Histological and parasitological distinctive findings in clinically-lesioned and normal-looking skin of dogs with different clinical stages of leishmaniosis

Normal-looking skin of dogs with leishmaniosis frequently shows microscopic lesions along with the presence of Leishmania amastigotes. However, histological lesions with or without detection of amastigotes might not occur in less severe clinical cases. In addition, comparative studies between paired clinically-lesioned and normal-looking skin samples from dogs with different disease severity are lacking. The objective of this study was to compare histological and parasitological findings by Leishmania immunohistochemistry (IHC) and quantitative PCR (qPCR) on paired clinically-lesioned and normal-looking skin biopsies from 25 dogs with different clinical stages of leishmaniosis, 11 with stage I-mild disease (papular dermatitis) and 14 with stage II-III (ulcerative or exfoliative dermatitis). The study demonstrated microscopic lesions in 14 out of 25 (56%) samples from normal-looking skin biopsies. In those samples, perivascular to interstitial dermatitis composed by macrophages with lymphocytes and plasma cells was observed mainly in the superficial and mid-dermis. The intensity of the dermatitis was mild to moderate and always less prominent than in the clinically-lesioned skin. In normal-looking skin samples, the presence of parasites was detected by histology, IHC and qPCR in 5/25 (20%), 8/25 (32%) and 18/25 (72%), respectively. Leishmania was encountered in 11/25 (44%), 23/25 (92%) and 25/25 (100%) of clinically-lesioned skin samples by histology, IHC and qPCR, respectively. Normal-looking skin from dogs with stage I-mild disease was less frequently inflamed (P = 0.0172). Furthermore, Leishmania was more easily demonstrated by histology (P = 0.0464), IHC (P = 0.0421) or qPCR (P = 0.0068) in normal-looking skin of dogs with stage II-III-moderate to severe disease. In addition, in the latter group, there was a significantly higher parasite load studied by means of qPCR than in dogs with less severe disease (P = 0.043). Clinically-lesioned skin from dogs with stage I disease was more frequently characterised by the nodular to diffuse pattern and granuloma formation (P = 0.0166) and by a lower parasite load studied by means of qPCR (P = 0.043) compared with more diseased dogs. Normal-looking skin from dogs with stage I is less likely to present histological lesions as well as harbour the parasite when compared with dogs with moderate to severe leishmaniosis

Erratum to: Leishmania infantum-specific production of IFN-γ and IL-10 in stimulated blood from dogs with clinical leishmaniosis

BACKGROUND: There is limited information available on cytokine profiles in dogs with different degrees of disease severity due to natural infection of Leishmania infantum. The aim of this study was to investigate L. infantum-specific IFN-γ and IL-10 production in blood from dogs with leishmaniosis at diagnosis and correlate these findings with disease severity, humoral immune response and blood parasitemia. METHODS: Sixty dogs were diagnosed based on physical examination, routine laboratory tests, L. infantum-specific antibody levels measured by quantitative ELISA and blood parasitemia by real-time PCR. Heparin whole blood was stimulated with L. infantum soluble antigen (LSA) and concanavalin A (ConA) and incubated for 5 days. IFN-γ and IL-10 concentrations were measured in supernatants with sandwich ELISAs. RESULTS: The majority of dogs (n = 36) were classified as LeishVet stage II (moderate disease). The rest of the dogs were classified as stage I (n = 10), III (n = 10) and IV (n = 4). Dogs classified with stage I and IIa presented significantly higher (P = 0.02) LSA IFN-γ concentrations, lower (P <0.0001) antibody levels and a tendency for lower blood parasitemia (P = 0.1) than dogs classified with stages IIb, III or IV while no differences in ConA IFN-γ or IL-10 concentrations were observed among groups. Thirty-five dogs produced significantly higher LSA IFN-γ (mean ± SD: 2320 ± 3960 pg/ml) and ConA IFN-γ (mean ± SD: 7887 ± 7273 pg/ml) when compared with 25 dogs that did not produce detectable LSA IFN-γ but produced ConA IFN-γ (mean ± SD: 4917 ± 5233 pg/ml). IFN-γ producer dogs presented lower (mean ± SD: 5750 ± 14,082 ELISA units (EU), P = 0.001) antibody levels and blood parasitemia (mean ± SD:   5 ± 10 parasites/ml, P = 0.001) when compared with IFN-γ non-producers (mean ± SD: 19,638 ± 28,596 EU and 1100 ± 5112 parasites/ml), respectively. LSA IL-10 was not detectable in 34 dogs while 49 dogs secreted ConA IL-10 (mean ± SD of 90 ± 103 pg/ml). LSA IFN-γ concentration was negatively correlated with blood parasitemia and antibody levels and positively correlated with ConA IFN-γ and LSA IL-10 concentrations. CONCLUSIONS: The results of this study demonstrate that sick dogs lacking L. infantum specific IFN-γ production in stimulated whole blood produce a strong humoral response, have a high blood parasitemia and severe clinical disease. IL-10 does not appear to be a marker of disease severity

Leishmania infantum-specific IFN-γ production in stimulated blood from dogs with clinical leishmaniosis at diagnosis and during treatment

