Live attenuated influenza viruses produced in a suspension process with avian AGE1.CR.pIX cells

Background

Neosporosis is an infectious disease primarily of cattle and dogs, caused by intracellular parasite, Neospora caninum. Neosporosis appears to be a major cause of abortion in dairy cattle worldwide and causes to huge economic loss to dairy industry.

Results

Recombinant surface associated antigen 1 (NcSAG1), NcSAG1 related sequence 2 (NcSRS2) and the dense granule antigen 2 (NcGRA2) of N. caninum were expressed either in silkworm or in Escherichia coli and purified. The purified recombinant proteins bound to the N. caninum-specific antibodies in serum samples from infected cattle as revealed by an enzyme-linked immunosorbent assay (ELISA). By co-immobilizing these recombinant proteins, a novel indirect ELISA was developed for detection of neosporosis. With the use of 32 serum samples, comprising 12 positive serum samples and 20 negative serum samples, the sensitivity and specificity of the assay were found to be 91.7 and 100%, respectively. Seventy-two serum samples from dairy farms were also tested and one was diagnosed with neosporasis with both this method and a commercial assay.

Conclusions

A diagnostic method employing recombinant proteins of N. caninum was developed. The method showed high sensitivity and specificity. Diagnostic test with field serum samples suggested its applicability to the practical diagnosis of neosporosis.     

Development of competitive ELISA for neosporosis by employing immunoproteomics

In this study, proteomics was used to explore the antigenic proteins that are involved in cross-reactivity during serodiagnosis between Neospora caninum (N. caninum) and Toxoplasma gondii (T. gondii). Competitive enzyme-linked immunosorbent assay (C-ELISA) developed by proteomics shed a new light on the infection of N. caninum. Cross-reactivity of antigenic proteins between N. caninum and T. gondii tachyzoites was explored by using the conventional sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) (1-DE) and two-dimensional gel electrophoresis (2-DE) immunoblot. The proteins were identified by matrix-assisted laser desorption/ionization-time of flight mass spectrometry. The protein expression patterns in the immunoblot profiles of N. caninum were similar to bovine, chicken, and rabbit anti-N. caninum serum, but they were not similar to rabbit anti-T. gondii serum. Band at 79 kDa, HSP70, and actin on immunoblot profiles reacted, in general, with bovine, chicken, and rabbit anti-N. caninum serum and also with rabbit anti-T. gondii serum, respectively. Whereas the band at 144 kDa, and NCDG-1 were detected on bovine, chicken, and rabbit anti-N. caninum immunoblot profiles, they were not observed on rabbit anti-T. gondii immunoblot profile. These specific antigenic proteins were recorded as species-specific proteins of N. caninum against T. gondii. Based on the proteome analysis, C-ELISA was developed to screen the cattle infected with N. caninum by using N. caninum tachyzoite lysate as a coating antigen and chicken anti-N. caninum immunoglobulin (Ig)Y as a competitor. C-ELISA was able to detect the antibody of N. caninum without cross-reactivity with T. gondii. Furthermore, it achieved a fine diagnostic performance in the cases of 162 bovine sera.     

Neospora caninum Recruits Host Cell Structures to Its Parasitophorous Vacuole and Salvages Lipids from Organelles

Toxoplasma gondii and Neospora caninum, which cause the diseases toxoplasmosis and neosporosis, respectively, are two closely related apicomplexan parasites. They have similar heteroxenous life cycles and conserved genomes and share many metabolic features. Despite these similarities, T. gondii and N. caninum differ in their transmission strategies and zoonotic potential. Comparative analyses of the two parasites are important to identify the unique biological features that underlie the basis of host preference and pathogenicity. T. gondii and N. caninum are obligate intravacuolar parasites; in contrast to T. gondii, events that occur during N. caninum infection remain largely uncharacterized. We examined the capability of N. caninum (Liverpool isolate) to interact with host organelles and scavenge nutrients in comparison to that of T. gondii (RH strain). N. caninum reorganizes the host microtubular cytoskeleton and attracts endoplasmic reticulum (ER), mitochondria, lysosomes, multivesicular bodies, and Golgi vesicles to its vacuole though with some notable differences from T. gondii. For example, the host ER gathers around the N. caninum parasitophorous vacuole (PV) but does not physically associate with the vacuolar membrane; the host Golgi apparatus surrounds the N. caninum PV but does not fragment into ministacks. N. caninum relies on plasma lipoproteins and scavenges cholesterol from NPC1-containing endocytic organelles. This parasite salvages sphingolipids from host Golgi Rab14 vesicles that it sequesters into its vacuole. Our data highlight a remarkable degree of conservation in the intracellular infection program of N. caninum and T. gondii. The minor differences between the two parasites related to the recruitment and rearrangement of host organelles around their vacuoles likely reflect divergent evolutionary paths.     

