Interacción hospedador/parásito en la toxoplasmosis ovina: influencia de la virulencia del parásito sobre la respuesta inmunitaria y la patogenia de la enfermedad

Abstract

[ES] La Equinococosis quística (CE) o Hidatidosis es una zoonosis cosmopolita crónica causada por el metacéstodo de un complejo de especies crípticas pertenecientes a Echinococcus granulosus s.l., siendo Echinococcus granulosus s.s. la especie causante de la mayor parte de los casos de CE humana y se encuentra distribuida en todo el mundo. Esta cestodiasis es un importante problema de salud pública y económico para las poblaciones afectadas, principalmente en zonas rurales y ganaderas. En Sudamérica, Chile es uno de los países en donde la Equinococosis quística es endémica, a pesar de esto y a los programas de intervención para controlar esta parasitosis en regiones y provincias rurales en donde se practica la ganadería caprina y ovina, esta parasitosis sigue presente, siendo las regiones de Aysén, Magallanes, La Araucanía y Coquimbo, las que poseen los índices de prevalencia más altos. En este estudio se determinó la prevalencia de la equinococosis quística y sus posibles factores de riesgo en cuatro comunas de la provincia de Limarí, región de Coquimbo, entre los años 2016-2017 y se evaluó la diversidad genética de E. granulosus s.s. en tres regiones endémicas, Coquimbo, La Araucanía y Magallanes, mediante la secuencia de la subunidad 1 del gen mitocondrial de la de la citocromo oxidasa (cox1). Un total de 1221 personas entre 5 - 88 años fueron examinados con el método de ELISA. Los factores de riesgo fueron evaluados por medio de un cuestionario en 908 personas mayores de 18 años. Se utilizó el método de regresión logística para determinar la probabilidad de asociación entre la presencia de la infección equinococócica y los factores de riesgo; y la escolaridad y los factores de riesgo. La diversidad genética y la estructura poblacional de metacéstodos de E. granulosus s.s. fue evaluada en tres regiones endémicas de Chile, mediante la secuencia de la sub unidad 1 del gen mitocondrial de la citocromo oxidasa (cox1). Se secuenciaron 46 quistes, 6 de la región de Coquimbo, 18 de la Araucanía y 22 de Magallanes, aislados de distintos hospederos intermediarios, bovinos, caprinos, ovinos y seres humanos. De este modo se observó que la prevalencia general de CE en la población analizada fue de 7,9% (97/1221), 6,71% (21/313) de menores de 18 años y el 8,4% (76/908) de los adultos, no se observaron diferencias significativas entre los grupos etarios analizados. La comuna de Punitaqui presenta la mayor frecuencia relativa 13.33% (12/90) de CE. En el análisis de los factores de riesgo para la CE, el tener agua potable en las casas y el haber tenido equinococosis quística en alguna etapa de su vida se asociaron con la mayor probabilidad de presentar equinococosis quística. Los resultados de este estudio muestran una alta endemicidad de Equinococosis quística en la provincia de Limarí. Así también, todos los quistes hidatídicos analizados fueron identificados como E. granulosus s.s.. Predominó la cepa EG01, detectando 4 haplotipos, EG01, EG1A, EG1D, EgCLOM16, con una baja diversidad de haplotipos (Hd=0,461±0,00637) y una baja diversidad de nucleótidos (0,00181±0,00036). La red de parsimonia de cox1 en la población chilena mostró forma de estrella, en donde el haplotipo EG01 se encontró en el centro. Los índices de neutralidad Tajima`s D y F de Fu`s fueron negativos para las poblaciones de Coquimbo (D= -0,93302; Fs= -0,003) y Magallanes (D= -0,17406; Fs= -0,121), lo que indica un exceso de sitios polimórficos raros, expansión poblacional y una desviación significativa de la neutralidad. Los índices D de Tajima y Fs de Fu fueron positivos para la población de La Araucanía (D= 0,8695; Fs= 1,039), mostrando niveles bajos de alelos raros o falta de alelos raros. El índice de fijación de Fisher por pares (Fst), muestran valores negativos en las poblaciones Coquimbo- La Araucanía (-0,08761). El valor de Fst más alto y significativo fue entre las poblaciones de La Araucanía- Magallanes (0,10703), indicando diferencias entre estas poblaciones. Nuestros resultados muestran que la estructura genética poblacional de E. granulosus s.s es compleja, debido a la presencia de diferentes haplotipos en las diferentes regiones a lo largo de Chile, la cual posiblemente se complejice aún más si se utilizan secuencias más largas, que permitan estudiar la microdiversidad de E. granulosus s.s. en los diferentes hospederos. De esta forma, se sugiere que para diseñar futuros programas de control de la Equinococosis quística (CE), se deben tener en cuenta la variabilidad de la cepa en cada una de las regiones endémicas.