There is limited data regarding Leishmania infantum specific T cell mediated immunity in naturally infected sick dogs at the time of diagnosis and during anti-Leishmania treatment. Our aim was to investigate the kinetics of L. infantum specific IFN-γ production in dogs with leishmaniosis at the time of diagnosis and during treatment and to correlate it with specific L. infantum antibodies, blood parasitemia and clinicopathological findings. Thirty-four dogs were diagnosed with leishmaniosis based on physical examination, routine laboratory tests and L. infantum-specific antibody levels by quantitative ELISA. Heparinized whole blood was stimulated with L. infantum soluble antigen (LSA) and concanavalin A (ConA) and incubated for 5 days. IFN-γ concentration was evaluated in supernatants of stimulated blood using a commercial sandwich ELISA. Leishmania real-time PCR was also performed for assessing blood parasitemia. Dogs were treated with meglumine antimoniate and allopurinol. Sixteen dogs were classified as IFN-γ non-producers after LSA stimulation (mean ± SD: 0 ± 0 pg/mL) and 18 dogs as IFN-γ producers (mean ± SD: 2885.3 ± 4436.1 pg/mL) at the time of diagnosis (P < 0.0001). IFN-γ non-producers were classified in a more severe clinical staging than IFN-γ producers that presented a mild to moderate clinical staging (P = 0.03). In the IFN-γ non-producer group, production of IFN-γ after LSA stimulation was significantly increased during treatment especially at day 365 (P = 0.018) together with clinical improvement when compared with day 0. In contrast, IFN-γ producers maintained their IFN-γ production after LSA stimulation and no statistically significant changes were found during treatment follow-up. At diagnosis, IFN-γ non-producers showed a significantly higher blood parasitemia versus IFN-γ -producers (P = 0.005). IFN-γ non-producers drastically reduced blood parasitemia to minimum values at day 365 when compared with day 0 (P = 0.017). No significant differences were found at day 365 in blood parasitemia of IFN-γ producers compared to pre-treatment. At diagnosis, L. infantum specific antibodies were higher in IFN-γ non-producers than IFN-γ producers (P = 0.014). A marked reduction of antibody levels was found at day 365 when compared with day 0 in IFN-γ non-producers (P = 0.005) and producers (P = 0.001). These results demonstrate that IFN-γ concentration increases with long-term anti-Leishmania treatment together with clinical improvement in dogs that do not produce IFN-γ at diagnosis. Together with clinical recovery, reduction in blood parasitemia and L. infantum specific antibodies, tracking IFN-γ concentration could constitute an important prognostic tool for immune monitoring in CanL

Exploring the Relationship Between Susceptibility to Canine Leishmaniosis and anti-Phlebotomus Perniciosus Saliva Antibodies in Ibizan Hounds and Dogs of Other Breeds in Mallorca, Spain

Background: Canine leishmaniosis caused by Leishmania infantum is a neglected zoonosis transmitted by sand flies like Phlebotomus perniciosus. Clinical signs and disease susceptibility vary according to various factors, including host immune response and breed. In particular, Ibizan hounds appear more resistant. This immunocompetence could be attributed to a more frequent exposure to uninfected sand flies, eliciting a stronger anti-sand fly saliva antibody response. Methods: This study aimed to investigate the prevalence of anti-P. perniciosus saliva antibodies in Ibizan hounds and dogs of other breeds in the Leishmania-endemic area of Mallorca, Spain, and to correlate these antibody levels with clinical, immunological and parasitological parameters. Anti-sand fly saliva IgG was examined in 47 Ibizan hounds and 45 dogs of other breeds using three methods: P. perniciosus whole salivary gland homogenate (SGH) ELISA; recombinant protein rSP03B ELISA; and rSP03B rapid tests (RT). Additionally, diagnostic performance was evaluated between methods. Results: Results indicate significantly higher anti-SGH antibodies (P = 0.0061) and a trend for more positive SGH ELISA and RT results in Ibizan hounds compared to other breeds. General linear model analysis also found breed to be a significant factor in SGH ELISA units and a marginally significant factor in RT result. Although infection rates were similar between groups, Ibizan hounds included significantly more IFN-γ producers (P = 0.0122) and papular dermatitis cases (P < 0.0001). Older age and L. infantum seropositivity were also considered significant factors in sand fly saliva antibody levels according to at least one test. Fair agreement was found between all three tests, with the highest value between SGH and rSP03B RT. Conclusions: To our knowledge, this is the first study elaborating the relationship between anti-P. perniciosus saliva antibodies and extensive clinical data in dogs in an endemic area. Our results suggest that Ibizan hounds experience a higher frequency of exposure to sand flies and have a stronger cellular immune response to L. infantum infection than other breed dogs. Additional sampling is needed to confirm results, but anti-P. perniciosus saliva antibodies appear to negatively correlate with susceptibility to L. infantum infection and could possibly contribute to the resistance observed in Ibizan hounds

Serological and molecular survey of Leishmania infection in dogs from Venezuela

Venezuela is a country where human and canine leishmaniosis due to Leishmania infantum, Leishmania braziliensis and other Leishmania spp. is endemic. However, only limited data is available on canine Leishmania infection in Venezuela. The aim of this cross-sectional study was to evaluate the prevalence of Leishmania infection in dogs (n = 152) from the states of Lara (n = 91) and Yaracuy (n = 61) in Venezuela by means of serological and molecular methods. Physical examination was performed and blood samples were collected from all dogs. Serology for antibodies reactive with L. infantum and L. braziliensis antigens was assessed by the enzyme-linked immunosorbent assay (ELISA) and detection of Leishmania DNA from blood samples was evaluated by kinetoplast Leishmania real-time polymerase chain reaction (RT-PCR). In addition, Leishmania internal transcribed spacer (ITS-1) RT-PCR was performed on the samples positive by kinetoplast RT-PCR. The prevalence of Leishmania infection based on serological and/or molecular techniques was 11.8%. The seroprevalence for L. infantum and L. braziliensis antigens were 2.1% (3/144) and 8.3% (12/144), respectively. All dogs from the state of Yaracuy were serologically negative to L. infantum while 4.6% (4/86) of the dogs were reactive to L. braziliensis antigen. Fourteen percent (8/58) of the dogs from the state of Lara were positive to L. infantum and 5.2% (3/58) to L. braziliensis antigen. Three dogs were positive to both Leishmania spp. antigens. By RT-PCR, 6.5% (4/61) and 4.4% (4/91) of the dog were positive for infection in the states of Lara and Yaracuy, respectively. The RT-PCR product of one dog from the state of Yaracuy was sequenced revealing a 100% identity with L. infantum. However, all RT-PCR positive dogs were seronegative to both Leishmania spp. antigens. In conclusion, the positivity for Leishmania spp. infections observed indicates that dogs are frequently infected by L. infantum, L. braziliensis or related Leishmania spp. in Venezuela

