Protective efficacy of the NcGRA7-deficient parasite as a live attenuated vaccine against Neospora caninum infection in mice

Abstract

Live vaccination is the most protective method against bovine neosporosis, which is the major cause of bovine abortion globally. Here, the Neospora caninum parenteral strain Nc1 and NcGRA7-deficient N. caninum (NcGRA7KO), which is less virulent in mice, were evaluated as potential live vaccines. BALB/c mice were subcutaneously inoculated with high (1 × 10⁵) or low (1 × 10⁴) doses of tachyzoites. At high doses, Nc1-inoculated female mice presented decreased body weight gain and increased clinical signs and died before challenge infection with green fluorescent protein (GFP)-expressing Nc1 (Nc1-GFP), whereas NcGRA7KO-inoculated animals exhibited increased survival before and after challenge infection. Although inoculation of female mice with Nc1 or NcGRA7KO resulted in a lower brain parasite number of challenged Nc1-GFP than in noninoculated animals, the total brain parasite burden in NcGRA7KO-infected mice decreased compared with that in Nc1-infetced animals. At low dose of NcGRA7KO, increased survival rates of mice and lower total brain parasite number were observed compared with high dose of NcGRA7KO. In male mice, a significant lower brain parasite burden of Nc1-GFP was observed in both high and low doses of NcGRA7-inoculated mice, and the total parasite number in the brains of low dose of NcGRA7KO-inoculated animals was lower than that in the brains of high dose of NcGRA7KO-inoculated or noninoculated animals. In conclusion, these results suggest that NcGRA7KO parasites have potential for use as a live vaccine against N. caninum infection.

INTRODUCTION

Neosporosis is a parasitic protozoan disease with a worldwide distribution [7]. Annually, neosporosis causes losses of approximately 1.3 billion US dollars in bovines through stillbirth, abortion, or the birth of weak calves [31]. Additionally, Neospora caninum infection can result in the delivery of healthy but persistently infected calves, which play a role in the persistence of the parasite through transplacental transmission to the next generation [10]. Neosporosis vaccine development has been challenging. An inactivated vaccine (Neoguard, Intervet International B.V., Boxmeer, the Netherlands) was developed and was commercially available until it was withdrawn from the market because only moderate protection against abortions was observed in field trials [35, 39]. Immunization with live tachyzoites has shown protective efficacy against fetal death in cattle [40] and decreases the abortion rate and transplacental transmission after infection in heifers before mating [38]. Another study revealed that immunization with live tachyzoites before pregnancy partially protected against fetal loss [34]. For commercially available options, the pathogenicity of live tachyzoites is the main obstacle in the development of a commercial vaccine.

Notably, an efficient vaccine against N. caninum infection should fulfill the following requirements: prevention of tachyzoite proliferation and dissemination in pregnant cattle (or other animals); prevention or minimization of oocyst shedding by dogs; and prevention of tissue cyst formation in animals that have been infected with oocysts or tissue cysts (to avoid horizontal parasite transmission to final hosts). A vaccine that stimulates both protective cellular immunity and antibody responses should achieve these goals [12, 40]. A summary of candidate vaccines against N. caninum composed of live tachyzoites or different antigens from parasite secretory organelles, such as micronemes, rhoptries, and dense granules (GRAs), that were tested in mice is available in the literature [10]. These vaccine studies revealed a significant degree of variation in the mouse strain, Neospora parasite strain, the time elapsed between vaccination and challenge infection, methods of challenge and vaccination, and vaccine adjuvants used in the studies. Additionally, vaccination with live or attenuated N. caninum tachyzoites is an efficient method of providing protection, in both mice and cattle, against acute N. caninum infection and preventing transplacental parasite transmission [10].

A subclinical N. caninum infection induces protective immunity in mice against challenge infection with a sufficient number of parasites to cause severe encephalitis. This study used the high-dose (1 × 10⁶) and low-dose (1 × 10⁴) parental strain Nc1 and parasite lysates [17]. The long-term passage of N. caninum tachyzoites can attenuate the virulence of the parasite when challenged in vivo, which may open the field for the use of the live attenuated vaccine from a long-passage parasite in tissue culture [6].

