Neospora caninum protein disulfide isomerase is involved in tachyzoite-host cell interaction

We have previously shown that treatment of Neospora caninum tachyzoites with the aspartyl protease inhibitor pepstatin A reduces host cell invasion [Naguleswaran, A., Muller, N., Hemphill, A., 2003. Neospora caninum and Toxoplasma gondii: a novel adhesion/invasion assay reveals distinct differences in tachyzoite-host cell interactions. Exp. Parasitol. 104, 149-158]. Pepstatin A-affinity-chromatography led to the isolation of a major band of approximately 52 kDa which was identified as a homologue of a previously described Toxoplasma gondii putative protein disulfide isomerase (TgPDI) through tandem mass spectrometry. A BLAST search against N. caninum expressed sequence tags (ESTs) on the ApiDots server using TgPDI cDNA as query sequence revealed a 2251 bp PDI-like consensus (NcPDI), which shows 94% identity to the T. gondii homologue. In N. caninum tachyzoites, NcPDI was found mainly in the soluble hydrophilic fraction. Immunofluorescence showed that expression of NcPDI was dramatically down-regulated in the bradyzoite stage, and immunogold-EM on tachyzoites localised the protein to the cytoplasm, mostly in close vicinity to the nuclear membrane, to the micronemes, and to the parasite cell surface. However, NcPDI was absent in rhoptries and dense granules. Preincubation of tachyzoites with the sulfhydryl blocker 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), p-chloromercuribenzoic acid (pCMBA), and with the PDI inhibitor bacitracin reduced adhesion of parasites to host cells. In addition, incubation of N. caninum tachyzoites with affinity-purified anti-NcPDI antibodies reduced host cell adhesion. PDIs catalyse the formation, reduction or isomerisation of disulfide bonds. Many major components of the adhesion and invasion machinery of apicomplexan parasites are cysteine-rich and dependent on correct folding via disulfide bond formation. Thus, our data points towards an important role for surface-associated NcPDI in Neospora-host cell interaction.     

Vero cell surface proteoglycan interaction with the microneme protein NcMIC(3) mediates adhesion of Neospora caninum tachyzoites to host cells unlike that in Toxoplasma gondii

Neospora caninum and Toxoplasma gondii are characterised by a very low host cell specificity, thus they are able to infect a wide range of different cells in vivo and in vitro. Infection of the host cell by tachyzoites is a process which is preceded by adhesion onto the host cell surface. The receptors on the host cell surface which would allow N. caninum to establish a physical interaction have not been investigated so far. Here we report the role of host cell surface proteoglycans as receptors for the adhesion of N. caninum tachyzoites to Vero cell monolayers. We found that N. caninum tachyzoites, similar to T. gondii tachyzoites, can bind to sulphated proteoglycans which naturally occur on the surface of mammalian cells, including heparin/heparan sulphate, chondroitin sulphates, as well as to the artificially sulphated glycosaminoglycan dextran sulphate. Although removal of heparan sulphate from the host cell surface results in decreased adhesion of T. gondii tachyzoites, binding of N. caninum tachyzoites is not affected by this treatment. Conversely, enzymatic removal of chondroitin sulphate A, B and C decreases N. caninum adhesion but does not affect T. gondii binding to Vero cells. Thus, T. gondii and N. caninum tachyzoites exhibit differential adhesive properties with regard to host cell surface glycosaminoglycans. Additional experiments employing Triton X-100 solubilised NcSRS2 and NcMIC3 showed that NcSRS2 binds to the host cell surface, but not through those sulphated glycosaminoglycans investigated in this study. In contrast, NcMIC3 binding to the host cell surface is dramatically influenced by these modifications. Further experiments showed that the NcMIC3 adhesive motif comprised of four consecutive epidermal growth factor-like domains expressed as a recombinant protein exhibits a high binding activity for sulphated glycosaminoglycans. These results suggest that host cell surface proteoglycan interaction of N. caninum differs from that observed for T. gondii, and that the epidermal growth factor-like adhesive motif in NcMIC3 could be involved in this process.     

