Efficacy of the bumped kinase inhibitor BKI-1708 against the cyst-forming apicomplexan parasites Toxoplasma gondii and Neospora caninum in vitro and in experimentally infected mice.

Toxoplasma gondii and Neospora caninum are major worldwide morbidity-causing pathogens. Bumped kinase inhibitors (BKIs) are a compound class that has been optimized to target the apicomplexan calcium-dependent protein kinase 1 (CDPK1) - and several members of this class have proven to be safe and highly active in vitro and in vivo. BKI-1708 is based on a 5-aminopyrazole-4-carboxamide scaffold, and exhibited in vitro IC50 values of 120 nM for T. gondii and 480 nM for N. caninum ß-galactosidase expressing strains, and did not affect human foreskin fibroblast (HFF) viability at concentrations up to 25 µM. Electron microscopy established that exposure of tachyzoite-infected fibroblasts to 2.5 µM BKI-1708 in vitro induced the formation of multinucleated schizont-like complexes (MNCs), characterized by continued nuclear division and harboring newly formed intracellular zoites that lack the outer plasma membrane. These zoites were unable to finalize cytokinesis to form infective tachyzoites. BKI-1708 did not affect zebrafish (Danio rerio) embryo development during the first 96 h following egg hatching at concentrations up to 2 µM. Treatments of mice with BKI-1708 at 20 mg/kg/day during five consecutive days resulted in drug plasma levels ranging from 0.14 to 4.95 µM. In vivo efficacy of BKI-1708 was evaluated by oral application of 20 mg/kg/day from day 9-13 of pregnancy in mice experimentally infected with N. caninum (NcSpain-7) tachyzoites or T. gondii (TgShSp1) oocysts. This resulted in significantly decreased cerebral parasite loads and reduced vertical transmission in both models without drug-induced pregnancy interference.

Study of specific immunodominant antigens in different stages of Neospora caninum, Toxoplasma gondii, Sarcocystis spp. and Hammondia spp.

The family Sarcocystidae includes several intracellular coccidial parasites such as Toxoplasma gondii, Neospora caninum, Sarcocystis spp. and Hammondia spp. with heteroxenous life cycles involving different parasitic stages (oocysts/sporocysts, tachyzoites and bradyzoites in tissue cysts). The aim of this work was to evaluate monoclonal antibodies (MAb) (anti NcSAG1, anti NcSAG4 and anti TgCC2) and/or polyclonal antibodies (PAb) (anti NcSAG4 and anti TgBAG1) to label specific immunodominant antigens in different parasitic stages of N. caninum (oocyst, bradyzoite and tachyzoite), T. gondii (oocyst, cyst and tachyzoite), H. heydorni (oocyst), S. cruzi (cyst and bradyzoite) and S. falcatula (sporocyst). It was observed that the MAb directed against NcSAG1 reacted exclusively with N. caninum tachyzoites. In contrast, the MAb directed against NcSAG4 did not react with any of the parasites tested at any stage. The MAb directed against NcSAG4 reacted with both N. caninum and T. gondii tachyzoites, T. gondii tissue cysts and S. cruzi tissue cysts and bradyzoites. As expected, the MAb directed against the T. gondii tissue cyst wall antigen TgCC2 reacted with T. gondii tissue cysts, N. caninum bradyzoites, but also with T. gondii and H. heydorni oocysts and S. falcatula sporocysts. Finally, the PAb directed against the T. gondii bradyzoite proteinTgBAG1 reacted with T. gondii tissue cysts, N. caninum bradyzoites, and also with S. cruzi tissue cysts and bradyzoites. These data reveal a wide range of cross-reactions between different species of protozoa and between different developmental stages, which should be taken into account in the design and evaluation of diagnostic tests, as well as in the assessment of vaccination and challenge studies.

In vitro regulation of gene expression of pregnancy-associated proteins and cytokines in bovine endometrial epithelial cells and bovine trophoblastic cells by infection with Neospora caninum.