Seroprevalence and associated risk factors of Toxoplasma gondii and Neospora caninum infections in cattle in Central India

Toxoplasma gondii and Neospora caninum are closely related cyst-forming parasites identified as important causes of reproductive failures in ruminants. While these parasites have been reported worldwide, seroprevalence and associated risk factors for cattle infections have not been determined in India. A total of 576 serum samples of cattle were analyzed for antibodies to T. gondii and N. caninum using enzyme-linked immunosorbent assay (ELISA), modified/Neospora agglutination test (MAT/NAT), and an indirect fluorescent antibody test (IFAT-tachyzoite and bradyzoite). Additionally, general information about cattle, movement of cats and dogs, the menace of rodents, management, and reproductive disorders were assessed to identify the potential risk factors. Overall, 32.9% (190/576) serum samples reacted positively to T. gondii and 24.8% (143/576) to N. caninum. The performance of the diagnostic tests showed excellent agreement between IFAT and ELISA (kappa [κ] = 0.98) and between MAT/NAT and ELISA (κ = 0.97). Combining both infections on avidity test, 94% sera had high-IgG avidity, and 3% had low-IgG avidity antibodies, indicating chronic infection in the majority of the cases. The identified risk factors (p < 0.05) for exposure to T. gondii were: increasing age (Odds Ratio [OR]: 2.02), movement of cat (OR: 4.8) and rodents (OR: 1.57) in the farm; and for N. caninum: increasing age (OR: 1.6), movement of dogs in the farm (OR: 2.07), drinking pond water (OR: 1.64) and abortion (OR: 1.82). These findings revealed that T. gondii and N. caninum infections are widespread in the study area and suggest conducting nationwide epidemiological studies owing to their economic importance.     

Generation and Immunity Testing of a Recombinant Adenovirus Expressing NcSRS2-NcGRA7 Fusion Protein of Bovine Neospora caninum

Neospora caninum is the etiologic agent of bovine neosporosis, which affects the reproductive performance of cattle worldwide. The transmembrane protein, NcSRS2, and dense-granule protein, NcGRA7, were identified as protective antigens based on their ability to induce significant protective immune responses in murine neosporosis models. In the current study, NcSRS2 and NcGRA7 genes were spliced by overlap-extension PCR in a recombinant adenovirus termed Ad5-NcSRS2-NcGRA 7, expressing the NcSRS2-NcGRA7 gene, and the efficacy was evaluated in mice. The results showed that the titer of the recombinant adenovirus was 10⁹TCID₅₀/ml. Three weeks post-boost immunization (w.p.b.i.), the IgG antibody titer in sera was as high as 1:4,096. IFN-γ and IL-4 levels were significantly different from the control group (P<0.01). This research established a solid foundation for the development of a recombinant adenovirus vaccine against bovine N. caninum.     

Neospora caninum: detection in wild rabbits and investigation of co-infection with Toxoplasma gondii by PCR analysis

Neospora caninum is an important pathogen of cattle causing significant economic loss. There is much current interest in wild animal reservoirs for this parasite. The role of the rabbit in this is currently unknown. DNA samples from the brains of wild rabbits (Oryctolagus cuniculus) collected from the Malham area of the Yorkshire dales were investigated by species-specific PCR for the presence of N. caninum and Toxoplasma gondii. We found prevalences of N. caninum of 10.5% (6/57) and T. gondii of 68.4% (39/57) with 8.8% (5/57) co-infected. Strain typing of T. gondii positive rabbits revealed strain types I-III were present in this population. Investigation of tissue distribution determined N. caninum DNA was most often detected in the brain and heart, less often in the tongue and not in the liver. To our knowledge this is the first report of N. caninum detection in naturally infected wild rabbits.     

