Epidemiology and control of canine leishmaniosis

[eng] Leishmaniosis is an important vector-borne zoonosis caused by Leishmania infantum. The disease is widespread across several continents and endemic in the Mediterranean region. The domestic dog is the main vertebrate reservoir for the parasite and control of canine leishmaniosis (CanL) is deemed to be essential for the control of human cases of the disease. Due to the heterogeneous distribution of infection in endemic areas, epidemiological surveillance should be carried out focally, including both screening of canine populations and vector detection, the two determinant factors for parasite survival and expansion. CanL control measures are usually directed at the canine reservoir through the detection and treatment of infected individuals, as well as disease prevention through insecticide treatments and/or canine immunoprophylaxis. Vaccination against CanL is relatively recent and evidence of its impact in infection control at the community level is still insufficient. This is also the case for CaniLeish® vaccine, the first CanL vaccine to be licensed in Europe, in 2011. Pre-licensing studies were performed exclusively in homogeneous populations of beagle dogs, experimentally infected or introduced in endemic areas, and very little is known regarding this vaccine’s performance in native and heterogeneous dog populations from L. infantum endemic areas. The study presented in this thesis is divided into two parts. The first consists of a CanL epidemiological study in Girona province, a previously uncharacterized region of north-eastern Spain. The results obtained confirmed the endemicity of CanL in Girona province, characterized by a high prevalence of L. infantum infection in dogs (19.5%), together with the detection of a significant proportion of asymptomatic infected individuals (93.2%). The increase of dogs’ age and lower altitude of the kennel location were identified as risk factors. The two antigens tested to assess dog exposure to Phlebotomus perniciosus (SGH and rSP03B salivary antigens) proved to be suitable, with specific antibodies showing a marked decrease during the non-transmission season, which allowed detection of recent host exposure to vectors. In addition, detected levels of antibodies against both SGH and rSP03B were associated with seropositivity to L. infantum. The second part of this thesis describes a one year field trial of CaniLeish® vaccine, performed in a native heterogeneous canine population from Girona province. These dogs were kept in their natural housing conditions throughout the study and were naturally exposed to an L. infantum transmission season. Results showed that CaniLeish® vaccine induces the production of non-specific antibodies interfering with the serological diagnosis of L. infantum infection in dogs and that this interference could have a greater impact between one and four months post-vaccination. Vaccine trial results did not confirm CaniLeish® reported efficacy in preventing active L. infantum infection or clinical disease in dogs during the first year post-vaccination. These results were supported by an apparently short-lived vaccine-induced cellular mediated immunity, assessed in this study through the quantification of gamma-interferon (IFN-γ) produced by trial dogs at one and nine months post-vaccination. The results presented in this thesis support the need for maintaining and extending epidemiological surveillance in CanL endemic areas, in order to better characterize current CanL distribution and to anticipate possible L. infantum expansion trends. Additionally, further CaniLeish® evaluation studies are needed, together with active vaccine surveillance, to definitely assess the utility of this vaccine in CanL control at the community level in L. infantum endemic areas.[spa] La leishmaniosis es una zoonosis de transmisión vectorial que en la región mediterránea está causada por Leishmania infantum y presenta al perro como principal reservorio. Su distribución heterogénea hace que la vigilancia epidemiológica deba realizarse de manera focalizada, abarcando tanto la detección de perros infectados como de los flebotomos vectores. Las medidas de control de la leishmaniosis canina (LCan) incluyen la vacunación de perros. En Europa, la primera vacuna para la LCan (CaniLeish®) se autorizó en 2011 y los estudios previos a la licencia se realizaron exclusivamente en poblaciones homogéneas de perros beagle, infectados experimentalmente o introducidos en áreas endémicas. La primera parte de la tesis incluye un estudio epidemiológico de la LCan en la provincia de Girona, previamente sin caracterizar. Los resultados obtenidos mostraron una alta prevalencia de infección por L. infantum (19,5%) y una proporción significativa de individuos infectados asintomáticos (93,2%). Se identificaron como factores de riesgo el aumento de la edad de los perros y la menor altitud de la ubicación de las perreras. El estudio de la exposición de los perros a los flebotomos a través del análisis de los antígenos salivales de Phlebotomus perniciosus (SGH y rSP03B) mostró ser útil. Los niveles de anticuerpos detectados mostraron una marcada disminución durante la temporada de no transmisión, lo que permitiría la detección de la exposición reciente a los vectores, y una asociación significativa con la seropositividad frente a L. infantum. La segunda parte describe un ensayo de campo de un año de CaniLeish®, realizado en una población canina heterogénea natural de Girona. Los perros se mantuvieron en condiciones habituales de alojamiento y estuvieron naturalmente expuestos a una temporada de transmisión. La vacuna indujo la producción de anticuerpos no específicos que interferirían en el diagnóstico serológico de la infección por L. infantum, con un impacto mayor entre uno y cuatro meses después de la vacunación. Los resultados no confirmaron la eficacia de CaniLeish® en la prevención de la infección activa por L. infantum o la enfermedad clínica en perros durante el primer año post-vacunación. Estos resultados fueron respaldados por una inmunidad mediada por células inducida por la vacuna aparentemente de corta duración, evaluada a través de la cuantificación del interferón gamma (IFN-γ)

