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J. P. Dubey - One of the best experts on this subject based on the ideXlab platform.
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shedding of Neospora caninum oocysts by dogs fed tissues from naturally infected water buffaloes bubalus bubalis from brazil
Veterinary Parasitology, 2004Co-Authors: Aline Aparecida Rezende Rodrigues, Daniel Moura De Aguiar, Solange Maria Gennari, C Sreekumar, D E Hill, Katarzyna B Miska, M C B Vianna, J. P. DubeyAbstract:Attempts were made to isolate Neospora caninum from naturally infected water buffaloes (Bubalus bubalis) from Brazil. Brains from six buffaloes with indirect fluorescent antibodies (>1:100) to N. caninum were used to isolate the parasite by bioassay in dogs and gerbils followed by in vitro culture. Shedding of Neospora-like oocysts was noticed in dogs fed brains from three buffaloes (isolate designation NcBrBuf-1, 2 and 4). Two more isolates (NcBrBuf-3 and 5) were obtained by in vitro culture of the brains of gerbils previously infected with brains of two other buffaloes. The identity of the isolates was confirmed by biological and molecular methods. The isolates were found to be non-pathogenic to gerbils. All five isolates amplified the gene 5 amplicons using Neospora-specific PCR assay. The sequences of gene 5 fragments and the common toxoplasmatiid ITS-1 fragments were analyzed. The dynamics of oocyst production in the dogs indicate that water buffaloes are natural intermediate hosts for N. caninum. This is the first report of isolation of N. caninum from water buffaloes.
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first isolation of Neospora caninum from the feces of a naturally infected dog
Journal of Parasitology, 2001Co-Authors: W Basso, O. C. H. Kwok, D E Hill, L Venturini, M C Venturini, S K Shen, J. P. DubeyAbstract:Neospora caninum is a major cause of abortion in cattle worldwide. Cattle become infected with N. caninum by ingesting oocysts from the environment or transplacentally from dam to fetus. Experimentally, dogs can act as definitive hosts, but dogs excrete few oocysts after ingesting tissue cysts. A natural definitive host was unknown until now. In the present study, N. caninum was isolated from the feces of a dog. Gerbils (Meriones unguiculatus) fed feces from the dog developed antibodies to N. caninum in the Neospora caninum agglutination test, and tissue cysts were found in their brains. Neospora caninum was isolated in cell culture and in gamma-interferon gene knockout mice inoculated with brain homogenates of infected gerbils. The DNA obtained from fecal oocysts of the dog, from the brains of gerbils fed dog feces, and from organisms isolated in cell cultures inoculated with gerbil brains was confirmed as N. caninum. The identification of N. caninum oocyst by bioassay and polymerase chain reaction demonstrates that the dog is a natural definitive host for N. caninum.
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characterization of the oregon isolate of Neospora hughesi from a horse
Journal of Parasitology, 2001Co-Authors: J. P. Dubey, D Mattson, Clarence A Speer, D K Howe, Susan Liddell, Mark C JenkinsAbstract:Neospora hughesi was isolated in cell cultures inoculated with homogenate of spinal cord from a horse in Oregon. Tachyzoites of this Oregon isolate of N. hughesi were maintained continuously by cell culture passage and tachyzoites were infective to immunosuppressed mice. Gamma interferon gene knockout (KO) mice injected with tachyzoites developed fatal myocarditis and numerous tachyzoites were seen in lesions. Gerbils (Meriones unguiculatus) inoculated with tachyzoites developed antibodies (≥1:500) as indicated by the Neospora caninum agglutination test but did not develop clinical signs, and Neospora organisms were not demonstrable in their tissues. Tissue cysts were not found in gerbils, nude mice, KO mice, immunosuppressed outbred Swiss Webster mice, or BALB/c mice injected with the Oregon isolate of N. hughesi. Ultrastructurally, tachyzoites of the Oregon isolate from the myocardium of infected KO mice and from cell culture were similar to N. caninum tachyzoites. Western blot analysis using NcSAG1 and...
