Javier Regidor-cerrillo - Academia.edu (original) (raw)
Papers by Javier Regidor-cerrillo
Veterinary Research, 2021
Toxoplasma gondii is an important zoonotic agent with high genetic diversity, complex epidemiolog... more Toxoplasma gondii is an important zoonotic agent with high genetic diversity, complex epidemiology, and variable clinical outcomes in animals and humans. In veterinary medicine, this apicomplexan parasite is considered one of the main infectious agents responsible for reproductive failure in small ruminants worldwide. The aim of this study was to phenotypically characterize 10 Spanish T. gondii isolates recently obtained from sheep in a normalized mouse model and in an ovine trophoblast cell line (AH-1) as infection target cells. The panel of isolates met selection criteria regarding such parameters as genetic diversity [types II (ToxoDB #1 and #3) and III (#2)], geographical location, and sample of origin (aborted foetal brain tissues or adult sheep myocardium). Evaluations of in vivo mortality, morbidity, parasite burden and histopathology were performed. Important variations between isolates were observed, although all isolates were classified as “nonvirulent” (< 30% cumulativ...
Pathogens
Despite the importance of bovine neosporosis, relevant knowledge gaps remain concerning the patho... more Despite the importance of bovine neosporosis, relevant knowledge gaps remain concerning the pathogenic mechanisms of Neospora caninum. Infection of the placenta is a crucial event in the pathogenesis of the disease; however, very little is known about the relation of the parasite with this target organ. Recent studies have shown that isolates with important variations in virulence also show different interactions with the bovine trophoblast cell line F3 in terms of proliferative capacity and transcriptome host cell modulation. Herein, we used the same model of infection to study the interaction of Neospora with these target cells at the proteomic level using LC-MS/MS over the course of the parasite lytic cycle. We also analysed the proteome differences between high- (Nc-Spain7) and low-virulence (Nc-Spain1H) isolates. The results showed that mitochondrial processes and metabolism were the main points of Neospora-host interactions. Interestingly, Nc-Spain1H infection showed a higher ...
Proceedings of the National Academy of Sciences
Neospora caninum, a cyst-forming apicomplexan parasite, is a leading cause of neuromuscular disea... more Neospora caninum, a cyst-forming apicomplexan parasite, is a leading cause of neuromuscular diseases in dogs as well as fetal abortion in cattle worldwide. The importance of the domestic and sylvatic life cycles of Neospora, and the role of vertical transmission in the expansion and transmission of infection in cattle, is not sufficiently understood. To elucidate the population genomics of Neospora, we genotyped 50 isolates collected worldwide from a wide range of hosts using 19 linked and unlinked genetic markers. Phylogenetic analysis and genetic distance indices resolved a single genotype of N. caninum. Whole-genome sequencing of 7 isolates from 2 different continents identified high linkage disequilibrium, significant structural variation, but only limited polymorphism genome-wide, with only 5,766 biallelic single nucleotide polymorphisms (SNPs) total. Greater than half of these SNPs (∼3,000) clustered into 6 distinct haploblocks and each block possessed limited allelic diversit...
Frontiers in Cellular and Infection Microbiology
Intraspecific differences in biological traits between Neospora caninum isolates have been widely... more Intraspecific differences in biological traits between Neospora caninum isolates have been widely described and associated with variations in virulence. However, the molecular basis underlying these differences has been poorly studied. We demonstrated previously that Nc-Spain7 and Nc-Spain1H, high-and low-virulence isolates, respectively, show different invasion, proliferation and survival capabilities in bovine macrophages (boMØs), a key cell in the immune response against Neospora, and modulate the cell immune response in different ways. Here, we demonstrate that these differences are related to specific tachyzoite gene expression profiles. Specifically, the low-virulence Nc-Spain1H isolate showed enhanced expression of genes encoding for surface antigens and genes related to the bradyzoite stage. Among the primary up-regulated genes in Nc-Spain7, genes involved in parasite growth and redox homeostasis are particularly noteworthy because of their correlation with the enhanced proliferation and survival rates of Nc-Spain7 in boMØs relative to Nc-Spain1H. Genes potentially implicated in induction of proinflammatory immune responses were found to be up-regulated in the low-virulence isolate, whereas the high-virulence isolate showed enhanced expression of genes that may be involved in immune evasion. These results represent a further step in understanding the parasite effector molecules that may be associated to virulence and thus to disease traits as abortion and transmission.