Epidemiology and control of canine leishmaniosis: characterization of a previously undescribed endemic area in Catalonia and CaniLeish® vaccine field trial

Leishmaniosis is an important vector-borne zoonosis caused by Leishmania infantum. The disease is widespread across several continents and endemic in the Mediterranean region. The domestic dog is the main vertebrate reservoir for the parasite and control of canine leishmaniosis (CanL) is deemed to be essential for the control of human cases of the disease. Due to the heterogeneous distribution of infection in endemic areas, epidemiological surveillance should be carried out focally, including both screening of canine populations and vector detection, the two determinant factors for parasite survival and expansion. CanL control measures are usually directed at the canine reservoir through the detection and treatment of infected individuals, as well as disease prevention through insecticide treatments and/or canine immunoprophylaxis. Vaccination against CanL is relatively recent and evidence of its impact in infection control at the community level is still insufficient. This is also the case for CaniLeish® vaccine, the first CanL vaccine to be licensed in Europe, in 2011. Pre-licensing studies were performed exclusively in homogeneous populations of beagle dogs, experimentally infected or introduced in endemic areas, and very little is known regarding this vaccine’s performance in native and heterogeneous dog populations from L. infantum endemic areas. The study presented in this thesis is divided into two parts. The first consists of a CanL epidemiological study in Girona province, a previously uncharacterized region of north-eastern Spain. The results obtained confirmed the endemicity of CanL in Girona province, characterized by a high prevalence of L. infantum infection in dogs (19.5%), together with the detection of a significant proportion of asymptomatic infected individuals (93.2%). The increase of dogs’ age and lower altitude of the kennel location were identified as risk factors. The two antigens tested to assess dog exposure to Phlebotomus perniciosus (SGH and rSP03B salivary antigens) proved to be suitable, with specific antibodies showing a marked decrease during the non-transmission season, which allowed detection of recent host exposure to vectors. In addition, detected levels of antibodies against both SGH and rSP03B were associated with seropositivity to L. infantum. The second part of this thesis describes a one year field trial of CaniLeish® vaccine, performed in a native heterogeneous canine population from Girona province. These dogs were kept in their natural housing conditions throughout the study and were naturally exposed to an L. infantum transmission season. Results showed that CaniLeish® vaccine induces the production of non-specific antibodies interfering with the serological diagnosis of L. infantum infection in dogs and that this interference could have a greater impact between one and four months post-vaccination. Vaccine trial results did not confirm CaniLeish® reported efficacy in preventing active L. infantum infection or clinical disease in dogs during the first year post-vaccination. These results were supported by an apparently short-lived vaccine-induced cellular mediated immunity, assessed in this study through the quantification of gamma-interferon (IFN-γ) produced by trial dogs at one and nine months post-vaccination. The results presented in this thesis support the need for maintaining and extending epidemiological surveillance in CanL endemic areas, in order to better characterize current CanL distribution and to anticipate possible L. infantum expansion trends. Additionally, further CaniLeish® evaluation studies are needed, together with active vaccine surveillance, to definitely assess the utility of this vaccine in CanL control at the community level in L. infantum endemic areas.La leishmaniosis es una zoonosis de transmisión vectorial que en la región mediterránea está causada por Leishmania infantum y presenta al perro como principal reservorio. Su distribución heterogénea hace que la vigilancia epidemiológica deba realizarse de manera focalizada, abarcando tanto la detección de perros infectados como de los flebotomos vectores. Las medidas de control de la leishmaniosis canina (LCan) incluyen la vacunación de perros. En Europa, la primera vacuna para la LCan (CaniLeish®) se autorizó en 2011 y los estudios previos a la licencia se realizaron exclusivamente en poblaciones homogéneas de perros beagle, infectados experimentalmente o introducidos en áreas endémicas. La primera parte de la tesis incluye un estudio epidemiológico de la LCan en la provincia de Girona, previamente sin caracterizar. Los resultados obtenidos mostraron una alta prevalencia de infección por L. infantum (19,5%) y una proporción significativa de individuos infectados asintomáticos (93,2%). Se identificaron como factores de riesgo el aumento de la edad de los perros y la menor altitud de la ubicación de las perreras. El estudio de la exposición de los perros a los flebotomos a través del análisis de los antígenos salivales de Phlebotomus perniciosus (SGH y rSP03B) mostró ser útil. Los niveles de anticuerpos detectados mostraron una marcada disminución durante la temporada de no transmisión, lo que permitiría la detección de la exposición reciente a los vectores, y una asociación significativa con la seropositividad frente a L. infantum. La segunda parte describe un ensayo de campo de un año de CaniLeish®, realizado en una población canina heterogénea natural de Girona. Los perros se mantuvieron en condiciones habituales de alojamiento y estuvieron naturalmente expuestos a una temporada de transmisión. La vacuna indujo la producción de anticuerpos no específicos que interferirían en el diagnóstico serológico de la infección por L. infantum, con un impacto mayor entre uno y cuatro meses después de la vacunación. Los resultados no confirmaron la eficacia de CaniLeish® en la prevención de la infección activa por L. infantum o la enfermedad clínica en perros durante el primer año post-vacunación. Estos resultados fueron respaldados por una inmunidad mediada por células inducida por la vacuna aparentemente de corta duración, evaluada a través de la cuantificación del interferón gamma (IFN-γ)

