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1.  Genetic Diversity and Geographic Population Structure of Bovine Neospora caninum Determined by Microsatellite Genotyping Analysis 
PLoS ONE  2013;8(8):e72678.
The cyst-forming protozoan parasite Neosporacaninum is one of the main causes of bovine abortion worldwide and is of great economic importance in the cattle industry. Recent studies have revealed extensive genetic variation among N. caninum isolates based on microsatellite sequences (MSs). MSs may be suitable molecular markers for inferring the diversity of parasite populations, molecular epidemiology and the basis for phenotypic variations in N. caninum, which have been poorly defined. In this study, we evaluated nine MS markers using a panel of 11 N. caninum-derived reference isolates from around the world and 96 N. caninum bovine clinical samples and one ovine clinical sample collected from four countries on two continents, including Spain, Argentina, Germany and Scotland, over a 10-year period. These markers were used as molecular tools to investigate the genetic diversity, geographic distribution and population structure of N. caninum. Multilocus microsatellite genotyping based on 7 loci demonstrated high levels of genetic diversity in the samples from all of the different countries, with 96 microsatellite multilocus genotypes (MLGs) identified from 108 N. caninum samples. Geographic sub-structuring was present in the country populations according to pairwise FST. Principal component analysis (PCA) and Neighbor Joining tree topologies also suggested MLG segregation partially associated with geographical origin. An analysis of the MLG relationships, using eBURST, confirmed that the close genetic relationship observed between the Spanish and Argentinean populations may be the result of parasite migration (i.e., the introduction of novel MLGs from Spain to South America) due to cattle movement. The eBURST relationships also revealed genetically different clusters associated with the abortion. The presence of linkage disequilibrium, the co-existence of specific MLGs to individual farms and eBURST MLG relationships suggest a predominant clonal propagation for Spanish N. caninum MLGs in cattle.
doi:10.1371/journal.pone.0072678
PMCID: PMC3735528  PMID: 23940816
2.  Infected Dendritic Cells Facilitate Systemic Dissemination and Transplacental Passage of the Obligate Intracellular Parasite Neospora caninum in Mice 
PLoS ONE  2012;7(3):e32123.
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.
doi:10.1371/journal.pone.0032123
PMCID: PMC3293873  PMID: 22403627
3.  Influence of Neospora caninum intra-specific variability in the outcome of infection in a pregnant BALB/c mouse model 
Veterinary Research  2010;41(4):52.
Previous assays in pregnant animals have demonstrated the effect of different host factors and timing of infection on the outcome of neosporosis during pregnancy. However, the influence of Neospora caninum isolate itself has been poorly investigated. Here, we compared the effects on clinical outcome and vertical transmission observed in a pregnant mouse model following infection with 10 different N. caninum isolates. The isolates in our study included the Nc-Liv isolate and nine N. caninum isolates obtained from calves. Female BALB/c mice were inoculated with 2 × 106 tachyzoites at day 7 of pregnancy. Morbidity and mortality, in both dams and offspring during the course of infection, and transmission to progeny at day 30 postpartum were evaluated. The serum IgG1 and IgG2a production in dams were also examined. All dams showed elevated IgG1 and IgG2a responses, confirming N. caninum infection, although signs of disease were only exhibited in dams infected with 4 of the 10 isolates (Nc-Spain 4H, Nc-Spain 5H, Nc-Spain 7 and Nc-Liv). In neonates, clinical signs were observed in all N. caninum-infected groups, and neonatal mortality rates varied from greater than 95% with the isolates mentioned above to less than 32.5% with the other isolates. Vertical transmission rates, as assessed by parasite PCR-detection in neonate brains, also varied from 50% to 100% according to the isolate implicated. These results confirm the wide pathogenic and transmission variability of N. caninum. The intra-specific variability observed herein could help us explain the differences in the outcome of the infection in the natural host.
doi:10.1051/vetres/2010024
PMCID: PMC2878169  PMID: 20416260
Neospora caninum; bovine isolate; pathogenic characterisation; vertical transmission; pregnant mouse model
4.  In Vitro and In Vivo Treatments of Echinococcus Protoscoleces and Metacestodes with Artemisinin and Artemisinin Derivatives▿  
In vitro treatment of Echinococcus multilocularis and Echinococcus granulosus larval stages with the antimalarials dihydroartemisinin and artesunate (10 to 40 μM) exhibited promising results, while 6 weeks of in vivo treatment of mice infected with E. multilocularis metacestodes (200 mg/kg of body weight/day) had no effect. However, combination treatments of both drugs with albendazole led to a substantial but statistically not significant reduction in parasite weight compared to results with albendazole alone.
doi:10.1128/AAC.00553-08
PMCID: PMC2533465  PMID: 18625777
5.  In Vitro Metacestodicidal Activities of Genistein and Other Isoflavones against Echinococcus multilocularis and Echinococcus granulosus▿  
Antimicrobial Agents and Chemotherapy  2006;50(11):3770-3778.