A recent study reported that the NcGRA7 antigen may play a role in decreasing the vertical transmission of N. caninum under both high and low infection doses in mice infected during early pregnancy [2]. A previously reported study revealed that NcGRA7KO strain is less virulent in non-pregnant mice [27], resulting in decreased vertical transmission of the parasites in pregnant mice model [2]. These data support the use of NcGRA7KO as a live vaccine candidate against N. caninum infection. Additionally, another recent study reported reduced virulence of the NcGRA7KO parasite in pregnant BALB/c mice [32]. One strategy for increasing the efficacy and safety of live N. caninum vaccines is the disruption of parasite virulence factors. Therefore, the aim of the present study was to evaluate the use of NcGRA7KO as a live attenuated vaccine against N. caninum infection in both female and male BALB/c mice.

MATERIALS AND METHODS

Ethics statement

This study was performed in strict accordance with the recommendations of the Guide for the Care and Use of Laboratory Animals of the Ministry of Education, Culture, Sports, Science and Technology, Japan. The study protocol was approved by the Committee on the Ethics of Animal Experiments at Obihiro University of Agriculture and Veterinary Medicine, Obihiro, Japan (permit numbers 21-30, 21-36). To decrease animal suffering, all surgical operations, blood collection, and cervical dislocation were performed under isoflurane anesthesia.

Mice

Female and male BALB/c mice, aged 10–12 weeks, were bred and housed under specific-pathogen-free conditions in the animal facility of the National Research Center for Protozoan Diseases at Obihiro University of Agriculture and Veterinary Medicine, Obihiro, Japan. The mice were initially obtained from Clea Japan (Tokyo, Japan). The animals were kept under standard laboratory conditions and provided commercial food and water ad libitum on a 12/12-hr light/dark cycle at 21°C under 40% relative humidity.

Parasites and cell cultures

NcGRA7KO, generated via the clustered regularly interspaced short palindromic repeats (CRISPR)-associated gene 9 (CRISPR/CAS9) system [27], and Nc1 and a green fluorescent protein (GFP)-expressing Nc1 strain of N. caninum (Nc1-GFP) were maintained in African green monkey kidney epithelial (Vero) cells as previously described [16, 23, 25]. The parasites were cultured in Eagle’s minimum essential medium (Sigma, St. Louis, MO, USA) supplemented with 8% heat-inactivated fetal bovine serum (Nichirei Biosciences, Tokyo, Japan), 100 U/mL penicillin, and 10 µg/mL streptomycin. Parasites and host cell debris were washed with cold phosphate-buffered saline (PBS), and the final pellet resuspended in cold PBS was passed through a 27-gauge needle and a 5.0-µm-pore size filter (Millipore, Bedford, MA, USA). After 72 hr, the parasites were washed with PBS by centrifugation at 1,500 × g for 10 min before being filtered again, and the number of parasites was then hemocytometrically counted for each experiment.

Experimental design

To estimate the virulence of the two parasite lines (Experiment 1, Table 1), Nc1 and NcGRA7KO, six mice per group were used. Female mice were inoculated subcutaneously with 0.1 mL of RPMI-1640 medium containing 1 × 10⁵ Nc1 or NcGRA7KO tachyzoites or with RPMI-1640 medium alone as noninoculated mice. At 45 days after inoculation, all survived mice were challenged intraperitoneally with 2 × 10⁶ Nc1-GFP tachyzoites in 0.4 mL/mouse, and postmortem examination was performed at 30 days post challenge infection (corresponding to 76 days after inoculation).

Table 1. Experimental design for vaccination and challenge with Neospora caninum in different mouse models.

