RESUMEN

Brucellosis is one of the most common and widespread bacterial zoonoses and is caused by Gram-negative bacteria belonging to the genus Brucella. These organisms are able to infect and replicate within the placenta, resulting in abortion, one of the main clinical signs of brucellosis. Although the mouse model is widely used to study Brucella virulence and, more recently, to evaluate the protection of new vaccines, there is no clear consensus on the experimental conditions (e.g., mouse strains, doses, routes of inoculation, infection/pregnancy time) and the natural host reproducibility of the pregnant mouse model for reproductive brucellosis. This lack of consensus calls for a review that integrates the major findings regarding the effect of Brucella wild-type and vaccine strains infections on mouse pregnancy. We found sufficient evidence on the utility of the pregnant mouse model to study Brucella-induced placentitis and abortion and propose suitable experimental conditions (dose, time of infection) and pregnancy outcome readouts for B. abortus and B. melitensis studies. Finally, we discuss the utility and limitations of the pregnant mouse as a predictive model for the abortifacient effect of live Brucella vaccines.

RESUMEN

One of the main causes of human brucellosis is Brucella melitensis infecting small ruminants. To date, Rev1 is the only vaccine successfully used to control ovine and caprine brucellosis. However, it is pathogenic for pregnant animals, resulting in abortions and vaginal and milk shedding, as well as being infectious for humans. Therefore, there is an urgent need to develop an effective vaccine that is safer than Rev1. In efforts to further attenuate Rev1, we recently used wzm inactivation to generate a rough mutant (Rev1Δwzm) that retains a complete antigenic O-polysaccharide in the bacterial cytoplasm. The aim of the present study was to evaluate the placental pathogenicity of Rev1Δwzm in trophoblastic cells, throughout pregnancy in mice, and in ewes inoculated in different trimesters of pregnancy. This mutant was evaluated in comparison with the homologous 16MΔwzm derived from a virulent strain of B. melitensis and the naturally rough sheep pathogen B. ovis. Our results show that both wzm mutants triggered reduced cytotoxic, pro-apoptotic, and pro-inflammatory signaling in Bewo trophoblasts, as well as reduced relative expression of apoptosis genes. In mice, both wzm mutants produced infection but were rapidly cleared from the placenta, in which only Rev1Δwzm induced a low relative expression of pro-apoptotic and pro-inflammatory genes. In the 66 inoculated ewes, Rev1Δwzm was safe and immunogenic, displaying a transient serological interference in standard RBT but not CFT S-LPS tests; this serological response was minimized by conjunctival administration. In conclusion, these results support that B. melitensis Rev1Δwzm is a promising vaccine candidate for use in pregnant ewes and its efficacy against B. melitensis and B. ovis infections in sheep warrants further study.

Asunto(s)

Brucella melitensis , Brucelosis , Placenta , Animales , Brucella melitensis/patogenicidad , Brucella melitensis/inmunología , Brucella melitensis/genética , Femenino , Ovinos , Brucelosis/prevención & control , Brucelosis/inmunología , Brucelosis/veterinaria , Embarazo , Placenta/microbiología , Ratones , Enfermedades de las Ovejas/prevención & control , Enfermedades de las Ovejas/inmunología , Enfermedades de las Ovejas/microbiología , Trofoblastos/inmunología , Trofoblastos/microbiología , Vacuna contra la Brucelosis/inmunología , Vacuna contra la Brucelosis/administración & dosificación , Vacuna contra la Brucelosis/genética , Humanos , Vacunas Atenuadas/inmunología , Vacunas Atenuadas/administración & dosificación