Veterinary Research
Early Neospora caninum infection dynamics were investigated in pregnant heifers intravenously ino... more Early Neospora caninum infection dynamics were investigated in pregnant heifers intravenously inoculated with PBS (G-Control) or 10 7 tachyzoites of high (G-NcSpain7)-or low (G-NcSpain1H)-virulence isolates at 110 days of gestation. Serial culling at 10 and 20 days post-infection (dpi) was performed. Fever was detected at 1 dpi in both infected groups (P < 0.0001), and a second peak was detected at 3 dpi only in G-NcSpain7 (P < 0.0001). At 10 dpi, Nc-Spain7 was detected in placental samples from one animal related to focal necrosis, and Nc-Spain7 transmission was observed, although no foetal lesions were associated with this finding. The presence of Nc-Spain1H in the placenta or foetuses, as well as lesions, were not detected at 10 dpi. At 20 dpi, G-NcSpain7 animals showed almost 100% positive placental tissues and severe focal necrosis as well as 100% transmission. Remarkably, foetal mortality was detected in two G-NcSpain7 heifers. Only one animal from G-NcSpain1H presented positive placental samples. No foetal mortality was detected, and lesions and parasite transmission to the foetus were not observed in this group. Finally, 100% of G-NcSpain7 heifers at 20 dpi presented specific antibodies, while only 60% of G-NcSpain1H animals presented specific antibodies at 20 dpi. In addition, earlier seroconversion in G-Nc-Spain7 was observed. In conclusion, tachyzoites from Nc-Spain7 reached the placenta earlier and multiplied, leading to lesion development, transmission to the foetus and foetal mortality, whereas Nc-Spain1H showed delayed infection of the placenta and no lesional development or transmission during early infection.
Veterinary Research
Parameters such as pathogen dose and inoculation route are paramount in animal models when studyi... more Parameters such as pathogen dose and inoculation route are paramount in animal models when studying disease pathogenesis. Here, clinical findings, including foetal mortality, parasite transmission rates and lesion severity, and immune responses were evaluated in Asturiana pregnant heifers at day 110 of gestation challenged with a virulent (Nc-Spain7) Neospora caninum isolate. Four different doses of parasite tachyzoites were inoculated intravenously (IV1, 10 7 parasites, n = 6; IV2, 10 5 , n = 6; IV3, 10 3 , n = 6; and IV4, 10 2 , n = 5), and the subcutaneous (SC) inoculation route was also assessed for the dose of 10 5 tachyzoites (SC, n = 6). In addition, a control group (n = 4 pregnant heifers) was evaluated. Foetal death was observed in all infected groups from 25 to 62 days post-infection, varying with the dose (IV1:4/6, IV2:3/6; IV4:2/5, IV3:1/6), and was three times less frequently associated with the SC route than IV inoculation (1/6 vs. 3/6). A dose-dependent effect for parasite loads in placental and foetal brain tissues was also detected. After SC challenge, a reduced number of tachyzoites were able to reach foetal brain tissues, and no lesions were observed. In calves, specific IgG responses in precolostral sera were mainly associated with high-dose groups (IV1 [100.0%] and IV2 [66.7%]), and cerebral parasite DNA detection was scarce (3/18). In dams, IFN-γ production and the dynamics of anti-N. caninum IgG antibodies varied with the dose, and the cell-mediated immune response was also found to be route-dependent. Our results confirm the influence of parasite dose and inoculation route on the outcome and dynamics of bovine neosporosis at mid-gestation.