Differential susceptibility of human trophoblastic (BeWo) and uterine cervical (HeLa) cells to Neospora caninum infection

Neospora caninum is an apicomplexan parasite, closely related to Toxoplasma gondii, and causes abortion and congenital neosporosis in cattle worldwide. Trophoblast cells act in mechanisms of innate immune defense at the fetal-maternal interface and no data are available about the interaction of Neospora with human trophoblasts. Thus, this study aimed to verify the susceptibility of human trophoblastic (BeWo) compared with uterine cervical (HeLa) cell lines to N. caninum. BeWo and HeLa cells were infected with different parasite:cell ratios of N. caninum tachyzoites and analyzed at different times after infection for cell viability using thiazolyl blue tetrazole and lactate dehydrogenase assays. Both cell lines were also evaluated for cytokine production and parasite infection/replication assays when pre-treated or not with Neospora lysate antigen (NLA) or human recombinant IFN-γ. Cell viability was increased up to 48 h of infection in both types of cells, suggesting that infection could inhibit early cell death and/or induce cell proliferation. Neospora infection induced up-regulation of the macrophage migration inhibitory factor (MIF), mainly in HeLa cells, which was enhanced by cell pre-treatment by NLA or IFN-γ. Conversely, parasite infection induced down-regulation of the transforming growth factor (TGF-β), mostly in BeWo cells, which was decreased with NLA or IFN-γ pre-treatment. HeLa cells were more susceptible to Neospora infection than BeWo cells and IFN-γ pre-treatment resulted in reduced infection indices in both cell lines. Control of parasite growth was mediated by IFN-γ through an indoleamine-2,3-dioxygenase-dependent mechanism in HeLa cells alone. Based on these results, we concluded that BeWo and HeLa cells are readily infected by N. caninum, although presenting differences in susceptibility to infection, cytokine production and cell viability. Thus, these host cells can be considered in comparative approaches to understand strategies used by N. caninum to survive at the maternal-fetal interface.     

Antibody reaction of human anti-Toxoplasma gondii positive and negative sera with Neospora caninum antigens

Anti-Neospora caninum antibody was detected in anti-Toxoplasma gondii positive and negative human sera by ELISA, western blot and immunofluorescence assay (IFA). Twelve cases out of 172 (6.7%) Toxoplasma-positive sera cross-reacted with both T. gondii and N. caninum antigens, and one out of 110 Toxoplasma-negative sera reacted with N. caninum antigen by ELISA. By western blot, all 12 sera reacted with T. gondii antigens with various banding patterns but specifically at 30 kDa (SAG1) and 22 kDa (SAG2) bands. With N. caninum antigen, the number of reactive bands was reduced, however a 43 kDa band reacted in three cases in Toxoplasma-positive sera in addition to one in Toxoplasma-negative control sera. All sera of the Toxoplasma-positive group labeled surface membrane of T. gondii, but reacted differently with N. caninum. Fluorescence was detected in surface membrane, subcellular organelles, or both in N. caninum. And one case in the Toxoplasma-negative group also reacted with N. caninum strongly in subcellular organelles. This suggested that the antibody against N. caninum may be present in human sera although the positive rate was very low in this study. The possibility of human infection with N. caninum remains to be evaluated further.     

Serosurvey of Toxoplasma gondii,Neospora caninum and Sarcocystis neurona in raptors and risk factor analysis

Raptors are carnivorous birds with great hunting ability. Toxoplasma gondii, Neospora caninum and Sarcocystis spp. are intracellular Apicomplexan protozoans which infect a wide range of intermediate hosts, including birds. The aims of this study were to evaluate the serological reactivity of captive raptors serum to T. gondii, N. caninum and S. neurona antigens and identify possible risk factors associated with the infection. From August 2014 to September 2015, blood samples from 72 raptors were collected and serum samples were tested by immunofluorescence antibody test (IFAT). Antigen slides were prepared using tachyzoites of T. gondii and N. caninum and using merozoites of S. neurona. Serum samples were tested at the following cut-off dilutions: 1:16 for T. gondii and 1:50 for N. caninum and S. neurona. An anti-chicken IgY antibody conjugated with FITC was used as a secondary antibody at 1:50 dilution. Out of the 72 raptors serum tested by IFAT, 2.7% reacted to N. caninum, 8.3% to T. gondii and 11.1% to S. neurona antigens. The region in which the sample was collected, the reason the raptors were kept in captivity and diet were statistically associated with seropositivity to T. gondii and the use of the birds and diet were statistically associated with seropositivity to N. caninum and S. neurona (p ≤ 0.05). We highlight the occurrence of these protozoans in birds of prey and the importance of good hygiene and feeding management of these birds in captivity to reduce the risk of protozoal infections.     

