We report the development of a real-time PCR assay for the quantitative detection ofNeospora caninumin 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 ofN. 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 10⁻¹tachyzoite per assay was observed with excellent linearity (R²= 0.998). Assay specificity was confirmed by using DNA from the closely related parasiteToxoplasma gondii. The applicability of the technique was successfully tested in a variety of host brain tissues: (i) aborted bovine fetuses classified into negative or positiveNeospora-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 ITS1Neosporanested 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 ofNeosporainfection.

The aim of this study was to investigate the need for different cut-off points, according to animal age and the purpose of testing, for two of the most widely used serological techniques in bovine neosporosis, IFAT and a crude antigen ELISA (Civtest, HIPRA). Therefore, the population reference sera used were defined using a combination of multiple criteria such as epidemiological/clinical and histopathological parameters and an immunoblot test. Firstly, foetuses and breeding cattle (heifers and cows) were considered as separate subpopulations for serological evaluation. Secondly, cut-off points for each serological technique (IFAT and ELISA) according to age group (foetuses and breeding cattle) and the different practical applications (detection of infection and abortion) were calculated following the receiver operating characteristics (ROC) analysis. Cut-off points were defined, for IFAT and ELISA for aborted breeding cattle and for IFAT alone in the case of the foetuses, assuming an equivalent cost of false positive and negative results. In infected breeding cattle, for IFAT and ELISA and in foetuses for ELISA, two possible cut-off values were obtained, one for a maximum sensitivity and one for a maximum specificity and the intervals of unclear results were defined. In this case, a cut-off value for equal sensitivity and specificity was also estimated. When cut-off points for infected breeding cattle, 1:100-1:250 for IFAT and 0.306-0.451 for ELISA were applied to a target population, optimal and similar negative and positive predictive values together with similar apparent and true prevalence results were observed suggesting the possibility of using both tests interchangeably.

SUMMARY: Bovine besnoitiosis is caused by the cyst-forming apicomplexan parasiteBesnoitia besnoiti.This disease progresses in two sequential phases: a febrile acute phase with oedemas and respiratory disorders, and a chronic phase characterized by the presence of subcutaneous tissue cysts and skin lesions. Serious consequences of the infection are poor body condition, sterility in bulls and eventual death. The role of host/parasite-dependent factors, which play a major role in the pathogenesis of the disease, is not yet fully elucidated. Isolate/strain virulence, parasite stage, dose and the route of parasite inoculation were studied under different experimental conditions, which make it difficult to compare the results. Data on host-dependent factors obtained from naturally infected cattle showed that (i) the seroprevalence of infection is similar in both sexes; (ii) seropositivity increases with age; (iii) both beef and dairy cattle are susceptible to the infection; and (iv) the cell-mediated immune response is likely to play a major role because a T cell response has been observed around several tissue cysts. Whether colostral antibodies are protective and to what extent the humoral immune response might reflect the disease/protection status require further research. Thus, a well-established experimental bovine model could help to clarify these important questions. The dynamics ofB. besnoitiinfection in cattle and available knowledge on relevant factors in the pathogenesis of the infection are reviewed in the present work.

In this study, we examined the in vitro invasion and proliferation capacities of the Nc-Liv and ten Spanish Neospora caninum isolates (Nc-Spain 1 H - Nc-Spain 10). The invasion rate was determined as the number of tachyzoites that completed their internalisation into MARC-145 cells at 2, 4, and 6 h post-inoculation (pi). The proliferation rate was evaluated by determining the doubling time during the exponential proliferation period. Significant differences in the invasion rates of these isolates were detected at 2 and 4 h pi (P < 0.0001, Kruskal-Wallis test). At 4 h pi, the Nc-Spain 4 H and Nc-Liv isolates displayed the highest, while the Nc-Spain 3 H and Nc-Spain 1 H isolates had the lowest invasion rates (by Dunn's test). Variations in the proliferation kinetics of these isolates were also observed. Between different isolates, the lag phase, which occurs before the exponential growth phase, ranged from 8 to 44 h, and the doubling time ranged from 9.8 to 14.1 h (P = 0.0016, ANOVA test). Tachyzoite yield, which combines invasion and proliferation data, was also assessed and confirmed marked differences between the highly and less prolific isolates. Interestingly, a direct correlation between the invasion rates and tachyzoite yields, and the severity of the disease that was exhibited by infected pregnant mice in previous works could be established for the isolates in this study (Spearman's coefficient > 0.62, P < 0.05). The results of this study may help us to explain the differences in the pathogenicity that are displayed by different isolates.