Examination of artificial oocyst excystation methods in the murine coccidium, Eimeria krijgsmanni

Abstract

Eimeria spp. cause coccidiosis characterized by diarrhea and induce serious economic losses in livestock industries. Although several anti-coccidial drugs are currently available, the emergence of resistant strains and drug residues is problematic; therefore, the development of new drugs is needed. Since sporozoites of Eimeria spp. invade host intestinal epithelial cells and numerous merozoites are formed, drugs that target sporozoites are expected to be useful. We previously used murine Eimeria krijgsmanni as a model to examine anti-coccidial drug susceptibility; however, few studies have conducted drug evaluations against sporozoites. The establishment of excystation protocols is essential for progress in in vitro experiments using sporozoites because oocysts must be isolated from feces using complex techniques before the excystation process. Various artificial excystation protocols have been reported for each Eimeria spp.; however, those for E. krijgsmanni have not yet been examined. Therefore, 4 protocols described in previous studies were herein conducted for E. krijgsmanni. Pepsin was important for excystation in rodent Eimeria spp., and this was also the case for E. krijgsmanni. Excystation rates were higher with the physical disruption of oocyst walls than with pepsin. An incubation in HBSS containing 0.25% (w/v) trypsin and 0.1% (w/v) sodium taurocholate after a physical treatment achieved higher and the most stable excystation rates. Modifications to this method were also examined, and no improvements were observed. The optimal excystation protocol for E. krijgsmanni was elucidated as of now.

INTRODUCTION

Eimeria spp. causing coccidiosis belong to the phylum Apicomplexa and more than 1,100 species in various hosts have been reported worldwide [8]. They exhibit strict host specificity [2, 6, 15] and are generally known as intestinal parasites [2]. Mixed infections or infections with highly pathogenetic species, such as Eimeria bovis, E. zuernii, E. tenella, and E. necatrix, in cattle and chicken often induce severe diarrhea and bloody feces [2, 12]; however, most Eimeria spp. have low pathogenicity. Coccidiosis is responsible for growth retardation and death in severe cases, which results in serious economic losses in livestock industries [1, 13, 17]. Several anti-coccidial drugs that attenuate clinical symptoms and reduce the number of oocysts discharged into feces are currently available [12]. The emergence of resistant strains and drug residues due to overuse has become problematic; therefore, the development of new drugs is needed [2, 10, 11, 13].

After the oral ingestion of mature oocysts by hosts, sporozoites are released from oocysts in the digestive tract and invade intestinal epithelial cells. Numerous merozoites are formed by repeated asexual reproduction in host cells followed by sexual reproduction, and immature oocysts are then excreted in feces. The prevention of sporozoite invasion into host intestinal epithelial cells may contribute to the attenuation of coccidiosis because merozoites are rapidly formed by asexual reproduction. Therefore, drugs that target sporozoites are expected to be effective. In vitro experiments are useful for accurately evaluating the effects of drugs against sporozoites. The isolation of oocysts from feces and the artificial excystation of oocysts are required as preparation for in vitro experiments. An efficient protocol for the collection of sporozoites will reduce costs and efforts for isolation and excystation procedures [9], thereby facilitating experiments to evaluate coccidiostats.

Eimeria spp. are important parasites in cattle and chicken; however, infection experiments using large animals, such as cattle, are difficult. Murine models have been utilized to evaluate the susceptibility of parasites to anticoccidial drugs [5, 10] because mice are widely used in experiments, are easy to manage and handle, and are microbiologically and genetically controlled. We previously examined the effects of various drugs in vivo using murine E. krijgsmanni [3, 14], and reported a high excystation rate [14]. This rate was calculated based on the number of oocysts before and after the excystation process. However, in an in vitro drug evaluation, it is important to establish an excystation protocol based on an accurate number of sporozoites obtained. Additionally, the most suitable excystation methods may vary for each Eimeria spp. because the enzymes needed for the in vivo excystation of oocysts have been shown to depend on the host species [2, 9]. In the present study, we examined the excystation rates of E. krijgsmanni using various protocols from previous studies on mice [18], rats [6, 7], and rabbits [4], which are rodents or closely-related host animals, and attempted to establish an optimal protocol for this species.