Neospora caninum in birds: A review

Neospora caninum is an obligate intracellular protozoan parasite that infects domestic and wild animals. Canids are considered to be definitive hosts since they may shed oocysts into the environment through their feces. The disease is recognized as one of the major causes of bovine abortion worldwide, leading to important economic losses in the dairy and beef cattle industries. Previous studies have reported N. caninum infection in different species of birds; infection in birds has been associated with increased seroprevalence and reproductive problems in dairy cattle. Although the role of birds in the epidemiological cycle of neosporosis is unknown, birds are exposed to infection because they feed on the ground and could thus contribute to parasite dissemination. This review is focused on the current state of knowledge of neosporosis in birds.     

Natural breeding with bulls experimentally infected with Neospora caninum failed to induce seroconversion in dams

Four bulls and 56 heifers seronegative to Neospora caninum were used to determine the feasibility of venereal transmission in bovine neosporosis under natural conditions. Bulls were experimentally infected with 10⁸ live N. caninum tachyzoites. Two of them with the Nc-1 isolate and the other two with the Nc-Spain-7 isolate. After 13 months of initial infection, each bull was re-infected with the same isolate and dose. The experiments were carried out from March to September during 2006 and 2007 where groups of cyclic heifers were naturally mated by the experimentally infected bulls. In year 2006, two bulls infected with different N. caninum isolate serviced 12 heifers each. In year 2007, the same bulls serviced the same heifers a second time (now primiparous) and six new heifers were also added to each group. In addition, the other two bulls serviced 10 additional heifers each. Experimental animals were monitored for 30 weeks and serum samples were collected weekly and fortnightly in years 2006 and 2007, respectively to evaluate the presence of specific antibodies to N. caninum. Experimentally infected bulls showed a significant increase of specific IgG antibodies from 13 (Nc-SP-7) and 21 (Nc-1) days post-infection. Serum IgG antibody responses of individual animals were similar in kinetics but slightly different in magnitude. Serum samples from heifers were all negative. Pregnant rates were 100% in heifers and 91% in primiparous animals. Calves did not show precolostral specific antibodies to N. caninum. Venereal transmission of bovine neosporosis under natural grazing conditions is unlikely to occur.     

Development of Two Murine Antibodies against Neospora caninum Using Phage Display Technology and Application on the Detection of N. caninum

Neosporosis, caused by an intracellular parasite, Neospora caninum, is an infectious disease primarily of cattle and dogs. It occurs worldwide and causes huge damages to dairy farms. In this study, we immunized mice with recombinant surface-associated protein 1 of N. caninum (rNcSAG1) and developed two novel monoclonal antibodies, A10 and H3, against NcSAG1 using phage-display technology. Both clones bound to purified rNcSAG1 and the half maximal inhibitory concentrations of A10 and H3 are 50 and 72 nM of rNcSAG1, respectively. In immunofluorescence assays, both A10 and H3 Fabs bound to N. caninum parasites. Direct detection of N. caninum parasites was developed firstly using an enzyme-linked immunosorbent assay (ELISA) with A10 and H3. Binding of A10 and H3 antibodies to rNcSAG1 was also inhibited by some certain anti-N. caninum antibodies in the neosporosis-positive cattle sera, suggesting they might bind to the same epitopes of NcSAG1 with those anti-N. caninum antibodies of bovine. These antibodies were demonstrated to have a potential for monitoring the N. caninum parasites in a dairy farm, which may lead to protect livestock from parasite-infection.     

Neospora caninum excreted/secreted antigens trigger CC-chemokine receptor 5-dependent cell migration

Neospora caninum, the causative agent of neosporosis, is an obligate intracellular parasite considered to be a major cause of abortion in cattle throughout the world. Most studies concerning N. caninum have focused on life cycle, seroepidemiology, pathology and vaccination, while data on host-parasite interaction, such as host cell migration, mechanisms of evasion and dissemination of this parasite during the early phase of infection are still poorly understood. Here we show the ability of excreted/secreted antigens from N. caninum (NcESAs) to attract monocytic cells to the site of primary infection in both in vitro and in vivo assays. Molecules from the family of cyclophilins present on the NcESAs were shown to work as chemokine-like proteins and NcESA-induced chemoattraction involved G_i protein signaling and participation of CC-chemokine receptor 5 (CCR5). Additionally, we demonstrate the ability of NcESAs to enhance the expression of CCR5 on monocytic cells and this increase occurred in parallel with the chemotactic activity of NcESAs by increasing cell migration. These results suggest that during the first days of infection, N. caninum produces molecules capable of inducing monocytic cell migration to the sites of infection, which will consequently enhance initial parasite invasion and proliferation. Altogether, these results help to clarify some key features involved in the process of cell migration and may reveal virulence factors and therapeutic targets to control neosporosis.     