Frontiers in Immunology
Neospora caninum, a protozoan parasite closely related to Toxoplasma gondii, represents one of th... more Neospora caninum, a protozoan parasite closely related to Toxoplasma gondii, represents one of the main causes of abortion in cattle. Macrophages (MØs) are mediators of the innate immune response against infection and likely one of the first cells encountered by the parasite during the host infection process. In this study, we investigated in vitro how high or low virulent isolates of N. caninum (Nc-Spain7 and Nc-Spain1H, respectively) interact with bovine monocyte-derived MØs and the influence of the isolate virulence on the subsequent cellular response. Both isolates actively invaded, survived and replicated in the MØs. However, Nc-Spain7 showed a higher invasion rate and a replication significantly faster, following an exponential growth model, whereas Nc-Spain1H presented a delayed replication and a lower growth rate without an exponential pattern. N. caninum infection induced a hypermigratory phenotype in bovine MØs that was characterized by enhanced motility and transmigration in vitro and was accompanied by morphological changes and abrogated extracellular matrix degradation. A significantly higher hypermotility was observed with the highly virulent isolate Nc-Spain7. Nc-Spain1H-infected MØs showed elevated reactive oxygen species (ROS) production and IL12p40 expression, which also resulted in increased IFN-γ release by lymphocytes, compared to cells infected with Nc-Spain7. Furthermore, IL-10 was upregulated in MØs infected with both isolates. Infected MØs exhibited lower expression of MHC Class II, CD86, and CD1b molecules than uninfected MØs, with non-significant differences between isolates. This work characterizes for the first time N. caninum replication in bovine monocyte-derived MØs and details isolate-dependent differences in host cell responses to the parasite.
Experimental Parasitology
Veterinary parasitology, Jan 15, 2018
Neospora caninum is an apicomplexan protozoan responsible for abortion in ruminants. The present ... more Neospora caninum is an apicomplexan protozoan responsible for abortion in ruminants. The present study aimed to diagnose an abortion from an Anglo Nubian goat from a dairy herd located in Buenos Aires province, Argentina. The goat delivered a fetus of approximately 3 months gestation that was studied by indirect fluorescent antibody test (IFAT), Immunoblot (IB), histopathology (HP), immunohistochemistry (IHC), and molecular assays (PCR, sequencing and microsatellite genotyping). Interferon gamma knock-out mice were inoculated with a pool of tissues for isolation attempts. The mother had IFAT titers of 1:3200 and 1:400 for N. caninum and Toxoplasma gondii, respectively, as well as positive IB reactions, whereas the fetus was seronegative to both parasites by IFAT and IB. The fetus had severe multifocal necrotizing myocarditis and hepatitis, moderate interstitial pneumonia, and nephritis. Myocardium sample resulted positive by IHC, evidencing clusters of N. caninum tachyzoites within ...
International journal for parasitology. Drugs and drug resistance, 2018
Neospora caninum is one of the main causes of abortion in cattle, and recent studies have highlig... more Neospora caninum is one of the main causes of abortion in cattle, and recent studies have highlighted its relevance as an abortifacient in small ruminants. Vaccines or drugs for the control of neosporosis are lacking. Bumped kinase inhibitors (BKIs), which are ATP-competitive inhibitors of calcium dependent protein kinase 1 (CDPK1), were shown to be highly efficacious against several apicomplexan parasites in vitro and in laboratory animal models. We here present the pharmacokinetics, safety and efficacy of BKI-1553 in pregnant ewes and foetuses using a pregnant sheep model of N. caninum infection. BKI-1553 showed exposure in pregnant ewes with trough concentrations of approximately 4 μM, and of 1 μM in foetuses. Subcutaneous BKI-1553 administration increased rectal temperatures shortly after treatment, and resulted in dermal nodules triggering a slight monocytosis after repeated doses at short intervals. BKI-1553 treatment decreased fever in infected pregnant ewes already after tw...
