Australian dingoes are definitive hosts of Neospora caninum

To provide objective data on the potential role of dingoes (Canis lupus dingo) in the life cycle of Neospora caninum in Australia, the production of N. caninum oocysts by experimentally infected canids was investigated. Three dingo pups raised in captivity and three domestic dogs were fed tissue from calves infected with an Australian isolate of N. caninum, Nc-Nowra. Oocysts of N. caninum, confirmed by species-specific PCR, were shed in low numbers by one dingo pup at 12-14 days p.i. The remaining animals did not shed oocysts. Furthermore, the blood from two out of three dingoes tested positive for DNA of N. caninum using PCR tests at 14 and 28 days p.i. Oocyst shedding from the intestinal tract of a dingo demonstrates that dingoes are definitive hosts of N. caninum and horizontal transmission of N. caninum from dingoes to farm animals and wildlife may occur in Australia.     

Diagnostic Potential of Anti-rNcp-43 Polyclonal Antibodies for the Detection of Neospora caninum

Neosporosis is a disease caused by the apicomplexan parasite Neospora caninum, which is closely related to Toxoplasma gondii. N. caninum infection represents an important cause of reproductive failure in sheep, goats, horses, and cattle worldwide. The diagnosis of neosporosis is based on the detection of pathogen-specific antibodies in animal sera or the presence of tissue cysts. However, morphological similarities and serological cross-reactivity between N. caninum and T. gondii can result in the misdiagnosis. In this study, the N. caninum tachyzoite surface protein Ncp-43 was expressed in a recombinant form to elicit polyclonal antibodies (pAb) response. The pAb was purified and conjugated to horseradish peroxidase (HRP) or fluorescein isothiocyanate (FITC) to detect the recombinant and native Ncp-43 proteins, respectively. The pAb and pAb/HRP were able to recognize rNcp-43 by dot blot and ELISA, and pAb/FITC immunolabeled the apical complex of tachyzoites. A blocking enzyme-linked immunosorbent assay (b-ELISA) was performed to evaluate pAb/HRP as a diagnostic tool. The mean percent inhibition for the positive and negative serum samples from cattle with neosporosis was significantly different (P < 0.0001). These results suggest that the pAb may bind to the same epitopes of Ncp-43 as anti-N. caninum antibodies in the positive samples tested. The b-ELISA using the pAb/HRP can facilitate diagnostic testing for neosporosis, since fewer steps are involved, and cross-reactivity with secondary antibodies is avoided. In summary, this report describes the production of antibodies against N. caninum, and evaluates the potential of these tools for the development of new diagnostic tests for neosporosis.     

Neospora caninum: Activity of cholinesterases during the acute and chronic phases of an experimental infection in gerbils

Neosporosis is an infectious disease primarily of dogs and cattle which has been found in many countries around the world. Neospora caninum causes an important immune response (cellular and humoral) in animals that it infects. Since the participation of the cholinergic system in the immune response is well documented, the aim of this study was to evaluate the relationship between N. caninum infection and activities of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) during the acute and chronic phase of infection. For that, tachyzoites of N. caninum (Nc-1 strain) were inoculated intraperitoneally in gerbils (Meriones unguiculatus), which were separated in two experiments, I and II, differing in infective doses of tachyzoites, aiming to reach an acute phase as well as chronic phase, respectively. Samples were collected on day 7 post infection (PI) for Experiment I and on days 15 and 30 PI for Experiment II. AChE activity was evaluated on whole blood and brain, while BChE was evaluated in plasma. On day 7 a reduction of AChE in total blood and brain was observed, along with reduction of BChE in plasma of infected animals when compared with non-infected. In Experiment II, AChE activity increased in total blood on day 30 PI; however, maintaining, during the same period, the AChE activity has a reduced in brain tissue. BChE activity was significantly increased on day 30 PI. Based on the results obtained, it was possible to observe a response of the cholinergic system, providing different grades of AChE and BChE activities, in response to the acute and chronic infection of gerbils experimentally infected with N. caninum. These results will serve as initial points to further studies of our research group about the relationship between the infection/disease and the cholinergic system.     

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.     