	Mouse group	Mouse model	Parasite vaccination dose	Number of animals used / group (initial mouse number)	Challenge by Nc1-GFP tachyzoites/mouse (dose)	Time of scarification	Parameters observed
Experiment 1 (Fig. 1)	Group A (non-inoculated)	Female	Non-inoculated	6 (6)	Yes (2 × 106)	Scarified at 76 days post-vaccination (30 days post-infection)	Body weight change Clinical score Survival rate Parasite DNA in the brain (vaccinated and challenged parasites)
	Group B (Nc1-inoculated)		1 × 105	3 (6)
	Group C (NcGRA7KO-inoculated)		1 × 105	5 (6)
	Group D (Nc1-inoculated)		1 × 105	6 (6)	No challenge
	Group E (NcGRA7KO-inoculated)		1 × 105	6 (6)
Experiment 2 (Fig. 2)	Group A (non-inoculated)		Not vaccinated	6 (6)	Yes (2 × 106)
	Group B (NcGRA7KO- inoculated)		1 × 104	6 (6)
	Group C (NcGRA7KO- inoculated)		1 × 105	6 (6)
Supplemental Experiment (Supplementary Fig. 1)	Group A (non-inoculated)		Not vaccinated	6	No challenge	Scarified at 115 days post-vaccination (51 days post-infection)
	Group B (Nc1-vaccinated)		1 × 104	6
	Group C (NcGRA7KO-vaccinated)		1 × 104	6
Experiment 3 (Fig. 3)	Group A (unvaccinated)	Male	Not vaccinated	6 (6)	Yes, (2 × 106)	Scarified at 76 days post-vaccination (30 days post-infection)
	Group B (NcGRA7KO-inoculated)		1 × 104	6 (6)
	Group C (NcGRA7KO- inoculated)		1 × 105	5 (6)

The number of mice, vaccination and challenge doses, and timing of mice sacrifice for each performed experiment. In Experiment 1, six female mice per group were vaccinated with the Neospora caninum parenteral strain Nc1 or NcGRA7-deficient N. caninum (NcGRA7KO) or were injected with RPMI-1640 medium as an unvaccinated control. Groups A–C were challenged, and groups D and E were not challenged. Daily observations were performed until 76 days after inoculation (30 days post-challenge infection [dpi]). The data are shown in Fig. 1. In Experiment 2, six female mice were used for each group, and all animals were vaccinated with 10⁵ or 10⁴ NcGRA7KO parasites. Daily observations were performed until 76 days after inoculation (30 dpi). Brain samples were collected from all mice at their time of death or scarification at 76 days after inoculation for use in qPCR to determine the N. caninum distribution, the data were shown in Fig. 2. In supplemental experiment, under chronic conditions; daily observations were performed until 115 days after inoculation (51 dpi), six female mice per group were inoculated with 10⁴ Nc1 or NcGRA7KO parasites or were injected with RPMI-1640 medium to check the virulence until 115 days after inoculation. Brain samples were collected from all animals at the time of death or at 115 days after inoculation (51 dpi) for use in quantitative (q) PCR to determine the N. caninum distribution. In Experiment 3, six male mice were used for each group, and all animals were vaccinated with 10⁵ or 10⁴ of NcGRA7KO parasites. Daily observations were performed until 76 days after inoculation (30 dpi). Brain samples were collected from all mice at their time of death or scarification at 76 days after inoculation for use in qPCR to determine the N. caninum distribution. The data are shown in Fig. 3.

To compare the effects of inoculation dose (Experiment 2, Table 1), female mice were inoculated subcutaneously with a low dose (1 × 10⁴) or high dose (1 × 10⁵) of NcGRA7KO tachyzoites in a 0.1 mL volume or with RPMI-1640 medium alone as noninoculated mice. The daily body weight and clinical score of each mouse were recorded from −2 (2 days before inoculation) to 76 days after inoculation.

Clinical scores were assigned according to the extent of hunching, piloerection, warm-seeking behavior, ptosis, sunken eyes, ataxia, the latency of movement, flaccidity, latent touch reflexes, skin and eye reflexes, and belly lying observed in the mice and were represented by a score ranging from 0 (no symptoms) to 10 (complete symptoms), in accordance with previous studies [3, 9, 13]. At 45 days after inoculation, all the mice were challenged intraperitoneally with 2 × 10⁶ Nc1-GFP tachyzoites, and a postmortem examination was performed at 30 days post challenge infection (76 days after inoculation). Brain tissues for measuring parasite burden were collected from all the mice. The animals were sacrificed in accordance with the ARRIVE guidelines 2.0 according to a reported study [36]. The use of the mouse model, type of used strain for vaccination, dose of vaccination and challenge, parameters, and analysis performed for each experiment was summarized in Table 1.