Clinical and diagnostic aspects of feline cutaneous leishmaniosis in Venezuela

Abstract Background: Venezuela is an endemic area for human and canine leishmaniosis due to Leishmania infantum and parasites of the Leishmania braziliensis and L. mexicana complexes. Limited data are available on feline leishmaniosis (FeL) in this region. The aim of this study was to describe clinical and diagnostic aspects of FeL in Venezuela. Results: Thirty-one domestic cats from urban areas of Lara State in Venezuela were enrolled. Twenty-five were healthy. Six other cats had solitary or multiple nodular lesions, which were located on the nose; ears; ears and nose; and nose, ears, tail and lower limbs. Cutaneous lesions were characterized by diffuse pyogranulomatous infiltrate in all sick cats with numerous intracellular and extracellular amastigotes, and immunohistochemistry was positive for Leishmania in five sick cats. All healthy cats were seronegative for L. infantum and L. braziliensis antigens by ELISA. Two out of five sick cats yielded a positive ELISA result to both Leishmania antigens with higher antibody levels to L. braziliensis compared to L. infantum. Significantly higher antibody levels by ELISA as well as a higher number of bands by Western blot (WB) were found for L. braziliensis when compared to L. infantum antigens in all sera from Venezuelan sick and healthy cats. All healthy cats were blood Leishmania spp. qPCR negative while three out of six sick cats were blood qPCR positive. All paraffin-embedded skin biopsies (n = 4) as well as cutaneous cytology (n = 3) were positive by Leishmania spp. qPCR in sick cats. Leishmania speciation was obtained only from the cutaneous lesion samples from cytological preparations of two out of three sick cats which were identified as infected with L. mexicana or a closely related specie. Conclusions: FeL should be included in the differential diagnosis list of nodular-ulcerative lesions. The most reliable diagnostic technique in sick cats is cytological or histopathological examination along with immunohistochemistry, since blood PCR and serology by ELISA might be negative. WB appears to be more sensitive in detecting infection. Cats with leishmaniosis from Venezuela are most likely infected with species of L. mexicana or a closely related species or the L. braziliensis species complex and not with L. infantum

Does co-infection with vector-borne pathogens play a role in clinical canine leishmaniosis?

The severity of canine leishmaniosis (CanL) due to Leishmania infantum might be affected by other vector-borne organisms that mimic its clinical signs and clinicopathological abnormalities. The aim of this study was to determine co-infections with other vector-borne pathogens based on serological and molecular techniques in dogs with clinical leishmaniosis living in Spain and to associate them with clinical signs and clinicopathological abnormalities as well as disease severity. Sixty-one dogs with clinical leishmaniosis and 16 apparently healthy dogs were tested for Rickettsia conorii, Ehrlichia canis, Anaplasma phagocytophilum and Bartonella henselae antigens by the immunofluorescence antibody test (IFAT) and for E. canis, Anaplasma spp., Hepatozoon spp., Babesia spp. and filarioid DNA by polymerase chain reaction (PCR). Among the dogs examined by IFAT, the seroprevalences were: 69% for R. conorii, 57% for E. canis, 44% for A. phagocytophilum and 37% for B. henselae ; while the prevalences found by PCR were: 8% for Ehrlichia / Anaplasma, 3% for Anaplasma platys and 1% for H. canis. No other pathogen DNA was detected. Statistical association was found between dogs with clinical leishmaniosis and seroreactivity to R. conorii antigen (Fisher's exact test: P = 0.025, OR = 4.1, 95% CI = 1-17) and A. phagocytophilum antigen (Fisher's exact test: P = 0.002, OR = 14.3, 95% CI = 2-626) and being positive to more than one serological or molecular tests (co-infections) (Mann-Whitney test: U = 243, Z = -2.6, n = 14, n = 61, P = 0.01) when compared with healthy dogs. Interestingly, a statistical association was found between the presence of R. conorii, E. canis, A. phagocytophilum and B. henselae antibodies in sick dogs and some clinicopathological abnormalities such as albumin and albumin/globulin ratio decrease and increase in serum globulins. Furthermore, seroreactivity with A. phagocytophilum antigens was statistically associated with CanL clinical stages III and IV. This study demonstrates that dogs with clinical leishmaniosis from Catalonia (Spain) have a higher rate of co-infections with other vector-borne pathogens when compared with healthy controls. Furthermore, positivity to some vector-borne pathogens was associated with more marked clinicopathological abnormalities as well as disease severity with CanL

Ex vivo and in vitro studies on Toll like receptors in canine Leishmania infection