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oral infection of calves with Neospora caninum oocysts from dogs humoral and cellular immune responses
International Journal for Parasitology, 1999Co-Authors: T De Marez, Susan Liddell, Mark C Jenkins, J. P. Dubey, Louis C GasbarreAbstract:Abstract Neospora caninum has been identified as a major cause of abortion in cattle in a number of countries throughout the world. Until the recent demonstration that dogs can serve as a definitive host of this parasite, it was not possible to study the infection in cattle orally exposed to oocysts. The aim of this study was to investigate the potential of N. caninum oocysts to infect calves, and to define initial immune responses that arise after oral infection. Seven calves were fed approximately 10 4 –10 5 N. caninum oocysts, three calves served as uninfected controls. Before infection, all calves were serologically negative for anti- Neospora antibodies and the calves were non-reactive to Neospora antigen in an in vitro lymphocyte proliferation assay. Peripheral blood lymphocytes from inoculated calves were able to mount in vitro proliferative responses to crude N. caninum antigen extract as early as 1 week p.i. Within 2 and 4 weeks p.i., Neospora -specific IgG1 and IgG2 antibodies were detected by IFAT and ELISA in serum from infected calves but not from sham-infected calves. The continued presence of reactive cells in the blood, spleen and mesenteric, inguinal, bronchial lymph nodes was seen as late as 2.5 months p.i., and parasite DNA was detected in the brain and spinal cord of the infected animals by PCR, indicating that the cattle were infected by oral inoculation of N. caninum oocysts collected from dogs, and that the animals were systematically sensitised by parasite antigen.
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recent advances in Neospora and neosporosis
Veterinary Parasitology, 1999Co-Authors: J. P. DubeyAbstract:Neospora caninum has emerged as a major cause of abortion in cattle in many countries. This paper reviews recent advances in the life cycle and biology of Neospora with the emphasis on bovine neosporosis. The role of the recently discovered oocyst stage of N. caninum in the epidemiology of neosporosis is discussed. Progress made in serologic diagnosis of N. caninum infection is discussed. There is no vaccine for preventing Neospora-induced abortions in cattle or to prevent oocyst shedding in dogs.
Patricia A Conrad - One of the best experts on this subject based on the ideXlab platform.
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differentiation of Neospora hughesi from Neospora caninum based on their immunodominant surface antigen sag1 and srs2
International Journal for Parasitology, 1999Co-Authors: Antoinette E Marsh, Daniel K Howe, N Cannon, Bradd C. Barr, G. Wang, Patricia A ConradAbstract:Abstract Neospora hughesi is a newly recognised parasite that is closely related to Neospora caninum , and is a cause of equine protozoal myeloencephalitis. We have characterised two N. hughesi immunodominant tachyzoite antigens which exhibit antigenic and molecular differences from the homologous tachyzoite antigens on N. caninum . These antigens on N. hughesi are referred to as NhSAG1 and NhSRS2, using the same mnemonics as used for the N. caninum antigens (NcSAG1 and NcSRS2), and are homologous to Toxoplasma gondii surface antigen 1 (SAG1) and SAG1-related sequence 2 (SRS2). The NcSAG1 and NcSRS2 were antigenically conserved in six different N. caninum isolates from cattle and dogs. The two equine-derived Neospora isolates, one designated as N. hughesi , were similar to each other but different from N. caninum . There was 6% difference in amino acid identity between NcSAG1 and NhSAG1, whereas there was a 9% difference when NcSRS2 and NhSRS2 were compared. The polymorphism of these genes and their corresponding proteins provide additional markers which can be used to distinguish N. caninum from N. hughesi .
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Description of a new Neospora species (Protozoa: Apicomplexa: Sarcocystidae).