Experimental parasitology, Jan 8, 2018
The purpose of this study was to perform genotypic characterization and to evaluate the virulence... more The purpose of this study was to perform genotypic characterization and to evaluate the virulence of Toxoplasma gondii obtained from aborted fetuses in an abortion outbreak in goats from northeastern Brazil. Brain samples from 32 fetuses were submitted to mouse bioassay for T. gondii isolation. Two isolates were obtained and subjected to genotypic characterization. Isolate virulence was evaluated using murine model in different doses (from 10 to 10 tachyzoites/mL). In genotyping, both isolates were classified as clonal lineage type II (genotype #1 ToxoDB) and showed to be virulent for mice. This is the first description of genotype #1 in cases of goat abortion, showing the circulation of virulent T. gondii isolate producing reproductive disorders in pregnant goat.
Parasites & vectors, Jan 10, 2017
Neospora caninum, one of the main causes of abortion in cattle, is very effective at crossing the... more Neospora caninum, one of the main causes of abortion in cattle, is very effective at crossing the placental barrier and placental damage is crucial in the pathogenesis of abortion. Bovine trophoblast and caruncular cell layers are key cellular components in the maternal-foetal interface in placentomes, playing a fundamental role in placental functionality. We studied tachyzoite adhesion, invasion, proliferation and egress of high- (Nc-Spain7) and low- (Nc-Spain1H) virulence N. caninum isolates in established cultures of bovine caruncular epithelial (BCEC-1) and trophoblast (F3) cells. The parasite invasion rate (pInvR) and the cell infection rate (cInfR) were determined by immunostaining plaque assay at different time points and multiplicities of infection (MOIs), respectively. In addition, tachyzoite growth kinetics were investigated using real-time PCR (qPCR) analysis and immunostaining plaque assay at different times. Neospora caninum invaded and proliferated in both cell lines. ...
International Journal for Parasitology
Besnoitia besnoiti is an apicomplexan parasite responsible for bovine besnoitiosis, a chronic and... more Besnoitia besnoiti is an apicomplexan parasite responsible for bovine besnoitiosis, a chronic and debilitating disease that causes systemic and skin manifestations and sterility in bulls. Neither treatments nor vaccines are currently available. In the search for therapeutic candidates, calcium-dependent protein kinases have arisen as promising drug targets in other apicomplexans (e.g. Neospora caninum, Toxoplasma gondii, Plasmodium spp. and Eimeria spp.) and are effectively targeted by bumped kinase inhibitors. In this study, we identified and cloned the gene coding for BbCDPK1. The impact of a library of nine bumped kinase inhibitor analogues on the activity of recombinant BbCDPK1 was assessed by luciferase assay. Afterwards, those were further screened for efficacy against Besnoitiabesnoiti tachyzoites grown in Marc-145 cells. Primary tests at 5µM revealed that eight compounds exhibited more than 90% inhibition of invasion and proliferation. The compounds BKI 1294, 1517, 1553 and 1571 were further characterised, and EC99 (1294: 2.38µM; 1517: 2.20µM; 1553: 3.34µM; 1571: 2.78µM) were determined by quantitative real-time polymerase chain reaction in 3-day proliferation assays. Exposure of infected cultures with EC99 concentrations of these drugs for up to 48h was not parasiticidal. The lack of parasiticidal action was confirmed by transmission electron microscopy, which showed that bumped kinase inhibitor treatment interfered with cell cycle regulation and non-disjunction of tachyzoites, resulting in the formation of large multi-nucleated complexes which co-existed with viable parasites within the parasitophorous vacuole. However, it is possible that, in the face of an active immune response, parasite clearance may occur. In summary, bumped kinase inhibitors may be effective drug candidates to control Besnoitiabesnoiti infection. Further in vivo experiments should be planned, as attainment and maintenance of therapeutic blood plasma levels in calves, without toxicity, has been demonstrated for BKIs 1294, 1517 and 1553.