To ensure the efficacy of the live parasite NcGRA7KO and compare the virulence of both Nc1 and NcGRA7KO under chronic conditions, female mice were inoculated subcutaneously with a low dose (1 × 10⁴) of both Nc1 and NcGRA7KO tachyzoites in a 0.1 mL volume or with RPMI-1640 medium alone as an unvaccinated control. The mice were observed until 115 days after inoculation (Table 1, Supplementary Fig. 1).

The same protocols performed in Experiment 2 were performed on male animals (Experiment 3; Table 1, Fig. 3).

DNA isolation and quantitative qPCR to determine N. caninum distribution in the brain

DNA was extracted from the brains of the mice from all the experiments. The Brain was thawed in 10 volumes of extraction buffer (0.1 M Tris-HCl [pH 9.0], 1% SDS, 0.1 M NaCl, 1 mM EDTA) and 20 µg/mL proteinase K at 50°C. The resulting DNA was purified via phenol‒chloroform extraction and ethanol precipitation. Parasite DNA was then amplified with N. caninum Nc5 gene-specific primers [29] (forward 5′-ACT GGA GGC ACG CTG AAC AC-3′ and reverse 5′-AAC AAT GCT TCG CAA GAG GAA-3 for the parasites used for vaccination and with GFP gene-specific primers (forward 5′-TGG CCC TGT CCT TTT ACC AGA-3′) and reverse 5′-TCC ATG CCA TGT GTA ATC CCA-3′) for the parasites used for challenge infection. Amplification, data acquisition, and data analysis were performed with the ABI Prism 7900HT sequence detection system (Applied Biosystems, Waltham, MA, USA), and the cycle threshold (Ct) values based on Nc5 gene and GFP gene were calculated as described previously [29]. Standard curves were constructed via 10-fold serial dilutions of N. caninum DNA extracted from 10⁵ parasites; thus, each curve ranged from 0.01 to 10,000 parasites. Parasite numbers were calculated from the standard curve.

Statistical analysis

GraphPad Prism 8.3.4 software (GraphPad Software Inc., La Jolla, CA, USA) was used for data analysis. The data are presented as the means ± standard deviations (SDs). Statistical analyses were performed via the Mann–Whitney U test and two-way analysis of variance (ANOVA), followed by the Tukey‒Kramer post hoc test for group comparisons. Survival rates and significant differences were assessed via a log-rank Mantel‒Cox test. Statistically significant differences, defined by P values of <0.05, are marked in the figures with asterisks and defined in each corresponding figure legend together with the name of the statistical test that was used.

RESULTS

Parasite virulence and comparative protective efficacy of 10⁵ inoculation with Nc1 and NcGRA7KO in female mice

To confirm parasite virulence, we first performed Experiment 1 (Table 1, Fig. 1). Female mice were inoculated with 1 × 10⁵ of either parental strain Nc1 or NcGRA7KO tachyzoites or with RPMI media as a noninoculated control. At 30 days post-inoculation, the mice in Groups D and E were sacrificed to assess the parasite burden in the brain (Fig. 1A). Three Nc1-inoculated mice in Group B and one NcGRA7KO-inoculated mouse in Group C died before the challenge, due to the virulence of live parasites. The parasite burden in the brains of NcGRA7KO-infected mice tended to be lower than that in the brains of Nc1-infected animals, but a significant difference was not detected (Fig. 1A). Furthermore, higher clinical scores and body weight loss were observed in Nc1-inoculated mice than in NcGRA7KO-inoculated animals 45 days after inoculation (Fig. 1C, 1D).

Fig. 1.