Journal of Parasitology, 1998Co-Authors: Antoinette E Marsh, Andrea E. Packham, Bradd C. Barr, Patricia A ConradAbstract:: Neospora hughesi n. sp. was isolated from the central nervous system tissue of an adult equine (Equus caballus) from California. The tachyzoites are crescent-shaped, approximately 2 x 5 microm (1.8-3.0 x 4.0-7.0 microm), with characteristic apical complex structures consisting of an anterior polar ring, conoid, numerous rhoptries filled with a uniform electron-dense material, and 22 microtubules extending posteriorly from the polar ring. Comparison of N. hughesi to canine and bovine Neospora caninum isolates showed phenotypic differences in immunoreactive proteins. Molecular analysis of the small subunit ribosomal RNA gene revealed no differences in the nucleotide sequence between N. hughesi and N. caninum isolates examined. However, the internal transcribed spacer I region revealed 7 nucleotide base differences between N. hughesi and N. caninum isolates (CN1 and BPA1) analyzed in this study. The existence of nucleotide base differences in the internal transcribed spacer regions suggests that this region may be a genetic marker for discriminating species within the genus Neospora. The ultrastructural, antigenic, and molecular data support distinction of N. hughesi as a new species, separate from N. caninum, the only recognized species in this genus.
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In vitro isolation and characterisation of a bovine Neospora species in Japan
Research in Veterinary Science, 1997Co-Authors: Itsuro Yamane, Karen W Sverlow, T. Kokuho, K. Shimura, Tomoyuki Shibahara, Makoto Haritani, Y. Ouchi, Patricia A ConradAbstract:Abstract Eleven aborted bovine fetuses and five calves suspected as having neosporosis were necropsied and tissues from these animals were inoculated into bovine cardiopulmonary aortic endothelial cells and monkey kidney cells and maintained at 37°C with 5 per cent CO 2 . Neospora tachyzoites were observed in one cell 49 days after inoculation. The isolated parasite ( JPA1 ) was morphologically identical to the previously reported bovine Neospora species ( BPA1 ) and confirmed by its strong antigenic reactivity with bovine control antisera to Neospora species and its lack of reactivity with Toxoplasma gondii and Sarcocystis cruzi antisera. This is the first bovine Neospora species isolate in Asia and further studies with this isolate are now expected.
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Detection of Neospora sp. from infected bovine tissues by PCR and probe hybridization.
Journal of Parasitology, 1997Co-Authors: Michael S. Y. Ho, Andrea E. Packham, Antoinette E Marsh, Bradd C. Barr, Mark L Anderson, Karen W Sverlow, Joan D Rowe, Patricia A ConradAbstract:: Neospora sp. can cause fetal abortion or neurological disease in congenitally infected calves. Latent tissue stages in infected cows may contribute to vertical transmission of Neospora sp. from dam to offspring in multiple pregnancies. In this investigation, the polymerase chain reaction (PCR) and Neospora-specific assay were employed to detect Neospora sp. by amplification of nuclear small-subunit rRNA gene sequences in infected cattle tissues. Tissues from 11 cattle, including 6 experimentally and 2 naturally infected cows, 1 naturally infected newborn calf, and 2 uninfected control cows, were evaluated in this study. Neospora-specific PCR products were amplified from DNAs of different bovine tissues, including brain, spinal cord, heart, lung, kidney, diaphragm, skeletal muscle, and placenta, as well as amniotic fluid samples of infected cattle. The PCR-based amplification and probe hybridization system proved useful in assessing the location of tissue-stage parasites in naturally and experimentally infected cattle, even when Neospora sp. antibody titers fall below normal cut-off values by an indirect immunofluorescent antibody test.