Reduction in transplacental transmission of Neospora caninum in outbred mice by vaccination

Infection with the protozoan parasite Neospora caninum is an important cause of abortion in cattle. A major source of infection is transplacental transfer of the parasite from mother to offspring during pregnancy. This study describes investigations on the immunisation of outbred Qs mice before pregnancy with live or a crude lysate of N. caninum (NC-Nowra isolate) to prevent transplacental transfer of a challenge infection administered during pregnancy. Parasites present in the brains of pups from mice challenged with N. caninum (NC-Liverpool) were detected by PCR. Injection of live NC-Nowra tachyzoites before pregnancy dramatically reduced transplacental transfer from 75 to 0.8% in one experiment and from 76 to 8% in a second experiment. Injection of a crude lysate of NC-Nowra tachyzoites reduced transplacental transfer from 67 to 53% in one experiment and from 76 to 63% in a second experiment. Analysis of N. caninum-specific IgG1 and IgG2a antibody levels prior to pregnancy and challenge showed that NC-Nowra lysate induced a response skewed towards IgG1 whereas live parasites induced both IgG1 and IgG2a antibodies. After pregnancy and a challenge infection, a similar IgG1/IgG2a response was seen in all challenged groups. These results provide further positive support for the hypothesis that transplacental transmission of this parasite is preventable by vaccination.     

Interspecific antagonism and virulence in hosts exposed to two parasite species

Co-infection of host organisms by multiple parasite species has evolutionary consequences for all participants in the symbiosis. In this study, we co-exposed aquatic-snails (Biomphalaria glabrata) to two of their trematode parasites, Schistosoma mansoni and Echinostoma caproni. In co-exposed snails, E. caproni prevalence was 63% compared to only 23% for S. mansoni. Co-exposed E. caproni-infected snails exhibited reduced fecundity, higher mortality, and higher parasite reproduction (higher virulence) compared to hosts exposed to echinostomes alone. Conversely, co-exposed S. mansoni-infected snails released fewer parasites and produced greater numbers of eggs compared to hosts exposed to S. mansoni alone. These results suggest that co-exposure not only influences the establishment (presence or absence) of particular parasite species, but also impacts host life history, parasite reproduction, and the virulence of the interaction.     

Leishmanicidal activity of synthetic antimicrobial peptides in an infection model with human dendritic cells

Different species of Leishmania are responsible for cutaneous, mucocutaneous or visceral leishmaniasis infections in millions of people around the world [14]. The adverse reactions caused by antileishmanial drugs, emergence of resistance and lack of a vaccine have motivated the search for new therapeutic options to control this disease. Different sources of antimicrobial molecules are under study as antileishmanial agents, including peptides with antimicrobial and/or immunomodulatory activity, which have been considered to be potentially active against diverse species of Leishmania [7] and [39]. This study evaluated the cytotoxicity on dendritic cells, hemolytic activity, leishmanicidal properties on Leishmania panamensis and Leishmania major promastigotes and effectiveness on parasite intracellular forms (dendritic cells infected with L. panamensis and L. major promastigotes), when each parasite in culture was exposed to different concentrations of a group of synthetic peptides with previously reported antimicrobial properties, which were synthesized based on their naturally occurring reported sequences. Dermaseptin, Pr-2 and Pr-3 showed inhibitory activity on the growth of L. panamensis promastigotes, while Andropin and Cecropin A (with a selectivity index of 4 and 40, respectively) showed specific activity against intracellular forms of this species. The activities of Andropin and Cecropin A were exclusively against the intracellular forms of the parasite, therefore indicating the relevance of these two peptides as potential antileishmanial agents. In the case of L. major promastigotes, Melittin and Dermaseptin showed inhibitory activity, the latter also showed a selectivity index of 8 against intracellular forms. These findings suggest Andropin, Cecropin A and Dermaseptin as potential therapeutic tools to treat New and Old World cutaneous leishmaniasis.     