Abortion caused by the parasite Neospora caninum is an important threat to the livestock industry worldwide. Trophoblasts and caruncular cells play major roles in initiating innate immune responses and controlling parasite infection at the fetal-maternal interface. In the present study, bovine uterine epithelial cells (BUECs) and bovine trophoblastic (BT) cells treated with bovine interferon-gamma (IFN-γ), IFN-alpha (IFN-α) and IFN-tau (IFN-τ) followed by infection with N. caninum were examined by measuring the mRNA expression levels of numerous pregnancy-associated proteins and observing parasite growth to elucidate the host-parasite interaction at the uteroplacental region. N. caninum infection increased the expression of prolactin-related protein 1 (PRP1), pregnancy-associated glycoprotein 1 (PAG1), and cytokines (TNF-α, IL-8 and IL-10) in BUECs and of IL-8 in BT cells. Bovine IFN-γ inhibited IL-8 and TNF-α expression in BUECs and IL-8 in BT cells. In contrast, the expression of the interferon-stimulated gene OAS1 was significantly increased by treatment of the infected BT cells with IFN-γ. However, treatment with bovine IFNs did not inhibit N. caninum growth in either cell line. In conclusion, our results suggest that bovine IFN-γ plays a crucial role in control of pathogenesis in uterus and induction of inflammatory response in the placental region following N. caninum infection, rather than growth inhibition of the parasites.

Infection with different Neospora caninum strains causes differences in the glycosylation pattern in the uteri and placentae of Neospora caninum-infected heifers.

Neospora caninum is an obligate intracellular parasite that causes abortion in ruminants. Different strains produce differences in the severity of disease outcomes. These differences may cause physiological or pathological changes in cells, modifying the intercellular interactions and intracellular transport pathways that could be evidenced by identifying the terminal sugars. This study aimed to characterize the oligosaccharide pattern in the bovine placenta and uterus after infection with tachyzoites of three different strains of N. caninum (Nc-1, Nc-6 Argentina and Nc Spain-7) during early gestation. Fourteen heifers were inoculated intravenously on day 70 of gestation with 2 × 108 N. caninum tachyzoites and samples of placentae and uteri were analysed by histology and lectin histochemistry. In the infected groups, severe placentitis was associated with changes in lectin binding in the vascular endothelium by Lens culinaris agglutinin (LCA), Pisum sativum agglutinin (PSA) and Ricinus communis I (RCA-I) lectins, in the epithelial cells of the endometrial glands by RCA-I, Dolichos biflorus agglutinin (DBA), succinylated wheat germ agglutinin, peanut agglutinin (PNA), concanavalin-A (CON-A), LCA, PSA and Phaseolus vulgaris erythroagglutinin (PHA-e), and in the trophoblast layer by PNA, CON-A, LCA, PSA, PHA-e, soybean agglutinin, RCA-I, DBA and Bandieraea simplicifolia agglutinin (BSA-I). The results suggest that N. caninum causes changes in the glycosylation pattern in the maternofetal interface tissues and might cause abortions in early gestation due to changes in the cellular structure of the placenta.

Are genetically modified protozoa eligible for ATMP status? Concerning the legal categorization of an oncolytic protozoan drug candidate.

Neospora caninum is an obligate intracellular protozoan that affects several animal species. It is not pathogenic for humans, and its ability to infect and lyse a variety of cells and stimulate the immune system makes it an interesting drug candidate in oncology. The intrinsic oncolytic properties of N. caninum have been confirmed in several preclinical models. Moreover, it can be modified to improve its safety and/or efficacy against cancer cells. In this study, we propose the legal categorization of this new biological drug candidate and the impact of modifications, notably the integration of a suicide gene, the deletion of a gene allowing its multiplication in healthy cells, and/or the insertion of a gene coding for a therapeutic protein into its genome. When unmodified, N. caninum can be categorized as a biological medicinal product, whereas modifications aimed at increasing its safety classify it as a Somatic Cell Therapy Medicinal Product, and modifications aiming to increase its efficacy or both safety and efficacy make it as a Gene Therapy Medicinal Product. This categorization is fundamental because it determines the guidelines applicable for preclinical development. These guidelines being numerous and complex, we have focused on the key requirements necessary for the development of the future medicinal product.

Construction of luciferase-expressing Neospora caninum and drug screening.

