Dose-dependent effects of experimental infection with the virulent Neospora caninum Nc-Spain7 isolate in a pregnant mouse model

Arranz-Solís, David; Aguado‐Martínez, Adriana; Müller, Joachim; Regidor‐Cerrillo, Javier; Ortega‐Mora, Luis Miguel; Hemphill, Andrew

doi:10.1016/j.vetpar.2015.05.021

Cited by 29 publications

(53 citation statements)

References 43 publications

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“…In order to assess the reproducibility of these findings using another virulent N. caninum isolate, another mode of infection, and another formulation of the compound, a second experiment was performed according to the recently standardized infection model established by Arranz-Solís et al (26). Mice were mated as described above and infected 7 days postmating by subcutaneous injection of 10 5 N. caninum Nc-Spain7 tachyzoites.…”

Section: Resultsmentioning

confidence: 99%

“…Subsequently, female mice were randomly distributed in 3 groups of 16 mice each. Mice from groups 1 and 2 were subcutaneously challenged with 10 5 tachyzoites of the Nc-Spain7 isolate at midgestation (days 7 after mating), while mice from group 3 were left unchallenged and received a culture medium inoculation (26). Two days after infection, all mice from group 1 were given the BKI-1294 (50 mg/kg/ day emulsified in 100 l corn oil) for a period of 6 days by gavage once a day, while the group 2 mice received corn oil alone.…”

Section: Methodsmentioning

confidence: 99%

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In Vitro and In Vivo Effects of the Bumped Kinase Inhibitor 1294 in the Related Cyst-Forming Apicomplexans Toxoplasma gondii and Neospora caninum

Winzer

Müller

Aguado‐Martínez

et al. 2015

Antimicrob Agents Chemother

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124

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eWe report on the in vitro effects of the bumped kinase inhibitor 1294 (BKI-1294) in cultures of virulent Neospora caninum isolates Nc-Liverpool (Nc-Liv) and Nc-Spain7 and in two strains of Toxoplasma gondii (RH and ME49), all grown in human foreskin fibroblasts. In these parasites, BKI-1294 acted with 50% inhibitory concentrations (IC 50 s) ranging from 20 nM (T. gondii RH) to 360 nM (N. caninum Nc-Liv), and exposure of intracellular stages to 1294 led to the nondisjunction of newly formed tachyzoites, resulting in the formation of multinucleated complexes similar to complexes previously observed in BKI-1294-treated N. caninum beta-galactosidase-expressing parasites. However, such complexes were not seen in a transgenic T. gondii strain that expressed CDPK1 harboring a mutation (G to M) in the gatekeeper residue. In T. gondii ME49 and N. caninum NcLiv, exposure of cultures to BKI-1294 resulted in the elevated expression of mRNA coding for the bradyzoite marker BAG1. Unlike in bradyzoites, SAG1 expression was not repressed. Immunofluorescence also showed that these multinucleated complexes expressed SAG1 and BAG1 and the monoclonal antibody CC2, which binds to a yet unidentified bradyzoite antigen, also exhibited increased labeling. In a pregnant mouse model, BKI-1294 efficiently inhibited vertical transmission in BALB/c mice experimentally infected with one of the two virulent isolates Nc-Liv or Nc-Spain7, demonstrating proof of concept that this compound protected offspring from vertical transmission and disease. The observed deregulated antigen expression effect may enhance the immune response during BKI-1294 therapy and will be the subject of future studies. N eospora caninum is a cyst-forming apicomplexan parasite that is closely related to Toxoplasma gondii but exhibits distinct differences in transmission patterns, virulence, host specificity, immunogenetic aspects, and the pathology it induces. T. gondii causes toxoplasmosis in humans and many domestic and wildlife animals, with great economic impact especially in sheep but also in many other animal species (1). Human toxoplasmosis causes serious pathology in immune-suppressed individuals. In addition, if a seronegative mother acquires primary infection during pregnancy, human toxoplasmosis can lead to abortion, microcephalus and hydrocephalus, and other fetal abnormalities causing intellectual disability (2). N. caninum is a veterinary health problem and represents one of the most important infectious causes of bovine abortion, stillbirth, and the birth of weak calves, with an economic impact of over $1.3 billion (3-5). In addition, N. caninum causes neuromuscular disease in dogs, and neosporosis has also been detected in a wide range of other species of livestock and wild animals worldwide.Despite their differences, an important common feature of these parasites is their ability to invade and replicate within a wide range of cell types and tissues, where they reside in an intracellular parasitophorous vacuole, surrounded by a parasitophorous vacuole me...