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evidence of vertical transmission of Neospora sp infection in dairy cattle
Javma-journal of The American Veterinary Medical Association, 1997Co-Authors: Mark L Anderson, Andrea E. Packham, Bradd C. Barr, Karen W Sverlow, James P Reynolds, Joan D Rowe, Patricia A ConradAbstract:OBJECTIVE: To determine whether heifers with naturally acquired congenital exposure to Neospora sp would transmit the infection to their offspring during gestation. DESIGN: Prospective cohort study. ANIMALS: Neonatal heifers on a dairy with a history of Neospora sp infections were selected for the study on the basis of their serum titers to Neospora sp, as determined by the use of indirect fluorescent antibody testing. Seropositive heifers (n = 25) had titers > or = 1:5,120 and seronegative heifers (25) had titers or = 1:1,280 to Neospora sp. All offspring from seropositive heifers that were necropsied had evidence of Neospora sp infection. All seronegative heifers and their offspring had titers < 1:80 to Neospora sp. CLINICAL IMPLICATIONS: Congenitally acquired Neospora sp infection can persist in clinically normal heifers and be transmitted transplacentally to their offspring. Vertical transmission can be a way by which neosporosis is maintained in herds.
Luis Miguel Ortegamora - One of the best experts on this subject based on the ideXlab platform.
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exposure to Neospora spp and besnoitia spp in wildlife from israel
International journal for parasitology. Parasites and wildlife, 2018Co-Authors: Monica L Mazuz, Gema Alvarezgarcia, Varda Shkap, Igor Savisky, Roni King, Luis Miguel Ortegamora, D GutierrezexpositoAbstract:Abstract Neosporosis and besnoitiosis, caused by cyst-forming protozoa Neospora caninum and Besnoitia besnoiti, respectively, are parasitic infestations of livestock in Israel. These parasites cause significant economic losses in cattle due to reproductive and productive disorders. Both parasites have been detected in several wild ruminant species throughout other regions of the world, while the existence of a sylvatic life cycle in Israel remains uncertain. Thus, a wide panel of 871 sera from two wild carnivores and nine wild ruminant species were tested. All sera were first analysed by MAT for an initial screening and positive samples were confirmed a posteriori by Western blot. Additionally, a complementary IFAT was used for the detection of antibodies against N. caninum. Neospora antibodies were present in six out of the 11 species investigated, whereas Besnoitia antibodies were undetected. Golden jackal, red fox, addax, Arabian oryx, Persian fallow deer, mouflon, mountain gazelle, Nubian ibex, scimitar horned oryx and water buffalo were seropositive against N. caninum infection by IFAT and/or MAT. Moreover, the presence of Neospora spp.-specific antibodies was confirmed by Western blot in golden jackal (6/189; 3.2%), red fox (1/75; 1.3%), Persian fallow deer (13/232; 5.6%), mouflon (1/15; 16.7%), Nubian ibex (22/55; 40%) and water buffalo (12/18; 66.7%). Addax (1/49) and water buffalo (1/18) were MAT-seropositive against B. besnoiti but were seronegative by Western blot. Hence, Neospora sylvatic cycle is present in Israel and may cross over to a domestic life cycle. In contrast, wildlife species investigated are unlikely to present a risk of transmitting Besnoitia to livestock in Israel.