BACKGROUND: Neospora caninum is an apicomplexan parasite that is particularly responsible for abortions in cattle and neuromuscular disease in dogs. Due to the limited effectiveness of currently available drugs, there is an urgent need for new therapeutic approaches to control neosporosis. Luciferase-based assays are potentially powerful tools in the search for antiprotozoal compounds, permitting the development of faster and more automated assays. The aim of this study was to construct a luciferase-expressing N. caninum and evaluate anti-N. caninum drugs. METHODS: Luciferase-expressing N. caninum (Nc1-Luc) was constructed using clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (CRISPR/Cas9). After testing the luciferase expression and phenotype of the Nc1-Luc strains, the drug sensitivity of Nc1-Luc strains was determined by treating them with known positive or negative drugs and calculating the half-maximal inhibitory concentration (IC50). The selective pan-rapidly accelerated fibrosarcoma (pan-RAF) inhibitor TAK-632 was then evaluated for anti-N. caninum effects using Nc1-Luc by luciferase activity reduction assay and other in vitro and in vivo studies. RESULTS: The phenotypes and drug sensitivity of Nc1-Luc strains were consistent with those of the parental strains Nc1, and Nc1-Luc strains can be used to determine the IC50 for anti-N. caninum drugs. Using the Nc1-Luc strains, TAK-632 showed promising activity against N. caninum, with an IC50 of 0.6131 µM and a selectivity index (SI) of 62.53. In vitro studies demonstrated that TAK-632 inhibited the invasion, proliferation, and division of N. caninum tachyzoites. In vivo studies showed that TAK-632 attenuated the virulence of N. caninum in mice and significantly reduced the parasite burden in the brain. CONCLUSIONS: In conclusion, a luciferase-expressing N. caninum strain was successfully constructed, which provides an effective tool for drug screening and related research on N. caninum. In addition, TAK-632 was found to inhibit the growth of N. caninum, which could be considered as a candidate lead compound for new therapeutics for neosporosis.

Protective effect of Inonotus obliquus polysaccharide on mice infected with Neospora caninum.

The study aimed to explore the protective effects of Inonotus obliquus polysaccharide (IOP) on Neospora caninum (N. caninum) infection. Our data showed that the survival rate of the mice was the highest and the survival time was the longest when the IOP was 2 mg/10 g. In agreement with these observations, IOP alleviated the pathological damage in the various organs and tissues of the mice. Compared with that in the Neosporidium infection model group, the content of N. caninum in the heart, liver, spleen, lung, kidney and brain, determined through HE staining, was significantly lower. In addition, IOP inhibited the levels of immunoglobulin G1 (IgG1) and immunoglobulin G2 (IgG2a) from the 21st to 42nd day of the administration group, whereas the levels of interleukin-12 (IL-12) and serum tumor necrosis factor alpha (TNF-α) were down-regulated at 7 d - 42 d. The production of CD4+ T lymphocytes was promoted, the number of CD8+ T lymphocytes were significantly lower and the CD4+/CD8+ ratio was significantly elevated. Furthermore, IOP effectively balanced the levels of hormones including gonadotropin-releasing hormone (GnRH), luteotropic hormone (LH) and testosterone (T) in male mice, and progesterone (PROG), estradiol (E2) and prolactin (PRL) in female mice. These findings demonstrate that IOP exerts protective effects against pathological damage caused by N. caninum infection in mice, and improve the immune function of the organism and regulate the secretion balance of sex hormones.

Serological and molecular detection of Toxoplasma gondii and Neospora caninum in free-ranging rats from Nagpur, India.

Toxoplasma gondii and Neospora caninum are cyst-forming coccidian parasites that infect both wild and domestic non-felids as intermediate hosts, with rodents serving as important reservoir hosts during their life cycles. This study was aimed at investigating T. gondii and N. caninum infections and identifying factors favouring T. gondii infection in free-ranging rats from India. A total of 181 rodents were trap-captured, and blood and brain samples were subsequently collected for serological and molecular examination of T. gondii and N. caninum. Antibodies against T. gondii and N. caninum were detected by MAT/NAT and IFAT in 13.8% (25/181) and 1.65% (3/181) of rodents, respectively. All three N. caninum samples positive by NAT/IFAT were also positive for ELISA, while for T. gondii, 19 of 25 MAT/IFAT positive samples were also positive for ELISA. The antibody titers (MAT/NAT/IFAT) of rodents seropositive for T. gondii ranged from 25 to 400, while those of rats seropositive for N. caninum ranged from 25 to 100. Also, using PCR, DNA from T. gondii (B1 gene) and N. caninum (NC5 gene) was found in 2.76% (5/181) of brain samples and 0.55% (1/181) of brain samples. All PCR positive samples were also seropositive. No mixed infections were observed in the serological and molecular detections. A Chi-square analysis revealed that older rats and rats living in urban areas are significantly associated with T. gondii infection; however, rodent species, gender, location, habitat types, and seasonality were statistically nonsignificant. Overall, this study demonstrated that T. gondii was widely distributed while N. caninum was less prevalent among free-ranging rats in the studied area.

Dysfunction, oxidative stress markers, and cytokine expression in the placentae of mice experimentally infected with Neospora caninum.

