scholarly journals Spatial distance between sites of sampling associated with genetic variation among Neospora caninum in aborted bovine foetuses from northern Italy

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Luca Villa ◽  
Pavlo Maksimov ◽  
Christine Luttermann ◽  
Mareen Tuschy ◽  
Alessia L. Gazzonis ◽  
...  
Keyword(s):  
Genetic Distance ◽  
Neospora Caninum ◽  
Geographic Distance ◽  
Rare Event ◽  
Northern Italy ◽  
Spatial Distance ◽  
Italian Regions ◽  
Bovine Abortion

Abstract Background Neospora caninum, a coccidian protozoan, represents an important cause of bovine abortion. Available N. caninum strains show considerable variation in vitro and in vivo, including different virulence in cattle. To which extent sexual recombination, which is possible in the intestines of domestic dogs and closely related carnivores as definitive hosts, contributes to this variation is not clear yet. Methods Aborted bovine foetuses were collected between 2015 and early 2019 from Italian Holstein Friesian dairy herds suffering from reproductive problems. A total of 198 samples were collected from 165 intensive farms located in Lombardy, northern Italy. N. caninum samples were subjected to multilocus-microsatellite genotyping using ten previously established microsatellite markers. In addition to our own data, those from a recent study providing data on five markers from other northern Italian regions were included and analysed. Results Of the 55 samples finally subjected to genotyping, 35 were typed at all or 9 out of 10 loci and their individual multilocus-microsatellite genotype (MLMG) determined. Linear regression revealed a statistically significant association between the spatial distance of the sampling sites with the genetic distance of N. caninum MLMGs (P < 0.001). Including data from this and a previous North Italian study into eBURST analysis revealed that several of N. caninum MLMGs from northern Italy separate into four groups; most of the samples from Lombardy clustered in one of these groups. Principle component analysis revealed similar clusters and confirmed MLMG groups identified by eBURST. Variations observed between MLMGs were not equally distributed over all loci, but predominantly observed in MS7, MS6A, or MS10. Conclusions Our findings confirm the concept of local N. caninum subpopulations. The geographic distance of sampling was associated with the genetic distance as determined by microsatellite typing. Results suggest that multi-parental recombination in N. caninum is a rare event, but does not exclude uniparental mating. More comprehensive studies on microsatellites in N. caninum and related species like Toxoplasma gondii should be undertaken, not only to improve genotyping capabilities, but also to understand possible functions of these regions in the genomes of these parasites.

scholarly journals Spatial Distance Between Sites of Sampling Associated With Genetic Variation Among Neospora Caninum in Aborted Bovine Foetuses From Northern Italy

2020 ◽  
Author(s):  
Luca Villa ◽  
Pavlo Maksimov ◽  
Christine Luttermann ◽  
Mareen Tuschy ◽  
Alessia L. Gazzonis ◽  
...  
Keyword(s):  
Genetic Distance ◽  
Neospora Caninum ◽  
Geographic Distance ◽  
Rare Event ◽  
Northern Italy ◽  
Spatial Distance ◽  
Italian Regions ◽  
Bovine Abortion

Abstract Background: Neospora caninum, a coccidian protozoan, represents an important cause of bovine abortion. While only a single genotype of N. caninum exists word-wide, available N. caninum strains show considerable variation in vitro and in vivo, including different virulence in cattle. To which extent sexual recombination, possible in the intestines of domestic dogs and closely related carnivores as definitive hosts, contribute to this variation is not clear, yet. Methods: Aborted bovine foetuses were collected between 2015 and early 2019 from Italian Holstein Friesian dairy herds suffering from reproductive problems. A total of 198 samples were collected from 165 intensive farms located in Lombardy, northern Italy. N. caninum samples were subjected to multilocus-microsatellite genotyping (MLMG) using ten previously established microsatellite markers. In addition to own data, those from a recent study providing data on five markers from other northern Italian regions were included and analyzed. Results: Of the 55 samples finally subjected to MLMG, 35 were typed at all or 9 out of 10 loci. Linear regression revealed a statistically significant association between the spatial distance of the sampling sites with the genetic distance of N. caninum MLMGs (P< 0.001). Including data from a previous North Italian study (eBURST analysis) revealed that part of N. caninum MLMGs from northern Italy separate into four groups; most of the samples from Lombardy clustered in one of these groups. Principle component analysis revealed similar clusters and confirmed MLMG groups identified by eBURST. Variations observed between MLMGs were not equally distributed over all loci, but predominantly observed in MS7, MS6A, or MS10.Conclusions: Our findings confirm the concept of local N. caninum subpopulations. The geographic distance of sampling was associated with the genetic distance as determined by MLMGs. Results suggest that multi-parental recombination in N. caninum is a rare event, but does not exclude uniparental mating. More comprehensive studies on microsatellites in N. caninum and related species like Toxoplasma gondii should be undertaken, not only to improve genotyping capabilities, but also to understand possible functions of these regions in the genomes of these parasites.