Parasite virulence and comparative protective efficacy of the inoculation of 10⁵Neospora caninum parenteral strain Nc1 and NcGRA7-deficient N. caninum (NcGRA7KO) in female mice. Female mice were inoculated with 1 × 10⁵ of either Nc1 or NcGRA7KO tachyzoites or with RPMI media as a noninoculated control. (A) Parasite burdens in the brains of the mice (n=6 per group) at 30 days after inoculation. The values represent the number of parasites in 50 ng of brain tissue DNA. Differences were analyzed for statistical significance via the Mann–Whitney U test, but no significant differences were found. (B) Mice inoculated with 1 × 10⁵ of either parental strain Nc1 or NcGRA7KO tachyzoites or RPMI media were challenged with green fluorescent protein (GFP)-expressing Nc1 (Nc1-GFP) at 45 days after inoculation. Kaplan–Meier survival curve of the surviving mice 76 days after inoculation were generated (survival rates: unvaccinated, 5/6, 83.3%; Nc1, 1/6, 16.67%; NcGRA7KO, 4/6, 66.67%). The statistical significance of differences was analyzed with a log-rank test; *P<0.05. (C) Clinical scores of the surviving mice 76 days after inoculation. (D) The body weight percentage (%) of the surviving mice was calculated from the initial body weight on Day 0 (inoculation day) and the daily body weight through 76 days after inoculation. (E, F) Parasite burdens in the brains of surviving mice at 76 days after inoculation and of the dead animals (black symbols). The values represent the number of total parasites detected by the Nc5 gene (E) and Nc1-GFP detected by the GFP gene (F) in 50 ng of brain tissue DNA. Differences were analyzed for statistical significance via two-way ANOVA plus the Tukey‒Kramer post hoc test; *P<0.05.

To investigate the protective efficacy of both strains Nc1 and NcGRA7KO against challenge infection with N. caninum, the mice from Groups A, B and C (Experiment 1, Table 1) were subsequently challenged with 2 × 10⁶ Nc1-GFP at 45 days after inoculation, and three and one Nc1-inoculated mice in Group B and NcGRA7KO-inoculated mouse in Group C died before challenge, respectively (Fig. 1B). The mortality of Nc1-inoculated mice was significantly greater than that of noninoculated mice, whereas the mortality of NcGRA7KO mice was similar to that of noninoculated animals (Fig. 1B). qPCR targeting of the Nc5 gene in both the first inoculated strain and the challenged strain confirmed a significantly greater number of parasites in the brains of Nc1-inoculated mice compared with control animals (Fig. 1E). However, when the number of challenged Nc1-GFP parasites was quantified, the results of qPCR targeting the GFP gene indicated that the number of Nc1-GFP parasites in Nc1- and NcGRA7KO-inoculated mice was significantly lower than that in noninoculated control animals (Fig. 1F).

Protective efficacy of high-dose and low-dose inoculation with NcGRA7KO in female mice

To further confirm the above results, we performed Experiment 2 to compare the protective efficacy of low- (1 × 10⁴ parasites) and high-dose (1 × 10⁵ parasites) inoculation with NcGRA7KO (Table 1, Fig. 2). Although the survival rates of both high- and low-dose-inoculated mice at 30 days before challenge were 100%, the highest mortality rate (50%) was observed in high-dose-inoculated mice following challenge infection (Fig. 2A). Importantly, no mortality was confirmed in low-dose-inoculated mice (Fig. 2A). In addition, mild clinical symptoms, such as lower clinical scores and fewer body weight changes, were observed in low-dose-inoculated mice than in high-dose-inoculated mice and noninoculated mice in Experiment 2 (Fig. 2B, 2C). The total parasite number in inoculated and challenged parasites from low-dose-inoculated mice tended to be lower than that in noninoculated mice and high-dose-inoculated mice, while no significant difference was observed (Fig. 2D). Additionally, the number of challenged parasites in both high- and low-dose-inoculated mice was lower than that in noninoculated mice, while no significant difference was seen (Fig. 2E).

Fig. 2.