Neosporosis is the major cause of abortion and reproductive failures in cattle, leading to significant economic losses. In this study, we evaluated the impact of Neospora caninum infection on oxidative stress (OS) markers and local cytokine mRNA expression at the placenta, as well as its effect on the progesterone (P4) serum levels and systemic cytokine profile in a pregnant mouse model. Infected pregnant mice (NC-1 group) showed increased percentages of fetal losses and IFN-γ serum levels, decreased serum progesterone, increased placental mRNA expression levels of both Th1-type (IFN-γ and TNF-α) and Th2-type (IL-4) cytokines, and inhibited expression of TGF-ß1 (Treg) compare to control dams (CONTROL group). In addition, lipid peroxidation and ROS were increased, whereas the antioxidant enzymes, superoxide dismutase (SOD), and catalase (CAT) activities were modified in the placentae of infected mice compared to control mice. These findings demonstrate that multiple factors, including placental OS, are involved in fetal losses associated with N. caninum infection in mice, thus OS contribution to the placental physiopathology of neosporosis in other hosts must not be ruled out.

Bovine leukosis virus, bovine viral diarrhea, and bovine neosporosis seroprevalence in specialized dairy herds in Antioquia-Colombia.

Enzootic bovine leukosis (EBL) is a chronic infectious disease caused by the bovine leukosis virus (BLV), a Deltaretrovirus. Bovine viral diarrhea (BVD) is an infectious disease caused by a pestivirus. Bovine neosporosis is caused by the obligate intracellular parasite Neospora caninum (Nc). These pathogens can have horizontal (postnatal) or vertical (transplacental) transmissions and affect the productive and reproductive performance of infected bovines. This work aimed to detect BLV, BVD, and Nc seroprevalence in specialized dairy cattle from the north, east, and Aburrá Valley regions of the Department of Antioquia, the highest in milk production regions in Antioquia. A total of 599 blood samples, obtained from 53 specialized dairy cattle herds, were evaluated by the ELISA test. The results revealed a seroprevalence of 41.13% for BLV (242/599), 28.48% (163/599) for Nc, and 22.7% (132/599) for BVD. Regarding the regional seroprevalence evaluation, BLV was found in 47.02% of the samples from the east, 36.87% from the north, and 46.02% from the Aburrá Valley. Nc was found in 31.03% of the samples from the east, 24.26% from the north, and 36.63% from Aburrá Valley. BVD was found in 21.62% of the samples from the east, 25.03% from the north region, and 10.39% of the samples from the Aburrá Valley. It is highlighted by these results that the north region, with the highest milk production in Antioquia, had the lowest BLV and Nc seroprevalences but the highest seroprevalence of BVD. BLV has increased in Antioquia in recent years, and as an immunosuppressive infection, opportunities for other pathogens are created by it. A significant statistical difference was found in the average prevalence of the pathogens according to the municipality, cattle breed, and region of origin of the sample. The seroprevalence of these pathogens in specialized dairy herds in Antioquia can be classified as medium-low. However, it is recommended that biosecurity practices should be maximized to avoid the spread of these pathogens due to the variability detected in the region, municipality, breed group, and herd age. The rapid and efficient diagnosis of these three pathogens through reliable methodologies will allow for the control of dissemination in dairy herds.

Characterization of Neospora caninum virulence factors NcGRA7 and NcROP40 in bovine target cells.

Bovine neosporosis is one of the major causes of reproductive failure in cattle worldwide, and differences in virulence between isolates have been widely shown. However, the molecular basis and mechanisms underlying virulence in Neospora caninum are mostly unknown. Recently, we demonstrated the involvement of NcGRA7 and NcROP40 in the virulence of N. caninum in a pregnant murine model using single knockout mutants in these genes generated by CRISR/Cas9 technology. In this study, the role of these proteins was investigated in two in vitro models using bovine target cells: trophoblast (F3 cell line) and monocyte-derived macrophages (BoMØ). The proliferation capacity of the single knockout mutant parasites was compared to the wild-type strain, the Nc-Spain7 isolate, using both cell populations. For the bovine trophoblast, no differences were observed in the growth of the defective parasites compared to the wild-type strain, neither in the proliferation kinetics nor in the competition assay. However, in naïve BoMØ, a significant decrease in the proliferation capacity of the mutant parasites was observed from 48 h pi onwards. Stimulation of BoMØ with IFN-γ showed a similar inhibition of tachyzoite growth in defective and wild-type strains in a dose-dependent manner. Finally, BoMØ infected with knockout parasites showed higher expression levels of TLR3, which is involved in pathogen recognition. These results suggest that NcGRA7 and NcROP40 may be involved in the manipulation of innate immune defense mechanisms against neosporosis and confirm the usefulness of the BoMØ model for the evaluation of N. caninum virulence mechanisms. However, the specific functions of these proteins remain unknown, opening the way for future research.

Nasal administration of recombinant Neospora caninum secreting IL-15/IL-15Rα inhibits metastatic melanoma development in lung.