RESUMEN

The intraspecific variability among Neospora caninum isolates in their in vitro behaviour and in vivo virulence has been widely studied. In particular, transcriptomic and proteomic analyses have shown a higher expression/abundance of specific genes/proteins in high-virulence isolates. Consequently, the dense granule protein NcGRA7 and the rhoptry protein NcROP40 were proposed as potential virulence factors. The objective of this study was to characterize the role of these proteins using CRISPR/Cas9 knockout (KO) parasites in a well-established pregnant BALB/c mouse model of N. caninum infection at midgestation. The deletion of NcGRA7 and NcROP40 was associated with a reduction of virulence, as infected dams displayed milder clinical signs, lower parasite burdens in the brain, and reduced mortality rates compared to those infected with the wild-type parasite (Nc-Spain7). Specifically, those infected with the NcGRA7 KO parasites displayed significantly milder clinical signs and a lower brain parasite burden. The median survival time of the pups from dams infected with the two KO parasites was significantly increased, but differences in neonatal mortality rates were not detected. Overall, the present study indicates that the disruption of NcGRA7 considerably impairs virulence in mice, while the impact of NcROP40 deletion was more modest. Further research is needed to understand the role of these virulence factors during N. caninum infection.

RESUMEN

BACKGROUND: Congenital disorders associated with prenatal vertical transmission of Zika virus (ZIKV) is well established since the 2016 outbreak in the Americas. However, despite clinical reports of similar mode of transmission for other flaviviruses such as dengue virus (DENV), the phenomenon has not been experimentally explored. METHODS: Pregnant AG129 mice were infected with DENV1 in the presence or absence of enhancing antibodies at different gestational time points. ZIKV was used for comparison. We quantified viral load in fetus and placentas and performed comprehensive gene expression profiling in the maternal (decidua) and fetal portion of placenta separately. FINDINGS: We demonstrate in a laboratory experimental setting that DENV can be transmitted vertically in a gestation stage-dependent manner similar to ZIKV, and this incidence drastically increases in the presence of enhancing antibodies. Interestingly, a high rate of DENV fetal infection occurs even though the placental viral load is significantly lower than that found in ZIKV-infected dams. Comprehensive gene expression profiling revealed DENV infection modulates a variety of inflammation-associated genes comparable to ZIKV in decidua and fetal placenta in early pregnancy. INTERPRETATION: Our findings suggest that the virus-induced modulation of host gene expression may facilitate DENV to cross the placental barrier in spite of lower viral burden compared to ZIKV. This mouse model may serve to identify the host determinants required for the vertical transmission of flaviviruses and develop appropriate countermeasures. FUNDING: National Medical Research Council/Open Fund Individual Research Grant MOH-000524 (SW), MOH-000086 and OFIRG20nov-0017 (SGV).

Asunto(s)

Virus del Dengue , Infección por el Virus Zika , Virus Zika , Animales , Anticuerpos Antivirales , Femenino , Humanos , Ratones , Placenta , Embarazo , Virus Zika/genética

RESUMEN

Zika virus (ZIKV) infection during pregnancy has been associated with adverse outcomes and birth defects such as microcephaly in newborn children. Congenital malformations associated with ZIKV are believed to occur via direct infection of the fetus. Unfortunately, there are no licensed therapeutic or preventative tools to block maternal-fetal transmission of ZIKV. In this study, we developed a mouse model of ZIKV infection that specifically establishes vertical maternal-fetal transmission of ZIKV in 40-60% of fetuses when the dams acquire ZIKV infection during pregnancy. This mouse model somewhat mirrors the experience in humans at the peak of the epidemic in the Americas. Using this model, we demonstrate that a well-documented directly acting antiviral (DAA) compound that targets flaviviral RNA synthesis can completely prevent fetal infection when the treatment is started at the time of infection. Notably, we show that the treatment commenced at the time of peak viremia is still able to reduce the risk of fetal infection concomitant with significant reduction in placental viral load. Our results show for the first time the potential for clinical development of antiviral drugs for preventing vertical maternal-fetal transmission of ZIKV.