La leishmaniosis canina (CanL) es una enfermedad causada por el protozoa Leishmania infantum. Las manifestaciones clínicas en el perro son muy variables, abarcan desde una infección subclínica a un cuadro severo, que puede ser fatal. Al ser una enfermedad compleja, es difícil de diagnosticar. La respuesta inmunitaria de cada individuo juega un papel muy importante, tanto para el diagnóstico como para su tratamiento. Los receptores tipo Toll (TLRs) son los principales iniciadores de la respuesta inmunitaria innata cuya función es frenar cualquier intrusión de agentes extraños al cuerpo. Sin embargo, poca información existe sobre el papel de estos receptores en la CanL. El objetivo general de esta tesis doctoral es investigar el papel que los TLRs tiene en la CanL, tanto en experimentos ex vivo como in vitro. En la presente tesis doctoral, se ha estudiado la expresión genética en sangre no estimulada de los TLR2 y TLR4, en el momento del diagnóstico y durante el tratamiento, demostrando un notable aumento de la transcripción del TLR2 en perros con leishmaniosis clinica. Cuando se compararon perros enfermos en diferentes estadíos clínicos, se observó una expresión más notoria del TLR2 en aquellos perros que padecían la enfermedad de forma más severa comparado con los perros con estadíos más leves o con dermatitis papular. En, estos últimos, se notó de forma característica que tenían una alta producción de la citoquina interferon-gamma (IFN-γ). En sangre estimulada con antígeno soluble de Leishmania (LSA), la expresión del TLR2 y TLR4 disminuye en los perros enfermos que son productores de IFN-γ. Estimulando la sangre con TLRs agonistas, se halló un aumento en la producción de las citoquinas IFN-γ, TNF-α y IL-6 particularmente después de la estimulación con los agonistas TLR4 y TLR7 en sangre de perros naturalmente infectados. En el estudio in vitro incluido en esta tesis doctoral, en el cual macrófagos caninos fueron infectados experimentalmente con L. infantum y tratados con diferentes fármacos convencionales y TLRs agonistas, solos o combinados entre ellos, se demostró que la tasa de infección y la intensidad de infección se redujeron después de la estimulación los agonistas de TLRs. Además, el agonista TLR4 parece tener un efecto sinérgico combinado con el alopurinol. Así pues, con estos resultados, se ha ampliado el conocimiento de los TLRs en la infección por L. infantum en el perro. Y se han obtenido resultados esperanzadores para seguir investigando en esta línea sobre cómo los TLRs agonistas podrían actuar como inmunomoduladores en el tratamiento de esta enfermedad o incluso como adyuvantes en vacunas.La leishmaniosi canina (CanL) és una malaltia zoonòtica causada pel paràsit Leishmania infantum, de distribució mundial i altament endèmica a la conca mediterrània. Aquest paràsit es transmet mitjançant la picada de les femelles de flebòtoms, sent el gos l'hoste i reservori principal. Les manifestacions clíniques de la infecció per L. infantum en el gos són molt variables, des d'una infecció subclínica crònica fins a una malaltia molt severa, que pot ser fatal. Deguda a la seva complexa patogènesi, és important el paper que juguen tant la resposta immunitària innata i adaptativa en aquesta infecció. No obstant això, hi ha més informació al gos sobre la resposta immunitària adaptativa que sobre la innata. Els receptors tipus Toll (TLRs) són primordials en la maquinària del sistema immunitari innat que faciliten la ràpida detenció de diverses infeccions així com l'activació de la cascada inflamatòria. No obstant això, el paper d'aquests receptors en la infecció per L. infantum en gossos no és molt coneguda i la informació que hi ha sobre els mateixos és molt limitada. La hipòtesi d'aquesta tesi doctoral és que els TLRs tenen un paper important en la infecció per L. infantum pel fet que estimulen la cascada inflamatòria. L'objectiu general de la present tesi doctoral va ser el d'investigar l'expressió de TLRs en la CanL comparant amb paràmetres parasitològics, clínics, bioquímics i immunològics així com avaluar el possible ús dels agonistes de TLRs en el tractament d'aquesta malaltia. Els objectius específics s'han desenvolupat en 5 estudis que es descriuen a continuació. El primer objectiu específic es descriu en el capítol 3.1. i va consistir en avaluar l'expressió dels receptors tipus Toll 2 (TLR2) i 4 (TLR4) en sang no estimulada de gossos amb leishmaniosi clínica moderada en el moment del diagnòstic i durant un any de tractament així com correlacionar els paràmetres clínics, bioquímics i parasitològics. En el capítol 3.2., es descriu el segon experiment on es va avaluar la quantificació relativa de l'TLR2 i TLR4 en gossos infectats per L. infantum en diferents estadis de la malaltia segons la classificació de LeishVet en el moment del diagnòstic. L'estudi va consistir a determinar a més els anticossos i la producció d'interferó gamma (IFN-γ) específiques enfront del paràsit així com la parasitèmia en estadi clínic I (gossos amb dermatitis papular) i en estadis clínics II-III (gossos amb dermatitis exfoliativa o ulcerativa). L'objectiu específic del tercer estudi (capítol 3.3.) Va ser el de determinar la transcripció dels gens TLR2, TLR4 i el lligant 1 de mort programada (PD-L1) en sang estimulada amb antigen soluble de L. infantum (LSA) i el mitogen Concanavalina a (Con A) en gossos malalts capaços de produïr IFN-γ i els no capaços i en gossos sans. El propòsit de l'estudi ex vivo 3.4. va ser determinar la producció de les citocines IFN-γ, TNF-α i IL-6 en sang estimulada amb LSA o agonistes TLRs (TLRsa) com el TLR3a, TLR4a i TLR7a sols o amb combinació de LSA i cadascun dels TLRa en gossos sans infectats naturalment per Leishmània. Finalment es va realitzar un experiment in vitro (estudi 3.5) on es va avaluar la susceptibilitat del paràsit a diferents fàrmacs convencionals anti-Leishmània (al·lopurinol, miltefosine o meglumine antimoniat) tan en promastigots com en amastigots en una línia cel·lular de macròfags canins així com amb el tractament dels agonistes de TLR2, 3, 4 i 7 sols o combinats amb els fàrmacs convencionals. Els estudis descriptius d'aquesta tesi han confirmat que els TLRs semblen tenir un paper en la malaltia. Els gossos infectats amb Leishmània malalts presenten diferent expressió dels TLRs comparat amb els individus sans, depenent directament també del perfil immunològic de l'animal i de l'estadi clínic. Així doncs, en aquesta tesi doctoral, es va observar la sobreexpressió del TLR2 en sang no estimulada en el moment del diagnòstic en gossos amb malaltia moderada (estadi II-III) en comparar amb els gossos sans. No obstant això, l'expressió de TLR2 en sang no estimulada va ser igual per als gossos amb malaltia lleu (estadi I) i els gossos sans. A més, es va produir una disminució de l'expressió del TLR2 durant un any de tractament i la millora clínica en els gossos amb malaltia moderada. No obstant això, no es van observar canvis en l'expressió de TLR4 en el diagnòstic ni durant el tractament en cap dels gossos estudiats. És important assenyalar també que l'expressió de TLR2 es va correlacionar amb paràmetres clínics, parasitològics i immunològics associats a malaltia de moderada a severa. A més, els gossos amb estadi lleu I i amb dermatitis papular tenien un perfil immunològic predominant Th1, en canvi, els animals classificats en estadis més severs predominava un perfil Th2. Els resultats obtinguts en sang estimulada amb LSA van ser molt interessants i correlacionats amb les troballes obtingudes en sang no estimulada. Es va observar una reducció en l'expressió gènica dels gens TLR2 i TLR4 en sang estimulada amb LSA en els gossos malalts que eren productors de IFN-γ comparat amb els gossos sans i una alta expressió de PD-L1 a tots els grups estudiats tant per LSA com per amb Con A. La sang estimulada amb TLRsa en gossos sans va resultar tenir una alta producció de les citoquines TNF-α i IL-6, comparades amb el medi sol. A més, es va observar un efecte sinèrgic pro-inflamatori quan es va estimular amb TLR4a i TLR7a en combinació amb LSA. En els estudis in vitro es va demostrar la susceptibilitat del paràsit als fàrmacs