BACKGROUND: Metastases are the leading cause of mortality in many cancer types and lungs are one of the most common sites of metastasis alongside the liver, brain, and bones. In melanoma, 85% of late-stage patients harbor lung metastases. A local administration could enhance the targeting of metastases while limiting the systemic cytotoxicity. Therefore, intranasal administration of immunotherapeutic agents seems to be a promising approach to preferentially target lung metastases and decrease their burden on cancer mortality. From observations that certain microorganisms induce an acute infection of the tumor microenvironment leading to a local reactivating immune response, microbial-mediated immunotherapy is a next-generation field of investigation in which immunotherapies are engineered to overcome immune surveillance and escape from microenvironmental cancer defenses. METHODS: The goal of our study is to evaluate the potential of the intranasal administration of Neospora caninum in a syngeneic C57BL6 mouse model of B16F10 melanoma lung metastases. It also compares the antitumoral properties of a wild-type N. caninum versus N. caninum secreting human interleukin (IL)-15 fused to the sushi domain of the IL-15 receptor α chain, a potent activator of cellular immune responses. RESULTS: The treatment of murine lung metastases by intranasal administration of an N. caninum engineered to secrete human IL-15 impairs lung metastases from further progression with only 0,08% of lung surface harboring metastases versus 4,4% in wild-type N. caninum treated mice and 36% in untreated mice. The control of tumor development is associated with a strong increase in numbers, within the lung, of natural killer cells, CD8+ T cells and macrophages, up to twofold, fivefold and sixfold, respectively. Analysis of expression levels of CD86 and CD206 on macrophages surface revealed a polarization of these macrophages towards an antitumoral M1 phenotype. CONCLUSION: Administration of IL-15/IL-15Rα-secreting N. caninum through intranasal administration, a non-invasive route, lend further support to N. caninum-demonstrated clear potential as an effective and safe immunotherapeutic approach for the treatment of metastatic solid cancers, whose existing therapeutic options are scarce. Combination of this armed protozoa with an intranasal route could reinforce the existing therapeutic arsenal against cancer and narrow the spectrum of incurable cancers.

Analytical sensitivity of a multiplex quantitative PCR for Toxoplasma gondii and Neospora caninum.

Cyst-forming coccidia, Toxoplasma gondii and Neospora caninum, are recognised as important causes of animal disease. Molecular diagnostics based on the presence of DNA in animal tissue are required to specifically detect T. gondii and N. caninum while achieving high levels of analytical sensitivity. We optimised available single-plex probe base qPCR assays into a multiplexed qPCR panel to detect cyst-forming coccidia, i.e. T. gondii and N. caninum. The T. gondii assay is based on a 529-bp repetitive (REP) element and the N. caninum assay on the NC5 repetitive region. Using target sequence synthetic DNA, the limit of detection (LOD) was determined to be 100 copies, that is less than a single tachyzoite of either T. gondii or N. caninum. The T. gondii and N. caninum multiplexed qPCR assay optimised in this study can be used to effectively detect parasite DNA for diagnostic purposes in animal tissue.

Neospora caninum and Toxoplasma gondii infections in one-humped camels (Camelus dromedarius) in central desert of Iran.

The protozoan parasite Neospora caninum infects carnivores as definitive and a wide range of mammals as intermediate hosts. This parasite is regarded as an important cause of abortion in cattle worldwide, causing significant economic losses. Although there is serological evidence of infection in Old World camelids, the significance of N. caninum in these animal species is still poorly understood. The aim of this study was to use molecular and histological methods to detect N. caninum in the blood and tissues of 100 slaughtered one-humped camels (Camelus dromedarius) in Iran. For this, genomic DNA was extracted from blood, brain, portal lymph node and liver of the camels, and nested-PCR assay followed by sequencing were performed. Besides, paraffin-embedded tissue sections were stained with hematoxylin and eosin (H&E) and studied microscopically. In addition, immunohistochemical staining for N. caninum was attempted on brain samples with positive PCR results. All animals were tested for antibodies against N. caninum and Toxoplasma gondii by whole tachyzoite-agglutination tests. N. caninum DNA was detected in blood, brain, and portal lymph node, but not in the liver of two (2%) camels. Histopathological examination revealed cysts resembling N. caninum in brain samples of one of these camels; however, immunohistochemical staining for N. caninum and T. gondii did not allow a morphological identification. IgG antibodies to N. caninum and T. gondii were detected in 36% and 35% of the camels, respectively. This study provides the first insight into direct detection of N. caninum in C. dromedarius in Iran. Further molecular studies on aborted fetuses, stillborn animals and cases of perinatal mortality are needed to understand the possible involvement of N. caninum in cases of reproductive failure. As the definitive hosts of N. caninum are domestic and wild canids, producers should be advised to monitor and limit exposure of their camelids to these species and their feces.