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a serosurvey of selected cystogenic coccidia in spanish equids first detection of anti besnoitia spp specific antibodies in europe
BMC Veterinary Research, 2017Co-Authors: D Gutierrezexposito, Michelle R Yeargan, Daniel K Howe, Antonio Arenasmontes, Sallyanne L Ness, Ignacio Garciabocanegra, Luis Miguel Ortegamora, Gema AlvarezgarciaAbstract:Equine besnoitiosis, caused by Besnoitia bennetti, and equine protozoal myeloencephalitis (EPM), caused by Sarcocystis neurona and Neospora hughesi are relevant equine diseases in the Americas that have been scarcely studied in Europe. Thus, a serosurvey of these cystogenic coccidia was carried out in Southern Spain. A cross-sectional study was performed and serum samples from horses (n = 553), donkeys (n = 85) and mules (n = 83) were included. An in-house enzyme-linked immunosorbent assay (ELISA) was employed to identify a Besnoitia spp. infection and positive results were confirmed by an a posteriori western blot. For Neospora spp. and Sarcocystis spp., infections were detected using in-house ELISAs based on the parasite surface antigens N. hughesi rNhSAG1 and S. neurona rSnSAG2/3/4. Risk factors associated with these protozoan infections were also investigated. Antibodies against Besnoitia spp., Neospora spp. and Sarcocystis spp. infections were detected in 51 (7.1%), 46 (6.4%) and 20 (2.8%) of 721 equids, respectively. The principal risk factors associated with a higher seroprevalence of Besnoitia spp. were the host species (mule or donkey), the absence of shelter and the absence of a rodent control programme. The presence of rodents was the only risk factor for Neospora spp. infection. This study was the first extensive serosurvey of Besnoitia spp. infection in European equids accomplished by two complementary tests and gives evidence of the presence of specific antibodies in these populations. However, the origin of the infection is still unclear. Further parasite detection and molecular genotyping are needed to identify the causative Besnoitia and Neospora species. Finally, cross-reactions with antibodies directed against other species of Sarcocystis might explain the positive reactions against the S. neurona antigens.
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pattern of recognition of Neospora caninum tachyzoite antigens by naturally infected pregnant cattle and aborted foetuses
Veterinary Parasitology, 2002Co-Authors: Gema Alvarezgarcia, J Pereirabueno, Mercedes Gomezbautista, Luis Miguel OrtegamoraAbstract:Abstract Different aspects of Neospora tachyzoite antigen recognition by Neospora -infected heifers and cows and aborted foetuses were studied. The pattern of antigen recognition and the relationship between IFAT titres and number of Neospora antigens detected, were evaluated. In addition, the tachyzoite antigens involved in the humoral immune response developed against infection in normal cows and cows that aborted were also characterised throughout pregnancy. Comparison of tachyzoite antigen recognition was carried out in 13 thoracic and/or abdominal fluids from Neospora aborted foetuses and 33 sera from Neospora infected cows that had aborted. The kinetics of Neospora -antigen recognition was studied in Neospora -infected heifers and cows that had aborted foetuses (7) or not (14) during pregnancy. Based on the frequency and intensity of recognition, four IDAs—17–18, 34–35, 37 and 60–62 kDa antigens—have been described. Moreover, a correlation was found between Western blot results and IFAT titres in both age groups. In relation to antigen recognition throughout pregnancy by seropositive cows that had aborted or not, the antibody fluctuations throughout pregnancy described in the literature could be due to differences in the intensity and frequency of recognition of particular antigens, especially the 17–18 kDa antigen. We emphasize the important role that the 17–18 kDa antigen could play in the serological diagnosis of Neospora infection in cattle as this was intensely detected in 100% of the animals.
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quantitative detection of Neospora caninum in bovine aborted fetuses and experimentally infected mice by real time pcr
Journal of Clinical Microbiology, 2002Co-Authors: Esther Collantesfernandez, Gema Alvarezgarcia, Angel Zaballos, Luis Miguel OrtegamoraAbstract: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.
Gema Alvarezgarcia - One of the best experts on this subject based on the ideXlab platform.