Seroprevalence of Cryptosporidium parvum and Neospora caninum in cattle in the southern Kyushu region of Japan

Cryptosporidium parvum and Neospora caninum are common parasites in domesticated cattle worldwide, including in Japan. We carried out a serological survey to detect C. parvum and N. caninum infection among cattle in the southern Kyushu region of Japan—including the small islands—by indirect enzyme-linked immunosorbent assay based on recombinant antigens. We found that total seropositivity in 570 Japanese black cattle was 96.3% for C. parvum and 18.4% for N. caninum. Although seroprevalence was correlated with cattle age, differences in the seroprevalence of C. parvum among age groups were not statistically significant. On the other hand, N. caninum seroprevalence increased with age, suggesting horizontal transmission through ingestion of food or water contaminated with oocysts. These findings underscore the importance of monitoring C. parvum and N. caninum in cattle and implementing measures to prevent the spread of infection to other livestock and to humans.     

Serological evidence of Toxoplasma gondii and Neospora caninum infection in black-boned sheep and goats in southwest China

Toxoplasma gondii and Neospora caninum are two closely related protozoan parasites which can cause abortion and significant economic losses in sheep and goats. However, it is yet to know whether black-bone sheep and goats are infected with T. gondii and N. caninum in China. In the present investigation, the seroprevalence and risk factors of T. gondii and N. caninum infections in black-boned sheep and goats were investigated in Yunnan Province, subtropical southwest China between July and August of 2017. A total of 481 serum samples were tested for T. gondii antibodies using the Modified Agglutination Test (MAT), and 468 serum samples were examined for N. caninum antibodies by indirect Enzyme-Linked Immunosorbent Assay (iELISA). The overall seroprevalence of T. gondii in black-boned sheep and goats was 36.80% (177/481, 95% CI 32.49–41.11), and 40 out of 468 serum samples were N. caninum-seropositive (8.55%, 95% CI 6.02–11.08). There was significant difference in the seroprevalence of T. gondii infection in different regions (χ² = 19.869, df = 2, P<0.01). As for the seroprevalence of N. caninum infection, region (χ² = 8.558, df = 2, P<0.05), age (χ² = 16.631, df = 3, P < 0.01), gender (χ² = 11.219, df = 1, P < 0.01) and species (χ² = 8.673, df = 1, P < 0.01) were the risk factors. In addition, the seroprevalence of coinfection of T. gondii and N. caninum in black-boned sheep and goats was 3.63% (17/468, 95% CI 1.94–5.32). To our knowledge, this is the first report of T. gondii and N. caninum seroprevalence in black-boned sheep and goats in China, which provided base-line data for the execution of control strategies and measures against T. gondii and N. caninum infection in black-boned sheep and goats.     

Evaluation of Neospora caninum serodiagnostic antigens for bovine neosporosis

Abortion and reproductive failure caused by Neospora caninum infection has a dramatic negative economic impact on the cattle industry. To date, no definitive serodiagnostic tool for assessing N. caninum abortion has been reported. In this study, we evaluated the diagnostic performance of numerous N. caninum antigens in relation to abortion in cattle. Five recombinant proteins with potential as diagnostic antigens (NcGRA6, NcGRA7, NcGRA14, NcCyP, and NcSAG1) were compared by indirect enzyme-linked immunosorbent assay (iELISA) using sera from mice and cattle experimentally infected with N. caninum. The best-performing three antigens (NcSAG1, NcGRA7, and NcGRA6) were evaluated by IgG-iELISAs to assess their utility in diagnosing Neospora abortion using sera from confirmed N. caninum-aborted dams based on immunohistochemical assays (IHC). Additionally, all samples were tested using a commercial N. caninum antibody competitive ELISA (cELISA). The iELISAs against both NcSAG1 and NcGRA7 could efficiently distinguish IHC positive and negative samples compared with iELISAs against NcGRA6 and the cELISA. Furthermore, antibody levels against NcSAG1 and NcGRA7 were significantly higher in aborting cows comparing with infected but non-aborted dams in a herd experiencing a Neospora abortion outbreak. Tracking the dynamics of antibody levels during pregnancy revealed a marked increase in NcSAG1- and NcGRA7-specific antibodies at the last trimester of pregnancy. In contrast, no marked differences in antibody levels against either antigen were noted in neurologically symptomatic calves compared with non-symptomatic infected calves. Our data suggests NcSAG1 and NcGRA7 as indicators for Neospora abortion.     