A preliminary study on placental damage associated to experimental neosporosis in BALB/c mice.

Neospora caninum is a protozoan parasite which can infect a range of animals, including dogs, cattle, and sheep. Bovine neosporosis, which mainly causes abortion in cattle, results in substantial economic losses worldwide. To study the effects of N. caninum infection on the placenta, a pregnant mouse model for N. caninum infection was established. The litter size (8.6 ± 1.5) and the number of live pups (6.4 ± 1.8) of infected dams were significantly lower compared with those of non-infected dams. Trophoblast cell shrinkage and a large number of apoptosomes were detected in the placentas of the infected group. The parasite load in the placental tissue was significantly higher with time after infection. Likewise, apoptosis of placental trophoblast cells significantly increased with time after infection. Among the 66 apoptotic genes detected in this study, eight genes, including Bcl-2, were significantly differentially expressed by about > tenfold in infected and uninfected mice. The expression of BAX and tumor necrosis factor-alpha (TNF-α) was upregulated in the placental cells of the infected mice, whereas the expression of BCL-2 was downregulated. Enzyme-linked immunosorbent assays (ELISAs) showed that apoptotic protease caspase-3 level was significantly increased in placental cell suspension, and insulin-like growth factor (IGF)-2 level was significantly reduced. Acetylcholine (ACH) and placental prolactin (PL) levels were initially decreased but eventually increased. In summary, infection of mice with N. caninum caused apoptotic damage to the placental tissues, cells, and genes and affected the normal physiological functions of placenta, which may largely explain the adverse pregnancy outcomes caused by N. caninum infection in mice.

Temporal transcriptomic changes in microRNAs involved in the host immune response and metabolism during Neospora caninum infection.

BACKGROUND: Neospora caninum infection is a major cause of abortion in cattle, which results in serious economic losses to the cattle industry. However, there are no effective drugs or vaccines for the control of N. caninum infections. There is increasing evidence that microRNAs (miRNAs) are involved in many physiological and pathological processes, and dysregulated expression of host miRNAs and the biological implications of this have been reported for infections by various protozoan parasites. However, to our knowledge, there is presently no published information on host miRNA expression during N. caninum infection. METHODS: The expression profiles of miRNAs were investigated by RNA sequencing (RNA-seq) in caprine endometrial epithelial cells (EECs) infected with N. caninum at 24 h post infection (pi) and 48 hpi, and the functions of differentially expressed (DE) miRNAs were predicted by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses. The transcriptome data were validated by using quantitative real-time polymerase chain reaction. One of the upregulated DEmiRNAs, namely chi-miR-146a, was selected to study the effect of DEmiRNAs on the propagation of N. caninum tachyzoites in caprine EECs. RESULTS: RNA-seq showed 18 (17 up- and one downregulated) and 79 (54 up- and 25 downregulated) DEmiRNAs at 24 hpi and 48 hpi, respectively. Quantitative real-time polymerase chain reaction analysis of 13 randomly selected DEmiRNAs (10 up- and three downregulated miRNAs) confirmed the validity of the RNA-seq data. A total of 7835 messenger RNAs were predicted to be potential targets for 66 DEmiRNAs, and GO and KEGG enrichment analysis of these predicted targets revealed that DEmiRNAs altered by N. caninum infection may be involved in host immune responses (e.g. Fc gamma R-mediated phagocytosis, Toll-like receptor signaling pathway, tumor necrosis factor signaling pathway, transforming growth factor-ß signaling pathway, mitogen-activated protein kinase signaling pathway) and metabolic pathways (e.g. lysine degradation, insulin signaling pathway, AMP-activated protein kinase signaling pathway, Rap1 signaling pathway, calcium signaling pathway). Upregulated chi-miR-146a was found to promote N. caninum propagation in caprine EECs. CONCLUSIONS: This is, to our knowledge, the first report on the expression profiles of host miRNAs during infection with N. caninum, and shows that chi-miR-146a may promote N. caninum propagation in host cells. The novel findings of the present study should help to elucidate the interactions between host cells and N. caninum.

Natural infection with Toxoplasma gondii, Neospora caninum and Sarcocystis species in domestic pigeons (Columba livia domestica) in Iran.