[EN] Toxoplasmosis is a protozoan infectious disease caused by the parasite Toxoplasma gondii. It is an important disease in both in Europe and globally due to its zoonotic potential and the reproductive failure it triggers in livestock, mainly in sheep, which results in heavy economic losses. Despite the fact that there are multiple methods for controlling this disease, such as management, vaccination or treatment; toxoplasmosis is still a very relevant animal health and public problem. There are still many unknown mechanisms of the pathogenesis of ovine toxoplasmosis, mainly regarding the occurrence of abortions. On the other hand, there are evidences that the variability of T. gondii isolates could influence the pathogenesis of the disease in murine models and humans’ observational studies. However, little is known regarding the influence of this variation in sheep. Bearing in mind that extrapolation of results between species is not always possible, it is necessary to study how the genotypic and phenotypic variability of T. gondii isolates could influence the development of the disease in pregnant sheep. For this, it is essential to use an experimental model of pregnant sheep, as the placenta and fetus are the main targets of the parasite. In order to clearly identified lesions caused by the disease, a detailed analysis of the morphological and physiological findings in both placenta and fetuses is also necessary.

Thus, the aim of this PhD Thesis is to analyze the influence of T. gondii isolates variability on parasite/host interaction in ovine toxoplasmosis and the consequences it may have on pregnant sheep. For this purpose, four studies have been conducted, focused in four specific objectives and based on two different experiments (Experiment 1 and 2). An in vitro assay with ovine macrophages derived from peripheral blood monocytes (OvMØs) was developed for the evaluation of the influence of isolates variability on these cells (Experiment 1). Then an in vivo experimental model with pregnant sheep was carried out, where the animals were orally infected with low doses of three genetic and phenotypically different T. gondii isolates (Experiment 2).

Chapter I is based on Experiment 1 with the specific aim of developing an in vitro model of OvMØs and study the influence that genetic variability of different T. gondii isolates may have on their response (objective 1). In this study, OvMØs were infected with six T. gondii isolates, obtained from Spanish sheep. Afterwards, the subsequent cell infections rates and transcript expression of cytokines and iNOS were evaluated. TgShSp24 isolate (Type III, ToxoDB#2) had a higher internalization/infection rate, followed by TgShSp2 (Type II, ToxoDB#1); compared with the rest of type II isolates: TgShSp1, TgShSp3, TgShSp11 and TgShSp16 (Type II, ToxoDB#3). Moreover, these two isolates (i.e. TgShSp24 and TgShSp2) also exhibited increments in cytokines that favored inflammation by a M1 macrophages polarization. TgShSp1 and TgShSp3 (Type II, ToxoDB#3 isolated from abortions) exhibited highest levels of anti-inflammatory cytokines. Isolates coming from abortions (fetal brains) triggered a higher iNOS expression. These results show intra- and inter-genotypic differences in the parasite/macrophage relationship, proving that more genotypic and phenotypic traits must be evaluated for the study of the virulence of T. gondii isolates. Besides, macrophages polarization could be affected depending on the infecting isolate.

Chapter II, III and IV are based in the results from Experiment 2, where seventy-three pregnant sheep were divided in four groups an orally inoculated at day 90 of gestation according to the following distribution: G1 infected with TgShSp1 isolate (type II, ToxoDB#3), G2 with TgShSp16 isolate (type II, ToxoDB#3), G3 with TgShSp24 isolate (type III, ToxoDB#2) and G4 of uninfected control sheep. Once inoculated clinical sings were evaluated and early and late abortions were noted. Five animals per group were culled at 14 and 28 dpi. The remaining sheep were left until delivery occurred (i.e. lambs or stillbirths).