Seasonal dynamics of canine antibody response to Phlebotomus perniciosus saliva in an endemic area of Leishmania infantum

Background: Canine leishmaniosis (CanL) is an important zoonotic parasitic disease, endemic in the Mediterranean basin. In this region, transmission of Leishmania infantum, the etiological agent of CanL, is through the bite of phlebotomine sand flies. Therefore, monitoring host- vector contact represents an important epidemiological tool, and could be used to assess the effectiveness of vector-control programmes in endemic areas. Previous studies have shown that canine antibodies against the saliva of phlebotomine sand flies are specific markers of exposure to Leishmania vectors. However, this method needs to be further validated in natural heterogeneous dog populations living in CanL endemic areas. Methods: In this study, 176 dogs living in 12 different locations of an L. infantum endemic area in north-east Spain were followed for 14 months. Blood samples were taken at 5 pre-determined time points (February, August and October 2016; January and April 2017) to assess the canine humoral immune response to whole salivary gland homogenate (SGH) and to the single salivary 43 kDa yellow-related recombinant protein (rSP03B) of Phlebotomus perniciosus, a proven vector of L. infantum naturally present in this region. Simultaneously, in all dogs, L. infantum infection status was assessed by serology. The relationship between anti-SGH and anti-rSP03B antibodies with the sampling month, L. infantum infection and the location was tested by fitting multilevel linear regression models. Results: The dynamics of canine anti-saliva IgG for both SGH and rSP03B followed the expected trends of P. perniciosus activity in the region. Statistically significant associations were detected for both salivary antigens between vector exposure and sampling month or dog seropositivity to L. infantum. The correlation between canine antibodies against SGH and rSP03B was moderate. Conclusions: Our results confirm the frequent presence of CanL vectors in the study area in Spain and support the applicability of SGH- and rSP03B-based ELISA tests to study canine exposure to P. perniciosus in L. infantum endemic areas. Keywords: Canine leishmaniosis, Phlebotomus perniciosus, Saliva proteins, Markers of exposure, Longitudinal study, Northeast Spai