Asunto(s)

Adenosina/análogos & derivados , Antivirales/uso terapéutico , Transmisión Vertical de Enfermedad Infecciosa , Malformaciones del Sistema Nervioso/virología , Infección por el Virus Zika , Adenosina/uso terapéutico , Animales , Modelos Animales de Enfermedad , Femenino , Feto/anomalías , Feto/virología , Humanos , Ratones , Microcefalia/virología , Malformaciones del Sistema Nervioso/tratamiento farmacológico , Malformaciones del Sistema Nervioso/prevención & control , Embarazo , Complicaciones Infecciosas del Embarazo , Carga Viral/efectos de los fármacos , Virus Zika/aislamiento & purificación , Infección por el Virus Zika/tratamiento farmacológico , Infección por el Virus Zika/transmisión

RESUMEN

Poxviruses cause many diseases in humans and animals worldwide, and there is a need for vaccines with improved safety and good efficacy. In addition, poxvirus vectors are widely used as recombinant vaccines for various infectious diseases and as recombinant and oncolytic vaccines for cancer. One concern with poxvirus vaccine vectors is that some poxviruses can infect a developing fetus and cause fetal loss or congenital disease. This can be an issue both for patients receiving a vaccine and for pregnant health care providers, including doctors, nurses, and veterinarians, who might receive accidental exposure to the poxvirus by injection or during patient care. We describe here a method for analyzing the safety of virus exposure in pregnant mammals using a mouse model testing vaccinia, canarypox, and raccoonpox virus vectors.

Asunto(s)

Infecciones por Poxviridae/diagnóstico , Poxviridae/patogenicidad , Animales , Chlorocebus aethiops , Femenino , Ratones , Orthopoxvirus/genética , Orthopoxvirus/patogenicidad , Orthopoxvirus/fisiología , Poxviridae/genética , Poxviridae/fisiología , Embarazo , Vaccinia/diagnóstico , Células Vero , Carga Viral , Vacunas Virales/genética , Replicación Viral

RESUMEN

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 (IC50s) of 0.05 ± 0.03 and 0.18 ± 0.03 µM, respectively. As assessed by an alamarBlue assay, fibroblast IC50s were above 20 µM; however, morphological changes occurred in cultures treated with >5 µM BKI-1517 after prolonged exposure (>6 days). Treatment of intracellular tachyzoites with 5 µM 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 µM 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.

Asunto(s)

Coccidiosis/tratamiento farmacológico , Coccidiostáticos/farmacología , Transmisión Vertical de Enfermedad Infecciosa/prevención & control , Estadios del Ciclo de Vida/efectos de los fármacos , Neospora/efectos de los fármacos , Inhibidores de Proteínas Quinasas/farmacología , Pirazoles/farmacología , Pirimidinas/farmacología , Quinolinas/farmacología , Animales , Encéfalo/efectos de los fármacos , Encéfalo/parasitología , Proliferación Celular/efectos de los fármacos , Coccidiosis/parasitología , Coccidiosis/transmisión , Coccidiostáticos/química , Femenino , Fertilidad/efectos de los fármacos , Fibroblastos/efectos de los fármacos , Fibroblastos/parasitología , Expresión Génica , Genes Reporteros , Humanos , Estadios del Ciclo de Vida/fisiología , Ratones , Ratones Endogámicos BALB C , Neospora/enzimología , Neospora/genética , Neospora/crecimiento & desarrollo , Oxazinas , Embarazo , Cultivo Primario de Células , Inhibidores de Proteínas Quinasas/química , Proteínas Quinasas/genética , Proteínas Quinasas/metabolismo , Proteínas Protozoarias/antagonistas & inhibidores , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismo , Pirazoles/química , Pirimidinas/química , Quinolinas/química , Xantenos , beta-Galactosidasa/genética , beta-Galactosidasa/metabolismo