MATERIALS AND METHODS

Parasites

E. krijgsmanni was obtained from the Division of Tropical Diseases and Parasitology, Department of Infectious Diseases, Kyorin University School of Medicine, and maintained by routine passaging through mice in the Laboratory of Parasitology, United Graduate School of Veterinary Science, Kagoshima University. Oocysts were collected and purified from the feces of infected mice using the sugar centrifugal floatation method, allowed to sporulate in 2% potassium dichromate solution at 25°C, and were then stored at 4°C until used.

Excystation protocols

The excystation rates of oocysts using experimental protocols from previous studies on protocols for Eimeria spp. in small mammals commonly used as experimental animals were examined. Protocol 1, protocol 2, protocol 3A and 3B, and protocol 4 were modified protocols for E. vermiformis [18] in mice, E. separata [6] and E. nieschulzi [7] in rats, and E. stiedai [4] in rabbits, respectively. These procedures were described in detail as follows and shown in Table 1. A total of 1.0 × 10⁶ oocysts was used and 10 mL of medium was prepared for each protocol. We attempted to identify the chemical agents and incubation times achieving higher excystation rates. In protocols 1 and 3 using a vortex mixer (VORTEX-GENIE 2 Mixer; M&S Instruments INC., Osaka, Japan) to disrupt oocyst walls, the optimal processing time was examined. The numbers of oocysts, sporocysts, and sporozoites were counted every 15 sec on a hemocytometer. The most suitable processing time was selected and applied to all protocols using the vortex mixer (protocols 1, 3, and 5 to 11).

Table 1. Excystation protocols 1 to 4.

	Temperature	0.4% pepsin		Vortexing		Trypsin + STC		2.5 mM MgCl2 + 0.75% STC		1% FBS
Protocol 1	37°C	-	→	+	→	0.25% + 0.1% (24 hr)
Protocol 2	40°C	+ (2 hr)	→	-	→	0.4% + 0.75% (3 hr)	→	+ (2 hr)	→	+ (10 hr)
Protocol 3A	37°C	-	→	+	→	0.25% + 0.75% (5 hr)
Protocol 3B	37°C	+ (3 hr)	→	+	→	0.25% +0.75% (3 hr)
Protocol 4	37°C	-	→	-		0.2% + 0.8% (5 hr)

Protocols 1, 2, 3A and 3B, and 4 were modified protocols originated from [18], [6], [7], and [4], respectively. FBS: foetal bovine serum, STC: sodium taurocholate. Differences between original reports and the present sutdy are as follows: Protocol 1: deoxycholic acid →STC; Protocol 3: sodium tauroglycocholate →STC.

Protocol 1: Four milliliters of an oocyst suspension containing 1.0 × 10⁶ oocysts were equally divided into 2 polypropylene tubes (5.0 × 10⁵ oocysts in 2 mL per tube) and 2 mL of 0.6 mm glass beads were added to each tube. The suspension was processed by the vortex mixer adjusted to the 8th speed on the dial gauge and then placed in a tube. The vortexed suspension was added to phosphate-buffered saline (PBS) and washed by inverting, and the resulting supernatant was aspirated to collect parasites. Glass beads were washed as described above 4 or 5 times. The supernatants collected were centrifuged at 1,700 × g for 5 min. The sediments were suspended in Hanks’ Balanced Salts Solution (HBSS) (Sigma-Aldrich, St. Louis, MO, USA) containing 0.25% (w/v) trypsin (powder, porcine, 1:250; Sigma-Aldrich; Cat. No. 85450C) and 0.1% (w/v) sodium taurocholate (Nacalai Tesque, Kyoto, Japan) and incubated at 37°C for 24 hr.

Protocol 2: Oocysts were suspended in distilled water (DW) containing 0.4% (w/v) pepsin (FUJIFILM Wako Chemicals, Osaka, Japan) adjusted at pH 3 with 2 M HCl (Nacalai Tesque) and incubated at 40°C for 2 hr. Samples were washed twice with PBS, suspended in RPMI1640 medium (Nacalai Tesque) containing 0.4% (w/v) trypsin and 0.75% (w/v) sodium taurocholate, and incubated at 40°C for 3 hr. After washing with PBS, oocysts were suspended in RPMI1640 medium containing 2.5 mM MgCl₂ (Nacalai Tesque) and 0.75% (w/v) sodium taurocholate and then incubated at 40°C for 2 hr with inversion mixing 2 to 3 times. After washing with PBS, oocysts were suspended in RPMI1640 medium containing 1% (w/v) fetal bovine serum (FBS; Cosmo Bio, Tokyo, Japan; Cat. No. CCP-FBS-BR-500) and then incubated stably at 40°C for 10 hr.

