Browsing by Subject "Neospora caninum"

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    A time-resolved fluorescence immunoassay for the detection of anti-Neospora caninum antibodies in sheep
    (Elsevier, 2019-11-18) Huertas López, Ana; Huertas-López, Ana; Martínez-Carrasco Pleite, Carlos; Cerón Madrigal, José Joaquín; Sánchez-Sánchez, Roberto; Vázquez-Calvo, Ángela; Álvarez-García, Gema; Martínez Subiela, Silvia; Sanidad Animal; Facultad de Veterinaria
    Neospora caninum is a protozoan parasite (Phylum Apicomplexa) that has been recently suggested as a relevant cause of reproductive disorders in small ruminants. The aim of the present study is to develop and validate a new serological test based on time resolved fluorescency using N. caninum GRA7 recombinant antigen (GRA7-TRFIA) for the detection of N. caninum antibodies in sheep. A total of 346 serum samples (208 from experimentally infected sheep, 117 from a dairy farm with a previous history of Neospora-associated abortion, and 21 negative sera) were used. The validation of the new assay was performed by the evaluation of assay precision, analytical sensitivity (Se), accuracy and cross reactivity. In the experimentally infected sheep, antibody kinetics was compared between GRA7-TRFIA and an in house N. caninum tachyzoite soluble extract-based ELISA (NcSALUVET ELISA) by Wilcoxon matched-pairs signed rank test. The cut-off and diagnostic Se and specificity (Sp) of GRA7-TRFIA was estimated by ROC analysis with field samples. In addition, concordance and correlation between GRA7-TRFIA and a commercial ELISA and NcSALUVET ELISA were assessed by kappa value and Spearman correlation coefficient, respectively. Overall, GRA7-TRFIA showed an adequate precision, analytical Se and accuracy to detect anti-N. caninum antibodies in ovine serum, and no cross reactivity with the closely related protozoan Toxoplasma gondii. In naturally infected sheep, 100% Se and 95.35% Sp were obtained for a cut-off point of 62.68 Units of Fluorometry for N. caninum (UFN). Moreover, GRA7-TRFIA allowed earlier detection of N. caninum infection than NcSALUVET ELISA in experimentally infected sheep.
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    Bottlenose dolphins (Tursiops truncatus) do also cast neutrophil extracellular traps against the apicomplexan parasite Neospora caninum
    (Elsevier, 2017-12) Villagra-Blanco, R.; Silva, L.M.R.; Aguilella-Segura, A.; Martínez Carrasco-Pleite, Carlos; Seipp, A.; Gärtner, U.; Ruiz de Ybáñez Carnero, María del Rocío; Taubert, A.; Hermosilla, C.; Arcenillas Hernández, Irene; Sanidad Animal
    Neutrophil extracellular traps (NETs) are web-like structures composed of nuclear DNA decorated with histones and cytoplasmic peptides which antiparasitic properties have not previously been investigated in cetaceans. Polymorphonuclear neutrophils (PMN) were isolated from healthy bottlenose dolphins (Tursiops truncatus), and stimulated with Neospora caninum tachyzoites and the NETs-agonist zymosan. In vitro interactions of PMN with the tachyzoites resulted in rapid extrusion of NETs. For the demonstration and quantification of cetacean NETs, extracellular DNA was stained by using either Sytox Orange® or Pico Green®. Scanning electron microscopy (SEM) and fluorescence analyses demonstrated PMN-derived release of NETs upon exposure to tachyzoites of N. caninum. Co-localization studies of N. caninum induced cetacean NETs proved the presence of DNA adorned with histones (H1, H2A/H2B, H3, H4), neutrophil elastase (NE), myeloperoxidase (MPO) and pentraxin (PTX) confirming the molecular properties of mammalian NETosis. Dolphin-derived N. caninum-NETosis were efficiently suppressed by DNase I and diphenyleneiodonium (DPI) treatments. Our results indicate that cetacean-derived NETs represent an ancient, conserved and relevant defense effector mechanism of the host innate immune system against N. caninum and probably other related neozoan parasites circulating in the marine environment.
