Neospora caninum: Early immune response of rat mixed glial cultures after tachyzoites infection

Neospora caninum causes neurologic disease in dogs and abortion in cattle. Little is known about the immune response of the CNS against this protozoan. The aim of this study was to evaluate production of IL-6, IL-10, TNF-α, IFN-γ, and NO in rat mixed glial cell cultures infected by N. caninum. IFN-γ was not observed. The mean cytokine released after 24 and 72 h of infection were 3.8 ± 0.6 and 3.7 ± 0.6 pg TNF-α/mg protein and 2.7 ± 0.69 and 4.1 ± 0.64 pg IL-10/mg protein, respectively, and more than 8.0 pg IL-6/mg protein for both time points. NO levels increased 24 h post-infection (2.3 ± 0.8 pg/mg protein) until 72 h (4.2 ± 1.1 pg/mg protein) and the number of tachyzoites reduced with the time. Our results show high levels of regulatory cytokines that may suppress the harmful effects of IFN-γ; high levels of TNF-α and NO may represent an effective response by infected glial cells against N. caninum.     

Neospora caninum: In vitro culture of tachyzoites in MCF-7 human breast carcinoma cells

The development of Neospora caninum tachyzoites, an apicomplexan protozoan parasite, was studied in vitro using the human breast carcinoma cell 7 (MCF-7) as the host cell line. The extracellular NC-1 tachyzoites in MCF-7 cells were observed and counted daily for 6 consecutive days post-infection to establish the growth curve. The intracellular parasites were observed by acridine orange staining using Laser scanning confocal microscope. The results indicated that NC-1 tachyzoites invaded MCF-7 cells and multiplied intracellularly. The number of extracellular NC-1 tachyzoites started to increase rapidly around day 3 and reached the maximum number around day 4. Results from the present study suggested that MCF-7 cells were susceptible to NC-1 tachyzoites and could be used as an alternative cell line for in vitro studies.     

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.     

Characterization of Neospora caninum macrophage migration inhibitory factor

The present study is the first characterization of Neospora caninum macrophage migration inhibitory factor (NcMIF). BLAST-N analysis of NcMIF revealed high similarity (87%) to the Toxoplasma gondii MIF. NcMIF was cloned and expressed in Escherichia coli in 3 forms, NcMIF (mature protein), NcMIFm (mutation of proline-2 to glycine), and NcMIFhis (addition of a polyhistidine tag at the N-terminus). None of these recombinant NcMIFs (rNcMIF) had tautomerase, oxidoreductase, or immunologic regulatory activities. rNcMIF was unable to compete with recombinant human MIF for a MIF receptor (CD74), suggesting that NcMIF does not bind to this MIF receptor. The glycine substitution for proline-2 of NcMIF resulted in increased retention time on SEC-HPLC and decreased formation of dimers and trimers. The addition of N-terminal HIS-tag led to increased formation of trimers. Immunofluorescence staining demonstrated that NcMIF was localized to the apical end of N. caninum tachyzoites. Immunoelectron microscopy further revealed that NcMIF was present in the micronemes, rhoptries, dense granules, and nuclei. NcMIF was abundant in the tachyzoite lysate and present in excretory and secretory antigen (ESAg) preparations. Total and secretory NcMIF was more abundant in a non-pathologic clone, Ncts-8, than in the wild type isolate (NC1). Furthermore, NcMIF release by the both isolates was increased in the presence of calcium ionophore. This differential production of NcMIF by the pathologic and non-pathologic isolates of N. caninum may suggest a critical role of this molecule in the infectious pathogenesis of this parasite.     

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.     