Cellular and immunological basis of the host-parasite relationship during infection withNeospora caninum

Parasitology ◽  
2006 ◽  
Vol 133 (3) ◽  
pp. 261-278 ◽  
Author(s):  
A. HEMPHILL ◽  
N. VONLAUFEN ◽  
A. NAGULESWARAN
Keyword(s):  
Host Cell ◽  
Cell Biology ◽  
Neospora Caninum ◽  
Host Cells ◽  
Term Survival ◽  
Prevention Of Infection ◽  
Parasite Relationship ◽  
Potential Applications

Neospora caninumis an apicomplexan parasite that is closely related toToxoplasma gondii, the causative agent of toxoplasmosis in humans and domestic animals. However, in contrast toT. gondii, N. caninumrepresents a major cause of abortion in cattle, pointing towards distinct differences in the biology of these two species. There are 3 distinct key features that represent potential targets for prevention of infection or intervention against disease caused byN. caninum. Firstly, tachyzoites are capable of infecting a large variety of host cellsin vitroandin vivo. Secondly, the parasite exploits its ability to respond to alterations in living conditions by converting into another stage (tachyzoite-to-bradyzoite orvice versa). Thirdly, by analogy withT. gondii, this parasite has evolved mechanisms that modulate its host cells according to its own requirements, and these must, especially in the case of the bradyzoite stage, involve mechanisms that ensure long-term survival of not only the parasite but also of the host cell. In order to elucidate the molecular and cellular bases of these important features ofN. caninum, cell culture-based approaches and laboratory animal models are being exploited. In this review, we will summarize the current achievements related to host cell and parasite cell biology, and will discuss potential applications for prevention of infection and/or disease by reviewing corresponding work performed in murine laboratory infection models and in cattle.

Long-term passage of tachyzoites in tissue culture can attenuate virulence of Neospora caninum in vivo

Parasitology ◽  
2006 ◽  
Vol 133 (4) ◽  
pp. 421-432 ◽  
Author(s):  
P. M. BARTLEY ◽  
S. WRIGHT ◽  
J. SALES ◽  
F. CHIANINI ◽  
D. BUXTON ◽  
...  
Keyword(s):  
Tissue Culture ◽  
Body Weight ◽  
Growth Rates ◽  
Clinical Symptoms ◽  
Neospora Caninum ◽  
High Passage ◽  
Low Passage

To determine whether prolonged in vitro passage would result in attenuation of virulence in vivo, Neospora caninum tachyzoites were passaged for different lengths of time in vitro and compared for their ability to cause disease in mice. Groups of Balb/c mice were inoculated intraperitoneally with 5×106 or 1×107 of low-passage or high-passage N. caninum tachyzoites. The mice were monitored for changes in their demeanour and body weight, and were culled when severe clinical symptoms of murine neosporosis were observed. Mice inoculated with the high-passage parasites survived longer (P<0·05), and showed fewer clinical symptoms of murine neosporosis, compared to the mice receiving the low-passage parasites. The parasite was detected in the brains of inoculated mice using immunohistochemistry and ITS1 PCR. Tissue cysts containing parasites were seen in mice inoculated with both low-passage and high-passage parasites. When the in vitro growth rates of the parasites were compared, the high-passage parasites initially multiplied more rapidly (P<0·001) than the low-passage parasites, suggesting that the high-passage parasites had become more adapted to tissue culture. These results would suggest that it is possible to attenuate the virulence of N. caninum tachyzoites in mice through prolonged in vitro passage.