convencionals sent el fàrmac més eficaç la miltefosina. A més, es va demostrar que els TLRs agonistes sols redueixen la infecció i es va observar també sinergia en la reducció de la infecció amb al·lopurinol i els agonistes per al TLR4. No obstant això, no es va detectar la producció de TNF-α ni de NO en els sobrenedants recollits a les 72 hores. No obstant això, sí que es va detectar la transcripció dels TLR2, 4 i 7 en totes les condicions estudiades. En general, es va demostrar una disminució de la transcripció de TLR2 o sense canvis en l'expressió de TLR4 i TLR7 amb la infecció . No obstant això, l'expressió de TLRs després del tractament amb fàrmacs anti-Leishmania convencionals sols o els agonistes de TLRs sols o combinacions dels dos va ser més variable. En conclusió, aquesta tesi doctoral ha demostrat que l'expressió de TLR2 en sang no estimulada és un marcador de malaltia de moderada a severa. No obstant això, el TLR4 no sembla ser un bon marcador per CanL en sang no estimulada. A més, la reducció de l'expressió dels TLR2 i 4 en sang estimulada amb LSA es va associar a gossos malalts productors d’IFN-γ els quals tenen un perfil més protector que gossos no productors d’IFN-γ. L'experiment in vitro ens va revelar que els fàrmacs combinats amb els TLRsa o fins i tot els TLRsa sols poden reduir la infecció. Per aquest motiu, els resultats trobats en aquesta tesi suggereixen que els TLRs podrien utilitzar-se com immunoteràpia, sols o combinats amb fàrmacs convencionals.Canine leishmaniosis (CanL) is a zoonotic disease caused by the protozoan Leishmania infantum, of worldwide distribution and highly endemic in the Mediterranean basin. This parasite is transmitted by the bite of sandfly females. The dog is the main host and reservoir. The clinical manifestations of L. infantum infection in the dog are very variable and range from a chronic subclinical infection to a very severe disease, which can be fatal. Due to its wide pathogenesis, the innate and adaptive immune responses play a role in this canine infection. However, there is much more information on the adaptive immune response than on the innate one. Toll-like receptors (TLR) are essential in the machinery of the immune system that facilitates the early arrest of several infections as well as the activation of the inflammatory cascade. However, the role of these receptors in L. infantum infection in dogs is not well known and the information is very limited. The hypothesis of this doctoral thesis is that TLRs have an important role in L. infantum infection in dogs because they stimulate the inflammatory cascade. The general objective of this doctoral thesis was to investigate the expression of TLRs in the CanL and compare with parasitological, clinical, biochemical and immunological parameters as well as to evaluate the possible use of TLR agonists in the treatment of this disease. The specific objectives have been developed in five studies and are described below. The first specific objective is described in chapter 3.1. and consisted in evaluating the expression of Toll-like receptors 2 (TLR2) and 4 (TLR4) in unstimulated blood of dogs with moderate clinical leishmaniosis at the time of diagnosis and during one year of treatment as well as correlating clinical, biochemical and parasitological parameters. In chapter 3.2., we described the second experiment where the relative quantification of TLR2 and TLR4 was evaluated in dogs infected by L. infantum in different stages of the disease according to the LeishVet classification at the time of diagnosis. This study also investigated the antibodies and the production of interferon gamma (IFN-γ) specific to the parasite as well as the parasitemia in clinical stage I (dogs with papular dermatitis) and in clinical stages II-III (dogs with exfoliative dermatitis or ulcerative) The specific objective of the third study (Chapter 3.3.) was to determine the transcription of the TLR2, TLR4 and programmed death ligand 1 (PD-L1) genes in blood stimulated with L. infantum soluble antigen (LSA) and the mitogen Concanavalin A (Con A) in sick dogs IFN-γ producers and non-IFN-γ producers and healthy dogs. The purpose of the study described in chapter 3.4. was to determine the production of the cytokines TNF-α and IL-6 in blood stimulated with LSA or TLRs agonists (TLR3, TLR4 and TLR7) alone or combined of dogs naturally infected by Leishmania. Finally, an in vitro experiment (Chapter 3.5) was carried out where the parasites’ susceptibility to different conventional anti-Leishmania drugs (allopurinol, miltefosine or meglumine antimonate) was evaluated in promastigote and amastigote assays in a canine macrophage cell line as well as in the treatment of TLRs agonists 2, 3, 4 and 7 (TLRsa) alone or in combination with conventional drugs. Descriptive studies of this thesis have confirmed that TLRs seem to have a role in the disease. Sick dogs infected with Leishmania present different expression of TLRs compared to healthy individuals, also directly depending on the immunological profile of the animal as well as the clinical stage. Thus, in this doctoral thesis, overexpression of TLR2 was obtained in unstimulated blood at the time of diagnosis in dogs with moderate disease (stage II-III) when compared with healthy dogs. However, expression of TLR2 in unstimulated blood was the same for dogs with mild disease (stage I) and healthy dogs. In addition, there was a decrease in the expression of TLR2 during one year of treatment and clinical improvement in dogs with moderate disease. However, no changes were observed in TLR4 expression at diagnosis or during treatment in any of the dogs studied. It is also important to highlight that the expression of TLR2 was correlated with clinical, parasitological and immunological parameters associated with moderate to severe disease. In addition, dogs with mild stage I and papular dermatitis had a predominantly Th1 immunological profile whereas animals classified in more severe stages had a Th2 profile predominant. The results obtained in blood stimulated with LSA were very interesting and correlated with the findings obtained in unstimulated blood. A reduction in gene expression of the TLR2 and TLR4 genes in blood stimulated with LSA was observed in sick dogs that were IFN-γ producers compared to healthy dogs and a high expression of PD-L1 in all the groups studied for both LSA as for Con A. The blood stimulated with TLRs agonists (TLRsa) and LSA of sick dogs turned out to have a high production of the cytokines IFN-γ, TNF-α and IL-6, compared with the medium alone. The combinations that gave the most production of cytokines of the Th1 profile are the agonists TLR4 and TLR7 each combined with LSA. In vitro studies demonstrated the susceptibility of the parasite to conventional drugs, being miltefosine the most effective drug. In addition, it was shown that TLRsa agonists alone reduced infection, and a synergistic effect was also observed in the reduction of infection with allopurinol and agonists for TLR4. However, the production of TNF-α or NO was not detected in the supernatants collected after 72 hours. However, the transcription of TLR2, 4 and 7 was detected in all the conditions studied. In general, a decrease in the transcription of TLR2s was demonstrated or no changes in the expression of TLR4 and TLR7 with infection. However, the expression of TLRs after treatment with conventional anti-Leishmania drugs alone or TLR agonists alone or combination of both was more variable. In conclusion, this doctoral thesis has shown that the expression of TLR2 in blood is not stimulated in a marker of moderate to severe disease. However, TLR4 does not appear to be a good marker for CanL in unstimulated blood. In addition, the reduced expression of TLR2 and 4 in blood stimulated with LSA was associated with sick dogs responding to IFN-γ which have a more protective profile than dogs not responding to IFN-γ. The in vitro study revealed that drugs combined with TLRsa or even TLRsa alone can reduce infection. For this reason, the findings found in this thesis are that TLRs can be used as immunotherapy or as adjuvants in future vaccines

