This study demonstrated the usefulness of an in vivo rodent D. immitis microfilaremia model. SCID mice could maintain microfilaremia in peripheral blood for up to seven months and at least 10 months in cardiac blood. These results suggest that SCID mice are effective for the long-term maintenance of D. immitis microfilaremia.
The lack of rodent models complicates canine heartworm studies. Establishing an in vivo model of D. immitis requires a well-equipped facility capable of rearing dogs or ferrets to isolate microfilariae. In 1983, Grieve et al. [12] developed a mouse model of D. immitis microfilaremia. They used immunodeficient BALB/cA mice and sublethal gamma-ray irradiation to induce microfilaremia in the peripheral blood that lasts for five weeks. This finding motivated us to investigate the potential of SCID mice for the long-term maintenance of D. immitis microfilariae. Furthermore, we investigated whether splenectomy could prolong microfilaremia, hypothesizing that the spleen, as a component of the immune system responsible for eliminating cellular waste from the blood, would play a role in this process [17]. Although we were unable to establish the efficacy of splenectomy in prolonging the duration of microfilaremia, SCID mice proved valuable for the long-term maintenance of microfilaremia, which is consistent with our hypothesis. The majority of microfilariae in dogs infected with D. immitis live in their pulmonary capillaries [18, 19]. Microfilariae were detected in SCID mice for a longer period of time in cardiac puncture blood than in peripheral blood. This shows that the dynamics of microfilariae in SCID mice are similar to those observed in dogs. Microfilaremia levels in SCID mice exhibited periodicity, with an increase at night. This trend was comparable to that seen in dogs [20]. These findings suggested that SCID mice can replicate the dynamics of microfilariae in their bodies, making them valuable models for studying the periodicity of D. immitis microfilariae. However, additional validation is required.
The normalcy of microfilariae is crucial when using microfilariae maintained in SCID mice to discover new drugs or study mosquito transmission mechanisms. We conducted in vitro cultivation, mosquito infectivity, L4 development, and ivermectin reactivity assays.
The in vitro cultivation of microfilariae is crucial for drug screening [21]. In our study, microfilariae isolated from SCID mice had a survival rate comparable to that of microfilariae isolated from infected dogs. This finding suggests that the lifespan of microfilariae was not shortened in SCID mice, implying that the microfilarial environment in SCID mice is comparable to that in dogs. In the ivermectin reactivity assay, microfilariae isolated from SCID mice reacted similarly to microfilariae recovered from dogs. This suggests that maintaining microfilariae in SCID mice does not affect their metabolism or homeostasis in response to ivermectin. As a result, SCID mice are regarded as a valuable source of microfilariae for drug screening or macrocyclic lactone resistance assays, although only one compound was tested in this study. This finding further supports the idea that the microfilarial conditions in SCID mice are nearly identical to those in dogs.
To determine the infectivity and developmental potential of microfilariae, we infected Ae. aegypti mosquitoes with microfilariae from SCID mice and counted the number of L3 larvae in each mosquito. Our results show that microfilariae isolated from SCID mice have normal infectivity when compared to those recovered from infected dogs. Additionally, L3 larvae derived from SCID mice displayed normal developmental ability to advance to the L4 stage in vitro. These results suggest that SCID mice can serve as an important source of microfilariae for mosquito infection experiments and L3 to L4 developmental tests for the development of innovative anti-heartworm preventive medicines.
The use of microfilariae derived from clinical samples is crucial for research purposes such as macrocyclic lactone resistance [22]. Maintaining the viability of microfilariae between clinical sample collection and the experimental setting is critical; however, long-term preservation presents challenges. Microfilariae can be cultured in vitro for up to 45 days [10]. Alternatively, cryopreservation may be used. However, the viability of cryopreserved microfilariae is frequently low. To address this, we used SCID mice to isolate and maintain microfilariae from veterinary clinical samples. We successfully established that SCID mice can be used to isolate and maintain microfilariae obtained from clinical samples, even when the blood sample is refrigerated for three days before being inoculated into the SCID mouse. Notably, ON1 microfilariae showed normal infectivity in mosquitoes and the developmental ability to proceed to the L4 stage. These findings highlight the efficacy of the SCID mouse model for isolating clinical microfilariae samples and suggest that it has the potential to improve the use of clinical sample-derived microfilariae in heartworm research.
However, the viability of cryopreserved microfilariae after thawing remains a substantial challenge [11]. In our earlier investigation, we found that thawed microfilariae had inferior fitness, despite approximately 80% of the microfilariae surviving three days after thawing in vitro [9]. Furthermore, the number of L3 larvae recovered from mosquitoes infected with cryopreserved microfilariae was approximately one-third of that recovered from mosquitoes infected with an equal number of non-cryopreserved fresh microfilariae [9]. In this study, we used SCID mice to recover the cryopreserved microfilariae. Notably, the cryopreserved microfilariae recovered from SCID mice had comparable infectivity and L4 developmental ability to fresh microfilariae collected from experimentally infected dogs. Furthermore, these cryopreserved microfilariae showed a similar survival curve in macrocyclic lactone resistance when compared to fresh microfilariae collected from experimentally infected dogs. These findings indicate that the fine microfilariae selected from cryopreserved samples retain their fitness and can be effectively recovered using SCID mice. This hypothesis is reinforced by the fact that cryopreserved microfilariae recovered from SCID mice survived longer in vitro than directly thawed cryopreserved microfilariae. Therefore, the combination of cryopreservation and recovery in SCID mice appears promising, particularly for research on macrocyclic resistance using microfilariae isolated from clinical samples. This approach effectively addresses the challenges of long-term preservation and microfilariae fitness in research involving clinical samples.
Recent studies have established mouse models for L4 development in D. immitis using NOD-, NSG-, or NXG immunodeficient mice [23, 24]. This study proposes a microfilaremia model for D. immitis using SCID mice. Developing a comprehensive rodent model of D. immitis infection shows potential for furthering heartworm research, particularly in terms of creating novel preventative strategies that overcome macrocyclic lactone resistance. Furthermore, these mouse models have the potential to improve animal welfare by minimizing the use of experimental companion animals, such as dogs and cats.
In this study, we established a dog heartworm microfilaremia model and demonstrated that our mouse model is useful for the source of microfilariae for the research, such as drug screening or transmission mechanism. For the drug research, in vivo infection model is useful. Our SCID mouse model may be useful for the in vivo study, although we could not determine in vivo drug administration test in this study. We hope that our SCID mouse model could contribute to the development of the novel anthelmintics in a future study.