J. Parasitol., 100(6), 2014, pp. 812–816 Ó American Society of Parasitologists 2014
FIRST ISOLATION OF NEOSPORA CANINUM FROM BLOOD OF A NATURALLY INFECTED ADULT DAIRY COW IN BEIJING, CHINA Pan Hao*, Na Yang*, Xia Cui, Jing Liu, Daoyu Yang, and Qun Liu Key Laboratory of Animal Epidemiology and Zoonosis, Ministry of Agriculture, National Animal Protozoa Laboratory, College of Veterinary Medicine, China Agricultural University, Beijing 100193, China. Correspondence should be sent to: [email protected] ABSTRACT: Neospora caninum is an important cause of abortion in cattle worldwide, but the isolation of a viable parasite from an abortus is difficult, and viable N. caninum has not been isolated from any host in China. In the present study, peripheral blood samples were collected from a jugular vein of an adult dairy cow that had aborted; the cow was seropositive to N. caninum antibodies by ELISA. White blood cells were separated and seeded onto Vero cell monolayer cultures for parasite isolation. Tachyzoites were first observed in cell culture on day 84 after initial inoculation. The parasite was confirmed to be N. caninum by gene sequencing and immunofluorescence, and by bioassays in BALB/c mice. The new N. caninum isolate (NC-Bj) has a unique pattern on microsatellite Cont-14. To our knowledge, this is the first successful isolation of N. caninum in China from any host.
monolayer cell culture. The tachyzoites obtained from the cell culture were sub-passaged on Vero cells and cryopreserved in liquid nitrogen.
Neospora caninum is an important obligate intracellular protozoan that can cause serious disease in cattle and dogs worldwide, and occasionally in other animals. Much has been published on the biology of N. caninum since it was described as a new genus and species in 1988, but neosporosis continues to be a major problem in cattle as it causes abortion and neonatal mortality (Dubey and Schares, 2011). Losses due to neosporosis in the cattle industry are considerable (Dubey et al., 2007; Dubey and Schares, 2011). Seroepidemiological surveys by us and others have shown that N. caninum infection is widely spread in dairy cattle in China (Liu et al., 2007). Additionally, N. caninum has been demonstrated in histological sections of an aborted bovine fetus in China (Zhang et al., 2007). In spite of many attempts, however, we have failed to isolate viable N. caninum in more than 80 aborted bovine fetal tissues. In the present paper, we report the first isolate of N. caninum in China obtained from the peripheral blood of a naturally infected cow and preliminary characterization of this isolate.
Identification of N. caninum DNA in the aborted fetus and cell cultures Genomic DNA was extracted from the brain of the fetus from the cow mentioned here using the phenol-chloroform method. Genomic DNA was also isolated from cell cultures collected 103 days post-inoculation using the blood, cell, and tissue genomic DNA fast extraction kit (Aidlab Biotechnologies, Beijing, China), according to the manufacturer’s instructions. A fragment of N. caninum–specific Nc5 gene (GenBank X84238) was amplified by nested-PCR. The external primer pairs were Np6/Np21, and the internal primers were Np9/Np10 (McInnes et al., 2006). To avoid the carryover of contaminating nucleic acids, each step of the procedure was performed in a different room, and aerosol-resistant pipette tips were used. The primary PCR was carried out in a total volume of 50 ll, which consisted of 25 ll 23 TransHiFi superMix I (Transgen, Beijing, China), 20 pM of each primer, 2 ll of the DNA template, and sterile ultrapure distilled water. NC-1 strain genomic DNA was used as the positive control (0.8 lg, 2 ll), and distilled water was used as the negative control. Toxoplasma gondii DNA (1 lg, 2 ll) was used to test the specificity of the nested PCR. The PCR program for both primary and secondary reaction included an initial pre-denaturation at 94 C for 5 min; 35 cycles of denaturation at 94 C for 30 sec, annealing at 63 C for 30 sec, and extension at 72 C for 30 sec; and a final extension at 72 C for 10 min. The templates for the secondary PCR were 0.5 ll of the primary reaction products. Another PCR was conducted to amplify the entire intergenic region between the 18S and 25S rRNA gene of approximately 1,000 bp in size. The primers NC 18S RNA (sense) 5 0 -TGCGGAAGGATCATTCACACG-3 0 and NC 25S RNA (antisense) 5 0 -CCGTTACTAAGGGAATCATAGTT-3 0 were used. The PCR reaction was performed as previously described (Vitale et al., 2008). Microsatellite DNA targets MS10 (Tand-3) and Cont-14 were amplified to confirm the identity of NC-Bj. Separate PCR for the 2 targets was performed, and the sizes of amplicons were monitored in agarose gels as described previously (Al-Qassab et al., 2010). All PCR products were visualized by electrophoresis in a 1% agarose/ GoldViewe gel in 13 TAE buffer. After purification using spin columns (Solarbio, Beijing, China), each amplicon was linked to pEASY-T1 simple vector (Transgen Biotech) for sequencing in a local company (Sunbiotech, Beijing, China).
MATERIALS AND METHODS Animal and serological analysis A 3-yr-old Holstein cow from a herd from a southwest suburb of Beijing, China, aborted at 254 days in her first gestation. Blood samples was obtained from a jugular vein the next day, centrifuged at 1,000 g for 15 min, and the serum was stored at 20 C until used. An ELISA was performed to detect antibodies against N. caninum as described by Liu et al. (2007). Parasite isolation from the blood Because the cow was found to have N. caninum antibodies by ELISA, another 10 ml sample of blood was collected in EDTA tubes 1 wk after the abortion. White blood cells were separated according to the method described by Bien et al. (2010). Briefly, the buffy coats were collected after the blood with EDTA was centrifuged. White blood cells with buffy coats were separated by Ficoll-paque (1.077 g/ml; M & C, Beijing, China) and centrifugation. The cells at the interface were collected, washed, and added to Vero cell monolayer cultures at 37 C with 5% CO2, in DMEM medium (M & C) supplemented with 2% fetal bovine serum, 100 U/ml penicillin, and 100 lg/ml streptomycin. Flasks containing cells were examined daily using an inverted microscope (Olympus CKX41, Tokyo, Japan). Every 2 days, the culture medium was changed, and every 2 wk, Vero cells were transferred to a new culture bottle. On each passage, the cultures were scraped, passed through a 27 gauge needle, and inoculated onto a new
Antigenic reactivities Reactivities of the Chinese bovine N. caninum isolate (NC-Bj) to N. caninum (NC-1) and T. gondii (RH) antisera were examined by indirect fluorescent antibody test (IFAT) as described previously (Yamane et al., 1997). Positive and negative control sera to either N. caninum or T. gondii were titrated at a 2-fold dilution from 100 to 200 and 400. The end-point titer was the last serum dilution that exhibited clear, whole parasite fluorescence.