RESUMEN

Administering monosodium glutamate (MSG) to neonatal rodents induces obesity and type 2 diabetes. In addition, several studies have shown that MSG administered to pregnant animals can cross the placenta and reach the foetus. The present study was performed to investigate the effects of administering MSG to pregnant ICR mice on dam and neonatal growth. Pregnant mice were treated with 60 or 120 mg MSG once daily from day 5 of pregnancy to one day before parturition by subcutaneous injection. In addition, the body weights of the neonates were determined until nine weeks of age. The birth weights of neonates were not different between the control and MSG-treated groups. However, MSG treatment resulted in a lower body weight gain of neonates during lactation. In addition, this underweight of the MSG-treated group at weaning returned to normal compared with the control group at five weeks of age. Cross-fostering experiments indicated that the lower body weight gain of neonates in the MSG-treated group during lactation was due to its effects on the dam. Serum prolactin levels and mammary gland development of the mice were examined next to determine the reasons for this lactation problem. Although there were no differences in prolactin levels, morphological analyses of the mammary glands revealed apparent differences, including low numbers and altered phenotype of alveoli, between the control and MSG-treated groups. Taken together, our results show that treating pregnant mice with excess MSG induced lower neonate body weight gain during lactation.

Asunto(s)

Aromatizantes/toxicidad , Complicaciones del Embarazo/fisiopatología , Glutamato de Sodio/toxicidad , Aumento de Peso/efectos de los fármacos , Animales , Animales Recién Nacidos , Relación Dosis-Respuesta a Droga , Femenino , Inyecciones Subcutáneas , Lactancia , Masculino , Ratones , Ratones Endogámicos ICR , Embarazo , Complicaciones del Embarazo/inducido químicamente , Prolactina/sangre , Glutamato de Sodio/administración & dosificación

RESUMEN

Pregnant BALB/c mice have been widely used as an in vivo model to study Neospora caninum infection biology and to provide proof-of-concept for assessments of drugs and vaccines against neosporosis. The fact that this model has been used with different isolates of variable virulence, varying infection routes and differing methods to prepare the parasites for infection, has rendered the comparison of results from different laboratories impossible. In most studies, mice were infected with similar number of parasites (2 × 10(6)) as employed in ruminant models (10(7) for cows and 10(6) for sheep), which seems inappropriate considering the enormous differences in the weight of these species. Thus, for achieving meaningful results in vaccination and drug efficacy experiments, a refinement and standardization of this experimental model is necessary. Thus, 2 × 10(6), 10(5), 10(4), 10(3) and 10(2) tachyzoites of the highly virulent and well-characterised Nc-Spain7 isolate were subcutaneously inoculated into mice at day 7 of pregnancy, and clinical outcome, vertical transmission, parasite burden and antibody responses were compared. Dams from all infected groups presented nervous signs and the percentage of surviving pups at day 30 postpartum was surprisingly low (24%) in mice infected with only 10(2) tachyzoites. Importantly, infection with 10(5) tachyzoites resulted in antibody levels, cerebral parasite burden in dams and 100% mortality rate in pups, which was identical to infection with 2 × 10(6) tachyzoites. Considering these results, it is reasonable to lower the challenge dose to 10(5) tachyzoites in further experiments when assessing drugs or vaccine candidates.

Asunto(s)

Coccidiosis/parasitología , Modelos Animales de Enfermedad , Neospora/clasificación , Neospora/patogenicidad , Animales , Anticuerpos Antiprotozoarios , Especificidad de Anticuerpos , Encéfalo/parasitología , Coccidiosis/inmunología , Coccidiosis/patología , ADN Protozoario/aislamiento & purificación , Femenino , Inmunoglobulina G/sangre , Tamaño de la Camada , Ratones , Ratones Endogámicos BALB C , Embarazo , Piel/patología , Virulencia