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    Detection of anti-Neospora caninum antibodies in sheep’s full-cream milk by time-resolved fluorescence immunoassay
    (Elsevier, 2021-12-25) Huertas-López, Ana; Sánchez-Sánchez, Roberto; Diezma-Díaz, Carlos; Alvarez-García, Gema; Martínez-Carrasco Pleite, Carlos; Martínez Subiela, Silvia; Cerón Madrigal, José Joaquín; Sanidad Animal; Facultad de Veterinaria
    Ovine neosporosis, caused by the Apicomplexan parasite Neospora caninum, leads to reproductive failure worldwide. Nowadays, there is a trend to develop diagnostic techniques using non-invasive samples, such as milk, in order to reduce animal stress, sample collection effort, and costs. The objective of this study was to develop and validate a highly sensitive and specific serological technique, based on a time resolved-fluorescence immunoassay using a N. caninum GRA7 antigen (GRA7-TRFIA), for the detection of anti-N. caninum immunoglobulins G on sheep’ full-cream milk samples. An analytical validation was performed, including intra- and inter-assay precision, analytical sensitivity and accuracy. The diagnostic performance of the assay was evaluated by studying the positive-negative discrimination by Mann Whitney U tests. In additon optimal cut-offs, diagnostic sensitivity and specificity, and areas under the curve were calculated by three Receiver Operating Curve (ROC) analyses, using GRA7-TRFIA and a N. caninum tachyzoite soluble extract-based ELISA (NcSALUVET-ELISA) in blood sera, and the coinciding results of both techniques, as reference techniques. Moreover, Spearman’s correlation of GRA7-TRFIA in milk with the techniques in sera and agreement (kappa values) were also estimated. GRA7-TRFIA for milk samples showed an adequate precision, with high analytical sensitivity and accuracy. Regarding ROC analyses, at the optimal cut-offs, the diagnostic sensitivity and specificity were more than 90 % in all cases. In addition, GRA7-TRFIA values in milk were more positively correlated to GRA7-TRFIA values in blood sera than in the case of values obtained with NcSALUVET-ELISA. GRA7-TRFIA in milk showed an almost perfect agreement with GRA7-TRFIA in blood sera (kappa = 0.98) and with the coinciding results of GRA7-TRFIA and NcSALUVET in blood sera (kappa = 1.00), while it has a substantial agreement with NcSALUVET-ELISA (kappa = 0.69). In the light of these results, GRA7-TRFIA in full-cream milk samples is a highly sensitive technique that could be used for screening anti-N. caninum antibodies in sheep flocks.
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    Epidemiological surveillance of Neospora caninum in sheep (Ovis aries) and European mouflon (Ovis aries musimon) in southern Spain
    (Elsevier, 2024-11-02) Jiménez-Martín, Débora; Huertas-López, Ana; Cerón Madrigal, José Joaquín; Martínez-Carrasco Pleite, Carlos; Martínez Subiela, Silvia; Prieto, Paloma; Almería, Sonia; Cano-Terriza, David; García-Bocanegra, Ignacio; Sanidad Animal; Facultad de Veterinaria
    Neospora caninum is a protozoan with a facultative heteroxenous life cycle, with canids as the definitive hosts and other mammals, mainly ruminants, acting as intermediate hosts. This parasite is recognized as one of the major abortifacient pathogens in cattle. Although reproductive disorders have also been reported in other domestic and wild ruminants, epidemiological data on N. caninum in ruminant species other than cattle is still limited. Here, we evaluate the seroprevalence and risk factors associated with N. caninum exposure in sheep (Ovis aries) and European mouflon (Ovis aries musimon) in southern Spain. Serum samples from 390 sheep and 387 free-ranging mouflons were tested for antibodies against N. caninum using in-house time-resolved fluorescence immunoassay based on NcGRA7 as a recombinant antigen. The individual seroprevalence was 26.2 % (95 %CI: 22.0–30.7) in sheep and 5.7 % (95 %CI: 3.8–8.5) in mouflons. At least one seropositive animal was detected in all the 26 sheep farms (100 %) and in eight of the 18 (44.4 %) hunting estates sampled. The presence of dogs on the farm (≥3) and the sheep breed (purebred) were potential risk factors associated with exposure to N. caninum in sheep. Our results indicate a high circulation of N. caninum in sheep farms in southern Spain. Control measures should be implemented to limit the exposure to this protozoan in sheep flocks. Although we have confirmed for the first time the presence of anti- N. caninum antibodies in European mouflon in Spain, and, despite the relatively low seroprevalence found, the impact of this parasite on mouflon populations should be further studied.