Pigeons are common birds around the world and may act as intermediate hosts of the tissue cyst-forming apicomplexan parasites Toxoplasma gondii, Neospora caninum and Sacrocystis spp. This study aimed to provide an overview on the prevalence of and exposure to these parasites in Iranian domestic rock pigeon (Columba livia domestica) through molecular, serological and histopathological examination. Blood and tissue samples (i.e., brain, heart, gizzard, neck, thigh, and pectoral muscles) were taken from 100 pigeons. Sera were screened by agglutination tests for detection of anti- T. gondii and N. caninum antibodies, genomic DNA from tissue samples were assessed by respective species-specific PCRs, and histopathological examination of tissues was carried out. A seroprevalence of 45 % to anti-T. gondii and 35 % to anti-N. caninum IgG was recorded. PCR detected T. gondii DNA in 28 pigeons. Sacrocystis spp. was detected in one animal, but sequencing of the 28 S rRNA gene product did not reveal the identity of the species. Histopathology revealed myocarditis, myositis, and gliosis in the heart, skeletal muscles, and brain, respectively. No Sarcocystis tissue-cysts were detected, but T. gondii tissue cyst-like structures in the brain (i.e., 4 %) and heart (i.e., 3 %) were found by histology. Data reported herein demonstrate that pigeons from Iran are infected with tissue cyst-forming apicomplexans, particularly T. gondii. Since domestic pigeons are in close contact with human populations, and consumption of their meat and egg is popular in different societies, control strategies for minimizing the risk of infection in both pigeons and humans are suggested.

Neospora caninum e toxoplasma gondii: revisão de literatura / Neospora caninum and toxoplasma gondii: review of literature / Neospora caninum y toxoplasma gondii: revisión de literatura

A Neospora caninum e a Toxoplasma gondii são os agentes etiológicos que causam a Neosporose e a Toxoplasmose, respectivamente. Estas duas doenças são consideradas de grande importância econômica e de distribuição mundial, que acometem tanto animais de produção quanto animais domésticos. Apresentam sinais clínicos inespecíficos, sendo a Neosporose frequentemente associada ao abortamento em fêmeas. Ambas enfermidades costumavam ser confundidas, dificultando o diagnóstico. São causadas por protozoários cosmopolitas de ciclos biológicos heteróxenos. O Toxoplasma gondii é responsável por doença clínica em cães e gatos, enquanto o Neospora caninum acomete somente cães. Além disso, não há, até o momento, relatos de Neosporose em humanos, diferente da Toxoplasmose. Ocasionalmente esta pode ocorrer em coiotes, suínos, ovinos, caprinos, equinos, cervídeos e bubalinos. Anticorpos contra Neospora tem sido descrito em raposas, camelos e felídeos. O objetivo da presente revisão, é elucidar a forma de transmissão, sinais clínicos, diagnóstico, tratamento e controle de ambas as doenças, mostrando suas semelhanças, afim de que se possa diagnosticá-las corretamente.(AU)

Neospora caninum and toxoplasma gondii are agents of great economic importance and worldwide distribution that affect production and domestic animals. They present nonspecific clinical signs, and neosporosis is a disease that frequently causes abortion in females, which is considered current, because both used to be confused, making diagnosis difficult. They are protozoan, cosmopolitan, of heterogeneous biological cycles. Toxoplasma gondii is responsible for clinical disease in dogs and cats, while Neospora caninum affects only dogs. Furthermore, there are no reports to date of neosporosis in humans, other than toxoplasmosis. Occasionally it may occur in coyotes, pigs, sheep, goats, horses, deer, and bubaline. Antibodies to Neospora have been described in foxes, camels, and felids. This review aims to elucidate the transmission, clinical signs, diagnosis, treatment, and control of both diseases, showing their similarities, so that they can be correctly diagnosed.(AU)

Neospora caninum y Toxoplasma gondii son los agentes etiológicos que causan Neosporosis y Toxoplasmosis, respectivamente. Estas dos enfermedades se consideran de gran importancia económica y de distribución mundial, afectando tanto al ganado como a los animales domésticos. Presentan signos clínicos inespecíficos y la neosporosis se asocia con frecuencia al aborto en mujeres. Ambas dolencias solían ser erróneas, lo que hacía difícil el diagnóstico. Son causados por protozoos cosmopolitas de ciclos biológicos heterogéneos. Toxoplasma gondii es responsable de enfermedades clínicas en perros y gatos, mientras que Neospora caninum sólo ataca a perros. Además, no se han notificado casos de Neosporosis en humanos hasta el momento, diferente de Toxoplasmosis. Ocasionalmente esto puede ocurrir en coyotes, cerdos, ovejas, cabras, caballos, ciervos y bubalinos. Se han notificado anticuerpos contra la Neospora en zorros, camellos y felinos. El propósito de esta revisión es dilucidar la forma de transmisión, los signos clínicos, el diagnóstico, el tratamiento y el control de ambas enfermedades, mostrando sus similitudes, de manera que puedan ser diagnosticadas correctamente.(AU)

Association of bovine viral diarrhea virus, bovine herpesvirus 1, and Neospora caninum with late embryonic losses in highly supplemented grazing dairy cows.