Veterinary Research, 2021
Toxoplasma gondii is an important zoonotic agent with high genetic diversity, complex epidemiolog... more Toxoplasma gondii is an important zoonotic agent with high genetic diversity, complex epidemiology, and variable clinical outcomes in animals and humans. In veterinary medicine, this apicomplexan parasite is considered one of the main infectious agents responsible for reproductive failure in small ruminants worldwide. The aim of this study was to phenotypically characterize 10 Spanish T. gondii isolates recently obtained from sheep in a normalized mouse model and in an ovine trophoblast cell line (AH-1) as infection target cells. The panel of isolates met selection criteria regarding such parameters as genetic diversity [types II (ToxoDB #1 and #3) and III (#2)], geographical location, and sample of origin (aborted foetal brain tissues or adult sheep myocardium). Evaluations of in vivo mortality, morbidity, parasite burden and histopathology were performed. Important variations between isolates were observed, although all isolates were classified as “nonvirulent” (< 30% cumulativ...
Pathogens
Despite the importance of bovine neosporosis, relevant knowledge gaps remain concerning the patho... more Despite the importance of bovine neosporosis, relevant knowledge gaps remain concerning the pathogenic mechanisms of Neospora caninum. Infection of the placenta is a crucial event in the pathogenesis of the disease; however, very little is known about the relation of the parasite with this target organ. Recent studies have shown that isolates with important variations in virulence also show different interactions with the bovine trophoblast cell line F3 in terms of proliferative capacity and transcriptome host cell modulation. Herein, we used the same model of infection to study the interaction of Neospora with these target cells at the proteomic level using LC-MS/MS over the course of the parasite lytic cycle. We also analysed the proteome differences between high- (Nc-Spain7) and low-virulence (Nc-Spain1H) isolates. The results showed that mitochondrial processes and metabolism were the main points of Neospora-host interactions. Interestingly, Nc-Spain1H infection showed a higher ...
Proceedings of the National Academy of Sciences
Neospora caninum, a cyst-forming apicomplexan parasite, is a leading cause of neuromuscular disea... more Neospora caninum, a cyst-forming apicomplexan parasite, is a leading cause of neuromuscular diseases in dogs as well as fetal abortion in cattle worldwide. The importance of the domestic and sylvatic life cycles of Neospora, and the role of vertical transmission in the expansion and transmission of infection in cattle, is not sufficiently understood. To elucidate the population genomics of Neospora, we genotyped 50 isolates collected worldwide from a wide range of hosts using 19 linked and unlinked genetic markers. Phylogenetic analysis and genetic distance indices resolved a single genotype of N. caninum. Whole-genome sequencing of 7 isolates from 2 different continents identified high linkage disequilibrium, significant structural variation, but only limited polymorphism genome-wide, with only 5,766 biallelic single nucleotide polymorphisms (SNPs) total. Greater than half of these SNPs (∼3,000) clustered into 6 distinct haploblocks and each block possessed limited allelic diversit...
Frontiers in Cellular and Infection Microbiology
Intraspecific differences in biological traits between Neospora caninum isolates have been widely... more Intraspecific differences in biological traits between Neospora caninum isolates have been widely described and associated with variations in virulence. However, the molecular basis underlying these differences has been poorly studied. We demonstrated previously that Nc-Spain7 and Nc-Spain1H, high-and low-virulence isolates, respectively, show different invasion, proliferation and survival capabilities in bovine macrophages (boMØs), a key cell in the immune response against Neospora, and modulate the cell immune response in different ways. Here, we demonstrate that these differences are related to specific tachyzoite gene expression profiles. Specifically, the low-virulence Nc-Spain1H isolate showed enhanced expression of genes encoding for surface antigens and genes related to the bradyzoite stage. Among the primary up-regulated genes in Nc-Spain7, genes involved in parasite growth and redox homeostasis are particularly noteworthy because of their correlation with the enhanced proliferation and survival rates of Nc-Spain7 in boMØs relative to Nc-Spain1H. Genes potentially implicated in induction of proinflammatory immune responses were found to be up-regulated in the low-virulence isolate, whereas the high-virulence isolate showed enhanced expression of genes that may be involved in immune evasion. These results represent a further step in understanding the parasite effector molecules that may be associated to virulence and thus to disease traits as abortion and transmission.