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exposure to Neospora spp and besnoitia spp in wildlife from israel
International journal for parasitology. Parasites and wildlife, 2018Co-Authors: Monica L Mazuz, Gema Alvarezgarcia, Varda Shkap, Igor Savisky, Roni King, Luis Miguel Ortegamora, D GutierrezexpositoAbstract:Abstract Neosporosis and besnoitiosis, caused by cyst-forming protozoa Neospora caninum and Besnoitia besnoiti, respectively, are parasitic infestations of livestock in Israel. These parasites cause significant economic losses in cattle due to reproductive and productive disorders. Both parasites have been detected in several wild ruminant species throughout other regions of the world, while the existence of a sylvatic life cycle in Israel remains uncertain. Thus, a wide panel of 871 sera from two wild carnivores and nine wild ruminant species were tested. All sera were first analysed by MAT for an initial screening and positive samples were confirmed a posteriori by Western blot. Additionally, a complementary IFAT was used for the detection of antibodies against N. caninum. Neospora antibodies were present in six out of the 11 species investigated, whereas Besnoitia antibodies were undetected. Golden jackal, red fox, addax, Arabian oryx, Persian fallow deer, mouflon, mountain gazelle, Nubian ibex, scimitar horned oryx and water buffalo were seropositive against N. caninum infection by IFAT and/or MAT. Moreover, the presence of Neospora spp.-specific antibodies was confirmed by Western blot in golden jackal (6/189; 3.2%), red fox (1/75; 1.3%), Persian fallow deer (13/232; 5.6%), mouflon (1/15; 16.7%), Nubian ibex (22/55; 40%) and water buffalo (12/18; 66.7%). Addax (1/49) and water buffalo (1/18) were MAT-seropositive against B. besnoiti but were seronegative by Western blot. Hence, Neospora sylvatic cycle is present in Israel and may cross over to a domestic life cycle. In contrast, wildlife species investigated are unlikely to present a risk of transmitting Besnoitia to livestock in Israel.
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first detection of anti besnoitia spp specific antibodies in horses and donkeys in italy
Parasitology International, 2018Co-Authors: Luca Villa, Gema Alvarezgarcia, Alessia Libera Gazzonis, Carlos Diezmadiaz, Sergio Aurelio Zanzani, M T ManfrediAbstract:Abstract Among Apicomplexa protozoa infecting equids, Besnoitia spp., Toxoplasma gondii and Neospora spp. represent important issues from a sanitary and zootechnical viewpoint. However, only scarce epidemiological data are available on the spread of the infections in horses and donkeys in Europe. Therefore, a serosurvey was planned to estimate the prevalence of these Sarcocystidae species in Italian equids. Serum samples from 268 horses and 18 donkeys raised in Italy were collected and serologically analyzed to detect anti-Besnoitia spp., anti-T. gondii and anti-Neospora spp. antibodies: an approach based on an initial screening by in-house ELISA followed by a confirmatory WB was used. Two horses (0.7%) and four donkeys (22.2%), showed antibodies anti-Besnoitia spp. Ten horses (3.7%) resulted positive to T. gondii and one of these (0.4%) was seropositive also to Neospora spp. This is the first detection of anti-Besnoitia spp. specific antibodies in Italian horses and donkeys. The study confirmed the circulation of Besnoitia spp. among equids in Europe. Low prevalence of T. gondii and Neospora spp. in horses raised in Italy was reported. Nevertheless, it is noteworthy to consider that consumption of horse meat could represent a source for human toxoplasmosis.
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a serosurvey of selected cystogenic coccidia in spanish equids first detection of anti besnoitia spp specific antibodies in europe
BMC Veterinary Research, 2017Co-Authors: D Gutierrezexposito, Michelle R Yeargan, Daniel K Howe, Antonio Arenasmontes, Sallyanne L Ness, Ignacio Garciabocanegra, Luis Miguel Ortegamora, Gema AlvarezgarciaAbstract:Equine besnoitiosis, caused by Besnoitia bennetti, and equine protozoal myeloencephalitis (EPM), caused by Sarcocystis neurona and Neospora hughesi are relevant equine diseases in the Americas that have been scarcely studied in Europe. Thus, a serosurvey of these cystogenic coccidia was carried out in Southern Spain. A cross-sectional study was performed and serum samples from horses (n = 553), donkeys (n = 85) and mules (n = 83) were included. An in-house enzyme-linked immunosorbent assay (ELISA) was employed to identify a Besnoitia spp. infection and positive results were confirmed by an a posteriori western blot. For Neospora spp. and Sarcocystis spp., infections were detected using in-house ELISAs based on the parasite surface antigens N. hughesi rNhSAG1 and S. neurona rSnSAG2/3/4. Risk factors associated with these protozoan infections were also investigated. Antibodies against Besnoitia spp., Neospora spp. and Sarcocystis spp. infections were detected in 51 (7.1%), 46 (6.4%) and 20 (2.8%) of 721 equids, respectively. The principal risk factors associated with a higher seroprevalence of Besnoitia spp. were the host species (mule or donkey), the absence of shelter and the absence of a rodent control programme. The presence of rodents was the only risk factor for Neospora spp. infection. This study was the first extensive serosurvey of Besnoitia spp. infection in European equids accomplished by two complementary tests and gives evidence of the presence of specific antibodies in these populations. However, the origin of the infection is still unclear. Further parasite detection and molecular genotyping are needed to identify the causative Besnoitia and Neospora species. Finally, cross-reactions with antibodies directed against other species of Sarcocystis might explain the positive reactions against the S. neurona antigens.