RESUMEN

Currently there are no effective vaccines for the control of bovine neosporosis. During the last years several subunit vaccines based on immunodominant antigens and other proteins involved in adhesion, invasion and intracellular proliferation of Neospora caninum have been evaluated as targets for vaccine development in experimental mouse infection models. Among them, the rhoptry antigen NcROP2 and the immunodominant NcGRA7 protein have been assessed with varying results. Recent studies have shown that another rhoptry component, NcROP40, and NcNTPase, a putative dense granule antigen, exhibit higher expression levels in tachyzoites of virulent N. caninum isolates, suggesting that these could be potential vaccine candidates to limit the effects of infection. In the present work, the safety and efficacy of these recombinant antigens formulated in Quil-A adjuvant as monovalent vaccines or pair-wise combinations (rNcROP40+rNcROP2 and rNcGRA7+rNcNTPase) were evaluated in a pregnant mouse model of neosporosis. All the vaccine formulations elicited a specific immune response against their respective native proteins after immunization. Mice vaccinated with rNcROP40 and rNcROP2 alone or in combination produced the highest levels of IFN-γ and exhibited low parasite burdens and low IgG antibody levels after the challenge. In addition, most of the vaccine formulations were able to increase the median survival time in the offspring. However, pup survival only ensued in the groups vaccinated with rNcROP40+rNcROP2 (16.2%) and rNcROP2 (6.3%). Interestingly, vertical transmission was not observed in those survivor pups immunized with rNcROP40+rNcROP2, as shown by PCR analyses. These results show a partial protection against N. caninum infection after vaccination with rNcROP40+rNcROP2, suggesting a synergistic effect of the two recombinant rhoptry antigens.

Asunto(s)

Coccidiosis/prevención & control , Neospora/fisiología , Proteínas Protozoarias/inmunología , Vacunas Antiprotozoos/inmunología , Animales , Anticuerpos Antiprotozoarios/sangre , Coccidiosis/mortalidad , Modelos Animales de Enfermedad , Femenino , Transmisión Vertical de Enfermedad Infecciosa/prevención & control , Ratones , Neospora/inmunología , Embarazo , Vacunas Antiprotozoos/normas , Proteínas Recombinantes/inmunología , Análisis de Supervivencia

RESUMEN

Abortion is the major clinical sign of brucellosis in animals but little is known about the underlying mechanisms. This study was designed to evaluate a pregnant mouse model for the vertical transmission of Brucella melitensis using four infectious doses: 10(3) colony-forming units (CFU), 10(4) CFU, 10(5) CFU, and 10(6) CFU. During the experimental period, no instances of abortion were recorded, but stillbirths were observed in the groups infected with doses of 10(4) CFU and higher. Regardless of whether the fetuses were stillborn or alive, transmission of bacteria to the fetus and bacterial replication in the cytoplasm of placental trophoblast giant cells were detected. A higher degree of bacterial colonization was found in the placenta than in the spleen or fetus. Doses of 10(5) CFU of B. melitensis or higher produced a severe, necrotizing placentitis similar to the pathological damage observed in ruminants. The data suggest that experimental murine brucellosis resembles ruminant brucellosis and represents a potential model for studying the pathogenic mechanisms of B. melitensis.

Asunto(s)

Brucella melitensis/fisiología , Brucelosis/transmisión , Transmisión Vertical de Enfermedad Infecciosa , Ratones , Modelos Animales , Animales , Técnicas Bacteriológicas , Brucelosis/microbiología , Femenino , Ratones Endogámicos ICR , Placenta/microbiología , Placenta/patología , Enfermedades Placentarias/microbiología , Enfermedades Placentarias/patología , Reacción en Cadena de la Polimerasa , Embarazo

RESUMEN

Objective To investigate the effects of maternal long-term exposure to low-dose trichlorfon on the serum paraoxonase (PON) activity of pregnant mice and development of embryos. Methods Female ICR mice (n=120) were randomly divided into control group and trichlorfon groups of different doses,and were managed by intragastric injection with trichlorfon of 0,2,10 and 50mg/kg,respectively. All the mice were managed once a day for a consecutive of 27 days,and were subjected to mating. The pregnant mice were continued to be managed with trichlorfon for 3 days,and were sacrificed on day 3 of gestation. The serum PON and acetylcholinesterase (AchE) activities were detected,and the development of embryos was evaluated. Results The serum PON activity of 2,10 and 50mg/kg trichlorfon group were (14.15?1.22),(12.78?1.80) and (10.45?1.95)IU/mL,respectively,and that of 50mg/kg trichlorfon group was significantly lower than that of control group [(13.37?2.31)IU/mL] (P0.05),while the the percentage of abnormal embryos of 50mg/kg trichlorfon group had an increased tendency. Conclusion Long-term exposure to low-dose trichlorfon can inhibit serum PON and AchE activity in pregnant mice without obvious effect on the development of embryos.