Infected dendritic cells facilitate systemic dissemination and transplacental passage of the obligate intracellular parasite Neospora caninum in mice

The obligate intracellular parasite Neospora caninum disseminates across the placenta and the blood-brain barrier, to reach sites where it causes severe pathology or establishes chronic persistent infections. The mechanisms used by N. caninum to breach restrictive biological barriers remain elusive. To examine the cellular basis of these processes, migration of different N. caninum isolates (Nc-1, Nc-Liverpool, Nc-SweB1 and the Spanish isolates: Nc-Spain 3H, Nc-Spain 4H, Nc-Spain 6, Nc-Spain 7 and Nc-Spain 9) was studied in an in vitro model based on a placental trophoblast-derived BeWo cell line. Here, we describe that infection of dendritic cells (DC) by N. caninum tachyzoites potentiated translocation of parasites across polarized cellular monolayers. In addition, powered by the parasite's own gliding motility, extracellular N. caninum tachyzoites were able to transmigrate across cellular monolayers. Altogether, the presented data provides evidence of two putative complementary pathways utilized by N. caninum, in an isolate-specific fashion, for passage of restrictive cellular barriers. Interestingly, adoptive transfer of tachyzoite-infected DC in mice resulted in increased parasitic loads in various organs, e.g. the central nervous system, compared to infections with free parasites. Inoculation of pregnant mice with infected DC resulted in an accentuated vertical transmission to the offspring with increased parasitic loads and neonatal mortality. These findings reveal that N. caninum exploits the natural cell trafficking pathways in the host to cross cellular barriers and disseminate to deep tissues. The findings are indicative of conserved dissemination strategies among coccidian apicomplexan parasites.     

Chickens (Gallus domesticus) are natural intermediate hosts of Neospora caninum

Neospora caninum naturally infects many mammal species, but has not previously been demonstrated in birds. We examined sera for N. caninum antibodies from 200 outdoor chickens and from 200 chickens confined indoors in the state of Bahia, Brazil. Seroprevalence was greater in outdoor chickens (23.5% versus 1.5%, P<0.001). PCR testing for N. caninum was positive in six of 10 seropositive chickens. Amplicons from two of these were sequenced and had 97-98% nucleotide identity with N. caninum. This finding extends the list of intermediate hosts of N. caninum to include birds and may have important epidemiological consequences.     

Structure-activity relationships from in vitro efficacies of the thiazolide series against the intracellular apicomplexan protozoan Neospora caninum

Nitazoxanide (2-acetolyloxy-N-(5-nitro 2-thiazolyl) benzamide; NTZ) represents the parent compound of a novel class of broad-spectrum anti-parasitic compounds named thiazolides. NTZ is active against a wide variety of intestinal and tissue-dwelling helminths, protozoa, enteric bacteria and a number of viruses infecting animals and humans. While potent, this poses a problem in practice, since this obvious non-selectivity can lead to undesired side effects in both humans and animals. In this study, we used real time PCR to determine the in vitro activities of 29 different thiazolides (NTZ-derivatives), which carry distinct modifications on both the thiazole- and the benzene moieties, against the tachyzoite stage of the intracellular protozoan Neospora caninum. The goal was to identify a highly active compound lacking the undesirable nitro group, which would have a more specific applicability, such as in food animals. By applying self-organizing molecular field analysis (SOMFA), these data were used to develop a predictive model for future drug design. SOMFA performs self-alignment of the molecules, and takes into account the steric and electrostatic properties, in order to determine 3D-quantitative structure activity relationship models. The best model was obtained by overlay of the thiazole moieties. Plotting of predicted versus experimentally determined activity produced an r2 value of 0.8052 and cross-validation using the "leave one out" methodology resulted in a q2 value of 0.7987. A master grid map showed that large steric groups at the R2 position, the nitrogen of the amide bond and position Y could greatly reduce activity, and the presence of large steric groups placed at positions X, R4 and surrounding the oxygen atom of the amide bond, may increase the activity of thiazolides against Neospora caninum tachyzoites. The model obtained here will be an important predictive tool for future development of this important class of drugs.     