Received 24 February 2014; revised 11 June 2014; accepted 11 June 2014. * These authors contributed equally to this work. DOI: 10.1645/14-498.1
Bioassay in mice Six-week-old female BALB/c mice were purchased from a local supplier (Laboratory Animal Center of Academy of Military Medical Sciences, 812
HAO ET AL.—FIRST ISOLATION OF N. CANINUM IN CHINA
Beijing, China) and acclimatized for 1 wk before the experiment. Mice were housed in groups of 5 in plastic box cages and provided with rodent feed and water ad libitum under standard conditions. The parasites were collected from 2-day-old growth cultures when most tachyzoites were still intracellular. The cultures were scraped from the flasks and passed through 27 gauge needles 3 times to fracture the cells and PV membranes and disperse the tachyzoites into individual cells. Tachyzoites were harvested by centrifugation at 1,100 g for 10 min and then washed twice in sterile PBS. Parasite viability was determined by trypan blue exclusion, followed by counting 3 aliquots in a Neubauer chamber. The mice were inoculated with 1 3 106 to 5 3 106 tachyzoites (200 ll) per mouse intraperitoneally. The control group was injected intraperitoneally with 200 ll of sterile PBS. Inoculated mice were monitored daily. The blood, brains, hearts, lungs, livers, and spleens from mice inoculated with 1 3 106 tachyzoites were sampled on days 1, 2, 4, 8, 16, 32, and 64 post-inoculation (PI) according to a well-established mouse model (Collantes-Ferna´ndez et al., 2006). Five mice in the infected group and 2 mice in the control group were sacrificed by CO2 inhalation and sampled at each time point. Mice inoculated with 5 3 106 tachyzoites were examined daily for illness from their inoculation until 32 days PI. Mice were killed by CO2 inhalation at the end of the experiment. All animal-handling procedures were conducted according to institutional guidelines for animal ethics. Kinetics of N. caninum DNA in target organs of inoculated mice Immediately after blood samples were collected from the orbital sinus, 100 ll aliquots of each sample were transferred into a new tube. The rest of the sample was kept for IgG detection. Genomic DNA was extracted from 100 ll of blood and 20 mg of target organs using the blood, cell, and tissue genomic DNA fast extraction kit (Aidlab Biotechnologies Co.) according to the manufacturer’s instructions. All DNA was extracted within 24 hr after samples were collected. A nested-PCR for the Nc5 gene was performed to detect whether N. caninum existed in the target tissues using Np6/Np21 and Np9/Np10 as external and internal primer pairs, respectively. One microliter of the DNA solution was used as the template in the primary amplification, and 0.5 ll of the primary products was used in the secondary amplification. The reaction system was 25 ll in both amplifications, and 23 Taq Mix (CWBIO, Beijing, China) was used. The other conditions were the same as mentioned above. Parasite load kinetics in the brain was done by real-time PCR as described previously (Collantes-Ferna´ndez et al., 2002). The PCRs for the Nc5 gene and 28S rRNA were carried out as separate assays. Amplifications were performed with an ABI PRISMt 7500 Real-time PCR system, using SYBRt Premix Ex TaqTM II Real Time kit (TaKaRa, Dalian, China) as recommended protocol. All standards and samples were analyzed in duplicate. The results were automatically analyzed with the 7500 Software (v2.0.5, Applied Biosystems, Foster City, California) and exported to Microsoft Excel to calculate the parasite load in brains, which is reported as the number of tachyzoites per 1 mg of host DNA. Serum IgG response in mice Blood was kept in collection tubes at 37 C for 2 hr and at 4 C overnight for serum separation. Sera were obtained by centrifugation at 1,500 g for 20 min and then aliquoted and preserved at 80 C before testing. Neospora caninum–specific serum total IgG and isotype IgG1 and IgG2a were determined using an ELISA. The 96 well microtiter plates were coated with soluble N. caninum tachyzoite antigen (0.5 lg in 100 ll/well) in 0.1 M sodium carbonate (pH 9.6). After incubation overnight at 4 C, blocking was performed with 5% skim milk in DMEM with 2% horse serum for 1 hr at 37 C. Serum samples, diluted 1:100 in PBS (1% BSA), were added to the wells and then incubated for 1 hr at 37 C. Anti-mouse IgG (HþL) HRP-conjugated secondary antibodies (1:5,000; Proteintech Group Inc., Chicago, Illinois) were added after washing the plates 3 times with PBST. IgG isotypes were analyzed using HRP-conjugated anti-mouse IgG1 or IgG2a (1:5,000; Proteintech Group Inc.). The absorbance was measured at 450 nm and 630 nm with an ELISA reader (Model 680, BIO-RAD, Hercules, California). Analysis of data The OD values for the total IgG and IgG isotypes were analyzed using a 1-way analysis of variance (ANOVA). The parasite burdens at each time point were compared by the Kruskal–Wallis H test. The Kaplan–Meier
813
survival method was applied to estimate the portion of all individuals surviving (not showing clinical signs) in the high dose infection experiment. A statistical analysis was carried out using SPSS software v. 18.0.0 (SPSS Inc., Chicago, Illinois) and GraphPad Prism 5 v. 5.01 (San Diego, California). A value of P , 0.05 was considered statistically significant.
