreseeable future, the Syrian golden hamster and Mongolian gerbil will continue to be critically important for the propagation and study of hookworms and H. contortus.

Conflict of Interest

The author(s) have no conflict(s) of interest to declare.

Funding

This work was internally funded.