Protective efficacy of high-dose and low-dose inoculation with NcGRA7-deficient N. caninum (NcGRA7KO) in female mice. Female mice were inoculated with 1 × 10⁴ (low dose) or 1 × 10⁵ (high dose) NcGRA7KO tachyzoites or RPMI media as a noninoculated control (n=6 per group). The mice were challenged with green fluorescent protein (GFP)-expressing Nc1 (Nc1-GFP) at 45 days after inoculation. (A) Kaplan‒Meier survival curve of the surviving mice 76 days after inoculation were generated (survival rates: unvaccinated, 5/6, 83.3%; low-dose, 6/6, 100%; high-dose, 3/6, 50.0%). The statistical significance of differences was analyzed with a log-rank test, but no significant differences were found. (B) Clinical scores of the surviving mice 76 days after inoculation. (C) The body weight percentage (%) of the surviving mice was calculated from the initial body weight on Day 0 (inoculation day) and the daily body weight through 76 days after inoculation. (D, E) Parasite burdens in the brains of surviving mice at 76 days after inoculation and of the dead animals (black symbols). The values represent the number of total parasites detected by the Nc5 gene (D) and Nc1-GFP detected by the GFP gene (E) in 50 ng of brain tissue DNA. Differences were analyzed for statistical significance via two-way ANOVA plus the Tukey‒Kramer post hoc test, but no significant differences were found.

When we monitored mouse virulence under low dose inoculation of Nc1 and NcGRA7KO until 115 days after inoculation, survival rate of NcGRA7KO-infected mice (4/6, 66.7%) was significantly higher than that of Nc1-infected animals (1/6, 16.7%) (Supplementary Fig. 1A). Furthermore, a lower mouse virulence of NcGRA7KO was confirmed based on lower clinical score, fewer body weight changes, and lower parasite number in the brain, compared with Nc1 (Supplementary Fig. 1B–D).

Protective efficacy of high- and low-dose inoculation with NcGRA7KO in male mice

The protective efficacy of NcGRA7KO inoculation was also evaluated in a male mouse model. Before the challenge infection, there were no significant differences in mouse survival, clinical scores, or body weight changes between the low-dose-inoculated and noninoculated animals, whereas the high-dose-inoculated animals died and exhibited a decrease in body weight compared with the unvaccinated animals (Fig. 3A‒C). Compared with that of the low-dose-inoculated animals, the body weight of the noninoculated animals decreased from 65–76 days after inoculation (Fig. 3C). The mice that received the low inoculation dose were the healthiest among all the experimental groups; the clinical scores of the inoculated animals were lower than those of the noninoculated animals at 49–73 days after inoculation (Fig. 3B). No significant difference was observed in the survival rate between the noninoculated animals (6/6, 100%) and either the high-dose-inoculated (5/6, 83.3%) or low-dose-inoculated (6/6, 100.0%) animals (Fig. 3A). The total parasite number from inoculated and challenged parasites of low-dose-inoculated mice tended to be lower than that of noninoculated mice and high-dose-inoculated mice, except for one animal (Fig. 3D). However, the number of challenged parasites in high- and low-dose-inoculated mice was significantly lower than that in noninoculated control mice (Fig. 3E).

Fig. 3.

Protective efficacy of high- and low-dose inoculation with NcGRA7-deficient N. caninum (NcGRA7KO) in male mice. Male mice were inoculated with 1 × 10⁴ (low dose) or 1 × 10⁵ (high dose) NcGRA7KO tachyzoites or RPMI media as a noninoculated control (n=6 per group). The mice were challenged with green fluorescent protein (GFP)-expressing Nc1 (Nc1-GFP) at 45 days after inoculation. (A) Kaplan‒Meier survival curves of the surviving mice 76 days after inoculation were generated (survival rates: uninoculated, 6/6, 100%; low-dose, 6/6, 100%; high-dose, 5/6, 83.3%). The statistical significance of differences was analyzed with a log-rank test, but no significant differences were found. (B) Clinical scores of the surviving mice 76 days after inoculation. (C) The body weight percentage (%) of the surviving mice was calculated from the initial body weight on Day 0 (inoculation day) and the daily body weight through 76 days after inoculation. (D, E) Parasite burdens in the brains of surviving mice at 76 days after inoculation and of the dead animals (black symbols). The values represent the number of total parasites detected by the Nc5 gene (D) and Nc1-GFP detected by the GFP gene (E) in 50 ng of brain tissue DNA. Differences were analyzed for statistical significance via two-way ANOVA plus the Tukey‒Kramer post hoc test; *P<0.05.