RESULTS Serology of the cow and fetal examination The serum of the cow reacted to N. caninum (the OD value was 1.69). The DNA sample extracted from brain tissue of the aborted fetus was positive for the N. caninum–specific gene 5 (Nc5) fragment by PCR. In vitro isolation and observation of N. caninum Tachyzoites in parasitophorous vacuoles were first observed in Vero cells after 5 passages on 84 days PI; on day 103 PI, more than 50% of the Vero cell layer was disrupted, requiring subculture of the parasite. The isolate designated as NC-Bj was continuously propagated and was maintained in Vero cells. Identification of N. caninum DNA An Nc5 fragment of the expected size (224 bp) was amplified from both the fetal brain and the NC-Bj isolate. Using N. caninum–specific primers for 18S and 25S rRNA gene, a 1,000 bp fragment was amplified. The Nc5 fragment sequences from the fetal brain and the isolated tachyzoites were the same, but different from that of NC-1 with 3 nucleotides. The sequences have been deposited in GenBank under the accession numbers JN634858 and JN634857, respectively. No DNA fragments were amplified from a DNA extract of T. gondii and the negative controls. Microsatellite DNA targets MS10 (Tand-3) and Cont-14 were successfully amplified, and fragments of expected size were observed in agarose gels. By sequencing, MS10 of NC-Bj showed its repeat units as (ACT)7(AGA)12(TGA)9, and Cont-14 showed 69 repeats of (GAA). The 2 sequences were deposited in the GenBank database under the accession numbers KC832778 and KC832779. Antigenic reactivities NC-Bj reacted only with positive anti–N. caninum sera and failed to react with N. caninum–negative sera or T. gondii antisera. Pathogenicity of NC-Bj in BALB/c mice In the mice infected with 1 3 106 tachyzoites, rough hair coats and apathy were observed in 1 infected mouse from day 29 PI. The mouse was killed on day 32 PI and necropsied. Of 30 mice inoculated with 5 3 106 tachyzoites, 13 mice died during the 32 day observation time (Fig. 1). Signs of illness such as rough coats, inactivity, or nervous system signs (hind limb weakness, head tilting, or walking in circles) were observed 1 or 2 days before death. Control mice remained clinically normal until the end of the experiment. The existence of parasites in the target organs was detected with nested-PCR, which is a sensitive method that can detect trace amount of parasite DNA as low as that equivalent to 1 tachyzoite,
814
THE JOURNAL OF PARASITOLOGY, VOL. 100, NO. 6, DECEMBER 2014
FIGURE 1. Kaplan–Meier survival curves for mice infected with 5 3 10 tachyzoites of NC-Bj. Each point represents the percentage of survival mice at that day. 6
as confirmed in an early experiment (data not shown). Parasite DNA was detected with high frequency in the blood between days 1 and 4 PI. In other target organs, N. caninum DNA was often detected during the early phase (days 1 to 8 PI) except in the hearts. No parasites could be detected after day 16 PI in any tissue (Table I). Parasite burdens in the brain from day 1 to 64 PI were calculated via real-time PCR. The highest parasite loads in the brains appeared on day 32 PI. However, the difference of brain parasite load was not significant from day 1 to 64 PI (P . 0.05 by the Kruskal–Wallis H test) (Fig. 2). The OD value of the total IgG from the infected group increased from day 2 PI, and the difference between the infected group and the control group was significant from day 4 to 64 PI (P , 0.05, 1-way ANOVA) (Fig. 3A). The IgG1 level did not show a significant increase until day 16 PI (Fig. 3B), while the IgG2a level increased significantly from day 4 PI (P , 0.01, 1-way ANOVA) (Fig. 3C). The infected mice had a mixed IgG1–IgG2a response, although the IgG2a response was predominant from day 4 PI onward (Fig. 3D). DISCUSSION The parasite in the present study was identified as N. caninum based on molecular, serological, and phenotypic characteristics. The parasite was mildly virulent to BALB/c mice, as were other previously published isolates. Viable parasites were isolated previously from the brains of 2 adult cows in Japan (Sawada et al., 2000) and New Zealand (Okeoma et al., 2004). Many attempts TABLE I. Neospora DNA detection by nested-PCR in the blood and target organs from BALB/c mice inoculated with NC-Bj. Days post-infection Organ
1
2
4
8
16
32
64
Blood Heart Liver Spleen Lung
5/5* 0/5 4/5 3/5 0/5
5/5 0/5 3/5 2/5 2/5
3/5 1/5 3/5 4/5 2/5
0/5 0/5 0/5 1/5 1/5
0/5 0/5 0/5 0/5 0/5
0/5 0/5 0/5 0/5 0/5
0/5 0/5 0/5 0/5 0/5
* Number of positive samples/number of total samples.
FIGURE 2. Box-plot graph representing the maximum and minimum values, lower and upper quartiles, and median values of the parasite burden in NC-Bj–infected mice.
to isolate viable N. caninum have been unsuccessful because most parasite stages die within the fetus when it succumbs to the infection (Dubey and Schares, 2006). As has been the experience of others, isolation of viable parasites from aborted fetal tissues is difficult, and the isolation of viable parasite from the blood of a living cow offers another approach to the cultivation of this parasite from cattle tissues. It is important to observe the cell culture flask for at least 2 mo because most N. caninum strains grow slowly, and tachyzoites may not be visible microscopically for at least 60 days from the time of inoculation (Dubey and Schares, 2006). Different growth patterns were observed in Vero cells after inoculation. In the present case, tachyzoites were first observed in cell cultures 84 days after inoculation. Therefore, it is difficult to discuss the differences in the propagation dynamics of N. caninum isolates in Vero cells. We can speculate that the time between the inoculation of Vero cells and the first visible tachyzoites in the culture depends on the number of tachyzoites in the sample (Bien et al., 2010). The 99% identity of the Nc5 gene fragment with NC-1 confirmed the isolate was N. caninum in genetics. Among different N. caninum isolates that have been characterized, unlike T. gondii (type I, II, and III) typing, there are no accepted standards for N. caninum typing according to the pathogenicity to mice or genotypes to date. However, multilocus microsatellite and minisatellite markers exhibited some differences among isolates, and they may be helpful to distinguish N. caninum isolates (AlQassab et al., 2010). Since MS10 (Tand-3) of NC-Bj showed an identical profile with NC-1 and Ncls491, we amplified another microsatellite Cont-14, which differs more greatly than the other loci (Al-Qassab et al., 2010). The number of GAA repeats in Cont-14 of NC-Bj was 69, which was different from that of any reported N. caninum isolates, indicating NC-Bj was a new isolate indeed. The BALB/c mouse has been used to study pathogenicity and vertical transmission of N. caninum because the outbred mice are not susceptible to N. caninum (Lindsay et al., 1995; CollantesFerna´ndez et al., 2006). The NC-Bj isolate was mildly virulent to BALB/c. Neurological signs compatible with neosporosis were usually a reflection of brain damage resulting from parasite proliferation and inflammatory reactions in the tissue. Although no significant differences in the parasite burden in the brain over time were
HAO ET AL.—FIRST ISOLATION OF N. CANINUM IN CHINA
815
FIGURE 3. Kinetics of total (A) IgG, (B) IgG1, and (C) IgG2a isotypes from BALB/c mice inoculated with NC-Bj at each time point. (D) IgG1/IgG2a ratios were calculated from day 4 PI because the IgG1 and IgG2a isotypes were not significantly different from the control group before day 2 PI. Each data point of IgG, IgG1, and IgG2a represents the average OD value, and the bars represent standard deviations.
observed throughout the experiment in either group (P . 0.05, Kruskal–Wallis H test), the highest parasite loads were detected on day 32 PI, which was near the time when symptoms developed. Neospora caninum DNA in target organs was frequently detected during the early phase of infection. With parasitemia from day 1 to 4 PI, the high detection rate in target organs, which always included some blood when sampling, might not be attributed to the tissue tropism of N. caninum. Levels of total IgG and isotypes IgG1 and IgG2a were detected to determine the immunological reaction stimulated by NC-Bj infection. The IgG1 and IgG2a levels followed a slightly different pattern of increase, which was consistent with previous findings in which Th1 response, as represented by IgG2a, emerged earlier than did the Th2 response, as represented by IgG1(Garc ´ıa-Melo et al., 2009). The IgG1/IgG2a ratio kinetics of NC-Bj–infected mice showed an obvious bias to IgG2a in the early phase and a slight predominance in the chronic phase, resembling Nc-Spain 3H and Nc-Spain 9 (Garc ´ıa-Melo et al., 2010), but predominance of the IgG1 response in the chronic phase was observed in mice infected with some other N. caninum isolates (Collantes-Ferna´ndez et al., 2006; Garc ´ıa-Melo et al.,
2009). The specific IgG2a seemed to emerge a little earlier, at 4 days PI in our experiment. However, the IgG2a levels didn’t greatly boost until day 8 PI, which was consistent with other reports (Collantes-Ferna´ndez et al., 2006; Aguado-Mart ´ınez et al., 2009). We found that the IgG1/IgG2a ratio patterns were almost the same as in studies where mice were infected with a lower parasite dose or attenuated parasites (Ramamoorthy et al., 2006; Rojo-Montejo et al., 2009). Given all of this, we conclude that NC-Bj has relatively low virulence but seems more virulent than at least Nc-Spain 1H. To the best of our knowledge, NC-Bj is the first successful isolation of N. caninum in China. The microsatellite DNA marker Cont-14 of NC-Bj showed a unique profile, although the pathogenicity in mice was similar to other isolates. The new isolate NC-Bj will contribute to further research on bovine neosporosis in China. ACKNOWLEDGMENTS This study was supported by the earmarked fund for Modern Agroindustry Technology Research System (CARS-37) and Research Fund for the Doctoral Program of Higher Education of China (20110008110006).