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

In Vitro and In Vivo Effects of the Bumped Kinase Inhibitor 1294 in the Related Cyst-Forming Apicomplexans Toxoplasma gondii and Neospora caninum

Winzer

Müller

Aguado‐Martínez

et al. 2015

Antimicrob Agents Chemother

Self Cite

124

View full text Add to dashboard Cite

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“…In this model, which uses the N. caninum Spain7 (Nc-Spain7) isolate, which is highly virulent in vitro and in vivo in mice (21,22) as well as in sheep (23), all pups of infected dams died within 3 weeks; pups of noninfected dams survived. Thus, the model is reproducible, as shown in several recent studies (17,20,24).…”

Section: Discussionmentioning

confidence: 99%

“…HFFs, Vero cells (30), and RH cells were cultured as described previously (31). N. caninum tachyzoites were maintained in Vero cells and prepared for infection of HFFs or mice as described previously (21).…”

Section: Methodsmentioning

confidence: 99%

“…For each experiment, female and male BALB/c mice were housed, and pregnancy was achieved after synchronization of estrus (21). Subsequently, female mice were randomly distributed into groups of 16 mice each and were subcutaneously infected with 10 5 tachyzoites of the Nc-Spain7 isolate at midgestation (days 5 to 8 postmating) or were left uninfected and received an inoculation of culture medium (21).…”

Section: Methodsmentioning

confidence: 99%

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Two Novel Calcium-Dependent Protein Kinase 1 Inhibitors Interfere with Vertical Transmission in Mice Infected with Neospora caninum Tachyzoites

Müller

Aguado‐Martínez

Balmer

et al. 2017

Antimicrob Agents Chemother

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We present the effects of two novel bumped kinase inhibitors, BKI-1517 and BKI-1553, against Neospora caninum tachyzoites in vitro and in experimentally infected pregnant mice. These compounds inhibited tachyzoite proliferation of a transgenic beta-galactosidase reporter strain cultured in human foreskin fibroblasts with 50% inhibitory concentrations (IC 50 s) of 0.05 Ϯ 0.03 and 0.18 Ϯ 0.03 M, respectively. As assessed by an alamarBlue assay, fibroblast IC 50 s were above 20 M; however, morphological changes occurred in cultures treated with Ͼ5 M BKI-1517 after prolonged exposure (Ͼ6 days). Treatment of intracellular tachyzoites with 5 M BKI-1553 for 6 days inhibited endodyogeny by interfering with the separation of newly formed zoites from a larger multinucleated parasite mass. In contrast, parasites treated with 5 M BKI-1517 did not form large complexes and showed much more evidence of cell death. However, after a treatment duration of 10 days in vitro, both compounds failed to completely prevent the regrowth of parasites from culture. BALB/c mice experimentally infected with N. caninum Spain7 (Nc-Spain7) and then treated during 6 days with BKI-1517 or BKI-1553 at different dosages showed a significant reduction of the cerebral parasite load. However, fertility was impaired by BKI-1517 when applied at 50 mg/kg of body weight/day. At 20 mg/kg/day, BKI-1517 significantly inhibited the vertical transmission of N. caninum to pups and increased the rate of survival of offspring. BKI-1553 was less detrimental to fertility and also provided significant but clearly less pronounced protection of dams and offspring. These results demonstrate that, when judiciously applied, this compound class protects offspring from vertical transmission and disease.KEYWORDS apicomplexan parasites, Neospora caninum, chemotherapy, vertical transmission, bumped kinase inhibitor, calcium-dependent protein kinase, pregnant mouse model, fertility N eospora caninum, a cyst-forming apicomplexan parasite that is closely related to Toxoplasma gondii, is one of the most important infectious causes of bovine abortion, stillbirth, and birth of weak calves, collectively named neosporosis (1). Furthermore, N. caninum infection may result in the birth of clinically healthy but persistently infected calves transmitting the parasite to the next generation. Neosporosis has also been described in other ruminants of economic importance, such as sheep and goats; was shown to cause neuromuscular disease in dogs; and has been detected in a wide range of other species and wild animals worldwide (1-3). In cattle, N. caninum