Veterinary Research
Early Neospora caninum infection dynamics were investigated in pregnant heifers intravenously ino... more Early Neospora caninum infection dynamics were investigated in pregnant heifers intravenously inoculated with PBS (G-Control) or 10 7 tachyzoites of high (G-NcSpain7)-or low (G-NcSpain1H)-virulence isolates at 110 days of gestation. Serial culling at 10 and 20 days post-infection (dpi) was performed. Fever was detected at 1 dpi in both infected groups (P < 0.0001), and a second peak was detected at 3 dpi only in G-NcSpain7 (P < 0.0001). At 10 dpi, Nc-Spain7 was detected in placental samples from one animal related to focal necrosis, and Nc-Spain7 transmission was observed, although no foetal lesions were associated with this finding. The presence of Nc-Spain1H in the placenta or foetuses, as well as lesions, were not detected at 10 dpi. At 20 dpi, G-NcSpain7 animals showed almost 100% positive placental tissues and severe focal necrosis as well as 100% transmission. Remarkably, foetal mortality was detected in two G-NcSpain7 heifers. Only one animal from G-NcSpain1H presented positive placental samples. No foetal mortality was detected, and lesions and parasite transmission to the foetus were not observed in this group. Finally, 100% of G-NcSpain7 heifers at 20 dpi presented specific antibodies, while only 60% of G-NcSpain1H animals presented specific antibodies at 20 dpi. In addition, earlier seroconversion in G-Nc-Spain7 was observed. In conclusion, tachyzoites from Nc-Spain7 reached the placenta earlier and multiplied, leading to lesion development, transmission to the foetus and foetal mortality, whereas Nc-Spain1H showed delayed infection of the placenta and no lesional development or transmission during early infection.
Veterinary Research
Parameters such as pathogen dose and inoculation route are paramount in animal models when studyi... more Parameters such as pathogen dose and inoculation route are paramount in animal models when studying disease pathogenesis. Here, clinical findings, including foetal mortality, parasite transmission rates and lesion severity, and immune responses were evaluated in Asturiana pregnant heifers at day 110 of gestation challenged with a virulent (Nc-Spain7) Neospora caninum isolate. Four different doses of parasite tachyzoites were inoculated intravenously (IV1, 10 7 parasites, n = 6; IV2, 10 5 , n = 6; IV3, 10 3 , n = 6; and IV4, 10 2 , n = 5), and the subcutaneous (SC) inoculation route was also assessed for the dose of 10 5 tachyzoites (SC, n = 6). In addition, a control group (n = 4 pregnant heifers) was evaluated. Foetal death was observed in all infected groups from 25 to 62 days post-infection, varying with the dose (IV1:4/6, IV2:3/6; IV4:2/5, IV3:1/6), and was three times less frequently associated with the SC route than IV inoculation (1/6 vs. 3/6). A dose-dependent effect for parasite loads in placental and foetal brain tissues was also detected. After SC challenge, a reduced number of tachyzoites were able to reach foetal brain tissues, and no lesions were observed. In calves, specific IgG responses in precolostral sera were mainly associated with high-dose groups (IV1 [100.0%] and IV2 [66.7%]), and cerebral parasite DNA detection was scarce (3/18). In dams, IFN-γ production and the dynamics of anti-N. caninum IgG antibodies varied with the dose, and the cell-mediated immune response was also found to be route-dependent. Our results confirm the influence of parasite dose and inoculation route on the outcome and dynamics of bovine neosporosis at mid-gestation.