Ex vivo and in vitro studies on Toll like receptors in canine Leishmania infection /

La leishmaniosis canina (CanL) es una enfermedad causada por el protozoa Leishmania infantum. Las manifestaciones clínicas en el perro son muy variables, abarcan desde una infección subclínica a un cuadro severo, que puede ser fatal. Al ser una enfermedad compleja, es difícil de diagnosticar. La respuesta inmunitaria de cada individuo juega un papel muy importante, tanto para el diagnóstico como para su tratamiento. Los receptores tipo Toll (TLRs) son los principales iniciadores de la respuesta inmunitaria innata cuya función es frenar cualquier intrusión de agentes extraños al cuerpo. Sin embargo, poca información existe sobre el papel de estos receptores en la CanL. El objetivo general de esta tesis doctoral es investigar el papel que los TLRs tiene en la CanL, tanto en experimentos ex vivo como in vitro. En la presente tesis doctoral, se ha estudiado la expresión genética en sangre no estimulada de los TLR2 y TLR4, en el momento del diagnóstico y durante el tratamiento, demostrando un notable aumento de la transcripción del TLR2 en perros con leishmaniosis clinica. Cuando se compararon perros enfermos en diferentes estadíos clínicos, se observó una expresión más notoria del TLR2 en aquellos perros que padecían la enfermedad de forma más severa comparado con los perros con estadíos más leves o con dermatitis papular. En, estos últimos, se notó de forma característica que tenían una alta producción de la citoquina interferon-gamma (IFN-γ). En sangre estimulada con antígeno soluble de Leishmania (LSA), la expresión del TLR2 y TLR4 disminuye en los perros enfermos que son productores de IFN-γ. Estimulando la sangre con TLRs agonistas, se halló un aumento en la producción de las citoquinas IFN-γ, TNF-α y IL-6 particularmente después de la estimulación con los agonistas TLR4 y TLR7 en sangre de perros naturalmente infectados. En el estudio in vitro incluido en esta tesis doctoral, en el cual macrófagos caninos fueron infectados experimentalmente con L. infantum y tratados con diferentes fármacos convencionales y TLRs agonistas, solos o combinados entre ellos, se demostró que la tasa de infección y la intensidad de infección se redujeron después de la estimulación los agonistas de TLRs. Además, el agonista TLR4 parece tener un efecto sinérgico combinado con el alopurinol. Así pues, con estos resultados, se ha ampliado el conocimiento de los TLRs en la infección por L. infantum en el perro. Y se han obtenido resultados esperanzadores para seguir investigando en esta línea sobre cómo los TLRs agonistas podrían actuar como inmunomoduladores en el tratamiento de esta enfermedad o incluso como adyuvantes en vacunas.La leishmaniosi canina (CanL) és una malaltia zoonòtica causada pel paràsit Leishmania infantum, de distribució mundial i altament endèmica a la conca mediterrània. Aquest paràsit es transmet mitjançant la picada de les femelles de flebòtoms, sent el gos l'hoste i reservori principal. Les manifestacions clíniques de la infecció per L. infantum en el gos són molt variables, des d'una infecció subclínica crònica fins a una malaltia molt severa, que pot ser fatal. Deguda a la seva complexa patogènesi, és important el paper que juguen tant la resposta immunitària innata i adaptativa en aquesta infecció. No obstant això, hi ha més informació al gos sobre la resposta immunitària adaptativa que sobre la innata. Els receptors tipus Toll (TLRs) són primordials en la maquinària del sistema immunitari innat que faciliten la ràpida detenció de diverses infeccions així com l'activació de la cascada inflamatòria. No obstant això, el paper d'aquests receptors en la infecció per L. infantum en gossos no és molt coneguda i la informació que hi ha sobre els mateixos és molt limitada. La hipòtesi d'aquesta tesi doctoral és que els TLRs tenen un paper important en la infecció per L. infantum pel fet que estimulen la cascada inflamatòria. L'objectiu general de la present tesi doctoral va ser el d'investigar l'expressió de TLRs en la CanL comparant amb paràmetres parasitològics, clínics, bioquímics i immunològics així com avaluar el possible ús dels agonistes de TLRs en el tractament d'aquesta malaltia. Els objectius específics s'han desenvolupat en 5 estudis que es descriuen a continuació. El primer objectiu específic es descriu en el capítol 3.1. i va consistir en avaluar l'expressió dels receptors tipus Toll 2 (TLR2) i 4 (TLR4) en sang no estimulada de gossos amb leishmaniosi clínica moderada en el moment del diagnòstic i durant un any de tractament així com correlacionar els paràmetres clínics, bioquímics i parasitològics. En el capítol 3.2., es descriu el segon experiment on es va avaluar la quantificació relativa de l'TLR2 i TLR4 en gossos infectats per L. infantum en diferents estadis de la malaltia segons la classificació de LeishVet en el moment del diagnòstic. L'estudi va consistir a determinar a més els anticossos i la producció d'interferó gamma (IFN-γ) específiques enfront del paràsit així com la parasitèmia en estadi clínic I (gossos amb dermatitis papular) i en estadis clínics II-III (gossos amb dermatitis exfoliativa o ulcerativa). L'objectiu específic del tercer estudi (capítol 3.3.) Va ser el de determinar la transcripció dels gens TLR2, TLR4 i el lligant 1 de mort programada (PD-L1) en sang estimulada amb antigen soluble de L. infantum (LSA) i el mitogen Concanavalina a (Con A) en gossos malalts capaços de