Summary

The aim of the present study was to attenuate Neospora caninum tachyzoites through continuous passage in murine macrophage and examine the virulence of attenuated tachyzoites in the mouse. The NC-1 reference isolate was 120 times passaged in the J774 cell line to produce attenuated strain. The virulence of tachyzoites was evaluated in a mouse model. Thirty-five female BALB/c mice randomly divided into seven groups. The first group, as control, was inoculated with culture media by subcutaneous injection. The second, third, and fourth groups received various dilutions (10×10⁶, 5×10⁶, 20×10⁶) of acute NC-1 tachyzoites. Other groups were inoculated by attenuated tachyzoites with the same dilutions. The skin test and ELISA assay were performed to assess the cellular and humoral immunity responses. The mortality and clinical changes were noted daily. Notably, the significant difference was observed in the mortality rate of mice inoculated by acute and attenuated tachyzoites. Besides, serum antibody titers for N.caninum were higher in mice that received attenuated N.caninum. Also, mice that received live attenuated NC-1 showed significant footpad swelling. In conclusion, the present findings indicated that the culture of N.caninum in the J774 cells led to decrease of virulence of NC-1 significantly, which is confirmed here by the response of humoral and cellular immunity. This attenuated tachyzoites might be used as a candidate for further research to develop live vaccines against N.caninum.

Keyword:Neospora caninum, murine macrophage, serial passages, Balb/c mice

Introduction

Neospora caninum was first recognized in 1984, which is an obligate, intracellular, coccidian parasite of the phylum Apicomplexa (Lindsay et al., 1996). Ithas a worldwide distribution, which causes infection in several animals such as cattle, sheep, goat, deer, horse, dog, cat, and mouse. Neonatal mortality and abortions are a significant obstacle in livestock operations, and neosporosis is a considerable cause of abortion in cattle.  (Dubey and Lindsay, 1996). N. caninum is known as one of the most efficiently trans-placental transmitted parasites (Dubey et al., 2007). The parasite seriously impacts the economic performance of the dairy and beef industries (Reichel and Ellis, 2006). Currently, there is no suitable chemotherapeutic strategy to prevent trans-placental transmission. Also, to eradicate the parasite in cattle, producing of an effective vaccine highly desirable. It is believed that vaccination may be a conceivable strategy for the control of bovine neosporosis.

A previous study showed the effective attenuation of N.caninum via serial passages (during three months) in the J774 cell line by chicken embryonated eggs, which dramatically reduced the mortality rate in chicken embryonated eggs (Khordadmehr et al., 2013). In the present study, we evaluated the virulence of attenuated N.caninum in the mouse model (in vivo) by increase the passage time, which has been previously performed in different cell lines (in vitro) and chicken embryonated eggs (in vivo) (Khordadmehr et al., 2013). The embryonated chicken is partly appropriate for N.caninum, Toxoplasma gondii, and Besnoitia (Namavari et al., 2012; Setasimy and Namavari, 2015; Namazi et al., 2010), and it is not convenient to produce immune responses. Since studies have shown the use of live attenuated strain as a vaccine has promising results (Monney and Hemphill, 2014), the present findings can be used for this purpose.

Materials and methods

Cell line and parasite-derived from J774

J774 cell line was obtained from Razi Vaccine and Serum Research Institute, which (Figure 1) was maintained in the condition of (37˚^C in an atmosphere of 95% air and 5% CO₂). The tachyzoites of NC-1 isolate were cultured in J774 cell line and they maintained in DMEM Medium (Sigma Co., USA), supplemented with 2% fetal calf serum, penicillin/ml (10,000U), streptomycin/ml (100μg) and streptomycin (25μg) (Khordadmehr et al., 2013). The whole development process of N. caninum tachyzoites in vitro in J774 cells was studied daily using an inverted microscope. Tachyzoites were taken from infected cell cultures when approximately 80–90% of the J774 host cells had lysed (Figure 2), as indicated by microscopic observation of cytopathic effects (CPE), which subsequently counted using a hemacytometer (Chamberland and Current, 1990). The incubation was continued until the maximum number of tachyzoites was released from host cells (Lv et al., 2010).

Fig. 1. J774 cell line ×100

Fig. 2. Released N. caninum tachyzoites from J774 cells ×400

Mice and experimental design

Seven groups of five female Balb/c mice were inoculated subcutaneously. As more details, the first group, as control, received culture media, the second, third, and fourth groups received acute NC-1 isolate with 5×10⁶, 10×10⁶, 20×10⁶ tachyzoites in 500μl DMEM. The fifth, sixth, and seventh groups were inoculated with 5×10⁶, 10×10⁶, and 20×10⁶ tachyzoites of attenuated NC-1 in 500μl DMEM. Mice were inspected daily for mortality and changes in clinical condition or behavior (Kargar et al., 2011; Lunden et al., 2002).

ELISA test

Three weeks after inoculation, the ELISA was used to establish the seroprevalence of anti-N. caninum antibody. ELISA was performed as previously described (Moore et al., 2005). Briefly, sera from each group were posited into 96-well plates coated with N.caninum tachyzoite antigen and diluted murine serum samples (1:50). Of note, all reactions were considered in duplicates. An ELISA reader was used (492 nm) for data analyses.