Protocol 3A: The disruption of oocyst walls with the vortex mixer was performed as described in protocol 1. Sediments were suspended in DMEM (Nacalai Tesque) containing 0.25% (w/v) trypsin and 0.75% (w/v) sodium taurocholate and then incubated at 37°C for 5 hr.

Protocol 3B: Oocysts were suspended in DW containing 0.4% (w/v) pepsin adjusted at pH 3 with 2 M HCl, and then incubated at 37°C for 3 hr before vortexing as described in protocol 1. Sediments were suspended in DMEM containing 0.25% (w/v) trypsin and 0.75% (w/v) sodium taurocholate and then incubated at 37°C for 5 hr.

Protocol 4: Oocysts were suspended in PBS containing 0.2% (w/v) trypsin and 0.8% sodium taurocholate and then incubated at 37°C for 5 hr.

Protocols 5 to 11: Seven modified protocols for protocol 1 showing higher and the most stable excystation rate among protocols 1 to 4 were established and examined to further improve the excystation rate. The procedures of protocols 5 to 11 are shown in Table 2. A total of 1.0 × 10⁶ oocysts in 10 mL of medium was used in each protocol. In protocols 5 and 6, oocysts were incubated at 39°C and 40°C, respectively, instead of 37°C. In protocol 7, oocysts were incubated in RPMI1640 medium containing 2.5 mM MgCl₂ and 0.75% (w/v) sodium taurocholate for 24 hr after an incubation in HBSS containing 0.25% (w/v) trypsin and 0.1% (w/v) sodium taurocholate for 1 hr. In protocol 8, oocysts were incubated in DMEM containing 0.25% (w/v) trypsin and 0.75% (w/v) sodium taurocholate for 1 hr before an incubation in RPMI1640 containing 2.5 mM MgCl₂ and 0.75% (w/v) sodium taurocholate for 24 hr. Protocols 9 to 11 used pancreatin (Nacalai Tesque), α-chymotrypsin (Nacalai Tesque), and protease (FUJIFILM Wako Chemicals; Cat. No. 165-19811), respectively, instead of trypsin and oocysts were incubated for 5 hr.

Table 2. Excystation protocols 5 to 11.

Protocol 5	0.25% trypsin + 0.1% STC in HBSS at 39°C for 24 hr
Protocol 6	0.25% trypsin + 0.1% STC in HBSS at 40°C for 24 hr
Protocol 7	0.25% trypsin + 0.1% STC in HBSS at 37°C for 1 hr	→	0.75% STC + 2.5 mM MgCl2 in RPMI medium at 37°C for 24 hr
Protocol 8	0.25% trypsin + 0.75% STC in DMEM at 37°C for 1 hr	→	0.75% STC + 2.5 mM MgCl2 in RPMI medium at 37°C for 24 hr
Protocol 9	0.25% pancreatin + 0.1% STC in HBSS at 37°C for 24 hr
Protocol 10	0.25% α-chymotrypsin + 0.1% STC in HBSS at 37°C for 24 hr
Protocol 11	0.25% protease + 0.1% STC in HBSS at 37°C for 24 hr

Protocols 5 to 11 were modified from protocol 1. In all protocols, vortexing with glass beads was conducted to disrupt oocyst walls as the first step of the excystation process. STC: sodium taurocholate.

Evaluation of excystation protocols

The number of sporozoites was counted on a hemocytometer after oocyst walls had been disrupted with glass beads, namely, before and during the incubation with each reagent. Counting was performed every hour up to 3 hr and at 5 and 24 hr in protocols 1, 4, 5, and 6, every hour up to 7 hr and at 17 hr in protocols 2, every hour up to 3 hr and at 5 hr in protocols 3A, 9, 10, and 11, every hour up to 6 hr in protocol 3B, and every hour up to 4 hr and at 6 and 25 hr in protocols 7 and 8. The results of counting were evaluated by calculating excystation rates using the following formula: Excystation rate (%)=the number of excysted sporozoites / the number of matured oocysts used ×8. The number 8 indicates the number of sporozoites that form in a mature oocyst of eimerian parasites. Sporozoite viability was assessed by trypan blue staining.