Rabbit antibodies against Toxoplasma Hsp20 are able to reduce parasite invasion and gliding motility in Toxoplasma gondii and parasite invasion in Neospora caninum

Toxoplasma gondii Hsp20 is a pellicle-associated functional chaperone whose biological role is still unknown. Hsp20 is present in different apicomplexan parasites, showing a high degree of conservation across the phylum, with Neospora caninum Hsp20 presenting an 82% identity to that of T. gondii. Hence rabbit anti-T. gondii Hsp20 serum was able to recognize the N. caninum counterpart. Interestingly, both N. caninum and T. gondii Hsp20 localized to the inner membrane complex and to the plasma membrane. Incubation of T. gondii and N. caninum tachyzoites with an anti-TgHsp20 serum reduced parasite invasion at rates of 57.23% and 54.7%, respectively. This anti-serum also reduced T. gondii gliding 48.7%. Together, all this data support a role for Hsp20 in parasite invasion and gliding motility.     

In vitro and in vivo effects of the phytohormone inhibitor fluridone against Neospora caninum infection

Neospora caninum causes abortion and stillbirth in cattle. Identification of effective drugs against this parasite remains a challenge. Previous studies have suggested that disruption of abscisic acid (ABA)-mediated signaling in apicomplexan parasites such as Toxoplasma gondii offers a new drug target. In this study, the ABA inhibitor, fluridone (FLU), was evaluated for its action against N. caninum. Production of endogenous ABA within N. caninum was confirmed by ultra-performance liquid chromatography–tandem quadruple mass spectrometry. Subsequently, FLU treatment efficacy was assessed using in vitro. Results revealed that FLU inhibited the growth of N. caninum and T. gondii in vitro (IC₅₀ 143.1 ± 43.96 μM and 330.6 ± 52.38 μM, respectively). However, FLU did not affect parasite replication at 24 h post-infection, but inhibited egress of N. caninum thereafter. To evaluate the effect of FLU in vivo, N. caninum-infected mice were treated with FLU for 15 days. FLU treatment appeared to ameliorate acute neosporosis induced by lethal parasite challenge. Together, our data shows that ABA might control egress in N. caninum. Therefore, FLU has potential as a candidate drug for the treatment of acute neosporosis.     

Neospora caninum: Cloning and expression of a gene coding for cytokine-inducing profilin

Profilins are actin-binding proteins that in Toxoplasma gondii stimulate innate immunity in mice by binding Toll-like receptors (TLR) on dendritic cells (DC) leading to release of inflammatory cytokines, primarily IL-12 and IFN-γ. The purpose of the present study was to characterize Neospora caninum profilin, termed NcProfilin. Recombinant NcProfilin was purified by affinity chromatography, and used to prepare specific antisera to allow characterization of native NcProfilin antigen in N. caninum tachyzoites. By immunoblotting, recombinant NcProfilin is 22 kDa, and is similar in size to the respective 22 kDa native protein. Immunofluorescence and immunoelectron microscopy localized native NcProfilin to the apical end of N. caninum tachyzoites. Incubation of recombinant NcProfilin with spleen cells from BALB/c mice induced release of IFN-γ. Also, injection of BALB/c mice with purified rNcProfilin elicited a strong IFN-γ and IL-12 responses at 6 and 24 h after injection indicating that NcProfilin may be an important protein in regulation of cytokine responses to N. caninum.     

Toxoplasma CRISPR/Cas9 constructs are functional for gene disruption in Neospora caninum

Herein we describe, to our knowledge for the first time the use of the clustered regularly interspaced short palindromic repeats/CRISPR-associated gene 9 (CRISPR/Cas9) system for genome editing of Neospora caninum, an apicomplexan parasite considered one of the main causes of abortion in cattle worldwide. By using plasmids containing the CRISPR/Cas9 components adapted to the closely related parasite Toxoplasma gondii, we successfully knocked out a green fluorescent protein (GFP) in an Nc-1 GFP-expressing strain, and efficiently disrupted the NcGRA7 gene in the Nc-Spain7 isolate by insertion of a pyrimethamine resistance cassette. The successful use of this technology in N. caninum lays the foundation for an efficient, targeted gene modification tool in this parasite.