DISCUSSION

An effective vaccine against neosporosis should aim not only to prevent abortion or vertical transmission in infected animals but also to prevent the subsequent infection of uninfected animals [14]. Multiple types of live vaccines against neosporosis have been developed in either mouse or cattle models, including less virulent strains, attenuated strains, or genetically modified strains [22].

Significant protection levels against vertical transmission in mice have been confirmed in previous studies [8, 20, 21, 33]. The summary of the vaccine studies in murine models performed against neosporosis in different mouse strains (C57/BL6 and BALB/c) has been summarized by Hemphill et al. [11]. Comparison among vaccine studies is difficult since the models employed exhibit a large degree of variation, not only concerning the tested vaccine but also concerning the mouse stains, the used Neospora strains, the time elapsed between vaccination and challenge, vaccination and challenge route, and vaccine dose [10, 11].

In experimental animal models, protective efficacy has been induced by immunization with live N. caninum tachyzoites. Recent studies have shown that the use of the parental strain NcLiv at a sublethal vaccination dose (1 × 10⁵) has protective effects and reduces vertical transmission of Nc-Sp7 in pregnant BALB/c mice [41]. Immunization of female BALB/c mice with naturally attenuated Nc-Spain 1 H tachyzoites induced a protective immunity after twice subcutaneously vaccination doses ranging from 5 × 10 to 5 × 10⁵ to control both congenital and cerebral neosporosis [33]. In our study, use of Nc1 under both high (1 × 10⁵) and low immunization doses (1 × 10⁴) showed high virulence in non-pregnant mice (Fig. 1, Supplementary Fig. 1). These results conclude that the efficiency of the Nc1 strain might differ from other strains in BALB/c mice. Our study together with the previously reported study [20] confirmed the use of a lower dose of live tachyzoite as an initial approach for manufacturing a vaccine against neosporosis. A study shows protective efficacy against a lethal challenge of N. caninum through inoculation with attenuated or virulent live tachyzoites. BALB/c mice were inoculated with either 1 × 10⁶ live virulent tachyzoites or 1 × 10⁶ live attenuated tachyzoites and were challenged 28 days later with 5 × 10⁶ virulent parasites. The parasite burden was lower in the brains of mice inoculated with the attenuated tachyzoites (56%) than those inoculated with live virulent tachyzoites (86%). This study shows that it is possible to protect against a lethal challenge of N. caninum through inoculation with live attenuated rather than live virulent tachyzoites [5]. Therefore, the use of the mutant strain of less virulence might be necessary to be used as a live vaccine candidate against the challenge infection with N. caninum in this study.

Currently, gene-editing technologies such as the CRISPR/Cas9 system are useful for reducing the virulence of N. caninum [4, 32]. Molecules involved in invasion and PV formation are targets for attenuation of parasites. Since the major surface antigen NcSAG1 plays a crucial role in parasite adhesion and invasion of host cells [26], NcSAG1 knockout parasites significantly decrease the infection rate and egress rate, resulting in reduced virulence in mice [1]. Additionally, deficiency of the rhoptry proteins of N. caninum, NcROP5 and NcROP16, causes reduced mortality in mice [18, 19]. Knockout of the dense granule protein NcGRA7 also reduces virulence in mice [27]. Thus, the deletion of a gene encoding a virulence factor is a promising strategy for the future development of a live attenuated vaccine.

Reported studies illustrate the use of transgenic live tachyzoites as a live vaccine against N. caninum infection were limited. A previous study using an attenuated live strain of Toxoplasma gondii, which is closely related intracellular protozoan parasites with N. caninum, deleted for the Mic1 and Mic3 genes (TgMic1-3KO), the outbred Swiss OF1 mice were immunized with 1 × 10⁴ Mic1-3KO parasites orally or 1 × 10⁶ parasites intraperitoneally and were then challenged with of 5 × 10⁷N. caninum tachyzoites (lethal dose) at 50 days after immunization. The vaccination with TgMic1-3KO induces protection and increases survival against N. caninum infection (Survival rates: 70 and 80%, respectively) [30]. Regarding N. caninum strains, Nc1 strain and 2 independent transgenic knock-in clones, Nc-1SAG4c1.1 and Nc-1SAG4c2.1, that express the bradyzoite stage-specific protein NcSAG4 in the tachyzoite stage have been tested for their protective efficacy against neosporosis in BALB/c mice [20]. The vaccination was performed twice subcutaneously with dose of 5 × 10⁵ tachyzoites of each strain and challenged with 2 × 10⁶ tachyzoites three weeks after the booster dose. Nc-1 SAG4c2.1 strain appears its high levels of protection against vertical transmission and its lower persistence in mice, suggesting a safe live vaccine.