Neospora caninum: comparative gene expression profiling of Neospora caninum wild type and a temperature sensitive clone

To understand the genetic basis of virulence, gene expression profiles of a temperature-sensitive clone (NCts-8, relatively avirulent) and its wild type (NC-1) of Neospora caninum were characterized and compared using a high-density microarray with approximately 63,000 distinct oligonucleotides. This microarray consists of 5692 unique N. caninum sequences, including 1980 Tentative Consensus sequences and 3712 singleton ESTs from the TIGR N. caninum Gene Index (NCGI, release 5.0). Each sequence was represented by 11 distinct 60mer oligonucleotides synthesized in situ on the microarray. The results showed that 111 genes were significantly repressed and no up-regulated genes were identified in the NCts-8 clone. The level of 10 randomly selected genes from the repressed genes was confirmed using real-time RT-PCR. Of the 111 repressed genes, 58 were hypothetical protein products and 53 were annotated genes. Over 70% of the repressed genes identified in this study are clustered on five chromosomes (I, VII, VIII, X and XII). These results suggest that the down-regulated genes may be in part responsible for the reduced pathogenesis of NCts-8; further characterization of the regulated genes may aid in understanding of molecular basis of virulence and development of countermeasures against neosporosis.     

The cell-mediated immune response to Neospora caninum during pregnancy in the mouse is associated with a bias towards production of interleukin-4

Neospora caninum is an obligate intracellular protozoan parasite which is efficiently transmitted transplacentally in cattle where it may cause abortion. A pregnant mouse model was used to characterise the immune response following N. caninum infection; the response in non-pregnant and pregnant mice was compared. Spleen cells from both infected/non-pregnant and infected/pregnant mice produced interferon-gamma, interleukin-12 and tumour necrosis factor alpha; however, the levels of these Th1 cytokines were lower in infected/pregnant mice. Infected/non-pregnant and infected/pregnant mice also produced the Th2 cytokine interleukin-10; however, there was no trend toward a decrease of this in pregnant mice. Interleukin-4 was exclusively produced at high levels by infected/pregnant mice and thus appears responsible for the observed decline in Th1 cytokine production in pregnant mice. A bias towards Th2 cytokines such as IL-4 and IL-10 is normally associated with the maintenance of a viable pregnancy, and not with the control of protozoal infections. Consequently, the importance and role of cytokines and cell-mediated immunity in the control of transplacental transmission and foetal loss due to N. caninum infection are discussed.     

Neosporacaninum: Application of apical membrane antigen 1 encapsulated in the oligomannose-coated liposomes for reduction of offspring mortality from infection in BALB/c mice

Liposomes coated with neoglycolipids constructed with mannopentaose and dipalmitoylphosphatidylethanolamine (M3-DPPE), referred to as M3-DPPE liposomes, have been shown to induce cellular immunity against antigens encapsulated therein. To evaluate whether these M3-DPPE liposomes have an adjuvant capacity against Neospora caninum infection, a novel immunization method utilizing soluble N. caninum apical membrane antigen 1 (NcAMA1) encapsulated in the M3-DPPE liposomes (M3-NcAMA1) was employed. The results revealed that a significant amount of interferon (IFN)-γ production was detected in culture supernatants of NcAMA1 protein- or N. caninum lysate-stimulated spleen cells obtained from the mice one week after the third immunization with M3-NcAMA1. The parasite burden in the dams’ brain tissue was decreased and the survival rate of offspring increased significantly in M3-NcAMA1-immunized mice. Thus, a parasite-specific Th1 immune response was successfully induced in the pregnant mice immunized with M3-NcAMA1, and an effective reduction of offspring mortality from N. caninum infection was triggered.     

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.     

Identification of an anticoagulant peptide that inhibits both fXIa and fVIIa/tissue factor from the blood-feeding nematode Ancylostoma caninum

Factor VIIa-tissue factor complex (fVIIa/TF) and factor XIa (fXIa) play important roles in the initiation and amplification of coagulation, respectively. They may be good targets for the development of novel anticoagulants to treat and prevent thromboembolic disease. In this study, we cloned, expressed and identified a novel anticoagulant peptide, AcaNAP10, from the blood-feeding nematode Ancylostoma caninum. AcaNAP10 showed potent anticoagulant activity and doubled the activated partial thromboplastin and prothrombin times at estimated concentrations of 92.9 nM and 28.8 nM, respectively. AcaNAP10 demonstrated distinct mechanisms of action compared with known anticoagulants. It inhibited fXIa and fVIIa/TF with IC₅₀ values of 25.76 ± 1.06 nM and 123.9 ± 1.71 nM, respectively. This is the first report on an anticoagulant that can inhibit both fXIa and fVIIa/TF. This anticoagulant peptide may be an alternative molecule for the development of novel anticoagulants.     