produïr IFN-γ i els no capaços i en gossos sans. El propòsit de l'estudi ex vivo 3.4. va ser determinar la producció de les citocines IFN-γ, TNF-α i IL-6 en sang estimulada amb LSA o agonistes TLRs (TLRsa) com el TLR3a, TLR4a i TLR7a sols o amb combinació de LSA i cadascun dels TLRa en gossos sans infectats naturalment per Leishmània. Finalment es va realitzar un experiment in vitro (estudi 3.5) on es va avaluar la susceptibilitat del paràsit a diferents fàrmacs convencionals anti-Leishmània (al·lopurinol, miltefosine o meglumine antimoniat) tan en promastigots com en amastigots en una línia cel·lular de macròfags canins així com amb el tractament dels agonistes de TLR2, 3, 4 i 7 sols o combinats amb els fàrmacs convencionals. Els estudis descriptius d'aquesta tesi han confirmat que els TLRs semblen tenir un paper en la malaltia. Els gossos infectats amb Leishmània malalts presenten diferent expressió dels TLRs comparat amb els individus sans, depenent directament també del perfil immunològic de l'animal i de l'estadi clínic. Així doncs, en aquesta tesi doctoral, es va observar la sobreexpressió del TLR2 en sang no estimulada en el moment del diagnòstic en gossos amb malaltia moderada (estadi II-III) en comparar amb els gossos sans. No obstant això, l'expressió de TLR2 en sang no estimulada va ser igual per als gossos amb malaltia lleu (estadi I) i els gossos sans. A més, es va produir una disminució de l'expressió del TLR2 durant un any de tractament i la millora clínica en els gossos amb malaltia moderada. No obstant això, no es van observar canvis en l'expressió de TLR4 en el diagnòstic ni durant el tractament en cap dels gossos estudiats. És important assenyalar també que l'expressió de TLR2 es va correlacionar amb paràmetres clínics, parasitològics i immunològics associats a malaltia de moderada a severa. A més, els gossos amb estadi lleu I i amb dermatitis papular tenien un perfil immunològic predominant Th1, en canvi, els animals classificats en estadis més severs predominava un perfil Th2. Els resultats obtinguts en sang estimulada amb LSA van ser molt interessants i correlacionats amb les troballes obtingudes en sang no estimulada. Es va observar una reducció en l'expressió gènica dels gens TLR2 i TLR4 en sang estimulada amb LSA en els gossos malalts que eren productors de IFN-γ comparat amb els gossos sans i una alta expressió de PD-L1 a tots els grups estudiats tant per LSA com per amb Con A. La sang estimulada amb TLRsa en gossos sans va resultar tenir una alta producció de les citoquines TNF-α i IL-6, comparades amb el medi sol. A més, es va observar un efecte sinèrgic pro-inflamatori quan es va estimular amb TLR4a i TLR7a en combinació amb LSA. En els estudis in vitro es va demostrar la susceptibilitat del paràsit als fàrmacs convencionals sent el fàrmac més eficaç la miltefosina. A més, es va demostrar que els TLRs agonistes sols redueixen la infecció i es va observar també sinergia en la reducció de la infecció amb al·lopurinol i els agonistes per al TLR4. No obstant això, no es va detectar la producció de TNF-α ni de NO en els sobrenedants recollits a les 72 hores. No obstant això, sí que es va detectar la transcripció dels TLR2, 4 i 7 en totes les condicions estudiades. En general, es va demostrar una disminució de la transcripció de TLR2 o sense canvis en l'expressió de TLR4 i TLR7 amb la infecció . No obstant això, l'expressió de TLRs després del tractament amb fàrmacs anti-Leishmania convencionals sols o els agonistes de TLRs sols o combinacions dels dos va ser més variable. En conclusió, aquesta tesi doctoral ha demostrat que l'expressió de TLR2 en sang no estimulada és un marcador de malaltia de moderada a severa. No obstant això, el TLR4 no sembla ser un bon marcador per CanL en sang no estimulada. A més, la reducció de l'expressió dels TLR2 i 4 en sang estimulada amb LSA es va associar a gossos malalts productors d'IFN-γ els quals tenen un perfil més protector que gossos no productors d'IFN-γ. L'experiment in vitro ens va revelar que els fàrmacs combinats amb els TLRsa o fins i tot els TLRsa sols poden reduir la infecció. Per aquest motiu, els resultats trobats en aquesta tesi suggereixen que els TLRs podrien utilitzar-se com immunoteràpia, sols o combinats amb fàrmacs convencionals.Canine leishmaniosis (CanL) is a zoonotic disease caused by the protozoan Leishmania infantum, of worldwide distribution and highly endemic in the Mediterranean basin. This parasite is transmitted by the bite of sandfly females. The dog is the main host and reservoir. The clinical manifestations of L. infantum infection in the dog are very variable and range from a chronic subclinical infection to a very severe disease, which can be fatal. Due to its wide pathogenesis, the innate and adaptive immune responses play a role in this canine infection. However, there is much more information on the adaptive immune response than on the innate one. Toll-like receptors (TLR) are essential in the machinery of the immune system that facilitates the early arrest of several infections as well as the activation of the inflammatory cascade. However, the role of these receptors in L. infantum infection in dogs is not well known and the information is very limited. The hypothesis of this doctoral thesis is that TLRs have an important role in L. infantum infection in dogs because they stimulate the inflammatory cascade. The general objective of this doctoral thesis was to investigate the expression of TLRs