Each experiment was performed in triplicate (three independent biological replicates).

RESULTS

The optimal processing time with glass beads for excystation of oocysts was examined (Fig. 1). The number of oocysts decreased, whereas those of sporocysts and sporozoites gradually increased until 60 sec. The number of sporocysts also decreased despite a continuous reduction in the number of oocysts. Furthermore, the number of sporozoites decreased after 90 sec or longer. Based on the results obtained, the disruption of oocyst walls with glass beads was performed for 60 sec in protocols 1, 3A, 3B, and 5 to 11 using the vortex mixer. Excystation rates in protocols 1 to 4 are shown in Fig. 2. In protocol 1, the excystation rate increased to approximately 6% after vortexing and the incubation with trypsin and sodium taurocholate for 1 hr, and was maintained 5 hr after the start of the incubation. In protocol 2, the excystation rate increased to approximately 4% after the incubation with pepsin for 2 hr, trypsin and sodium taurocholate for 3 hr, and MgCl₂ and sodium taurocholate for 1 hr (6 hr after the start of treatment) and did not subsequently change. In protocol 3A, the excystation rate increased to approximately 7% after vortexing and the incubation with trypsin and sodium taurocholate and was maintained 5 hr after the start of treatment. However, the dispersion of data was very large. In protocol 3B, the excystation rate did not increase during the incubation with pepsin for 3 hr. It then increased to approximately 5% after vortexing and the incubation with trypsin and sodium taurocholate for 1 hr. In protocol 4, the excystation rate did not increase. The maximum excystation rates in protocols 1, 2, 3A, 3B, and 4 were 6.4 ± 0.45, 4.4 ± 0.72, 7.7 ± 2.6, 4.8 ± 0.62, and 0.13 ± 0.18%, respectively. Protocol 4 showed a significantly low excystation rate (Tukey-Kramer, P<0.05), while the excystation rates of protocols 1 and 3 increased after a shorter time than in other protocols. In protocols 1 and 3A showing high excystation rates, protocol 1 consisted of easier procedure and showed smaller standard deviation (SD) than those of protocol 3A. Protocol 1 was regarded as the optimal among protocols 1 to 4. Protocols 5 to 11, with modifications to the incubation time and temperature of protocol 1, were performed to achieve improvements in excystation rates (Fig. 3). Excystation rates peaked with an incubation for 1 hr in each protocol. The maximum excystation rates in protocols 5, 6, 7, 8, 9, 10, and 11 were 5.4 ± 1.0, 6.0 ± 0.96, 4.3 ± 1.5, 4.1 ± 1.0, 2.9 ± 0.79, 5.2 ± 1.3, and 3.6 ± 0.47%, respectively. No significant differences were observed in excystation rates between protocols 1 and 5 to 11 (Fig. 4). The incubation times for protocols 3A and 3B were up to 5 and 6 hr, respectively. In other protocols incubated for longer than 6 hr, no further increase in the excystation rate was observed. Therefore, as shown in Figs. 2 and 3, we have presented the data up to 6 hr. Protocol 1 was optimal in the present study and its highest excystation rate was 6.4 ± 0.45%. We also verified sporozoite viability in these protocols; the rates were as follows. Protocol 1: 100% at 1 hr, 99.7% at 2 hr, 98.7% at 3 hr, 99.3% at 5 hr, and 99.0% at 24 hr; protocol 2: 99.3% at 6 hr, 98.3% at 7 hr, and 51.7% at 17 hr; protocol 3A: 99% at 1 hr and remained at 100% up to 5 hr; protocol 3B: 100% at 5 hr and 100% at 6 hr; protocol 4: viability was not measured as its excystation rate did not improve; protocol 5: 99% at 1 hr, 99% at 2 hr, and 100% at 3 and 5 hr; protocol 6: 99% at 1 hr, 99% at 2 hr, 100% at 3 hr, and 99% at 5 hr; protocol 7: 99% at 1 hr, 100% at 2 hr, 99% at 3 hr, 100% at 4 hr, 99% at 6 hr, and 100% at 25 hr; protocol 8: 99% at 1 hr, 99% at 2 hr, 99% at 3 hr, 100% at 4 and 6 hr, and 98% at 25 hr; protocol 9: 99% at 1 hr and remained at 100% up to 5 hr; protocol 10: 100% at 1 hr, 99% at 2 and 3 hr, and 100% at 5 hr; protocol 11: 100% at 1 hr and remained at 100% up to 5 hr. Sporozoite viability remained sufficiently high in all protocols up to 6 hr of incubation, and no remarkable differences were observed among them.