First epidemiological study on exposure to Neospora caninum in different canine populations in the Algiers District (Algeria)

Neospora caninum is an important cause of abortion in cattle worldwide. Dogs act as final hosts shedding oocysts in the environment. They can also harbour the extraintestinal stage of the parasite and this may be associated with a fairly rare neuromuscular condition. The sera of 781 dogs from the Algiers District were screened by IFAT for the presence of anti-N. caninum antibodies. These dogs were distributed into four populations: local stray dogs, police dogs, dogs from breeding kennels and farm dogs. The overall seroprevalence was 21.90%. Significant differences were observed between the different populations, the highest prevalence being observed in farm (44.44%) and stray dogs (22.55%). Additionally, the highest titres were observed in farm dogs. Among studied epidemiological parameters, breed, dog origin, season and vaccination status were significantly associated with IFAT results. Additionally, a recently described real time PCR was used on the blood of 100 pound dogs and the results were compared with the serological data. A higher proportion of dogs was found to be positive by PCR when compared to the IFAT results. There was only a fairly low agreement between PCR and IFAT results which suggests that these techniques measured different aspects of the host–parasite relationship. This study indicates that the level of exposure of the canine population of Algiers area to N. caninum is very high. This would indicate a potentially high risk for N. caninum induced abortion in cattle in this region and in Algeria.     

Real-time PCR assays for monitoring benzimidazole resistance-associated mutations in Ancylostoma caninum

Frequent and broad application of anthelmintic drugs for treatment of intestinal parasite infection has led to drug resistance that often renders whole populations of livestock unresponsive to treatment. Therefore, it is important to detect mutations associated with drug resistance before it becomes clinically manifest. To monitor developing drug resistance against benzimidazoles (BZ), we developed real-time PCR assays and applied them to analyse the beta-tubulin isotype-1 gene of the hookworm Ancylostoma caninum, an important parasite of dogs. Previously, we developed PCR assays to monitor codon positions 167 and 200. Here, we describe an assay which is able to detect resistance alleles in codon 198. These real-time PCR assays were subsequently applied to screen hookworm specimens recovered from dogs in Georgia. No elevated levels of polymorphisms at the investigated loci were found, suggesting that selection for resistance in the tested samples did not occur.     

Ectopic Repo suppresses expression of castor gene in Drosophila central nervous system

The ventral nerve cord (VNC) of the Drosophila embryo is derived from neuroblasts (NBs). NBs divide in a stem cell lineage to generate a series of ganglion mother cells (GMCs), each of which divides once to produce a pair of neurons or glial cells. One of the NB genes, castor (cas), is expressed in a subset of NBs and has never been identified in neurons and the peripheral nervous system; cas plays a role in axonogenesis. But its limited expression along the dorsal-ventral axis within the central nervous system has not been investigated yet. In the present study, we examined the expression patterns of both genes using confocal microscopy to determine the effects of repo mutation on cas expression. Cas was mainly expressed in layers different from repo-expressed layers during early embryogenesis: repo was expressed mostly from deep to mid layers, while cas, from mid to superficial layers. Loss-of-function of repo did not result in an ectopic expression of cas, but rather, a scattering of cas-expressing cells. However, repo gain-of-function mutation caused repression of cas. In addition, repo-expressing cells seemed to block the migration of cas-expressing cells.     

