Desarrollo de vacunas frente a la neosporosis bovina utilizando aislados de "Neospora caninum" inactivados y atenuados

Neospora caninum es un parásito intracelular formador de quistes considerado mundialmente como una de las principales causas de aborto en el ganado bovino.La magnitud de las pérdidas económicas asociadas a la neosporosis bovina, junto con la ausencia de medidas económicamente viables para su control, han conducido a que uno de los principales retos en la investigación sobre esta enfermedad sea el desarrollo de una vacuna eficaz, segura y rentable frente al aborto y la transmisión del parásito. En los últimos años, se han llevado a cabo numerosos estudios para el desarrollo de vacunas frente a las neosporosis bovina, incluyendo vacunas inactivadas, vivas, y de nueva generación basadas en proteínas recombinantes, en vectores vivos o en vacunas de ADN. Las vacunas de nueva generación ofrecen alternativas interesantes para el futuro pero los resultados de protección obtenidos hasta el momento son insuficientes. Por el contrario, las únicas vacunas que hasta la fecha se han probado en la especie de destino con cierto éxito utilizan parásitos inactivados y vivos atenuados, hecho que dio pie a la realización de la presente tesis doctoral. En la primera parte del estudio, se evaluó el efecto protector de formulaciones empleando parásito entero inactivado como antígeno. En este tipo de vacunas, la selección apropiada de determinadas variables, como el tipo de antígeno o el adyuvante, puede ser determinante para la inducción de una respuesta inmunoprotectora frente a la infección por N. caninum. Por ello, en un primer experimento se evaluó la influencia de tres adyuvantes (emulsión oleosa de agua en aceite -W/O- e hidróxido de aluminio combinado con CpG -Al/CpG- o extracto de Ginseng -Al/G-) en combinación con tres dosis diferentes de taquizoítos enteros inactivados (105, 5×105 y 106) en un modelo murino de neosporosis cerebral (Capítulo II). En este estudio se puso de manifiesto la influencia del adyuvante en la eficacia protectora de la vacunación. Así, la inmunización de W/O combinado con 5×105 taquizoítos limitó la presencia del parásito en el cerebro en la fase crónica y, por tanto, la posible cronificación y persistencia de la infección en este órgano, mientras que el adyuvante Al/G combinado con 5×105 taquizoítos redujo la parasitemia durante la fase aguda. La dosis de antígeno afectó, aunque de forma menos notable que el adyuvante, la eficacia protectora de la vacunación, de manera que en los grupos inmunizados con Al/G, la frecuencia y cargas parasitarias en el cerebro se incrementaron con la dosis. Para el siguiente estudio (Capítulo III), se seleccionaron los adyuvantes y dosis de antígeno que mejores niveles de protección mostraron en el modelo cerebral, y se estudió su capacidad protectora en un modelo de infección congénita. Por un lado, se seleccionaron el adyuvante W/O y la dosis de 5×105 taquizoítos, ya que dicha combinación redujo la infección cerebral, sugiriendo una posible protección frente a la cronificación de la infección. Por otro lado, se probó el adyuvante Al/G y la dosis de 5×105 taquizoítos frente a la infección congénita, ya que sus efectos reductores de la parasitemia en la fase aguda podrían también ayudar a controlar la transmisión transplacentaria del parásito a la descendencia tras una reactivación o una primoinfección en la hembra gestante. Adicionalmente, se comparó el efecto entre estas formulaciones y aquellas elaboradas a partir de una mezcla de antígenos procedentes de los estadios de taquizoíto y bradizoíto (TZ-BZ). La hipótesis de partida fue que estas últimas pudieran conferir protección frente a los procesos de reactivación en los que está implicado el estadio de bradizoíto. Sin embargo, la inmunización con la mezcla de TZ-BZ mostró los peores resultados de protección, originando una exacerbación de las infecciones cerebral y congénita, asociada a un probable desequilibrio de la respuesta inmunitaria caracterizado por una sobreexpresión de