816
THE JOURNAL OF PARASITOLOGY, VOL. 100, NO. 6, DECEMBER 2014
LITERATURE CITED ´ ´ , S. AGUADO-MARTINEZ , A., L. M. ORTEGA-MORA, G. A´ LVAREZ-GARCIA ´ -MARCO, V. RISCO-CASTILLO, V. MARUGA´N-HERNA´NDEZ, RODRIGUEZ ´ . 2009. Stage-specific expression of AND A. FERNA´NDEZ-GARCIA NcSAG4 as a marker of chronic Neospora caninum infection in a mouse model. Parasitology 136: 757–764. AL-QASSAB, S., M. P. REICHEL, AND J. T. ELLIS. 2010. A second generation multiplex PCR for typing strains of Neospora caninum using six DNA targets. Molecular and Cellular Probes 24: 20–26. BIEN, J., B. MOSKWA, AND W. CABAJ. 2010. In vitro isolation and identification of the first Neospora caninum isolate from European bison (Bison bonasus bonasus L.). Veterinary Parasitology 173: 200– 205. ´ , AND L. COLLANTES-FERNA´NDEZ, E., I. LOPEZ-PEREZ, G. A´ LVAREZ-GARCIA M. ORTEGA-MORA. 2006. Temporal distribution and parasite load kinetics in blood and tissues during Neospora caninum infection in mice. Infection and Immunity 74: 2491–2494. ´ , AND L. M. ORTEGA-MORA. ———, A. ZABALLOS, G. A´ LVAREZ-GARCIA 2002. Quantitative detection of Neospora caninum in bovine aborted fetuses and experimentally infected mice by real-time PCR. Journal of Clinical Microbiology 40: 1194–1198. DUBEY, J. P., AND G. SCHARES. 2006. Diagnosis of bovine neosporosis. Veterinary Parasitology 140: 1–34. ———, AND ———. 2011. Neosporosis in animals—The last five years. Veterinary Parasitology 180: 90–108. ———, ———, AND L. M. ORTEGA-MORA. 2007. Epidemiology and control of neosporosis and Neospora caninum. Clinical Microbiology Reviews 20: 323–367. ´ -MELO, D. P., J. REGIDOR-CERRILLO, E. COLLANTES-FERNA´NDEZ, A. GARCIA ´ MEZ´ , M. GO ´ , I. DEL POZO, E. MINGUIJON AGUADO-MARTINEZ BAUTISTA, G. ADURIZ, AND L. M. ORTEGA-MORA. 2010. Pathogenic characterization in mice of Neospora caninum isolates obtained from asymptomatic calves. Parasitology 137: 1057–1068. ———, ———, L. M. ORTEGA-MORA, E. COLLANTES-FERNA´NDEZ, V. S. F. OLIVEIRA, M. A. P. OLIVEIRA, AND A. C. SILVA. 2009. Isolation and biological characterisation of a new isolate of Neospora caninum from an asymptomatic calf in Brazil. Acta Parasitologica 54: 180–185.
LINDSAY, D. S., S. D. LENZ, R. A. COLE, J. P. DUBEY, AND B. L. BLAGBURN. 1995. Mouse model for central nervous system Neospora caninum infections. Journal of Parasitology 81: 313–315. LIU, J., J. YU, M. WANG, Q. LIU, W. ZHANG, C. DENG, AND J. DING. 2007. Serodiagnosis of Neospora caninum infection in cattle using a recombinant tNcSRS2 protein-based ELISA. Veterinary Parasitology 143: 358–363. MCINNES, L. M., P. IRWIN, D. G. PALMER, AND U. M. RYAN. 2006. In vitro isolation and characterisation of the first canine Neospora caninum isolate in Australia. Veterinary Parasitology 137: 355–363. OKEOMA, C. M., N. B. WILLIAMSON, W. E. POMROY, K. M. STOWELL, AND L. M. GILLESPIE. 2004. Isolation and molecular characterisation of Neospora caninum in cattle in New Zealand. New Zealand Veterinary Journal 52: 364–370. RAMAMOORTHY, S., D. S. LINDSAY, G. G. SCHURIG, S. M. BOYLE, R. B. DUNCAN, R. VEMULAPALLI, AND N. SRIRANGANATHAN. 2006. Vaccination with gamma-irradiated Neospora caninum tachyzoites protects mice against acute challenge with N. caninum. The Journal of Eukaryotic Microbiology 53: 151–156. ROJO-MONTEJO, S., E. COLLANTES-FERNA´NDEZ, J. REGIDOR-CERRILLO, G. ´ , V. MARUGAN-HERNA´NDEZ, S. PEDRAZA-DI´ AZ, J. A´ LVAREZ-GARCIA BLANCO-MURCIA, A. PRENAFETA, AND L. M. ORTEGA-MORA. 2009. Isolation and characterization of a bovine isolate of Neospora caninum with low virulence. Veterinary Parasitology 159: 7–16. SAWADA, M., H. KONDO, Y. TOMIOKA, C. PARK, T. MORITA, A. SHIMADA, AND T. UMEMURA. 2000. Isolation of Neospora caninum from the brain of a naturally infected adult dairy cow. Veterinary Parasitology 90: 247–252. VITALE, M., V. CURRO, M. L. GIGLIA, F. VITALE, K. GOZDZIK, AND SANTO CARACAPPA. 2008. A nested PCR for Toxoplasma gondii detection from different animal samples. Wiadomos´ i Parazytologiczne 54: supplement 19. YAMANE, I., T. KOKUHO, K. SHIMURA, M. ETO, T. SHIBAHARA, M. HARITANI, Y. OUCHI, K. SVERLOW, AND P. A. CONRAD. 1997. In vitro isolation and characterisation of a bovine Neospora species in Japan. Research in Veterinary Science 63: 77–80. ZHANG, W., C. DENG, Q. LIU, J. LIU, M. WANG, K. G. TIAN, X. L. YU, AND D. M. HU. 2007. First identification of Neospora caninum infection in aborted bovine foetuses in China. Veterinary Parasitology 149: 72–76.