Cloning of casbene and neocembrene synthases from Euphorbiaceae plants and expression in Saccharomycescerevisiae

A large number of diterpenes have been isolated from Euphorbiaceae plants, many of which are of interest due to toxicity or potential therapeutic activity. Specific Euphorbiaceae diterpenes of medical interest include the latent HIV-1 activator prostratin (and related 12-deoxyphorbol esters), the analgesic resiniferatoxin, and the anticancer drug candidate ingenol 3-angelate. In spite of the large number of diterpenes isolated from these plants and the similarity of their core structures, there is little known about their biosynthetic pathways. Other than the enzymes involved in gibberellin biosynthesis, the only diterpene synthase isolated to date from the Euphorbiaceae has been casbene synthase, responsible for biosynthesis of a macrocyclic diterpene in the castor bean (Ricinus communis). Here, we have selected five Euphorbiaceae species in which to investigate terpene biosynthesis and report on the distribution of diterpene synthases within this family. We have discovered genes encoding putative casbene synthases in all of our selected Euphorbiaceae species and have demonstrated high-level casbene production through expression of four of these genes in a metabolically engineered strain of Saccharomyces cerevisiae. The only other diterpene synthase found among the five plants was a neocembrene synthase from R. communis (this being the first report of a neocembrene synthase gene). Based on the prevalence of casbene synthases, the lack of other candidates, and the structure of the casbene skeleton, we consider it likely that casbene is the precursor to a large number of Euphorbiaceae diterpenes. Casbene production levels of 31 mg/L were achieved in S. cerevisiae and we discuss strategies to further increase production by maximizing flux through the mevalonate pathway.     

br regulates the expression of the ecdysone biosynthesis gene npc1

The growth and metamorphosis of insects are regulated by ecdysteroid hormones produced in the ring gland. Ecdysone biosynthesis-related genes are both highly and specifically expressed in the ring gland. However, the intrinsic regulation of ecdysone biosynthesis has received little attention. Here we used the Drosophila npc1 gene to study the mechanism of ring gland-specific gene expression. npc1 is important for sterol trafficking in the ring gland during ecdysone biosynthesis. We have identified a conserved ring gland-specific cis-regulatory element (RSE) in the npc1 promoter using promoter fusion reporter analysis. Furthermore, genetic loss-of-function analysis and in vitro electrophoretic mobility shift assays revealed that the ecdysone early response gene broad complex (br) is a vital factor in the positive regulation of npc1 ring gland expression. Moreover, br also affects the ring gland expression of many other ecdysone biosynthetic genes as well as torso and InR, two key factors in the regulation of ecdysone biosynthesis. These results imply that ecdysone could potentially act through its early response gene br to achieve positive feedback regulation of ecdysone biosynthesis during development.     

Differential gene expression of the honey bees Apis mellifera and A. cerana induced by Varroa destructor infection

Varroa destructor mite is currently the most serious threat to the world bee industry. Differences in mite tolerance are reported between two honey bee species Apis mellifera and Apis cerana. Differential gene expression of two honey bee species induced by V. destructor infection was investigated by constructing two suppression subtractive hybridization (SSH) libraries, as first steps toward elucidating molecular mechanisms of Varroa tolerance. From the SSH libraries, we obtained 289 high quality sequences which clustered into 132 unique sequences grouped in 26 contigs and 106 singlets where 49 consisted in A. cerana subtracted library and 83 in A. mellifera. Using BLAST, we found that 85% sequences had counterpart known genes whereas 15% were undescribed. A Gene Ontology analysis classified 51 unique sequences into different functional categories. Eight of these differentially expressed genes, representative of different regulation patterns, were confirmed by qRT-PCR. Upon the mite induction, the differentially expressed genes from both bee species were different, except hex 110 gene, which was up-regulated in A. cerana but down-regulated in A. mellifera, and Npy-r gene, which was down-regulated in both species. In general, most of the differential expression genes were involved in metabolic processes and nerve signaling. The results provide information on the molecular response of these two bee species to Varroa infection.     

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.