in the CanL and compare with parasitological, clinical, biochemical and immunological parameters as well as to evaluate the possible use of TLR agonists in the treatment of this disease. The specific objectives have been developed in five studies and are described below. The first specific objective is described in chapter 3.1. and consisted in evaluating the expression of Toll-like receptors 2 (TLR2) and 4 (TLR4) in unstimulated blood of dogs with moderate clinical leishmaniosis at the time of diagnosis and during one year of treatment as well as correlating clinical, biochemical and parasitological parameters. In chapter 3.2., we described the second experiment where the relative quantification of TLR2 and TLR4 was evaluated in dogs infected by L. infantum in different stages of the disease according to the LeishVet classification at the time of diagnosis. This study also investigated the antibodies and the production of interferon gamma (IFN-γ) specific to the parasite as well as the parasitemia in clinical stage I (dogs with papular dermatitis) and in clinical stages II-III (dogs with exfoliative dermatitis or ulcerative) The specific objective of the third study (Chapter 3.3.) was to determine the transcription of the TLR2, TLR4 and programmed death ligand 1 (PD-L1) genes in blood stimulated with L. infantum soluble antigen (LSA) and the mitogen Concanavalin A (Con A) in sick dogs IFN-γ producers and non-IFN-γ producers and healthy dogs. The purpose of the study described in chapter 3.4. was to determine the production of the cytokines TNF-α and IL-6 in blood stimulated with LSA or TLRs agonists (TLR3, TLR4 and TLR7) alone or combined of dogs naturally infected by Leishmania. Finally, an in vitro experiment (Chapter 3.5) was carried out where the parasites' susceptibility to different conventional anti-Leishmania drugs (allopurinol, miltefosine or meglumine antimonate) was evaluated in promastigote and amastigote assays in a canine macrophage cell line as well as in the treatment of TLRs agonists 2, 3, 4 and 7 (TLRsa) alone or in combination with conventional drugs. Descriptive studies of this thesis have confirmed that TLRs seem to have a role in the disease. Sick dogs infected with Leishmania present different expression of TLRs compared to healthy individuals, also directly depending on the immunological profile of the animal as well as the clinical stage. Thus, in this doctoral thesis, overexpression of TLR2 was obtained in unstimulated blood at the time of diagnosis in dogs with moderate disease (stage II-III) when compared with healthy dogs. However, expression of TLR2 in unstimulated blood was the same for dogs with mild disease (stage I) and healthy dogs. In addition, there was a decrease in the expression of TLR2 during one year of treatment and clinical improvement in dogs with moderate disease. However, no changes were observed in TLR4 expression at diagnosis or during treatment in any of the dogs studied. It is also important to highlight that the expression of TLR2 was correlated with clinical, parasitological and immunological parameters associated with moderate to severe disease. In addition, dogs with mild stage I and papular dermatitis had a predominantly Th1 immunological profile whereas animals classified in more severe stages had a Th2 profile predominant. The results obtained in blood stimulated with LSA were very interesting and correlated with the findings obtained in unstimulated blood. A reduction in gene expression of the TLR2 and TLR4 genes in blood stimulated with LSA was observed in sick dogs that were IFN-γ producers compared to healthy dogs and a high expression of PD-L1 in all the groups studied for both LSA as for Con A. The blood stimulated with TLRs agonists (TLRsa) and LSA of sick dogs turned out to have a high production of the cytokines IFN-γ, TNF-α and IL-6, compared with the medium alone. The combinations that gave the most production of cytokines of the Th1 profile are the agonists TLR4 and TLR7 each combined with LSA. In vitro studies demonstrated the susceptibility of the parasite to conventional drugs, being miltefosine the most effective drug. In addition, it was shown that TLRsa agonists alone reduced infection, and a synergistic effect was also observed in the reduction of infection with allopurinol and agonists for TLR4. However, the production of TNF-α or NO was not detected in the supernatants collected after 72 hours. However, the transcription of TLR2, 4 and 7 was detected in all the conditions studied. In general, a decrease in the transcription of TLR2s was demonstrated or no changes in the expression of TLR4 and TLR7 with infection. However, the expression of TLRs after treatment with conventional anti-Leishmania drugs alone or TLR agonists alone or combination of both was more variable. In conclusion, this doctoral thesis has shown that the expression of TLR2 in blood is not stimulated in a marker of moderate to severe disease. However, TLR4 does not appear to be a good marker for CanL in unstimulated blood. In addition, the reduced expression of TLR2 and 4 in blood stimulated with LSA was associated with sick dogs responding to IFN-γ which have a more protective profile than dogs not responding to IFN-γ. The in vitro study revealed that drugs combined with TLRsa or even TLRsa alone can reduce infection. For this reason, the findings found in this thesis are that TLRs can be used as immunotherapy or as adjuvants in future vaccines