FIRST ISOLATION OF NEOSPORA CANINUM FROM BLOOD OF A NATURALLY INFECTED ADULT DAIRY COW IN BEIJING, CHINA Pan Hao*, Na Yang*, Xia Cui, Jing Liu, Daoyu Yang, and Qun Liu Key Laboratory of Animal Epidemiology and Zoonosis, Ministry of Agriculture, National Animal Protozoa Laboratory, College of Veterinary Medicine, China Agricultural University, Beijing 100193, China. Correspondence should be sent to: [email protected] ABSTRACT: Neospora caninum is an important cause of abortion in cattle worldwide, but the isolation of a viable parasite from an abortus is difficult, and viable N. caninum has not been isolated from any host in China. In the present study, peripheral blood samples were collected from a jugular vein of an adult dairy cow that had aborted; the cow was seropositive to N. caninum antibodies by ELISA. White blood cells were separated and seeded onto Vero cell monolayer cultures for parasite isolation. Tachyzoites were first observed in cell culture on day 84 after initial inoculation. The parasite was confirmed to be N. caninum by gene sequencing and immunofluorescence, and by bioassays in BALB/c mice. The new N. caninum isolate (NC-Bj) has a unique pattern on microsatellite Cont-14. To our knowledge, this is the first successful isolation of N. caninum in China from any host.
monolayer cell culture. The tachyzoites obtained from the cell culture were sub-passaged on Vero cells and cryopreserved in liquid nitrogen.
Neospora caninum is an important obligate intracellular protozoan that can cause serious disease in cattle and dogs worldwide, and occasionally in other animals. Much has been published on the biology of N. caninum since it was described as a new genus and species in 1988, but neosporosis continues to be a major problem in cattle as it causes abortion and neonatal mortality (Dubey and Schares, 2011). Losses due to neosporosis in the cattle industry are considerable (Dubey et al., 2007; Dubey and Schares, 2011). Seroepidemiological surveys by us and others have shown that N. caninum infection is widely spread in dairy cattle in China (Liu et al., 2007). Additionally, N. caninum has been demonstrated in histological sections of an aborted bovine fetus in China (Zhang et al., 2007). In spite of many attempts, however, we have failed to isolate viable N. caninum in more than 80 aborted bovine fetal tissues. In the present paper, we report the first isolate of N. caninum in China obtained from the peripheral blood of a naturally infected cow and preliminary characterization of this isolate.
Identification of N. caninum DNA in the aborted fetus and cell cultures Genomic DNA was extracted from the brain of the fetus from the cow mentioned here using the phenol-chloroform method. Genomic DNA was also isolated from cell cultures collected 103 days post-inoculation using the blood, cell, and tissue genomic DNA fast extraction kit (Aidlab Biotechnologies, Beijing, China), according to the manufacturer’s instructions. A fragment of N. caninum–specific Nc5 gene (GenBank X84238) was amplified by nested-PCR. The external primer pairs were Np6/Np21, and the internal primers were Np9/Np10 (McInnes et al., 2006). To avoid the carryover of contaminating nucleic acids, each step of the procedure was performed in a different room, and aerosol-resistant pipette tips were used. The primary PCR was carried out in a total volume of 50 ll, which consisted of 25 ll 23 TransHiFi superMix I (Transgen, Beijing, China), 20 pM of each primer, 2 ll of the DNA template, and sterile ultrapure distilled water. NC-1 strain genomic DNA was used as the positive control (0.8 lg, 2 ll), and distilled water was used as the negative control. Toxoplasma gondii DNA (1 lg, 2 ll) was used to test the specificity of the nested PCR. The PCR program for both primary and secondary reaction included an initial pre-denaturation at 94 C for 5 min; 35 cycles of denaturation at 94 C for 30 sec, annealing at 63 C for 30 sec, and extension at 72 C for 30 sec; and a final extension at 72 C for 10 min. The templates for the secondary PCR were 0.5 ll of the primary reaction products. Another PCR was conducted to amplify the entire intergenic region between the 18S and 25S rRNA gene of approximately 1,000 bp in size. The primers NC 18S RNA (sense) 5 0 -TGCGGAAGGATCATTCACACG-3 0 and NC 25S RNA (antisense) 5 0 -CCGTTACTAAGGGAATCATAGTT-3 0 were used. The PCR reaction was performed as previously described (Vitale et al., 2008). Microsatellite DNA targets MS10 (Tand-3) and Cont-14 were amplified to confirm the identity of NC-Bj. Separate PCR for the 2 targets was performed, and the sizes of amplicons were monitored in agarose gels as described previously (Al-Qassab et al., 2010). All PCR products were visualized by electrophoresis in a 1% agarose/ GoldViewe gel in 13 TAE buffer. After purification using spin columns (Solarbio, Beijing, China), each amplicon was linked to pEASY-T1 simple vector (Transgen Biotech) for sequencing in a local company (Sunbiotech, Beijing, China).
MATERIALS AND METHODS Animal and serological analysis A 3-yr-old Holstein cow from a herd from a southwest suburb of Beijing, China, aborted at 254 days in her first gestation. Blood samples was obtained from a jugular vein the next day, centrifuged at 1,000 g for 15 min, and the serum was stored at 20 C until used. An ELISA was performed to detect antibodies against N. caninum as described by Liu et al. (2007). Parasite isolation from the blood Because the cow was found to have N. caninum antibodies by ELISA, another 10 ml sample of blood was collected in EDTA tubes 1 wk after the abortion. White blood cells were separated according to the method described by Bien et al. (2010). Briefly, the buffy coats were collected after the blood with EDTA was centrifuged. White blood cells with buffy coats were separated by Ficoll-paque (1.077 g/ml; M & C, Beijing, China) and centrifugation. The cells at the interface were collected, washed, and added to Vero cell monolayer cultures at 37 C with 5% CO2, in DMEM medium (M & C) supplemented with 2% fetal bovine serum, 100 U/ml penicillin, and 100 lg/ml streptomycin. Flasks containing cells were examined daily using an inverted microscope (Olympus CKX41, Tokyo, Japan). Every 2 days, the culture medium was changed, and every 2 wk, Vero cells were transferred to a new culture bottle. On each passage, the cultures were scraped, passed through a 27 gauge needle, and inoculated onto a new
Antigenic reactivities Reactivities of the Chinese bovine N. caninum isolate (NC-Bj) to N. caninum (NC-1) and T. gondii (RH) antisera were examined by indirect fluorescent antibody test (IFAT) as described previously (Yamane et al., 1997). Positive and negative control sera to either N. caninum or T. gondii were titrated at a 2-fold dilution from 100 to 200 and 400. The end-point titer was the last serum dilution that exhibited clear, whole parasite fluorescence.