Frontiers in Immunology
Neospora caninum, a protozoan parasite closely related to Toxoplasma gondii, represents one of th... more Neospora caninum, a protozoan parasite closely related to Toxoplasma gondii, represents one of the main causes of abortion in cattle. Macrophages (MØs) are mediators of the innate immune response against infection and likely one of the first cells encountered by the parasite during the host infection process. In this study, we investigated in vitro how high or low virulent isolates of N. caninum (Nc-Spain7 and Nc-Spain1H, respectively) interact with bovine monocyte-derived MØs and the influence of the isolate virulence on the subsequent cellular response. Both isolates actively invaded, survived and replicated in the MØs. However, Nc-Spain7 showed a higher invasion rate and a replication significantly faster, following an exponential growth model, whereas Nc-Spain1H presented a delayed replication and a lower growth rate without an exponential pattern. N. caninum infection induced a hypermigratory phenotype in bovine MØs that was characterized by enhanced motility and transmigration in vitro and was accompanied by morphological changes and abrogated extracellular matrix degradation. A significantly higher hypermotility was observed with the highly virulent isolate Nc-Spain7. Nc-Spain1H-infected MØs showed elevated reactive oxygen species (ROS) production and IL12p40 expression, which also resulted in increased IFN-γ release by lymphocytes, compared to cells infected with Nc-Spain7. Furthermore, IL-10 was upregulated in MØs infected with both isolates. Infected MØs exhibited lower expression of MHC Class II, CD86, and CD1b molecules than uninfected MØs, with non-significant differences between isolates. This work characterizes for the first time N. caninum replication in bovine monocyte-derived MØs and details isolate-dependent differences in host cell responses to the parasite.
Experimental Parasitology
Veterinary parasitology, Jan 15, 2018
Neospora caninum is an apicomplexan protozoan responsible for abortion in ruminants. The present ... more Neospora caninum is an apicomplexan protozoan responsible for abortion in ruminants. The present study aimed to diagnose an abortion from an Anglo Nubian goat from a dairy herd located in Buenos Aires province, Argentina. The goat delivered a fetus of approximately 3 months gestation that was studied by indirect fluorescent antibody test (IFAT), Immunoblot (IB), histopathology (HP), immunohistochemistry (IHC), and molecular assays (PCR, sequencing and microsatellite genotyping). Interferon gamma knock-out mice were inoculated with a pool of tissues for isolation attempts. The mother had IFAT titers of 1:3200 and 1:400 for N. caninum and Toxoplasma gondii, respectively, as well as positive IB reactions, whereas the fetus was seronegative to both parasites by IFAT and IB. The fetus had severe multifocal necrotizing myocarditis and hepatitis, moderate interstitial pneumonia, and nephritis. Myocardium sample resulted positive by IHC, evidencing clusters of N. caninum tachyzoites within ...
International journal for parasitology. Drugs and drug resistance, 2018
Neospora caninum is one of the main causes of abortion in cattle, and recent studies have highlig... more Neospora caninum is one of the main causes of abortion in cattle, and recent studies have highlighted its relevance as an abortifacient in small ruminants. Vaccines or drugs for the control of neosporosis are lacking. Bumped kinase inhibitors (BKIs), which are ATP-competitive inhibitors of calcium dependent protein kinase 1 (CDPK1), were shown to be highly efficacious against several apicomplexan parasites in vitro and in laboratory animal models. We here present the pharmacokinetics, safety and efficacy of BKI-1553 in pregnant ewes and foetuses using a pregnant sheep model of N. caninum infection. BKI-1553 showed exposure in pregnant ewes with trough concentrations of approximately 4 μM, and of 1 μM in foetuses. Subcutaneous BKI-1553 administration increased rectal temperatures shortly after treatment, and resulted in dermal nodules triggering a slight monocytosis after repeated doses at short intervals. BKI-1553 treatment decreased fever in infected pregnant ewes already after tw...