Neospora caninum: In vivo and in vitro treatment with artemisone

2012 ◽  
Vol 187 (1-2) ◽  
pp. 99-104 ◽  
Author(s):  
Monica L. Mazuz ◽  
Richard Haynes ◽  
Varda Shkap ◽  
Leah Fish ◽  
Ricardo Wollkomirsky ◽  
...  
Keyword(s):  
Neospora Caninum ◽  
In Vitro Treatment

scholarly journals Neospora caninum: Structure and Fate of Multinucleated Complexes Induced by the Bumped Kinase Inhibitor BKI-1294

Pathogens ◽  
2020 ◽  
Vol 9 (5) ◽  
pp. 382 ◽  
Author(s):  
Pablo Winzer ◽  
Nicoleta Anghel ◽  
Dennis Imhof ◽  
Vreni Balmer ◽  
Luis-Miguel Ortega-Mora ◽  
...  
Keyword(s):  
Drug Exposure ◽  
Kinase Inhibitors ◽  
Kinase Inhibitor ◽  
Neospora Caninum ◽  
Farm Animals ◽  
In Vivo Studies ◽  
Drug Pressure ◽  
Drug Removal

Background: Bumped kinase inhibitors (BKIs) are potential drugs for neosporosis treatment in farm animals. BKI-1294 exposure results in the formation of multinucleated complexes (MNCs), which remain viable in vitro under constant drug pressure. We investigated the formation of BKI-1294 induced MNCs, the re-emergence of viable tachyzoites following drug removal, and the localization of CDPK1, the molecular target of BKIs. Methods: N. caninum tachyzoites and MNCs were studied by TEM and immunofluorescence using antibodies directed against CDPK1, and against NcSAG1 and IMC1 as markers for tachyzoites and newly formed zoites, respectively. Results: After six days of drug exposure, MNCs lacked SAG1 surface expression but remained intracellular, and formed numerous zoites incapable of disjoining from each other. Following drug removal, proliferation continued, and zoites lacking NcSAG1 emerged from the periphery of these complexes, forming infective tachyzoites after 10 days. In intracellular tachyzoites, CDPK1 was evenly distributed but shifted towards the apical part once parasites were extracellular. This shift was not affected by BKI-1294. Conclusions: CDPK1 has a dynamic distribution depending on whether parasites are located within a host cell or outside. During MNC-to-tachyzoite reconversion newly formed tachyzoites are generated directly from MNCs through zoites of unknown surface antigen composition. Further in vivo studies are needed to determine if MNCs could lead to a persistent reservoir of infection after BKI treatment.

scholarly journals Disruption of Dense Granular Protein 2 (GRA2) Decreases the Virulence of Neospora caninum

2021 ◽  
Vol 8 ◽  
Author(s):  
Jingquan Dong ◽  
Nan Zhang ◽  
Panpan Zhao ◽  
Jianhua Li ◽  
Lili Cao ◽  
...  
Keyword(s):  
Neospora Caninum ◽  
Quantitative Polymerase Chain Reaction ◽  
Parasite Burden ◽  
Site Directed Mutagenesis ◽  
Cell Fractionation ◽  
Fractionation Analysis ◽  
Protein Functions ◽  
Localization Analysis

Neospora caninum causes abortions in cattle and nervous system dysfunction in dogs. Dense granular proteins (GRAs) play important roles in virulence; however, studies on NcGRA functions are limited. In the present study, multiple methods, including site-directed mutagenesis; CRISPR/Cas9 gene editing; Western blotting; quantitative polymerase chain reaction; confocal microscopy; plaque, invasion, egress, and replication assays; animal assays of survival rate and parasite burden; and hematoxylin–eosin staining, were used to characterize the NcGRA2 protein, construct an NcGRA2 gene disruption (ΔNcGRA2) strain, and explore its virulence in vivo and vitro. The results showed that NcGRA2 shared 31.31% homology with TgGRA2 and was colocalized with NcGRA6 at the posterior end of tachyzoites and the intravacuolar network of parasitophorous vacuoles (PVs). Cell fractionation analysis showed that NcGRA2 behaved as a transmembrane and membrane-coupled protein. The ΔNcGRA2 strain was constructed by coelectroporation of the NcGRA2-targeting CRISPR plasmid (pNc-SAG1-Cas9:U6-SgGRA2) and DHFR-TS DNA donor and verified at the protein, genome, and transcriptional levels and by immunofluorescence localization analysis. The in vitro virulence results showed that the ΔNcGRA2 strain displayed smaller plaques, similar invasion and egress abilities, and slower intracellular growth. The in vivo virulence results showed a prolonged survival time, lower parasite burden, and mild histopathological changes. Overall, the present study indicates that NcGRA2, as a dense granular protein, forms the intravacuolar network structure of PVs and weakens N. caninum virulence by slowing proliferation. These data highlight the roles of NcGRA2 and provide a foundation for research on other protein functions in N. caninum.