Coyotes (Canis latrans) are definitive hosts of Neospora caninum

Four captive-raised coyote pups consumed tissues from Neospora caninum-infected calves. Faeces were examined from 4 days before to 28 days after infection. One pup shed N. caninum-like oocysts, which tested positive for N. caninum and negative for Hammondia heydorni using PCR tests. Coyotes are the second discovered definitive host of N. caninum, after dogs. In North America, the expanding coyote ranges and population increase the probability of contact with domestic livestock. To reduce the risk of transmission of N. caninum to intensively farmed cattle, we recommend protection of feedstuffs using canid-proof fences, and careful disposal of dead stock.     

Toxoplasma gondii-derived heat shock protein 70 stimulates the maturation of human monocyte-derived dendritic cells

We investigated the role of Toxoplasma gondii-derived heat shock protein 70 (TgHSP70) as a dendritic cell (DC) maturation-inducing molecule. TgHSP70 induced the maturation of human monocyte-derived dendritic cells as determined by increased levels of surface markers, namely, CD40, CD80, CD86, and HLA-DR. Moreover, TgHSP70 also reduced phagocytic activity and increased the allostimulatory capacity of DCs, suggesting the functional maturation of DCs by TgHSP70. Maturation of DCs by TgHSP70 also elicited a significant increase in IL-12 production in a polymyxin B-insensitive manner. TgHSP70 also stimulated extracellular signal-regulated kinase and p38 kinase pathways in DCs, and TgHSP70-induced IL-12 production was inhibited by SB203580 but not by PD98059, thus indicating the role of p38 kinase in the maturation of DCs by TgHSP70. This study demonstrates the role of TgHSP70 in the functional maturation of DCs and suggests TgHSP70 as a useful molecule for the development of a vaccine against T. gondii.     

Toxoplasma gondii: Impaired maturation and pro-inflammatory response of dendritic cells in MIF-deficient mice favors susceptibility to infection

Macrophage migration inhibitory factor (MIF) has been found to be involved in host resistance to several parasitic infections. To determine the mechanisms of MIF-dependent responses to Toxoplasma gondii, we investigated host resistance in MIF−/− mice (BALB/c background) during natural oral infection. We focused on the potential involvement of MIF in Dendritic Cell (DC) maturation and IL-12 production. Following oral T. gondii infection, wild type mice developed a strong IL-12 response with an adequate maturation of their draining mesenteric lymph node DC (MLNDC) population and were resistant to challenge with either 40 or 100 cysts (ME49 strain). In contrast, similarly infected MIF−/− mice mounted a weak IL-12 response, displayed immature MLNDCs in the early phases of infection and rapidly succumbed to both type of challenges. Lack of maturation and IL-12 production of DCs in response to T. gondii antigens was confirmed by in vitro studies, and these effects were reversed following treatment with recombinant MIF. These findings demonstrate that MIF-induced early DC maturation and IL-12 production mediate resistance to T. gondii infection.     

Mycobacterium bovis BCG decreases MHC-II expression in vivo on murine lung macrophages and dendritic cells during aerosol infection

Mycobacterium tuberculosis and M. bovis BCG infect APCs. In vitro, mycobacteria inhibit IFN-gamma-induced MHC-II expression by macrophages, but the effects of mycobacteria on lung APCs in vivo remain unclear. To assess MHC-II expression on APCs infected in vivo, mice were aerosol-infected with GFP-expressing BCG. At 28 d, ∼1% of lung APCs were GFP+ by flow cytometry and CFU data. Most GFP+ cells were CD11b^high/CD11c^neg-mid lung macrophages (58-68%) or CD11b^high/CD11c^high DCs (28-31%). Lung APC MHC-II expression was higher in infected mice than naïve mice. Within infected lungs, however, MHC-II expression was lower in GFP+ cells than GFP− cells for both macrophages and DCs. MHC-II expression was also inhibited on purified lung macrophages and DCs that were infected with BCG in vitro. Thus, lung APCs that harbor mycobacteria in vivo have decreased MHC-II expression relative to uninfected APCs from the same lung, possibly contributing to evasion of T cell responses.     