The objectives of this study were: 1- to evaluate the association of Bovine Viral Diarrhea Virus (BVDV), Bovine Herpes Virus 1 (BoHV-1), and Neospora caninum (N. caninum) with the risk for Late Embryonic Loss (LEL) in grazing dairy cows, 2- to evaluate blood progesterone concentration at the time of LEL occurrence, and 3- to describe a novel ultrasound-guided technique for conceptus sampling. We run a prospective cohort study involving 92 cows (46 LEL and 46 NLEL). An LEL cow was that having an embryo with no heartbeat, detached membranes, or floating structures, including embryo remnants detected at pregnancy check by ultrasonography (US) 28-42 days post-AI, whereas an NLEL cow was that with embryo heartbeats detectable by US at pregnancy check 28-42 d post-IA. We took two blood samples from every cow at pregnancy check by US (the day of LEL detection) and 28 d later to perform serological diagnosis of BVDV, BoHV-1, and N. caninum; and to measure blood progesterone concentration at pregnancy check (28-42 d post-AI). We also sampled the conceptus from all the LEL cows. We performed PCR to detect BVDV, BoHV-1, and N. caninum in sampled conceptuses from LEL cows. Finally, we evaluated the associations of risk factors (serological titers, seroconversion, and progesterone) with LEL odds with logistic models. The risk for LEL was associated with serological titers to BVDV (P = 0.03) and tended to be associated with seroconversion to BVDV, given that 19.6% (9/46) in LEL and 6.5% (3/46) in NLEL cows seroconverted to BVDV (P = 0.09). In addition, BVDV was detected in conceptuses from LEL cows that seroconverted to BVDV but not in LEL cows that did not seroconvert. Conversely, the risk for LEL was not associated with the titers or seroconversion to BoHV-1 and N. caninum. BoHV-1 and N. caninum were not identified in any of the conceptuses. Finally, blood progesterone concentration was similar in LEL and NLEL cows, and it was not associated with the risk for LEL (P = 0.54). In conclusion, BVDV infection is a risk factor for LEL in dairy cows.

Neospora caninum inhibits tumor development by activating the immune response and destroying tumor cells in a B16F10 melanoma model.

BACKGROUND: Melanoma is a malignant tumor with a high mortality rate. Some microorganisms have been shown to activate the immune system and limit cancer progression. The objective of this study is to evaluate the anti-melanoma effect of Neospora caninum, a livestock pathogen with no pathogenic activity in humans. METHODS: Neospora caninum tachyzoites were inoculated into a C57BL/6 mouse melanoma model by intratumoral and distal subcutaneous injections. Tumor volumes were measured, and cell death areas were visualized by hematoxylin and eosin staining and quantified. Apoptosis in cell cultures and whole tumors was detected by propidium iodide (PI) and TUNEL staining, respectively. Cytokine and tumor-associated factor levels in tumors and spleens were detected by real-time quantitative polymerase chain reaction. Infiltration of macrophages and CD8+ T cells in the tumor microenvironment (TME) were detected by immunohistochemistry with anti-CD68 and anti-CD8 antibodies, respectively. Finally, 16S rRNA sequencing of mice cecal contents was performed to evaluate the effect of N. caninum on gut microbial diversity. RESULTS: Intratumoral and distal subcutaneous injections of N. caninum resulted in significant inhibition of tumor growth (P < 0.001), and more than 50% of tumor cells were dead without signs of apoptosis. Neospora caninum treatment significantly increased the mRNA expression levels of IL-12, IFN-γ, IL-2, IL-10, TNF-α, and PD-L1 in the TME, and IL-12 and IFN-γ in the spleen of tumor-bearing mice (P < 0.05). An increase in the infiltration of CD8+ T cells and macrophages in the TME was observed with these cytokine changes. Neospora caninum also restored the abundance of gut microbiota Lactobacillus, Lachnospiraceae, Adlercreutzia, and Prevotellaceae associated with tumor growth, but the changes were not significant. CONCLUSION: Neospora caninum inhibits B16F10 melanoma by activating potent immune responses and directly destroying the cancer cells. The stable, non-toxic, and efficacious properties of N. caninum demonstrate the potential for its use as a cancer treatment.