scholarly journals A motogenic GABAergic system of mononuclear phagocytes facilitates dissemination of coccidian parasites

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Amol K Bhandage ◽  
Gabriela C Olivera ◽  
Sachie Kanatani ◽  
Elizabeth Thompson ◽  
Karin Loré ◽  
...  
Keyword(s):  
Neospora Caninum ◽  
Mononuclear Phagocytes ◽  
Gamma Aminobutyric Acid ◽  
Gaba A ◽  
Voltage Dependent ◽  
Coccidian Parasites ◽  
Voltage Dependent Calcium Channels

Gamma-aminobutyric acid (GABA) serves diverse biological functions in prokaryotes and eukaryotes, including neurotransmission in vertebrates. Yet, the role of GABA in the immune system has remained elusive. Here, a comprehensive characterization of human and murine myeloid mononuclear phagocytes revealed the presence of a conserved and tightly regulated GABAergic machinery with expression of GABA metabolic enzymes and transporters, GABA-A receptors and regulators, and voltage-dependent calcium channels. Infection challenge with the common coccidian parasites Toxoplasma gondii and Neospora caninum activated GABAergic signaling in phagocytes. Using gene silencing and pharmacological modulators in vitro and in vivo in mice, we identify the functional determinants of GABAergic signaling in parasitized phagocytes and demonstrate a link to calcium responses and migratory activation. The findings reveal a regulatory role for a GABAergic signaling machinery in the host-pathogen interplay between phagocytes and invasive coccidian parasites. The co-option of GABA underlies colonization of the host by a Trojan horse mechanism.

scholarly journals NcGRA2as a molecular target to assess the parasiticidal activity of toltrazuril againstNeospora caninum

Parasitology ◽  
2008 ◽  
Vol 135 (9) ◽  
pp. 1065-1073 ◽  
Author(s):  
M. STROHBUSCH ◽  
N. MÜLLER ◽  
A. HEMPHILL ◽  
G. GREIF ◽  
B. GOTTSTEIN
Keyword(s):  
Real Time ◽  
Neospora Caninum ◽  
Day Treatment ◽  
Mrna Level ◽  
Live Cells ◽  
Wide Range ◽  
Biological Criteria ◽  
Infected Cells

SUMMARYThe treatment ofNeospora caninuminfection in the bovine host is still at an experimental stage. In contrast to thein vivosituation, a wide range of compounds have been intensively investigated in cell-culture-based assays. Tools to demonstrate efficacy of treatment have remained conventional including morphological and cell biological criteria. In this work, we present a molecular assay that allows the distinction between live and dead parasites. Live parasites can be detected by measuring the mRNA level of specific genes, making use of the specific mRNA available in live cells. TheNcGra2gene ofN. caninum, which is known to be expressed in both tachyzoites and bradyzoites, was used to establish a quantitative real-time RT-PCR, for monitoring parasite viability. Validation of the systemin vitrowas achieved usingNeospora-infected cells that had been treated for 2–20 days with 30 μg/ml toltrazuril.NcGRA2-RT-real time PCR demonstrated that a 10-day toltrazuril-treatment exerted parasitostatic activity, as assessed by the presence ofNcGRA2-transcripts, whereas after a 14-day treatment period noNcGRA2-transcripts were detected, showing that the parasites were no longer viable. Concurrently, extended culture for a period of 4 weeks in the absence of the drug following the 14-day toltrazuril treatment did not lead to further parasite proliferation, confirming the parasiticidal effect of the treatment. This assay has the potential to be widely used in the development of novel drugs againstN. caninum, with a view to distinguishing between parasiticidal and parasitostatic efficacy of given compounds.

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