Field study of the improved rapid sand fly exposure test in areas endemic for canine leishmaniasis

BACKGROUND: Canine leishmaniasis (CanL) is a severe chronic disease caused by Leishmania infantum and transmitted by sand flies of which the main vector in the Western part of the Mediterranean basin is Phlebotomus perniciosus. Previously, an immunochromatographic test (ICT) was proposed to allow rapid evaluation of dog exposure to P. perniciosus. In the present study, we optimized the prototype and evaluated the detection accuracy of the ICT in field conditions. Possible cross-reactions with other hematophagous arthropods were also assessed. METHODOLOGY/PRINCIPAL FINDINGS: The ICT was optimized by expressing the rSP03B protein in a HEK293 cell line, which delivered an increased specificity (94.92%). The ICT showed an excellent reproducibility and inter-person reliability, and was optimized for use with whole canine blood which rendered an excellent degree of agreement with the use of serum. Field detectability of the ICT was assessed by screening 186 dogs from different CanL endemic areas with both the SGH-ELISA and the ICT, and 154 longitudinally sampled dogs only with the ICT. The ICT results corresponded to the SGH-ELISA for most areas, depending on the statistical measure used. Furthermore, the ICT was able to show a clear seasonal fluctuation in the proportion of bitten dogs. Finally, we excluded cross-reactions between non-vector species and confirmed favorable cross-reactions with other L. infantum vectors belonging to the subgenus Larroussius. CONCLUSIONS/SIGNIFICANCE: We have successfully optimized the ICT, now also suitable to be used with whole canine blood. The test is able to reflect the seasonal fluctuation in dog exposure and showed a good detectability in a field population of naturally exposed dogs, particularly in areas with a high seroprevalence of bitten dogs. Furthermore, our study showed the existence of favorable cross-reactions with other sand fly vectors thereby expanding its use in the field

Evaluation of canine leishmaniosis vaccine CaniLeish® under field conditions in native dog populations from an endemic Mediterranean area - a randomized controlled trial.

Dog vaccination is considered an effective way of reducing Leishmania infantum infection incidence in the canine population, as well as its transmission to humans. However, the use of partially effective vaccines can have the detrimental effect of 'masking' vaccinated asymptomatic carriers, capable of harbouring the parasite and transmitting it to naïve individuals. After eight years on the European market, few studies have been released on CaniLeish® vaccine safety and efficacy. The present study, a one-year randomized CaniLeish® vaccine field trial, was performed in a canine leishmaniosis endemic area and included animals selected from a native dog population (n=168). No severe adverse reactions were observed in vaccinated dogs (n=85). Cases of active L. infantum infection were detected by serological, molecular and clinical follow-up of dogs. One-year post-vaccination, no differences in number or severity of L. infantum active infections were observed between study groups (n=4 in each group). Vaccine-induced cellular immunity, assessed through interferon-γ quantification, showed significantly higher levels of this cytokine one-month post-vaccination in the vaccine group (p<0.001), but no differences were observed after nine months between trial groups (p=0.078). These results fail to support the reported CaniLeish® efficacy in the prevention of active L. infantum infection in dogs from endemic areas and naturally exposed to the parasite

Seroprevalence of canine Leishmania infantum infection in the Mediterranean region and identification of risk factors: The example of North-Eastern and Pyrenean areas of Spain