citoquinas de tipo Th1 tras la inmunización y de tipo Th2 tras el desafío. Por el contrario, la combinación de W/O y taquizoítos indujo un equilibrio de la respuesta inmunitaria caracterizado por una elevada producción de anticuerpos, predominantemente de tipo Th2, y con una sobreexpresión de IFN-γ, que controló parcialmente la infección cerebral y la transmisión vertical. En la segunda parte del estudio, se investigó el desarrollo de una vacuna viva atenuada frente a la infección por N. caninum. Para ello, la estrategia de partida fue la obtención y caracterización de un aislado a partir de un animal asintomático que mostrara cierta atenuación en su virulencia (Capítulos IV y V). Como fruto de esta aproximación, se obtuvo el aislado Nc-Spain 1H de N. caninum a partir de un ternero clínicamente sano pero congénitamente infectado. Dicho aislado se identificó genéticamente mediante el análisis de 13 secuencias microsatélites previamente identificadas en el genoma de N. caninum, mostrando un patrón definido y exclusivo. Los ensayos de caracterización in vitro mostraron un bajo rendimiento en la producción de taquizoítos y una menor capacidad de infección del aislado Nc-Spain 1H en comparación con el aislado de referencia Nc-1. Durante su caracterización biológica in vivo, el aislado Nc-Spain 1H mostró una virulencia notablemente reducida en un modelo murino de infección cerebral y congénita, caracterizada por una menor persistencia en el cerebro de ratones y una virulencia reducida con ausencia de signos clínicos y bajas tasas de mortalidad y transmisión vertical de la infección a las crías. En un modelo bovino gestante, dicho aislado no produjo mortalidad fetal y no se detectó presencia del parásito en los tejidos fetales. [ABSTRACT]Neospora caninum is an intracellular cyst-forming parasite known as a major cause of bovine abortion worldwide. Due to the negative economic impact of bovine neosporosis, together with the lack of cost-effective measures for its control, the development of an economically viable, efficacious and safe vaccine against abortion and parasite transmission has become one of the main challenges for the research of this disease. In the last few years, many studies have been carried out with the aim of developing vaccines against bovine neosporosis, including live, inactivated, and new generation protein recombinant, live vector or DNA vaccines. Up to now, new generation vaccines offer an interesting potential for the future, but have shown insufficient protection results. Contrarily, inactivated and live attenuated vaccines are the only ones that have been tested in bovine models with some success so far. This fact motivated the present Doctoral Thesis. The first part of the study evaluated the protection conferred by vaccines containing nactivated whole parasites as antigen. In these types of formulations, an appropriate selection of certain variables, such as the type of antigen or the adjuvant, may determine the induction of a protective immune response against N. caninum infection. Therefore, the first experiment examined the role of three different adjuvants (water-in-oil emulsion -W/O- and aluminum hydroxide plus CpG-ODN -Al/CpG- or plus ginseng extract -Al/G) combined with three different doses of whole inactivated tachyzoites (105, 5×105 and 106) using a mouse model of cerebral neosporosis (Chapter I). The results highlighted the influence of the adjuvant on the protective efficacy of the vaccine. Thus, immunization with W/O combined with 5×105 tachyzoites limited parasite presence in the brain during the chronic stage of the infection and, consequently, the possibility that the infection becomes chronic and persistent in this organ. Contrarily, Al/G plus 5×105 tachyzoites reduced parasitaemia during the acute stage of the infection. The antigen dose played a role in the protective efficacy of the vaccine, although it was less significant than the adjuvant’s: among the groups immunized with Al/G, parasite frequency and load in the brain rised as the dose was increased