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Immunisation with Neospora caninum subunits rsNcSAG4 and rsNcGRA1 (NcSAG4 and NcGRA1 epitopes construct) in BALB/c mice: the profile of the immune response and controlling the vertical transmission

Fernandes Fidelis,

Silva de Araújo,

Prates-Patarroyo

et al. 2023

Parasitol Res

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Neospora caninum is an apicomplexan protozoan that causes neosporosis, which has a high economic impact on cattle herds with no available vaccine. During infection, the secretion of dense granules and the expression of surface antigens play an important role in hosting immunomodulation. However, some epitopes of those antigens are immunogenic, and using these fractions could improve the subunit antigens in vaccine design. This study evaluates the recombinant peptides rsNcGRA1 and rsNcSAG4 derived from NcGRA1 and NcSAG4 native antigens as vaccine candidates produced by a fermentative process in the yeast culture system of Komagataella phaffii strain Km71, confirmed by colony PCR, SDS-PAGE, and western blotting. The assay was conducted in BALB/c mice using the peptides at low (25 μg) and standard (50 μg) dosages in monovalent and combined administrations at three time points with saponin as an adjuvant assessing the immunogenicity by antibodies response and cytokine production. We challenge the females after pregnancy confirmation using 2 × 105 NC-1 tachyzoites previously propagated in Vero cells. We assessed the chronic infection in dams and vertical transmission in the offspring by PCR and histopathology. Mice, especially those immunised with combined peptides and monovalent rsNcGRA1 at a standard dose, controlling the chronic infection in dams with the absence of clinical manifestations, showed an immune response with induction of IgG1, a proper balance between Th1/Th2 cytokines and reduced vertical transmission in the pups. In contrast, dams inoculated with a placebo vaccine showed clinical signs, low-scored brain lesions, augmented chronic infection with 80% positivity, 31% mortality in pups, and 81% vertical transmission. These findings indicate that rsNcGRA1 peptides in monovalent and combined with rsNCSAG4 at standard dose are potential vaccine candidates and improve the protective immune response against neosporosis in mice.

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Dose-dependent effects of experimental infection with the virulent Neospora caninum Nc-Spain7 isolate in a pregnant mouse model

Cited by 29 publications

References 43 publications

In Vitro and In Vivo Effects of the Bumped Kinase Inhibitor 1294 in the Related Cyst-Forming Apicomplexans Toxoplasma gondii and Neospora caninum

In Vitro and In Vivo Effects of the Bumped Kinase Inhibitor 1294 in the Related Cyst-Forming Apicomplexans Toxoplasma gondii and Neospora caninum

Two Novel Calcium-Dependent Protein Kinase 1 Inhibitors Interfere with Vertical Transmission in Mice Infected with Neospora caninum Tachyzoites

Immunisation with Neospora caninum subunits rsNcSAG4 and rsNcGRA1 (NcSAG4 and NcGRA1 epitopes construct) in BALB/c mice: the profile of the immune response and controlling the vertical transmission

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