Proteome of Aedes aegypti larvae in response to infection by the intracellular parasite Vavraia culicis

We report on the modification of the Aedes aegypti larval proteome following infection by the microsporidian parasite Vavraia culicis. Mosquito larvae were sampled at 5 and 15 days of age to compare the effects of infection when the parasite was in two different developmental stages. Modifications of the host proteome due to the stress of infection were distinguished from those of a more general nature by treatments involving hypoxia. We found that the major reaction to stress was the suppression of particular protein spots. Older (15 days) larvae reacted more strongly to infection by V. culicis (46% of the total number of spots affected; 17% for 5 days larvae), while the strongest reaction of younger (5 days) larvae was to hypoxia for pH range 5-8 and to combined effects of infection and hypoxia for pH range 3-6. MALDI-TOF results indicate that proteins induced or suppressed by infection are involved directly or indirectly in defense against microorganisms. Finally, our MALDI-TOF results suggest that A. aegypti larvae try to control or clear V. culicis infection and also that V. culicis probably impairs the immune defense of this host via arginases-NOS competition.     

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.     

Seroprevalence of antibodies to Neospora caninum in dogs and raccoon dogs in Korea

Neospora caninum is an important cause of abortion in cattle, and dogs are its only known definitive host. Its seroprevalence among domestic urban and rural dogs and feral raccoon dogs (Nyctereutes procyonoides koreensis) in Korea was studied by indirect fluorescent antibody test (IFAT) and by the neospora agglutination test (NAT), respectively. Antibodies to N. caninum were found in 8.3% of urban dogs and in 21.6% of dogs at dairy farms. Antibody titers ranged from 1:50 to 1:400. Antibodies to N. caninum were found in six (23%) of 26 raccoon dogs. However, the potential role of raccoon dogs as a source of horizontal transmission of bovine neosporosis needs further investigation. The results of this study suggest that there is a close relationship between N. caninum infection among dairy farm dogs and cattle in Korea. This study reports for the first time upon the seroprevalence of N. caninum infection in raccoon dogs in Korea.     

Induction of tachyzoite egress from cells infected with the protozoan Neospora caninum by nitro- and bromo-thiazolides, a class of broad-spectrum anti-parasitic drugs

Neospora caninum represents an important pathogen causing stillbirth and abortion in cattle and neuromuscular disease in dogs. Nitazoxanide (NTZ) and its deacetylated metabolite tizoxanide (TIZ) are nitro-thiazolyl-salicylamide drugs with a broad-spectrum anti-parasitic activity in vitro and in vivo. In order to generate compounds potentially applicable in food and breeding animals, the nitro group was removed, and the thiazole-moiety was modified by other functional groups. We had shown earlier that replacement of the nitro-group by a bromo-moiety did not notably affect in vitro efficacy of the drugs against N. caninum. In this study we report on the characterization of two bromo-derivatives, namely Rm4822 and its de-acetylated putative metabolite Rm4847 in relation to the nitro-compounds NTZ and TIZ. IC(50) values for proliferation inhibition were 4.23 and 4.14 microM for NTZ and TIZ, and 14.75 and 13.68 microM for Rm4822 and Rm4847, respectively. Complete inhibition (IC(99)) was achieved at 19.52 and 22.38 microM for NTZ and TIZ, and 18.21 and 17.66 microM for Rm4822 and Rm4847, respectively. However, in order to exert a true parasiticidal effect in vitro, continuous culture of infected fibroblasts in the presence of the bromo-thiazolide Rm4847 was required for a period of 3 days, while the nitro-compound TIZ required 5 days continuous drug exposure. Both thiazolides induced rapid egress of N. caninum tachyzoites from their host cells, and egress was inhibited by the cell membrane permeable Ca(2+)-chelator BAPTA-AM. Host cell entry by N. caninum tachyzoites was inhibited by Rm4847 but not by TIZ. Upon release from their host cells, TIZ-treated parasites remained associated with the fibroblast monolayer, re-invaded neighboring host cells and resumed proliferation in the absence of the drug. In contrast, Rm4847 inhibited host cell invasion and respective treated tachyzoites did not proliferate further. This demonstrated that bromo- and nitro-thiazolides exhibit differential effects against the intracellular protozoan N. caninum and bromo-thiazolides could represent a valuable alternative to the nitro-thiazolyl-salicylamide drugs.