Received 24 February 2014; revised 11 June 2014; accepted 11 June 2014. * These authors contributed equally to this work. DOI: 10.1645/14-498.1
Bioassay in mice Six-week-old female BALB/c mice were purchased from a local supplier (Laboratory Animal Center of Academy of Military Medical Sciences, 812
HAO ET AL.—FIRST ISOLATION OF N. CANINUM IN CHINA
Beijing, China) and acclimatized for 1 wk before the experiment. Mice were housed in groups of 5 in plastic box cages and provided with rodent feed and water ad libitum under standard conditions. The parasites were collected from 2-day-old growth cultures when most tachyzoites were still intracellular. The cultures were scraped from the flasks and passed through 27 gauge needles 3 times to fracture the cells and PV membranes and disperse the tachyzoites into individual cells. Tachyzoites were harvested by centrifugation at 1,100 g for 10 min and then washed twice in sterile PBS. Parasite viability was determined by trypan blue exclusion, followed by counting 3 aliquots in a Neubauer chamber. The mice were inoculated with 1 3 106 to 5 3 106 tachyzoites (200 ll) per mouse intraperitoneally. The control group was injected intraperitoneally with 200 ll of sterile PBS. Inoculated mice were monitored daily. The blood, brains, hearts, lungs, livers, and spleens from mice inoculated with 1 3 106 tachyzoites were sampled on days 1, 2, 4, 8, 16, 32, and 64 post-inoculation (PI) according to a well-established mouse model (Collantes-Ferna´ndez et al., 2006). Five mice in the infected group and 2 mice in the control group were sacrificed by CO2 inhalation and sampled at each time point. Mice inoculated with 5 3 106 tachyzoites were examined daily for illness from their inoculation until 32 days PI. Mice were killed by CO2 inhalation at the end of the experiment. All animal-handling procedures were conducted according to institutional guidelines for animal ethics. Kinetics of N. caninum DNA in target organs of inoculated mice Immediately after blood samples were collected from the orbital sinus, 100 ll aliquots of each sample were transferred into a new tube. The rest of the sample was kept for IgG detection. Genomic DNA was extracted from 100 ll of blood and 20 mg of target organs using the blood, cell, and tissue genomic DNA fast extraction kit (Aidlab Biotechnologies Co.) according to the manufacturer’s instructions. All DNA was extracted within 24 hr after samples were collected. A nested-PCR for the Nc5 gene was performed to detect whether N. caninum existed in the target tissues using Np6/Np21 and Np9/Np10 as external and internal primer pairs, respectively. One microliter of the DNA solution was used as the template in the primary amplification, and 0.5 ll of the primary products was used in the secondary amplification. The reaction system was 25 ll in both amplifications, and 23 Taq Mix (CWBIO, Beijing, China) was used. The other conditions were the same as mentioned above. Parasite load kinetics in the brain was done by real-time PCR as described previously (Collantes-Ferna´ndez et al., 2002). The PCRs for the Nc5 gene and 28S rRNA were carried out as separate assays. Amplifications were performed with an ABI PRISMt 7500 Real-time PCR system, using SYBRt Premix Ex TaqTM II Real Time kit (TaKaRa, Dalian, China) as recommended protocol. All standards and samples were analyzed in duplicate. The results were automatically analyzed with the 7500 Software (v2.0.5, Applied Biosystems, Foster City, California) and exported to Microsoft Excel to calculate the parasite load in brains, which is reported as the number of tachyzoites per 1 mg of host DNA. Serum IgG response in mice Blood was kept in collection tubes at 37 C for 2 hr and at 4 C overnight for serum separation. Sera were obtained by centrifugation at 1,500 g for 20 min and then aliquoted and preserved at 80 C before testing. Neospora caninum–specific serum total IgG and isotype IgG1 and IgG2a were determined using an ELISA. The 96 well microtiter plates were coated with soluble N. caninum tachyzoite antigen (0.5 lg in 100 ll/well) in 0.1 M sodium carbonate (pH 9.6). After incubation overnight at 4 C, blocking was performed with 5% skim milk in DMEM with 2% horse serum for 1 hr at 37 C. Serum samples, diluted 1:100 in PBS (1% BSA), were added to the wells and then incubated for 1 hr at 37 C. Anti-mouse IgG (HþL) HRP-conjugated secondary antibodies (1:5,000; Proteintech Group Inc., Chicago, Illinois) were added after washing the plates 3 times with PBST. IgG isotypes were analyzed using HRP-conjugated anti-mouse IgG1 or IgG2a (1:5,000; Proteintech Group Inc.). The absorbance was measured at 450 nm and 630 nm with an ELISA reader (Model 680, BIO-RAD, Hercules, California). Analysis of data The OD values for the total IgG and IgG isotypes were analyzed using a 1-way analysis of variance (ANOVA). The parasite burdens at each time point were compared by the Kruskal–Wallis H test. The Kaplan–Meier
813
survival method was applied to estimate the portion of all individuals surviving (not showing clinical signs) in the high dose infection experiment. A statistical analysis was carried out using SPSS software v. 18.0.0 (SPSS Inc., Chicago, Illinois) and GraphPad Prism 5 v. 5.01 (San Diego, California). A value of P , 0.05 was considered statistically significant.