Experimental infection with a low virulence isolate of Neospora caninum at 70 days gestation in cattle did not result in foetopathy

The Nc-Spain 1H isolate of Neospora caninum, which was newly obtained from the brain of a congenitally asymptomatic infected calf, demonstrated a reduced in vitro tachyzoite yield and viability rate, as well as low virulence in mouse models. The objective of the present study was to determine the ability of this isolate to induce foetal death in a pregnant bovine model. For this purpose, 13 naïve pregnant heifers were divided into three groups and were experimentally challenged with either 107 tachyzoites of Nc-1 (group 1, n = 5), Nc-Spain 1H (group 2, n = 5) isolates or phosphate-buffered saline (group 3, n = 3) intravenously at 70 days of gestation. After inoculation, pregnancy was monitored and dams were sacrificed when foetal death was detected. The remaining animals were slaughtered at 45 days post-infection. Maternal and foetal samples were collected for examination by histology and parasite DNA detection. Parasitaemia, specific anti-N. caninum IgG and interferon γ responses were also studied. At 3–4 weeks after infection, foetal death was detected in 3 out of 5 Nc-1-infected dams. However, no evidence of foetal death was observed in either Nc-Spain 1H-infected or control groups during the period studied. The most severe histopathological lesions were observed in the placenta and foetal organs from Nc-1-infected cattle that exhibited foetal death. It was in these animals that N. caninum DNA was more frequently detected. Parasitaemia was observed in all Nc-1-infected dams and in only 3 out of 5 Nc-Spain 1H-infected animals. The magnitude of the immune response was significantly higher in the Nc-1-inoculated group than in the group inoculated with the Nc-Spain 1H isolate. These data reveal the reduced virulence of the Nc-Spain 1H isolate in cattle

Effects of challenge dose and inoculation route of the virulent Neospora caninum Nc-Spain7 isolate in pregnant cattle at mid-gestation

International audienceAbstractParameters 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, 107 parasites, n = 6; IV2, 105, n = 6; IV3, 103, n = 6; and IV4, 102, n = 5), and the subcutaneous (SC) inoculation route was also assessed for the dose of 105 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

Parasitología Interactiva

Desarrollo de una guía interactiva donde se muestran los ciclos biológicos,imágenes y dibujos de los estadios evolutivos de los principales géneros y especies de parásitos protozoos relevantes en al ámbito veterinario

HTLV-1 infection in solid organ transplant donors and recipients in Spain

HTLV-1 infection is a neglected disease, despite infecting 10-15 million people worldwide and severe illnesses develop in 10% of carriers lifelong. Acknowledging a greater risk for developing HTLV-1 associated illnesses due to immunosuppression, screening is being widely considered in the transplantation setting. Herein, we report the experience with universal HTLV testing of donors and recipients of solid organ transplants in a survey conducted in Spain. All hospitals belonging to the Spanish HTLV network were invited to participate in the study. Briefly, HTLV antibody screening was performed retrospectively in all specimens collected from solid organ donors and recipients attended since the year 2008. A total of 5751 individuals were tested for HTLV antibodies at 8 sites. Donors represented 2312 (42.2%), of whom 17 (0.3%) were living kidney donors. The remaining 3439 (59.8%) were recipients. Spaniards represented nearly 80%. Overall, 9 individuals (0.16%) were initially reactive for HTLV antibodies. Six were donors and 3 were recipients. Using confirmatory tests, HTLV-1 could be confirmed in only two donors, one Spaniard and another from Colombia. Both kidneys of the Spaniard were inadvertently transplanted. Subacute myelopathy developed within 1 year in one recipient. The second recipient seroconverted for HTLV-1 but the kidney had to be removed soon due to rejection. Immunosuppression was stopped and 3 years later the patient remains in dialysis but otherwise asymptomatic. The rate of HTLV-1 is low but not negligible in donors/recipients of solid organ transplants in Spain. Universal HTLV screening should be recommended in all donor and recipients of solid organ transplantation in Spain. Evidence is overwhelming for very high virus transmission and increased risk along with the rapid development of subacute myelopathy

Evaluation of the protection conferred by a naturally attenuated <it>Neospora caninum</it> isolate against congenital and cerebral neosporosis in mice