Specifically, Chapter II is based on the description of the main macroscopic and microscopic findings in healthy non-infected ovine placentas and fetal viscera from the control group, together with the evaluation of the main cell populations found in the placentomes (objective 2). In order to achieve this, control placentas and fetuses were examined macroscopically and microscopically by H-E. Besides, immunohistochemical evaluation of macrophages (Iba1, calprotectin), B (CD20) and T cells (CD3), Natural killer (NK) cells and caspase-3 was carried out on the placentomes. Macroscopically, the placentas showed placentomes with variations in terms of shape and size. The presence of amniotic plaques and allantoic calculi was fairly common too. Regarding histological evaluation, all placentomes showed hyalinization of caruncular septa and intracytoplasmic pigments in the trophoblasts at the hematomata arcade. Moreover, almost half of the placentomes evaluated showed hemorrhages, foci of cellular debris, protein extravasation and mineralization. Moderate levels of T cells and Iba1 positive macrophages, and scarce B cells were present in the interdigitating zone of the placentome. The most striking finding was the absence of NK cells in the placentome. Cellular debris and areas of hyalinization were not immunolabelled for caspase-3. In fetal organs the most common findings were hematopoiesis, lymphocyte infiltrates and vacuolation of the epithelial cells of the eye, esophagus, intestine and kidney. It is relevant to establish that all these macroscopic and histological findings are found under normal conditions, as they might lead to misinterpretations when analyzing samples from an experimental model of pregnant sheep.

Chapter III is aimed to study the influence of the genetic and phenotypic variability of previously mentioned three T. gondii isolates on the development of toxoplasmosis in pregnant sheep at mid gestation (objective 3), and it is based on the examination of all the animals from Experiment 2. The isolates used in this experiment had shown intra- and inter-genotypic variations in previous in vitro and in vivo experimental studies. In this study, the clinical outcome of the infection up to the end of gestation was investigated, together with the occurrence of histological lesions, the parasite distribution and burden at 14 and 28 dpi. Reproductive failure including early or late abortions and stillbirths was similar for all the groups. However, the onset of fever and seroconversion occurred later for sheep infected with TgShSp1 Isolate compared with those infected with TgShSp16 isolate and TgShSp24 isolate. Regarding serial culling, T. gondii-DNA was only detected in few placentomes from TgShSp1 infected sheep at 14 dpi, while at 28 dpi, the parasite was detected in all the groups in at least one animal. However, at 28 dpi, parasite detection and burdens were higher for the TgShSp1 infected group, followed by the infected with TgShSp24. Histological lesions in placentomes and fetal lesions from euthanized animals were only detected at 28 dpi and were more frequent at the TgShSp1 infected group. Infection with TgShSp16 isolate was associated with the lower values of all of these phenotypic traits. Regardless the isolate inoculated into the sheep, vascular lesions (i.e. vasculitis and thrombi) previously unacknowledged in placentomes at 28 dpi were found. These findings suggest that the variability in the infecting isolates could influence the pathogenesis of the disease. However, the outcome of the disease (i.e. reproductive failure) is similar in all the groups, despite the isolate used for infection.

Chapter IV is focused on the characterization of the histological lesions and the changes affecting the resident cell populations in the CNS of fetuses in ovine toxoplasmosis (objective 4). For this study, fetal brains from selected animals of Experiment 2 were used. Those fetal brains showing histological lesions consistent with T. gondii infection were characterized by digital analysis in H-E stained sections. Furthermore, the same region of the brain of five fetuses from each group was immunohistochemically labeled for the detection of neurons (NeuN), astrocytes (GFAP), macrophages and microglial cells (Iba1) and recently recruited macrophages (calprotectin). Together with the detection of apoptosis (caspase-3) and axonal damage (APP). Lesions caused by the TgShSp1 isolate in the brain were larger and more numerous and were associated to a larger percentage of damage area in the CNS. These lesions were mainly characterized by glial foci, with or without necrosis that together with the perivascular cuffs, were mainly formed by microglial cells. Moreover, histological lesions were also associated with a higher number of astrocytes, microglial cells and caspase-3 mediated apoptosis in the midbrain. These findings were accompanied by a reduction in the number of neurons. These results demonstrate that the variability of T. gondii isolates could influence the severity of the histological lesions in fetal brains. Besides, it seems that there is relation between the severity of histological lesions and the occurrence of microgliosis, astrocytosis, caspase-3 apoptosis and reduction in the number of neurons.

Taken all together, the results of this PhD Thesis demonstrate that the inter- and intra-genotype variability inherent to recently obtained T. gondii isolates could influence the host/parasite relationship in ovine toxoplasmosis, and also that findings from experimental models in given species might not be directly extrapolated to others. An experimental model based on OvMØs has demonstrated to be adequate for the investigation of isolate virulence. Besides, macrophages polarization is influenced by the isolate of T. gondii. Furthermore, this PhD Thesis provides an advance in the understanding of ovine toxoplasmosis specially regarding abortions, as previously unknown vascular changes were observed early after infection (i.e. 28dpi) and, regarding fetal nervous lesions, as the previous information on brain lesions and resident cell population changes was scarce.