RESULTS Serology of the cow and fetal examination The serum of the cow reacted to N. caninum (the OD value was 1.69). The DNA sample extracted from brain tissue of the aborted fetus was positive for the N. caninum–specific gene 5 (Nc5) fragment by PCR. In vitro isolation and observation of N. caninum Tachyzoites in parasitophorous vacuoles were first observed in Vero cells after 5 passages on 84 days PI; on day 103 PI, more than 50% of the Vero cell layer was disrupted, requiring subculture of the parasite. The isolate designated as NC-Bj was continuously propagated and was maintained in Vero cells. Identification of N. caninum DNA An Nc5 fragment of the expected size (224 bp) was amplified from both the fetal brain and the NC-Bj isolate. Using N. caninum–specific primers for 18S and 25S rRNA gene, a 1,000 bp fragment was amplified. The Nc5 fragment sequences from the fetal brain and the isolated tachyzoites were the same, but different from that of NC-1 with 3 nucleotides. The sequences have been deposited in GenBank under the accession numbers JN634858 and JN634857, respectively. No DNA fragments were amplified from a DNA extract of T. gondii and the negative controls. Microsatellite DNA targets MS10 (Tand-3) and Cont-14 were successfully amplified, and fragments of expected size were observed in agarose gels. By sequencing, MS10 of NC-Bj showed its repeat units as (ACT)7(AGA)12(TGA)9, and Cont-14 showed 69 repeats of (GAA). The 2 sequences were deposited in the GenBank database under the accession numbers KC832778 and KC832779. Antigenic reactivities NC-Bj reacted only with positive anti–N. caninum sera and failed to react with N. caninum–negative sera or T. gondii antisera. Pathogenicity of NC-Bj in BALB/c mice In the mice infected with 1 3 106 tachyzoites, rough hair coats and apathy were observed in 1 infected mouse from day 29 PI. The mouse was killed on day 32 PI and necropsied. Of 30 mice inoculated with 5 3 106 tachyzoites, 13 mice died during the 32 day observation time (Fig. 1). Signs of illness such as rough coats, inactivity, or nervous system signs (hind limb weakness, head tilting, or walking in circles) were observed 1 or 2 days before death. Control mice remained clinically normal until the end of the experiment. The existence of parasites in the target organs was detected with nested-PCR, which is a sensitive method that can detect trace amount of parasite DNA as low as that equivalent to 1 tachyzoite,
814
THE JOURNAL OF PARASITOLOGY, VOL. 100, NO. 6, DECEMBER 2014
FIGURE 1. Kaplan–Meier survival curves for mice infected with 5 3 10 tachyzoites of NC-Bj. Each point represents the percentage of survival mice at that day. 6
as confirmed in an early experiment (data not shown). Parasite DNA was detected with high frequency in the blood between days 1 and 4 PI. In other target organs, N. caninum DNA was often detected during the early phase (days 1 to 8 PI) except in the hearts. No parasites could be detected after day 16 PI in any tissue (Table I). Parasite burdens in the brain from day 1 to 64 PI were calculated via real-time PCR. The highest parasite loads in the brains appeared on day 32 PI. However, the difference of brain parasite load was not significant from day 1 to 64 PI (P . 0.05 by the Kruskal–Wallis H test) (Fig. 2). The OD value of the total IgG from the infected group increased from day 2 PI, and the difference between the infected group and the control group was significant from day 4 to 64 PI (P , 0.05, 1-way ANOVA) (Fig. 3A). The IgG1 level did not show a significant increase until day 16 PI (Fig. 3B), while the IgG2a level increased significantly from day 4 PI (P , 0.01, 1-way ANOVA) (Fig. 3C). The infected mice had a mixed IgG1–IgG2a response, although the IgG2a response was predominant from day 4 PI onward (Fig. 3D). DISCUSSION The parasite in the present study was identified as N. caninum based on molecular, serological, and phenotypic characteristics. The parasite was mildly virulent to BALB/c mice, as were other previously published isolates. Viable parasites were isolated previously from the brains of 2 adult cows in Japan (Sawada et al., 2000) and New Zealand (Okeoma et al., 2004). Many attempts TABLE I. Neospora DNA detection by nested-PCR in the blood and target organs from BALB/c mice inoculated with NC-Bj. Days post-infection Organ
1
2
4
8
16
32
64
Blood Heart Liver Spleen Lung
5/5* 0/5 4/5 3/5 0/5
5/5 0/5 3/5 2/5 2/5
3/5 1/5 3/5 4/5 2/5
0/5 0/5 0/5 1/5 1/5
0/5 0/5 0/5 0/5 0/5
0/5 0/5 0/5 0/5 0/5
0/5 0/5 0/5 0/5 0/5
* Number of positive samples/number of total samples.
FIGURE 2. Box-plot graph representing the maximum and minimum values, lower and upper quartiles, and median values of the parasite burden in NC-Bj–infected mice.
to isolate viable N. caninum have been unsuccessful because most parasite stages die within the fetus when it succumbs to the infection (Dubey and Schares, 2006). As has been the experience of others, isolation of viable parasites from aborted fetal tissues is difficult, and the isolation of viable parasite from the blood of a living cow offers another approach to the cultivation of this parasite from cattle tissues. It is important to observe the cell culture flask for at least 2 mo because most N. caninum strains grow slowly, and tachyzoites may not be visible microscopically for at least 60 days from the time of inoculation (Dubey and Schares, 2006). Different growth patterns were observed in Vero cells after inoculation. In the present case, tachyzoites were first observed in cell cultures 84 days after inoculation. Therefore, it is difficult to discuss the differences in the propagation dynamics of N. caninum isolates in Vero cells. We can speculate that the time between the inoculation of Vero cells and the first visible tachyzoites in the culture depends on the number of tachyzoites in the sample (Bien et al., 2010). The 99% identity of the Nc5 gene fragment with NC-1 confirmed the isolate was N. caninum in genetics. Among different N. caninum isolates that have been characterized, unlike T. gondii (type I, II, and III) typing, there are no accepted standards for N. caninum typing according to the pathogenicity to mice or genotypes to date. However, multilocus microsatellite and minisatellite markers exhibited some differences among isolates, and they may be helpful to distinguish N. caninum isolates (AlQassab et al., 2010). Since MS10 (Tand-3) of NC-Bj showed an identical profile with NC-1 and Ncls491, we amplified another microsatellite Cont-14, which differs more greatly than the other loci (Al-Qassab et al., 2010). The number of GAA repeats in Cont-14 of NC-Bj was 69, which was different from that of any reported N. caninum isolates, indicating NC-Bj was a new isolate indeed. The BALB/c mouse has been used to study pathogenicity and vertical transmission of N. caninum because the outbred mice are not susceptible to N. caninum (Lindsay et al., 1995; CollantesFerna´ndez et al., 2006). The NC-Bj isolate was mildly virulent to BALB/c. Neurological signs compatible with neosporosis were usually a reflection of brain damage resulting from parasite proliferation and inflammatory reactions in the tissue. Although no significant differences in the parasite burden in the brain over time were
HAO ET AL.—FIRST ISOLATION OF N. CANINUM IN CHINA
815
FIGURE 3. Kinetics of total (A) IgG, (B) IgG1, and (C) IgG2a isotypes from BALB/c mice inoculated with NC-Bj at each time point. (D) IgG1/IgG2a ratios were calculated from day 4 PI because the IgG1 and IgG2a isotypes were not significantly different from the control group before day 2 PI. Each data point of IgG, IgG1, and IgG2a represents the average OD value, and the bars represent standard deviations.
observed throughout the experiment in either group (P . 0.05, Kruskal–Wallis H test), the highest parasite loads were detected on day 32 PI, which was near the time when symptoms developed. Neospora caninum DNA in target organs was frequently detected during the early phase of infection. With parasitemia from day 1 to 4 PI, the high detection rate in target organs, which always included some blood when sampling, might not be attributed to the tissue tropism of N. caninum. Levels of total IgG and isotypes IgG1 and IgG2a were detected to determine the immunological reaction stimulated by NC-Bj infection. The IgG1 and IgG2a levels followed a slightly different pattern of increase, which was consistent with previous findings in which Th1 response, as represented by IgG2a, emerged earlier than did the Th2 response, as represented by IgG1(Garc ´ıa-Melo et al., 2009). The IgG1/IgG2a ratio kinetics of NC-Bj–infected mice showed an obvious bias to IgG2a in the early phase and a slight predominance in the chronic phase, resembling Nc-Spain 3H and Nc-Spain 9 (Garc ´ıa-Melo et al., 2010), but predominance of the IgG1 response in the chronic phase was observed in mice infected with some other N. caninum isolates (Collantes-Ferna´ndez et al., 2006; Garc ´ıa-Melo et al.,
2009). The specific IgG2a seemed to emerge a little earlier, at 4 days PI in our experiment. However, the IgG2a levels didn’t greatly boost until day 8 PI, which was consistent with other reports (Collantes-Ferna´ndez et al., 2006; Aguado-Mart ´ınez et al., 2009). We found that the IgG1/IgG2a ratio patterns were almost the same as in studies where mice were infected with a lower parasite dose or attenuated parasites (Ramamoorthy et al., 2006; Rojo-Montejo et al., 2009). Given all of this, we conclude that NC-Bj has relatively low virulence but seems more virulent than at least Nc-Spain 1H. To the best of our knowledge, NC-Bj is the first successful isolation of N. caninum in China. The microsatellite DNA marker Cont-14 of NC-Bj showed a unique profile, although the pathogenicity in mice was similar to other isolates. The new isolate NC-Bj will contribute to further research on bovine neosporosis in China. ACKNOWLEDGMENTS This study was supported by the earmarked fund for Modern Agroindustry Technology Research System (CARS-37) and Research Fund for the Doctoral Program of Higher Education of China (20110008110006).