Abstract The parasite Neospora caninum is an important abortifacient agent in cattle worldwide. At present, the development of an effective and safe vaccine against bovine neosporosis is of great relevance. Recently, a new isolate of N. caninum (Nc-Spain 1 H) which was obtained from the brain of an asymptomatic congenitally infected calf, exhibited non-virulent behaviour in mouse and bovine infection models. The aim of this study was to determine the safety and efficacy of Nc-Spain 1 H when used as a vaccinal isolate in well-established BALB/c models of congenital and cerebral neosporosis. Mice were subcutaneously immunised twice at 3-week intervals and were challenged with 2 × 106 tachyzoites of the virulent Nc-Liv isolate. After immunisation with live Nc-Spain 1 H tachyzoites, no parasitic DNA was detected in the dams’ brains before challenge and microsatellite analysis performed in PCR-positive mice showed that the profiles corresponded to the challenge isolate Nc-Liv, indicating the Nc-Spain 1 H isolate to be a safe vaccine candidate. The efficacy of the live vaccine was evaluated in the first experiment after the immunisation of mice with 5 × 105 live Nc-Spain 1 H tachyzoites. This immunisation protocol significantly reduced the neonatal mortality to 2.4%, reduced the vertical transmission from 89.1% to 2.3% and completely limited the cerebral infection. These results were associated with a Th1-type immune response. In the second experiment, the effect of various immunising doses was established using ten-fold dilutions of the tachyzoites (from 5 × 105 to 5 × 10). In all the cases, congenital protection rates above 60% were observed, and the mice that were immunised with the lowest dose (5 × 10) presented the highest protection rate (86%). Moreover, low immunising doses of Nc-Spain 1 H induced an IgG2a response, and high parasitic doses induced an IgG1 response. These results evidence the safety and the efficient protection that was conferred by Nc-Spain 1 H against congenital neosporosis, even when the mice were immunised with low parasitic doses.</p

Tritrichomonas foetus infection in cats with diarrhea from denselyhoused origins

Tritrichomonas foetus is a protozoan parasite that has been recently identified as a causative agent of chronic diarrhea in domestic cats. Transmission of infection occurs by the fecal-oral route through direct contact among animals. Consequently, feline trichomonosis (FT) is more likely to be present in multi-cat environments. The objective of this work was to study the presence of T. foetus and some associated risk factors in cats from densely housed origins and with a reported history of chronic diarrhea. Animals enrolled in this study were family cats (n = 15) acquired from pet shops, shelters or breeding centers and cattery cats belonging to one breeding center (n = 28) and two cat shelters (A and B, n = 25 each). In the catteries, a follow-up analysis for a period of up to 2 months was also performed to determine the parasite shedding pattern in feces and the incidence of infection. Fecal samples were analyzed using in vitro culture and a PCR technique. T. foetus was detected in a total of 38.7% (36/93) of the cats with chronic diarrhea. Parasite infection was similarly detected in family cats and cattery animals (40% versus 38.4%). In the catteries, the parasite was detected in 50%, 44% and 20% of the animals from the breeding center and shelters A and B, respectively. The follow-up analysis showed that 58.3% of infected cats intermittently shed trophozoites in their feces, with an incidence of 23.1%. Investigation of potential risk factors showed that cats ≤1 year old were more likely to be infected than older cats (57.1% versus 27.3%; P < 0.05). No significant differences were found when sex and breed factors were studied. These results confirm the importance of FT as a cause of chronic diarrhea in cats and highlight the relevance of close contact conditions for T. foetus transmission.Depto. de Sanidad AnimalCAI Ciencias de la Tierra y ArqueometríaTRUEpu

First description of naturally acquired Tritrichomonas foetus infection in a Persian cattery in Spain

Tritrichomonas foetus has been identified as the causative agent of feline intestinal trichomonosis, characterized by clinical signs of chronic large bowel diarrhoea. This disease has been reported in cats from the USA, Europe and Australia. However, its epidemiology is still unclear. The aim of the present study was to describe T. foetus infection in a Persian cattery in Spain. T. foetus infection was sequentially diagnosed in 20 cats by direct faecal smear examined under the microscope, specific culture (In Pouch TF medium) and PCR. A standard coprological sedimentation method was also performed in order to screen for other intestinal parasites in all the cats included. In addition, sera were tested for IgG antibodies against Leishmania infantum, Toxoplasma gondii, and for the detection of feline immunodeficiency virus (FIV) and feline leukaemia virus (FeLV). Five out of 20 cats were positive for T. foetus (25%), two of them by microscopy, culture and PCR and three by culture and PCR. No association was found between T. foetus infection and age or sex. L. infantum and T. gondii seroprevalence rates were 15% and 10%, respectively. The prevalence of FeLV p27 antigen and of FIV antibodies in the study population was zero. Cystoisospora spp. oocysts were detected in one cat. These preliminary results show that the transmission of T. foetus infection in cluster conditions may occur between asymptomatic cats and young or immunocompromised animalsDepto. de Sanidad AnimalFac. de VeterinariaTRUEpu