Experimental parasitology, Jan 8, 2018
The purpose of this study was to perform genotypic characterization and to evaluate the virulence... more The purpose of this study was to perform genotypic characterization and to evaluate the virulence of Toxoplasma gondii obtained from aborted fetuses in an abortion outbreak in goats from northeastern Brazil. Brain samples from 32 fetuses were submitted to mouse bioassay for T. gondii isolation. Two isolates were obtained and subjected to genotypic characterization. Isolate virulence was evaluated using murine model in different doses (from 10 to 10 tachyzoites/mL). In genotyping, both isolates were classified as clonal lineage type II (genotype #1 ToxoDB) and showed to be virulent for mice. This is the first description of genotype #1 in cases of goat abortion, showing the circulation of virulent T. gondii isolate producing reproductive disorders in pregnant goat.
Parasites & vectors, Jan 10, 2017
Neospora caninum, one of the main causes of abortion in cattle, is very effective at crossing the... more Neospora caninum, one of the main causes of abortion in cattle, is very effective at crossing the placental barrier and placental damage is crucial in the pathogenesis of abortion. Bovine trophoblast and caruncular cell layers are key cellular components in the maternal-foetal interface in placentomes, playing a fundamental role in placental functionality. We studied tachyzoite adhesion, invasion, proliferation and egress of high- (Nc-Spain7) and low- (Nc-Spain1H) virulence N. caninum isolates in established cultures of bovine caruncular epithelial (BCEC-1) and trophoblast (F3) cells. The parasite invasion rate (pInvR) and the cell infection rate (cInfR) were determined by immunostaining plaque assay at different time points and multiplicities of infection (MOIs), respectively. In addition, tachyzoite growth kinetics were investigated using real-time PCR (qPCR) analysis and immunostaining plaque assay at different times. Neospora caninum invaded and proliferated in both cell lines. ...
International Journal for Parasitology
Besnoitia besnoiti is an apicomplexan parasite responsible for bovine besnoitiosis, a chronic and... more Besnoitia besnoiti is an apicomplexan parasite responsible for bovine besnoitiosis, a chronic and debilitating disease that causes systemic and skin manifestations and sterility in bulls. Neither treatments nor vaccines are currently available. In the search for therapeutic candidates, calcium-dependent protein kinases have arisen as promising drug targets in other apicomplexans (e.g. Neospora caninum, Toxoplasma gondii, Plasmodium spp. and Eimeria spp.) and are effectively targeted by bumped kinase inhibitors. In this study, we identified and cloned the gene coding for BbCDPK1. The impact of a library of nine bumped kinase inhibitor analogues on the activity of recombinant BbCDPK1 was assessed by luciferase assay. Afterwards, those were further screened for efficacy against Besnoitiabesnoiti tachyzoites grown in Marc-145 cells. Primary tests at 5µM revealed that eight compounds exhibited more than 90% inhibition of invasion and proliferation. The compounds BKI 1294, 1517, 1553 and 1571 were further characterised, and EC99 (1294: 2.38µM; 1517: 2.20µM; 1553: 3.34µM; 1571: 2.78µM) were determined by quantitative real-time polymerase chain reaction in 3-day proliferation assays. Exposure of infected cultures with EC99 concentrations of these drugs for up to 48h was not parasiticidal. The lack of parasiticidal action was confirmed by transmission electron microscopy, which showed that bumped kinase inhibitor treatment interfered with cell cycle regulation and non-disjunction of tachyzoites, resulting in the formation of large multi-nucleated complexes which co-existed with viable parasites within the parasitophorous vacuole. However, it is possible that, in the face of an active immune response, parasite clearance may occur. In summary, bumped kinase inhibitors may be effective drug candidates to control Besnoitiabesnoiti infection. Further in vivo experiments should be planned, as attainment and maintenance of therapeutic blood plasma levels in calves, without toxicity, has been demonstrated for BKIs 1294, 1517 and 1553.