Fig. 1.

Relationships between vortexing times and densities of oocysts, sporocysts, and sporozoites. A suspension containing 5.0 × 10⁵ sporulated oocysts in 2 mL of medium was vortexed with 2 mL of 0.6 mm glass beads for 0–210 sec. The numbers of oocysts, sporocysts, and sporozoites were counted every 15 sec on a hemocytometer. Data are shown as the mean ± SD.

Fig. 2.

Excystation rates of protocols 1 to 4 over time. In protocols 1 and 3A, the excystation rate increased after 1 hr of incubation, while in protocols 2 and 3B, it increased after 4 hr and 6 hr, respectively. After the time shown in the graph, no further increase was observed in any of the protocols. Data are shown as the mean ± SD.

Fig. 3.

Excystation rates of protocols 5 to 11 (modifications of protocol 1) over time. In protocols 5, 6, 7, 8, 9, 10, and 11, the excystation rates reached a peak at 3, 5, 3, 4, 3, 5, and 5 hr after the start of the incubation, respectively. After the time shown in the graph, no further increase was observed for any of the protocols. Data are shown as the mean (n=3).

Fig. 4.

Maximum excystation rates of protocol 1 and its modified protocols (protocols 5 to 11). None of the other protocols achieved a higher maximum excystation rate than the original protocol 1. Data are shown as mean ± SD. P1: protocol 1, P5: protocol 5, P6: protocol 6, P7: protocol 7, P8: protocol 8, P9: protocol 9, P10: protocol 10, P11: protocol 11.

DISCUSSION

In the present study, the excystation rates of E. krijgsmanni using various protocols were examined in order to find an efficient protocol for the collection of sporozoites. Vortexing with glass beads affected the number of oocysts, sporocysts, and sporozoites obtained. It is considered that the physical disruption treatment destroyed oocyst walls and facilitated the release of sporozoites. We assumed that oocyst walls were effectively disrupted by 60 sec of vortexing; however, sporocyst walls and sporozoites were also ruptured by prolonged vortexing. The excessive physical disruption of oocyst and sporocyst walls reduced the yield of sporozoites as previously reported [7]. In protocols 2 and 4 without disruption using the vortex mixer, the excystation rate was higher in protocol 2, which included a treatment with pepsin. Therefore, an incubation with not only trypsin and sodium taurocholate, but also pepsin appears to be necessary unless the physical disruption treatment is applied for the excystation of E. krijgsmanni. This result is consistent with previous findings on murine Eimeria showing that pepsin digested the oocyst wall, leading to its collapse [16]. There were no significant differences between protocols 1 and 5 to 11. Improvements in the excystation rates of E. krijgsmanni in the present study were not dependent on the application of pancreatin, chymotrypsin, and protease as an alternative to trypsin, the concentration of sodium taurocholate, the treatment with MgCl₂ and FBS, or temperature in the present study.

Excystation rates, based on the number of sporozoites released from oocysts, were reported only in the original protocols 3A and 3B for E. nieschulzi among protocols 1 to 4, with rates of 12.75% and 56.38%, respectively [7]. In this study, after 60 sec of vortexing, the oocyst-to-sporocyst efficiency was 12.3%, as shown in Fig. 1. The excystation rates only by chemical treatment were generally more than 50%. However, the excystation rates from oocyst to sporozoite were relatively low because loss of collected parasites was also observed during washing process prior to incubation. Therefore, the type and quantity of glass beads, as well as methods for washing and parasite collection, could be further optimized. To examine sporozoites, feces are collected from host animals and oocysts are then isolated using complex procedures before the excystation process. Therefore, the establishment of an efficient excystation method is essential for progress in in vitro studies using sporozoites requiring time-consuming preparations. Excystation rates in the present study were lower than those in previous studies. However, excystation protocols for E. krijgsmanni as a useful experimental model using mice were attempted and evaluated for the first time herein, and the optimal excystation protocol was elucidated as of now.

CONFLICT OF INTEREST

The authors declare that they have no conflict of interest.