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    Seronegativity of Iberian ibex (Capra pyrenaica) against Toxoplasma gondii and Neospora caninum is consistent with eco-epidemiological and environmental features in Mediterranean mountainous areas
    (Elsevier, 2025-01-07) Cano Manuel, Alejandro; Granados, José Enrique; Álvarez García, Gema; Huertas López, Ana; Diezma Díaz, Carlos; Cano Manuel, Francisco Javier; Ortega Mora, Luis Miguel; Fandos, Paulino; Mentaberre, Gregorio; López Olvera, Jorge Ramón; Martínez-Carrasco Pleite, Carlos; Sanidad Animal; Facultades de la UMU::Facultad de Veterinaria
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    Serosurvey of selected reproductive pathogens in domestic ruminants from Upper Egypt
    (Frontiers Media, 2023-10-23) Farag, Shimaa Ismail; Cano Terriza, David; Gonzálvez Juan, Moisés; Salman, Doaa; Aref, Nasr-Eldin M.; Mubaraki, Murad A.; Jiménez Martín, Débora; García Bocanegra, Ignacio; Elmahallawy, Ehab Kotb; Sanidad Animal; Facultades de la UMU::Facultad de Veterinaria
    Toxoplasmosis, neosporosis, and Q fever are among the most important abortifacient diseases in ruminants worldwide. These diseases result in huge economic losses in livestock besides the fact that some of are of public health concern. The present study aimed to update the data about the current seroepidemiological situation of these diseases in Upper Egypt. A total of 411 blood samples were collected from small and large ruminants and serologically tested against the presence of T. gondii, N. caninum, and C. burnetii. Generalized estimating equation (GEE) models were performed to assess the potential risk factors associated with the exposure to these pathogens. The overall seroprevalence of T. gondii was 47.9% (197/411) with an individual seropositivity of 59.4% (63/106), 58.6% (17/29), 38.8% (54/139) and 46% (63/137) in cattle, buffalo, sheep and goats, respectively. Meanwhile, 9.7% (38/411) of the examined animals were tested positive for anti-N. caninum antibodies, with an individual seropositivity of 13.2% (12/106), 34.5% (10/29), 8.6% (12/139) and 2.9% (4/137) in cattle, buffalo, sheep and goats, respectively. Furthermore, the overall prevalence of antibodies against C. burnetii was 17.3% (63/411), and exposure to this pathogen was detected in 4.7% (5/106) of cattle, 19.3% (20/129) of sheep, 29.2% (38/130) of goats but none of the examined buffalo were found to be seropositive. A total of 12.1% (50/411) of the examined animals showed co-exposure to at least two of the tested pathogens. Regarding the potential risk factors, there were statistically significant differences among species in the frequency of exposure to the three tested pathogens. Age (> 6 months) was also shown to be a significant risk factor associated with T. gondii exposure. The results obtained provided updated information about the occurrence of three of the main reproductive pathogens in Upper Egypt. The high seropositivity values found for the tested zoonotic pathogens in most of the analyzed ruminant species suggest the necessity of performing additional in-depth studies to evaluate the epidemiology of these pathogens in the study area.