Pampas fox (Lycalopex gymnocercus) of the Argentine Pampas as intermediate host for Neospora caninum

The Pampas fox (Lycalopex gymnocercus) is the most abundant wild canid from South America. This wild canid inhabits grasslands, open woodlands, and areas highly modified by extensive ranching and agricultural activities. We aimed to evaluate Neospora caninum infection in tissues from the Pampas fox from Argentina. A total of 41 free-living Pampas foxes were sampled in rural areas located in the Humid Pampas region, Argentina. Brain tissue and different muscles were assessed by histologic and molecular methods. No N. caninum cysts were observed in brain and muscle tissue samples analyzed by histology and immunohistochemistry. Molecular N. caninum identification from brain tissue was based on amplification by PCR of Nc-5 gene and ITS1 rRNA fragments and subsequent sequencing. The presence of N. caninum DNA was 74% (23/31) for the Nc-5 gene and was confirmed by a second ITS1 PCR in 55% (17/31) of the brain tested. Thirteen ITS1 consensus sequences were obtained, and all have a 99.58–100% similarity with N. caninum reference sequences. Only 4% (1/23) of muscles samples analyzed were positive for the Nc-5 gene of N. caninum. This study demonstrated a high prevalence of N. caninum DNA in brain from free-ranging Pampas fox of the Pampa Argentine, thus confirming that this wild canid is a wide distributed intermediate host.     

Detection of Neospora caninum DNA by polymerase chain reaction in bats from Southern China

Neospora caninum is an intracellular protozoan that infects many domestic and wild animals. Domestic dogs and other canids function as definitive hosts, while other mammals serve as natural intermediate hosts. In the present study, the brain tissues of bats collected in Yunnan Province, Southern China were tested by N. caninum specific-nested PCR, targeting the Nc-5 gene and the internal transcribed spacer 1 (ITS1) region of the ribosomal DNA to determine whether bats could be infected with N. caninum. N. caninum DNA was detected in 1.8% (4/227) of bats, i.e., 1.7% (1/60) in Rousettus leschenaultia, 1.7% (1/58) in Hipposideros pomona, 2.9% (2/69) in Rhinolophus pusillus, and none (0/40) in Myotis daubentoniid. The findings of the present study are only the first indication that bats could serve as an intermediate host, and further studies are necessary to confirm whether bats are involved in the transmission of N. caninum infections.     

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.     

Experimental Neospora caninum infection in chickens (Gallus gallus domesticus) with oocysts and tachyzoites of two recent isolates reveals resistance to infection

The importance of birds in the biological cycle of Neospora caninum is not clear. We report unsuccessful Neospora infection in chickens (Gallus gallus domesticus) using two isolates of N. caninum. In experiment #1, 30 White Leghorn chickens were orally inoculated with viable N. caninum oocysts (NC-SP1 isolate, 200 oocysts per bird) via the crop at 21 days of age. Groups of three birds were euthanised at intervals of 7 days (a total of 9 weeks) and one group was challenged with the same oocyst dose at 37 days p.i. and observed for 11 weeks. Blood samples were collected weekly, and sera were tested using IFAT. Chicken tissues were collected for PCR, quantitative PCR and immunohistochemistry. Two dogs approximately 45 days of age were fed with tissues from chickens euthanised at 138 and 159 days p.i. The results indicated that the chickens were resistant to neosporosis as revealed by failure to seroconvert, to detect parasite DNA or N. caninum antigen by immunohistochemistry in inoculated bird tissues, and by no oocyst excretion by the dogs fed avian tissues. Similar results were obtained in experiment #2, in which 34 1-week-old chickens were each s.c. inoculated with 100,000 tachyzoites of the NcWTDMn1 isolate of N. caninum. The chickens were euthanised on days 7, 15, 22, 28, 36 and 60 p.i. At necropsy, all tissues and serum from each bird were collected. All chickens remained asymptomatic, and N. caninum antigen was not detected by immunohistochemistry. Seven chickens euthanised at day 60 p.i. demonstrated low (1:25 dilution) levels of antibodies by using the Neospora agglutination test. Two 12-week-old dogs fed tissues pooled from 10 inoculated chickens euthanised at day 60 p.i. did not excrete N. caninum oocysts. This investigation indicates that chickens are resistant to experimental infection by 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.     

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 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.     

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.     

Detection of IgG antibody against Neospora caninum in cattle in Korea

A total of 492 cattle sera was screened by IgG-ELISA against Neospora caninum (Nc-1 strain and a Korean isolate, KBA-2) and Toxoplasma gondii. Out of 492, 113 sera (23.0%) reacted positively to either Nc-1 or KBA-2 strains of N. caninum. Among the 113 positive sera, 92 sera (81.4%) reacted with antigens of both strains, but 6 sera (5.3%) with Nc-1 and 15 sera (13.3%) with KBA-2 strain only. And with T. gondii antigen, 6 sera (1.2%) were positive but all reacted with N. caninum antigen also. Western blot revealed typical binding pattern according to ELISA values, such that high OD group reacted specifically to the major surface proteins including 43 kDa protein. Seroprevalence of 23.0% indicates that neosporosis seemed to be one of major causes of abortion in cattle. It is suggested here to establish more epidemiological researches nationwide systematically.     