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pattern of recognition of Neospora caninum tachyzoite antigens by naturally infected pregnant cattle and aborted foetuses
Veterinary Parasitology, 2002Co-Authors: Gema Alvarezgarcia, J Pereirabueno, Mercedes Gomezbautista, Luis Miguel OrtegamoraAbstract:Abstract Different aspects of Neospora tachyzoite antigen recognition by Neospora -infected heifers and cows and aborted foetuses were studied. The pattern of antigen recognition and the relationship between IFAT titres and number of Neospora antigens detected, were evaluated. In addition, the tachyzoite antigens involved in the humoral immune response developed against infection in normal cows and cows that aborted were also characterised throughout pregnancy. Comparison of tachyzoite antigen recognition was carried out in 13 thoracic and/or abdominal fluids from Neospora aborted foetuses and 33 sera from Neospora infected cows that had aborted. The kinetics of Neospora -antigen recognition was studied in Neospora -infected heifers and cows that had aborted foetuses (7) or not (14) during pregnancy. Based on the frequency and intensity of recognition, four IDAs—17–18, 34–35, 37 and 60–62 kDa antigens—have been described. Moreover, a correlation was found between Western blot results and IFAT titres in both age groups. In relation to antigen recognition throughout pregnancy by seropositive cows that had aborted or not, the antibody fluctuations throughout pregnancy described in the literature could be due to differences in the intensity and frequency of recognition of particular antigens, especially the 17–18 kDa antigen. We emphasize the important role that the 17–18 kDa antigen could play in the serological diagnosis of Neospora infection in cattle as this was intensely detected in 100% of the animals.
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quantitative detection of Neospora caninum in bovine aborted fetuses and experimentally infected mice by real time pcr
Journal of Clinical Microbiology, 2002Co-Authors: Esther Collantesfernandez, Gema Alvarezgarcia, Angel Zaballos, Luis Miguel OrtegamoraAbstract: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.
Solange Maria Gennari - One of the best experts on this subject based on the ideXlab platform.
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Prevalence of antibodies against Neospora spp. and Sarcocystis neurona in donkeys from northeastern Brazil
Revista Brasileira De Parasitologia Veterinaria, 2016Co-Authors: Solange Maria Gennari, Marcos Gomes Lopes, Herbert Sousa Soares, S.n. Vitaliano, David S Lindsay, Aline Diniz Cabral, Hilda Fatima De Jesus Pena, Marcos AmakuAbstract:Sarcocystis neurona and Neospora hughesi are coccidian protozoa that can cause neurological illness in horses in America. In this study we report seroprevalence of Neospora spp. andS. neurona in sera of 333 donkeys from the northeastern region of Brazil. Antibodies to Neospora spp. were detected in 2% (7 donkeys) of 333 sera tested by the indirect fluorescent antibody test (IFAT) with a cut-off dilution of 1:40. Antibodies to S. neurona were found in 3% (10 donkeys) of the samples tested by IFAT (cut-off ≥50) and 21% (69 donkeys) by the direct agglutination test (SAT ≥50). The SAT and IFAT results for S. neurona showed a poor concordance (value of Kappa=0.051). This is the first report ofNeospora spp. antibodies in Brazilian donkeys and the first detection of antibodies against S. neurona in this animal species.