In our study, the comparative efficacy of inoculation with the parental strains Nc1 or the knock-out strain NcGRA7KO was evaluated in female and male mice to check the safety of the live tachyzoites as a vaccine. In female mice, inoculation with 1 × 10⁵ NcGRA7KO was safer than inoculation with 1 × 10⁵ Nc1, as demonstrated by the higher survival rate of mice (Nc1: 3/6, 50.0%), NcGRA7KO: 5/6, 83.3%) and lower number of brain parasites in the inoculated female mice before challenge infection (Fig. 1A, 1B). Similar results were obtained from inoculation with 1 × 10⁴ NcGRA7KO at the chronic stage of infection, indicating a higher survival rate and lower parasite burden in the brains of the inoculated female mice (Supplementary Fig. 1A, 1D). Our findings support the effective protective immune responses induced by reduced virulence of NcGRA7KO under low inoculation dose. This was confirmed by the lower total parasite DNA in the low-vaccinated female animals than in the uninoculated animals and high-dose-vaccinated animals (Fig. 2D). Our previous study reported that NcGRA7 mainly regulates host cytokine and chemokine production, which play essential roles in N. caninum pathogenesis [27]. Furthermore, NcGRA7 may prevent the aggregation of the cellular immune factor, immunity-related GTPase family member a6 (IRGa6) at the parasitophorous vacuole membrane (PVM) for intracellular survival [37]. However, the immune response induced by NcGRA7 via oligomannose-coated liposomes [24] or recombinant adenovirus expressing the NcSRS2-NcGRA7 fusion protein confers protection against N. caninum infection in mouse models [15]. In an infection model using male Holstein calves, vaccination with oligomannose-coated liposome-entrapped NcGRA7 recombinant protein caused the induction of N. caninum-specific immunity, resulting in a decreased brain parasite load [28]. According to published papers, NcGRA7-specific immunity plays a crucial role in the inhibition of brain infection and vertical transmission of N. caninum. Thus, the immune response induced by infection and that induced by antigen inoculation may play different roles.

In cattle, immunization with Nc-Spain 1H live tachyzoites also induced a protective immune response, which partially reduced the rates of N. caninum-associated abortions and transplacental transmission of the parasite [34]. The vaccination of heifers with live Nc-Nowra tachyzoites before breeding induces protection in 55.6% to 85.2% of vaccinated animals, depending on the immunization route (subcutaneous or intravenous) and vaccine strain growth conditions; this vaccination also reduces the rates of abortion and the presence of parasite DNA in calves, as determined by PCR [38]. Another study reported that the immunization of cattle with live tachyzoites from the Nc-Nowra strain before pregnancy conferred 100% protection from fetal death, whereas vaccination with parasite lysate failed to provide any protection [40]. Thus, the use of a live attenuated vaccine has great potential for practical application against bovine neosporosis.

Live vaccination is the most effective method for protecting against bovine neosporosis, a leading cause of bovine abortion worldwide. In this study, the parenteral Nc1 strain and the less virulent NcGRA7KO strain were evaluated as potential live vaccines. The results demonstrate that NcGRA7KO live parasites exhibit reduced virulence in mice while inducing protective efficacy in male and non-pregnant female animals. These findings highlight the potential of live attenuated tachyzoites from NcGRA7KO parasites as a less virulent vaccine against N. caninum infection. However, inoculation of NcGRA7KO under low inoculation dose killed mice from 80 days after the injection, suggesting remaining pathogenicity. Thus, further studies are needed to develop more less virulent strain based on NcGRA7KO such as multiple-knock out strain of virulent genes.

CONFLICT OF INTEREST

The authors declare that they have no financial or competing interests concerning this study.