The Mediterranean basin is an endemic region for canine leishmaniosis (CanL), where it represents a major veterinary problem and raises human health concerns. However, the distribution of the disease is heterogeneous and not all countries and locations have been equally studied and characterized. This work describes the situation of CanL in Girona province (Catalonia, Spain), for which no data has been previously reported, and presents a relevant study to exemplify other areas with similar characteristics across the region. Four cross-sectional seroprevalence surveys were performed from 2012 to 2016 throughout the province, including 36 sampling stations in 26 localities and a total of 593 dogs. For each animal, individual and location variables were also collected. Additionally, each dog owner answered a questionnaire about their knowledge of CanL and preventive methods used. Blood samples were analysed by an in-house ELISA and a mixed logistic regressio nmodel was used to assess the relationship between pre-determined variables and dog seropositivity. A Spearman's correlation was used to assess the association between dog owners'perceived risk of CanL an dLeishmania infantums eropositivity in dogs at a given location. The overall true seroprevalence estimated forGirona province was 19.5% (95%CI: 15.5-23.5), of which only 6.8% (10/146) were considered symptomatic. Age of the dog [OR = 1.21 (95%CI: 1.11-1.31); p < 0.001] and altitude [OR = 0.02 (95%CI: 0.001-0.19);p = 0.001] were identified as risk factors for the infection. The results obtained in this study are expected to aid in the implementation of directed control programmes in CanL endemic areas throughout Europe, as well as to provide suitable data for the design of better risk assessment maps of the diseas

Epidemiology and control of canine leishmaniosis: characterization of a previously undescribed endemic area in Catalonia and CaniLeish® vaccine field trial

Leishmaniosis is an important vector-borne zoonosis caused by Leishmania infantum. The disease is widespread across several continents and endemic in the Mediterranean region. The domestic dog is the main vertebrate reservoir for the parasite and control of canine leishmaniosis (CanL) is deemed to be essential for the control of human cases of the disease. Due to the heterogeneous distribution of infection in endemic areas, epidemiological surveillance should be carried out focally, including both screening of canine populations and vector detection, the two determinant factors for parasite survival and expansion. CanL control measures are usually directed at the canine reservoir through the detection and treatment of infected individuals, as well as disease prevention through insecticide treatments and/or canine immunoprophylaxis. Vaccination against CanL is relatively recent and evidence of its impact in infection control at the community level is still insufficient. This is also the case for CaniLeish® vaccine, the first CanL vaccine to be licensed in Europe, in 2011. Pre-licensing studies were performed exclusively in homogeneous populations of beagle dogs, experimentally infected or introduced in endemic areas, and very little is known regarding this vaccine’s performance in native and heterogeneous dog populations from L. infantum endemic areas. The study presented in this thesis is divided into two parts. The first consists of a CanL epidemiological study in Girona province, a previously uncharacterized region of north-eastern Spain. The results obtained confirmed the endemicity of CanL in Girona province, characterized by a high prevalence of L. infantum infection in dogs (19.5%), together with the detection of a significant proportion of asymptomatic infected individuals (93.2%). The increase of dogs’ age and lower altitude of the kennel location were identified as risk factors. The two antigens tested to assess dog exposure to Phlebotomus perniciosus (SGH and rSP03B salivary antigens) proved to be suitable, with specific antibodies showing a marked decrease during the non-transmission season, which allowed detection of recent host exposure to vectors. In addition, detected levels of antibodies against both SGH and rSP03B were associated with seropositivity to L. infantum. The second part of this thesis describes a one year field trial of CaniLeish® vaccine, performed in a native heterogeneous canine population from Girona province. These dogs were kept in their natural housing conditions throughout the study and were naturally exposed to an L. infantum transmission season. Results showed that CaniLeish® vaccine induces the production of non-specific antibodies interfering with the serological diagnosis of L. infantum infection in dogs and that this interference could have a greater impact between one and four months post-vaccination. Vaccine trial results did not confirm CaniLeish® reported efficacy in preventing active L. infantum infection or clinical disease in dogs during the first year post-vaccination. These results were supported by an apparently short-lived vaccine-induced cellular mediated immunity, assessed in this study through the quantification of gamma-interferon (IFN-γ) produced by trial dogs at one and nine months post-vaccination. The results presented in this thesis support the need for maintaining and extending epidemiological surveillance in CanL endemic areas, in order to better characterize current CanL distribution and to anticipate possible L. infantum expansion trends. Additionally, further CaniLeish® evaluation studies are needed, together with active vaccine surveillance, to definitely assess the utility of this vaccine in CanL control at the community level in L. infantum endemic areas.La leishmaniosis es una zoonosis de transmisión vectorial que en la región mediterránea está causada por Leishmania infantum y presenta al perro como principal reservorio. Su distribución heterogénea hace que la vigilancia epidemiológica deba realizarse de manera focalizada, abarcando tanto la detección de perros infectados como de los flebotomos vectores. Las medidas de control de la leishmaniosis canina (LCan) incluyen la vacunación de perros. En Europa, la primera vacuna para la LCan (CaniLeish®) se autorizó en 2011 y los estudios previos a la licencia se realizaron exclusivamente en poblaciones homogéneas de perros beagle, infectados experimentalmente o introducidos en áreas endémicas. La primera parte de la tesis incluye un estudio epidemiológico de la LCan en la provincia de Girona, previamente sin caracterizar. Los resultados obtenidos mostraron una alta prevalencia de infección por L. infantum (19,5%) y una proporción significativa de individuos infectados asintomáticos (93,2%). Se identificaron como factores de riesgo el aumento de la edad de los perros y la menor altitud de la ubicación de las perreras. El estudio de la exposición de los perros a los flebotomos a través del análisis de los antígenos salivales de Phlebotomus perniciosus (SGH y rSP03B) mostró ser útil. Los niveles de anticuerpos detectados mostraron una marcada disminución durante la temporada de no transmisión, lo que permitiría la detección de la exposición reciente a los vectores, y una asociación significativa con la seropositividad frente a L. infantum. La segunda parte describe un ensayo de campo de un año de CaniLeish®, realizado en una población canina heterogénea natural de Girona. Los perros se mantuvieron en condiciones habituales de alojamiento y estuvieron naturalmente expuestos a una temporada de transmisión. La vacuna indujo la producción de anticuerpos no específicos que interferirían en el diagnóstico serológico de la infección por L. infantum, con un impacto mayor entre uno y cuatro meses después de la vacunación. Los resultados no confirmaron la eficacia de CaniLeish® en la prevención de la infección activa por L. infantum o la enfermedad clínica en perros durante el primer año post-vacunación. Estos resultados fueron respaldados por una inmunidad mediada por células inducida por la vacuna aparentemente de corta duración, evaluada a través de la cuantificación del interferón gamma (IFN-γ)