816
THE JOURNAL OF PARASITOLOGY, VOL. 100, NO. 6, DECEMBER 2014
LITERATURE CITED ´ ´ , S. AGUADO-MARTINEZ , A., L. M. ORTEGA-MORA, G. A´ LVAREZ-GARCIA ´ -MARCO, V. RISCO-CASTILLO, V. MARUGA´N-HERNA´NDEZ, RODRIGUEZ ´ . 2009. Stage-specific expression of AND A. FERNA´NDEZ-GARCIA NcSAG4 as a marker of chronic Neospora caninum infection in a mouse model. Parasitology 136: 757–764. AL-QASSAB, S., M. P. REICHEL, AND J. T. ELLIS. 2010. A second generation multiplex PCR for typing strains of Neospora caninum using six DNA targets. Molecular and Cellular Probes 24: 20–26. BIEN, J., B. MOSKWA, AND W. CABAJ. 2010. In vitro isolation and identification of the first Neospora caninum isolate from European bison (Bison bonasus bonasus L.). Veterinary Parasitology 173: 200– 205. ´ , AND L. COLLANTES-FERNA´NDEZ, E., I. LOPEZ-PEREZ, G. A´ LVAREZ-GARCIA M. ORTEGA-MORA. 2006. Temporal distribution and parasite load kinetics in blood and tissues during Neospora caninum infection in mice. Infection and Immunity 74: 2491–2494. ´ , AND L. M. ORTEGA-MORA. ———, A. ZABALLOS, G. A´ LVAREZ-GARCIA 2002. Quantitative detection of Neospora caninum in bovine aborted fetuses and experimentally infected mice by real-time PCR. Journal of Clinical Microbiology 40: 1194–1198. DUBEY, J. P., AND G. SCHARES. 2006. Diagnosis of bovine neosporosis. Veterinary Parasitology 140: 1–34. ———, AND ———. 2011. Neosporosis in animals—The last five years. Veterinary Parasitology 180: 90–108. ———, ———, AND L. M. ORTEGA-MORA. 2007. Epidemiology and control of neosporosis and Neospora caninum. Clinical Microbiology Reviews 20: 323–367. ´ -MELO, D. P., J. REGIDOR-CERRILLO, E. COLLANTES-FERNA´NDEZ, A. GARCIA ´ MEZ´ , M. GO ´ , I. DEL POZO, E. MINGUIJON AGUADO-MARTINEZ BAUTISTA, G. ADURIZ, AND L. M. ORTEGA-MORA. 2010. Pathogenic characterization in mice of Neospora caninum isolates obtained from asymptomatic calves. Parasitology 137: 1057–1068. ———, ———, L. M. ORTEGA-MORA, E. COLLANTES-FERNA´NDEZ, V. S. F. OLIVEIRA, M. A. P. OLIVEIRA, AND A. C. SILVA. 2009. Isolation and biological characterisation of a new isolate of Neospora caninum from an asymptomatic calf in Brazil. Acta Parasitologica 54: 180–185.
LINDSAY, D. S., S. D. LENZ, R. A. COLE, J. P. DUBEY, AND B. L. BLAGBURN. 1995. Mouse model for central nervous system Neospora caninum infections. Journal of Parasitology 81: 313–315. LIU, J., J. YU, M. WANG, Q. LIU, W. ZHANG, C. DENG, AND J. DING. 2007. Serodiagnosis of Neospora caninum infection in cattle using a recombinant tNcSRS2 protein-based ELISA. Veterinary Parasitology 143: 358–363. MCINNES, L. M., P. IRWIN, D. G. PALMER, AND U. M. RYAN. 2006. In vitro isolation and characterisation of the first canine Neospora caninum isolate in Australia. Veterinary Parasitology 137: 355–363. OKEOMA, C. M., N. B. WILLIAMSON, W. E. POMROY, K. M. STOWELL, AND L. M. GILLESPIE. 2004. Isolation and molecular characterisation of Neospora caninum in cattle in New Zealand. New Zealand Veterinary Journal 52: 364–370. RAMAMOORTHY, S., D. S. LINDSAY, G. G. SCHURIG, S. M. BOYLE, R. B. DUNCAN, R. VEMULAPALLI, AND N. SRIRANGANATHAN. 2006. Vaccination with gamma-irradiated Neospora caninum tachyzoites protects mice against acute challenge with N. caninum. The Journal of Eukaryotic Microbiology 53: 151–156. ROJO-MONTEJO, S., E. COLLANTES-FERNA´NDEZ, J. REGIDOR-CERRILLO, G. ´ , V. MARUGAN-HERNA´NDEZ, S. PEDRAZA-DI´ AZ, J. A´ LVAREZ-GARCIA BLANCO-MURCIA, A. PRENAFETA, AND L. M. ORTEGA-MORA. 2009. Isolation and characterization of a bovine isolate of Neospora caninum with low virulence. Veterinary Parasitology 159: 7–16. SAWADA, M., H. KONDO, Y. TOMIOKA, C. PARK, T. MORITA, A. SHIMADA, AND T. UMEMURA. 2000. Isolation of Neospora caninum from the brain of a naturally infected adult dairy cow. Veterinary Parasitology 90: 247–252. VITALE, M., V. CURRO, M. L. GIGLIA, F. VITALE, K. GOZDZIK, AND SANTO CARACAPPA. 2008. A nested PCR for Toxoplasma gondii detection from different animal samples. Wiadomos´ i Parazytologiczne 54: supplement 19. YAMANE, I., T. KOKUHO, K. SHIMURA, M. ETO, T. SHIBAHARA, M. HARITANI, Y. OUCHI, K. SVERLOW, AND P. A. CONRAD. 1997. In vitro isolation and characterisation of a bovine Neospora species in Japan. Research in Veterinary Science 63: 77–80. ZHANG, W., C. DENG, Q. LIU, J. LIU, M. WANG, K. G. TIAN, X. L. YU, AND D. M. HU. 2007. First identification of Neospora caninum infection in aborted bovine foetuses in China. Veterinary Parasitology 149: 72–76.