Echinococcus multilocularis and Echinococcus granulosus metacestode infections in humans cause alveolar echinococcosis and cystic echinococcosis, respectively, in which metacestode development in visceral organs often results in particular organ failure. Further, cystic hydatidosis in farm animals causes severe economic losses. Although benzimidazole derivatives such as mebendazole and albendazole are being used as therapeutic agents, there is often no complete recovery after treatment. Hence, in searching for novel treatment options, we examined the in vitro efficacies of a number of isoflavones against Echinococcus metacestodes and protoscoleces. The most prominent isoflavone, genistein, exhibits significant metacestodicidal activity in vitro. However, genistein binds to the estrogen receptor and can thus induce estrogenic effects, which is a major concern during long-term chemotherapy. We have therefore investigated the activities of a number of synthetic genistein derivatives carrying a modified estrogen receptor binding site. One of these, Rm6423, induced dramatic breakdown of the structural integrity of the metacestode germinal layer of both species within 5 to 7 days of in vitro treatment. Further, examination of the culture medium revealed increased leakage of parasite proteins into the medium during treatment, but zymography demonstrated a decrease in the activity of metalloproteases. Moreover, two of the genistein derivatives, Rm6423 and Rm6426, induced considerable damage in E. granulosus protoscoleces, rendering them nonviable. These findings demonstrate that synthetic isoflavones exhibit distinct in vitro effects on Echinococcus metacestodes and protoscoleces, which could potentially be exploited further for the development of novel chemotherapeutical tools against larval-stage Echinococcus infection.
doi:10.1128/AAC.00578-06
PMCID: PMC1635224  PMID: 16954323
6.  Temporal Distribution and Parasite Load Kinetics in Blood and Tissues during Neospora caninum Infection in Mice  
Infection and Immunity  2006;74(4):2491-2494.
The kinetics of Neospora caninum loads in mice inoculated with NC-Liv or NC-1 isolates were studied. The acute phase was characterized by parasitemia and the detection of parasite DNA in several organs, whereas during the chronic phase, the parasite was detected mainly in the brain. Mice infected with NC-Liv developed clinical signs, showing higher brain parasite burdens than NC-1-infected mice.
doi:10.1128/IAI.74.4.2491-2494.2006
PMCID: PMC1418903  PMID: 16552086
7.  Quantitative Detection of Neospora caninum in Bovine Aborted Fetuses and Experimentally Infected Mice by Real-Time PCR 
Journal of Clinical Microbiology  2002;40(4):1194-1198.
We report the development of a real-time PCR assay for the quantitative detection of Neospora caninum in infected host tissues. The assay uses the double-stranded DNA-binding dye SYBR Green I to continuously monitor product formation. Oligonucleotide primers were designed to amplify a 76-bp DNA fragment corresponding to the Nc5 sequence of N. caninum. A similar method was developed to quantify the 28S rRNA host gene in order to compare the parasite load of different samples and to correct for the presence of potential PCR-inhibiting compounds in the DNA samples. A linear quantitative detection range of 6 logs with a calculated detection limit of 10−1 tachyzoite per assay was observed with excellent linearity (R2 = 0.998). Assay specificity was confirmed by using DNA from the closely related parasite Toxoplasma gondii. The applicability of the technique was successfully tested in a variety of host brain tissues: (i) aborted bovine fetuses classified into negative or positive Neospora-infected animals according to the observation of compatible lesions by histopathological study and (ii) experimentally infected BALB/c mice, divided into three groups, inoculated animals with or without compatible lesions and negative controls. All samples were also tested by ITS1 Neospora nested PCR and a high degree of agreement was shown between both PCR techniques (κ = 0.86). This technique represents a useful quantitative diagnostic tool to be used in the study of the pathogenicity, immunoprophylaxis, and treatment of Neospora infection.
doi:10.1128/JCM.40.4.1194-1198.2002
PMCID: PMC140374  PMID: 11923330

Results 1-7 (7)