The impact of canine leishmaniosis vaccination with CaniLeish® in Leishmania infantum infection seroprevalence studies

Effective vaccines against Leishmania parasites are a goal for the scientific community working with both canine and human leishmaniosis. However, possible side effects of vaccination should also be considered and evaluated, preferably before vaccine licensing and marketing. One of these possible effects is the cross-reaction of vaccine-induced antibodies with standard serological tests for detection of Leishmania infantum infection. Longitudinal studies were performed on the type of humoral profile induced by Brazilian marketed canine leishmaniosis vaccines, but little is known regarding the European situation. In this study, an annual follow-up of 85 CaniLeish® vaccinated dogs and 83 non-vaccinated control dogs was performed. Blood samples were taken for all animals at pre-determined time points: before vaccination; immediately before each one of the two following vaccine doses (at 21 days intervals); and then one, four, six, nine and 12 months after finishing the vaccination course. All samples were tested by an in-house ELISA, using a whole promastigote antigen, for the presence of anti-L. infantum antibodies. Humoral response detectable by the used serological diagnostic method was significantly higher in the vaccine group when compared with the control group (p < 0.01) until one-month post-vaccination. Results show that CaniLeish® vaccine-induced antibodies cross-react with a commonly used serological test for diagnosis of L. infantum natural infection. Implications of this interference are discussed, with special emphasis on a possible negative impact on canine leishmaniosis surveillance studies