Role of dense granule antigen 7 in vertical transmission of Neospora caninum in C57BL/6 mice infected during early pregnancy

Neosporosis is a parasitic disease affecting the health of dogs and cattle worldwide. It is caused by Neospora caninum, an obligate intracellular apicomplexan parasite. Dogs are its definitive host, it mostly infects livestock animals, especially cattle that acts as intermediate host. It is necessary to have well-established models of abortion and vertical transmission in experimental animals, in order to determine basic control measures for the N. caninum infection. We evaluated the role of N. caninum dense granule antigen 7 (NcGRA7) in the vertical transmission of N. caninum using the C57BL/6 pregnant mouse model. We inoculated mice on day 3.5 of pregnancy with parental Nc-1 or NcGRA7-deficient parasites (NcGRA7KO). Post-mortem analyses were performed on day 30 after birth and the surviving pups were kept until day 30 postpartum. The number of parasites in the brain tissues of offspring from NcGRA7KO-infected dams was significantly lower than that of the Nc-1-infected dams under two infection doses (1 × 10⁶ and 1 × 10⁵ tachyzoites/mouse). The vertical transmission rates in the NcGRA7KO-infected group were significantly lower than those of the Nc1-infected group. To understand the mechanism by which the lack of NcGRA7 decreases the vertical transmission, pregnant mice were sacrificed on day 13.5 of pregnancy (10 days after infection), although parasite DNA was detected in the placentas, no significant difference was found between the two parasite lines. Histopathological analysis revealed a greater inflammatory response in the placentas from NcGRA7KO-infected dams than in those from the parental strain. This finding correlates with upregulated chemokine mRNA expression for CCL2, CCL8, and CXCL9 in the placentas from the NcGRA7KO-infected mice. In conclusion, these results suggest that loss of NcGRA7 triggers an inflammatory response in the placenta, resulting in decreased vertical transmission of N. caninum.     

Fecal and Molecular Survey of Neospora caninum in Farm and Household Dogs in Mashhad Area, Khorasan Province, Iran

Neospora caninum is an important cause of abortion in dairy cattle worldwide. Dog is the definitive host for N. caninum and can infect dairy cattle. The aim of this study is to determine the prevalence of Neospora oocysts in feces of dogs from dairy farms. A total of 174 fecal samples was collected from 89 farm dogs and 85 household dogs during 2006 and 2008. Fecal samples of dogs were microscopically examined for detecting Hammondia Neospora-like oocysts (HNLO) by Mini Parasep®SF fecal parasite concentrator. HNLO were microscopically detected in 4 fecal samples (2.2%). The fecal samples with HNLO were examined by N. caninum-specific PCR. Two of the samples were positive for N. caninum. The 2 positive fecal samples were selected for inoculation to calves. Two inoculated calves were seronegative by ELISA for 4 months post-infection. This is the first report of finding N. caninum DNA in feces of farm dogs in Mashhad area, Iran.     

Effects of re-infection with Neospora caninum in bulls on parasite detection in semen and blood and immunological responses

Three bulls with experimentally induced primary infection with Neospora caninum were re-infected intravenously with 10(8) live N. caninum tachyzoites of the NC-1 isolate at 300 days post-infection to investigate the presence of N. caninum in semen and blood, and the associated immune responses. In parallel, three bulls with experimentally induced primary infection with N. caninum and three non-infected bulls were also monitored. Re-infected and infected bulls showed an intermittent presence of N. caninum DNA in semen with a parasite load ranging from 0.1 to 15.6 (mean 4.4) and 0.1 to 11.1 (mean 4.1) parasites/ml, respectively. Re-infected bulls showed significant and persistent serum-specific IgM and IgG antibody responses. Specific IgG levels were detected in seminal plasma of all infected bulls, but the magnitude of the response was significantly higher in re-infected rather than in chronically infected animals. The mean specific IFN-gamma levels in re-infected bulls were significantly increased as early as 3 and 7 days after experimental infection when compared to bulls in other groups. This study showed that the intermittent presence and parasite load of N. caninum in the semen of re-infected bulls is very similar to that reported in chronically infected animals. The protozoa could not be isolated from BALB/c nu/nu mice inoculated with PCR-positive semen samples and inseminated heifers with pooled semen samples did not show seroconversion. Plasma IFN-gamma level seems to be a good indicator of a recent N. caninum infection.     

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.     

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.     

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.