Skin test

Footpad reaction was carried four weeks after inoculation according to Omata’s method (Omata et al., 2006). Mice in each group were injected in the footpad with 50μl of PBS containing paraformaldehyde-fixed tachyzoites and the same volume of PBS injected to oppose the footpad. The average thickness of the hind footpad measured using a thickness gauge, 6 and 24 hours after injection (Omata et al., 2006).

Molecular study

For the molecular study, tissue samples (50 mg) from the brain, liver, and heart tissues of mice (after euthanizing) were removed and stored at -70˚C. DNA was extracted from all samples using the DNP™ Kit (Cinna-Gene, Iran) based on the manufacturer’s instructions. Then, the extracted DNA was stored at -70˚C for further analyses. Np21/Np6 primer pairs was used for amplification of the Nc-1 gene as follow (Kang et al., 2009; Okeoma et al., 2004):

Np21:5'-CCCAGTGCGTCCAATCCTGTAAC-3'

Np6:5'-CTCGCCAGTCAACCTACGTCTTCT-3'

The expected size of the amplicon with this primer pair is 328 bp (Kang et al., 2009; Okeoma et al., 2004). Each 25μl PCR reaction included (BioNeer, Korea):

• 2.5μl PCR buffer
• 3 mM MgCl₂
• 200μM of dNTP
• 400 nM of each primer
• 4μl of DNA

Thermal cycling was conducted as follows:

• 94˚C for 4 min (as initial denaturing)
• 25–30 cycles of 94˚C for 50 s
• 53˚C for 50 s
• 72˚C for 1 min
• 5 min heating at 72˚C (as the final extension)

Finally, six μl of PCR product was electrophoresed on 2% agarose gel.

Statistical analyses

For statistical analysis of the present data, GraphPad Prism software (GraphPad Prism 4.0, San Diego, CA) was used. The statistically significant differences between the experimental groups were evaluated by Student’s t-test (ELISA data) and Fisher exact test (for mortality rate and PCR data), and a p < 0.05 was considered statistically significant.

Results

Parasite attenuation

The present results presented rapid attenuation of virulence of N. caninum tachyzoites by continuous passages of parasites in J774 suspension cell line after 120 times during six months. These results suggest that it is possible to attenuate the virulence of tachyzoites through prolonged in vitro cultivation in J774 cell line.

Mortality rate

All mice of the control group and groups that received attenuated NC-1 remained alive. Mice that received 10×10⁶ and 20×10⁶ acute NC-1 had been died up to the second week after inoculate. Only the group that inoculated with 5×10⁶ survived but showed infection symptoms, such as reluctance to move and displayed rough hair coat (Figure 3). On the other hand, all mice in groups inoculated with 5×10⁶, 10×10⁶, and 20×10⁶ tachyzoites of attenuated NC-1 stayed alive until the end of the experiment, and there were no symptoms of infection in them (Table 1). This significant difference in mortality rates clearly demonstrated the effect of the J774 cell line on the attenuation of virulence of N. caninum tachyzoites.

Fig. 3. Balb/c mouse infected experimentally by N. caninum which showed the infection symptoms such rough hair coat

Table 1. Mortality results in different experimental groups (n = 5).

ELISA findings

Serum antibody titers for N.caninum were higher in mice that received attenuated N.caninum than control mice or mice that received acute NC-1. Based on the t-test examination, a significant difference (P ˂ 0.0001) was found between high passage tachyzoites and acute tachyzoites groups (figure 4).

Fig. 4. Results of the ELISA test. 1) Control group, 2) 5×10⁶ (Acute tachyzoite), 3) 5×10⁶ (Attenuated tachyzoite), 4) 10×10⁶ (Acute tachyzoite), 5) 20×10⁶ (Attenuated tachyzoite). The minimum and maximum of OD were observed in the control and 10×10⁶ Attenuated tachyzoite groups, respectively.

Skin test

Mice that received live attenuated NC-1 showed significant footpad swelling, while there was no significant swelling in the mice that received acute NC-1. In all mice, the mean increase in the thickness of the swelling peaked at 24h, about 1.2-fold of the thickness after injection (Figure 5).

Fig. 5. The results of the footpad thickness 24h after skin test.1) 5×10⁶ (Attenuated tachyzoite), 2) 10×10⁶ (Attenuated tachyzoite), 3) 20×10⁶ (Attenuated tachyzoite), 4) 5×10⁶ (Acute tachyzoite), 5) Control group. (Mice in other groups have died before skin test). The thickness of swelling in group received 10×10⁶ Attenuated tachyzoites is significantly higher than other groups.

Molecular finding

The results of the molecular study demonstrated the presence of parasites in the heart, liver, and brain of mice that received acute NC-1.  A significant reduction of the presence of parasite DNA in the brain, liver, and heart samples were detected in all groups that received live attenuated NC-1 in comparison with other groups. 93% of mice inoculated with attenuated tachyzoites showed negative results in PCR assay, while in groups inoculated with acute tachyzoites, only 32% were considered as negative for the presence of the parasite genome (figure 6). There was a significant difference between acute and attenuated tachyzoites groups (P ˂ 0.05).

Fig. 6. The results of the PCR test in acute and attenuated tachyzoites groups. A significant difference was observed between two groups (P ˂ 0.05).