Seasonal dynamics of canine antibody response to Phlebotomus perniciosus saliva in an endemic area of Leishmania infantum

Background: Canine leishmaniosis (CanL) is an important zoonotic parasitic disease, endemic in the Mediterranean basin. In this region, transmission of Leishmania infantum, the etiological agent of CanL, is through the bite of phlebotomine sand flies. Therefore, monitoring host- vector contact represents an important epidemiological tool, and could be used to assess the effectiveness of vector-control programmes in endemic areas. Previous studies have shown that canine antibodies against the saliva of phlebotomine sand flies are specific markers of exposure to Leishmania vectors. However, this method needs to be further validated in natural heterogeneous dog populations living in CanL endemic areas. Methods: In this study, 176 dogs living in 12 different locations of an L. infantum endemic area in north-east Spain were followed for 14 months. Blood samples were taken at 5 pre-determined time points (February, August and October 2016; January and April 2017) to assess the canine humoral immune response to whole salivary gland homogenate (SGH) and to the single salivary 43 kDa yellow-related recombinant protein (rSP03B) of Phlebotomus perniciosus, a proven vector of L. infantum naturally present in this region. Simultaneously, in all dogs, L. infantum infection status was assessed by serology. The relationship between anti-SGH and anti-rSP03B antibodies with the sampling month, L. infantum infection and the location was tested by fitting multilevel linear regression models. Results: The dynamics of canine anti-saliva IgG for both SGH and rSP03B followed the expected trends of P. perniciosus activity in the region. Statistically significant associations were detected for both salivary antigens between vector exposure and sampling month or dog seropositivity to L. infantum. The correlation between canine antibodies against SGH and rSP03B was moderate. Conclusions: Our results confirm the frequent presence of CanL vectors in the study area in Spain and support the applicability of SGH- and rSP03B-based ELISA tests to study canine exposure to P. perniciosus in L. infantum endemic areas. Keywords: Canine leishmaniosis, Phlebotomus perniciosus, Saliva proteins, Markers of exposure, Longitudinal study, Northeast Spai

Seasonal dynamics of canine antibody response to Phlebotomus perniciosus saliva in an endemic area of Leishmania infantum

Background: Canine leishmaniosis (CanL) is an important zoonotic parasitic disease, endemic in the Mediterranean basin. In this region, transmission of Leishmania infantum, the etiological agent of CanL, is through the bite of phlebotomine sand flies. Therefore, monitoring host- vector contact represents an important epidemiological tool, and could be used to assess the effectiveness of vector-control programmes in endemic areas. Previous studies have shown that canine antibodies against the saliva of phlebotomine sand flies are specific markers of exposure to Leishmania vectors. However, this method needs to be further validated in natural heterogeneous dog populations living in CanL endemic areas. Methods: In this study, 176 dogs living in 12 different locations of an L. infantum endemic area in north-east Spain were followed for 14 months. Blood samples were taken at 5 pre-determined time points (February, August and October 2016; January and April 2017) to assess the canine humoral immune response to whole salivary gland homogenate (SGH) and to the single salivary 43 kDa yellow-related recombinant protein (rSP03B) of Phlebotomus perniciosus, a proven vector of L. infantum naturally present in this region. Simultaneously, in all dogs, L. infantum infection status was assessed by serology. The relationship between anti-SGH and anti-rSP03B antibodies with the sampling month, L. infantum infection and the location was tested by fitting multilevel linear regression models. Results: The dynamics of canine anti-saliva IgG for both SGH and rSP03B followed the expected trends of P. perniciosus activity in the region. Statistically significant associations were detected for both salivary antigens between vector exposure and sampling month or dog seropositivity to L. infantum. The correlation between canine antibodies against SGH and rSP03B was moderate. Conclusions: Our results confirm the frequent presence of CanL vectors in the study area in Spain and support the applicability of SGH- and rSP03B-based ELISA tests to study canine exposure to P. perniciosus in L. infantum endemic areas. Keywords: Canine leishmaniosis, Phlebotomus perniciosus, Saliva proteins, Markers of exposure, Longitudinal study, Northeast Spai