RESUMEN

AIM: To develop a murine model for investigating the effects of prenatal cocaine exposure on the development of fetal nerve system. METHODS: A nutritionally paired control group of dams injected with saline and pair-fed with the COC dams were set up. Another two groups were COC groups injected with cocaine HC1 and SAL group administrated with saline. After injection twice daily during gestation days 8-17, mice were decapitated on E17 and blood and brain samples were collected for pharmacological analysis and neurotransmitter analysis by HPLC. RESULTS: Pharmacological analysis revealed that cocaine was found in maternal and fetal plasma at 15 min following ip administration to embryonic day E17 pregnant mice. Though COC dams and SPF dams had the same feeding condition, compared with the latter, the former had higher maternal concentrations of DA and 5-HT, lower fetal weight, brain weight, striatum weight and higher concentrations of DA and 5-HT in striatum, P < 0.01. But fetuses of all groups had almost the same brain/body weight ratio and striatum/brain weight ratio. CONCLUSION: Prenatal cocaine exposure can lead to fetal developmental retardation. The brain/body weight ratio and striatum/brain weight ratio were only slightly altered for the COC fetuses in comparison with SAL and SPF fetuses, suggesting a symmetrical weight reduction with little sparing of the brain. And it seems that the alteration of body weight, brain weight, striatum weight and neurotransmitters in COC fetuses show no relationship to the maternal malnutrition, but to the pharmacological and toxicological effect of cocaine.

RESUMEN

Objective To study the effect of rhu-IFN-? on immune state and vertical transmission in pregnant mice infected with Toxoplasma gondii. Methods Sixty pregnant BALB/c mice were randomly divided into three groups: a control group,infected group and treatment group. In the infected group and treatment group,each mouse was injected with T.gondii tachyzoites peritoneally on the day 8 of gestation. In the treatment group,each mouse was treated with 1 000 U rhu-IFN-? on the day 7,8,9 of gestation. Blood was collected from the tail veins of all the mice on the day 10,12 of gestation. The levels of blood CD4+ and CD8+ T cells were detected by flow cytometry. Meanwhile,on the day 12 of gestation,all mice were anatomized to observe live embryo rate and the infection status in fetal brain tissue. Results Compared with the control group,the levels of CD4+ T cells in the infected group and treatment group were low,and the levels of CD8+ T cells high on the day 10,12 of gestation,so the ratio of CD4+/CD8+ was inversed. However,compared with the infected group,the levels of CD4+ T cells in the treatment group were high and the levels of CD8+ T cells low on the day 10,12 of gestation,so the ratios of CD4+/CD8+ were high on the day 10,12 of gestation. Meanwhile,the live embryo rate was high and the infection rate of intrauterine embryonic low. Conclusion A proper dose of rhu-IFN-? could improve the function of immunity system and reduce the vertical transmission probability in pregnant mice infected with T.gondii.

RESUMEN

AIM To develop a murine model for investigating the effects of prenatal cocaine exposure on the development of fetal nerve system. METHODS A nutritionally paired control group of dams injected with saline and pair fed with the COC dams were set up. Another two groups were COC groups injected with cocaine HCl and SAL group administrated with saline. After injection twice daily during gestation days 8～17,mice were decapitated on E17 and blood and brain samples were collected for pharmacological analysis and neurotransmitter analysis by HPLC.RESULTS Pharmacological analysis revealed that cocaine was found in maternal and fetal plasma at 15 min following ip administration to embryonic day E17 pregnant mice. Though COC dams and SPF dams had the same feeding condition, compared with the latter, the former had higher maternal concentrations of DA and 5 HT, lower fetal weight, brain weight, striatum weight and higher concentrations of DA and 5 HT in striatum, P