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Differential diagnosis of oocysts of Hammondia-like organisms of dogs and cats by PCR-RFLP analysis of 70-kilodalton heat shock protein (HSP70) gene
Parasitology Research, 2008Co-Authors: Renata Molina Monteiro, Solange Maria Gennari, Hilda Fátima De Jesus Pena, Sheila Oliveira Souza, Leonardo José Richtzenhain, Rodrigo Martins SoaresAbstract:Organisms of the genera Toxoplasma , Hammondia and Neospora , the Hammondia -like organisms, are closely related coccidian with similarly sized oocysts. Therefore, a diagnosis based on microscopy of oocysts in feces is not a method of choice for species identification of these important parasites. In this paper, we present a polymerase chain reaction coupled with restriction fragment length polymorphism (PCR-RFLP) method to differentially diagnose oocysts of Toxoplasma gondii from oocyst of Hammondia hammondi . Another PCR-RFLP was designed to differentiate oocysts of Hammondia heydorni from oocysts of Neospora spp. Both PCR-RFLP are based on nucleotide sequences of the Hsp70 coding gene. In conclusion, we presented two alternative molecular diagnostic assays that can be successfully applied for the differentiation of oocysts of Hammondia -like organisms shed by felids and canids.
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Prevalence of Sarcocystis neurona and Neospora spp. infection in horses from Brazil based on presence of serum antibodies to parasite surface antigen
Veterinary Parasitology, 2005Co-Authors: Jessica S Hoane, Daniel Moura De Aguiar, Guacyara Tenório Cavalcante, Gérson L. Bonesi, Alexandre Secorun Borges, Marcio Garcia Ribeiro, Solange Maria Gennari, Jitender P. Dubey, Daniel K HoweAbstract:Sera from 961 horses from Brazil were tested for antibodies against the major surface antigens SnSAG4 and NhSAG1 to determine the seroprevalence of Sarcocystis neurona and Neospora hughesi, respectively. Antibodies against SnSAG4 were detected in 669 (69.6%) of the horses, while antibodies against NhSAG1 were detected in only 24 (2.5%) of the horses. These serologic results suggest that there is a high concentration of S. neurona in the environment of Brazil, which results in marked exposure of horses to this parasite. Additionally, the data further confirm that infection with Neospora spp. is relatively uncommon in horses.
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shedding of Neospora caninum oocysts by dogs fed tissues from naturally infected water buffaloes bubalus bubalis from brazil
Veterinary Parasitology, 2004Co-Authors: Aline Aparecida Rezende Rodrigues, Daniel Moura De Aguiar, Solange Maria Gennari, C Sreekumar, D E Hill, Katarzyna B Miska, M C B Vianna, J. P. DubeyAbstract:Attempts were made to isolate Neospora caninum from naturally infected water buffaloes (Bubalus bubalis) from Brazil. Brains from six buffaloes with indirect fluorescent antibodies (>1:100) to N. caninum were used to isolate the parasite by bioassay in dogs and gerbils followed by in vitro culture. Shedding of Neospora-like oocysts was noticed in dogs fed brains from three buffaloes (isolate designation NcBrBuf-1, 2 and 4). Two more isolates (NcBrBuf-3 and 5) were obtained by in vitro culture of the brains of gerbils previously infected with brains of two other buffaloes. The identity of the isolates was confirmed by biological and molecular methods. The isolates were found to be non-pathogenic to gerbils. All five isolates amplified the gene 5 amplicons using Neospora-specific PCR assay. The sequences of gene 5 fragments and the common toxoplasmatiid ITS-1 fragments were analyzed. The dynamics of oocyst production in the dogs indicate that water buffaloes are natural intermediate hosts for N. caninum. This is the first report of isolation of N. caninum from water buffaloes.