Presence of anti-Leishmania infantum antibodies in sheep (Ovis aries) in Spain

Sandflies are the primary transmission vector for Leishmania spp parasite in endemic regions. The role of other animals, different from the dog, is under discussion in the leishmaniosis endemic countries. A limited number of reports have been published on the possible role of livestock in European countries for Leishmania maintenance and diffusion. The aim of the present study was to perform a serosurvey on sheep in areas of Spain that are endemic for zoonotic leishmaniosis and establish the possible role of sheep regarding Leishmania infantum infection in endemic areas. Three hundred and two serum samples were obtained from sheep and were evaluated for serological survey to detect L. infantum by using the in-house ELISA technique. Twenty-eight out of the 302 samples included in this study, were positive for L. infantum antibodies (9.27%). In the present study, a significant association was found between adult age and seropositivity (p = 0.006) and female gender and seropositivity (p = 0.02). This association has not been previously described in other European studies related to L. infantum infection in sheep. Our study reveals that domestic sheep in a European Mediterranean country are exposed to L. infantum. To our knowledge, this study demonstrates the presence of seropositive sheep in different regions of Spain for the first time. Further epidemiological studies focus on evaluating the rural cycle of this parasite to know if livestock could act as a potential reservoir are needed.


Introduction
Leishmania parasites are transmitted through the bites of infected female phlebotomine sandflies in endemic regions.Some 70 animal species, including humans, can be the source of Leishmania parasites (WHO 2023).According to al. 2021).Up to now, information on the role of sheep and leishmaniosis is limited globally; nevertheless, available data suggests that sheep may have a role in the epidemiology of Leishmania spp.due to the asymptomatic nature of infection in this species (Mukhtar et al. 2000;Portús et al. 2002;Rohousova et al. 2015;Han et al. 2018).
The aim of the present study was to perform a serosurvey on sheep in areas of Spain that are endemic for zoonotic leishmaniosis and establish the possible role of sheep regarding Leishmania infantum infection in endemic areas.

Animals and sample collection
Three hundred and two residual serum samples were obtained from different sheep for diagnostic purposes at the Veterinary Faculty of Zaragoza (41°39′24.6276″N, 0°52′45.912″W).Based on an expected seroprevalence of 10% (the canine seroprevalence in Spain) (Baxarias et al. 2023), an accepted 5% deviation from the true prevalence and a confidence level of 95%, the sample size necessary to estimate the seroprevalence was calculated to be 139 animals (De Blas 2006).It is essential to highlight that these animals belong to the university farm.In the Ruminant Clinical Service (SCRUM), a small healthy, productive flock is maintained, and many referred cases are received from different farms in the northwestern area of Spain.The sera come from animals that arrive at the SCRUM as clinical cases with different diseases.They all come from sheep farms in the area of influence of the Faculty of Veterinary Medicine in Zaragoza: Aragon, the Valencian Community, the Basque Country and Navarra.All the animals come from commercial farms with a semi-intensive production system, in which they for graze an essential part of the year.
Serum samples were obtained of each sheep once the referred case was received in the SCRUM facilities.Serum samples were collected associated with routine healthcare check-ups from autumn 2020 to winter 2022.The separated residual sera were stored at − 35 °C until they were processed.
Before sampling, information was obtained about each animal regarding age (lambs (< 12 months), adults (from ≥ 1 year to ≤ 6 years), or seniors (> 6 years), gender, elevation of farm location (mountains with an altitude between 792 and 1171 m, semi-mountains with an altitude between 208 and 685 m, and flat areas, with an altitude between 80 and 181 m) and the season of the year in which the blood was taken.In addition, a complete physical examination was carried out to detect the presence of potential skin lesions compatible with clinical leishmaniosis because dermatological lesions are the most common clinical signs detected in animals due to L. infantum (Cardoso et al. 2021).
Finally, infected sheep by some other pathogens (n = 25) were included to evaluate cross-reactivity and diagnostic specificity of the antibody test in this study.For this purpose, the pathogens included were the most prevalent in Spain, such as Anaplasma ovis (n = 5), Coxiella burnetii (n = 5), Babesia ovis (n = 5), Babesia motasi (n = 5) and Theileria ovis (n = 5).These serum samples were from the serum bank of the Clinical Immunology Laboratory, Veterinary Faculty, University of Zaragoza and a private laboratory.

Detection of L. infantum antibodies by a quantitative ELISA
The ELISA was performed on all sera as described previously (Alcover et al. 2021), with some modifications.For the in-house ELISA (sensitivity of 99.37% and specificity of 97.50%), the crude antigen (strain MHOM/FR/78/LEM75 belonging to L. infantum zimodeme MON-1) was adjusted to a concentration of 20 µg/ml with phosphate-buffered saline (PBS).Briefly, each plate was coated with 100 µl/ well of the 20 µg/ml antigen solution in 0.1 M carbonate/ bicarbonate buffer and incubated overnight at 4 ºC.A 100µl aliquot of sheep sera, diluted 1:100 in PBS containing 0.05% Tween 20 (PBST) and 1% dry skimmed milk (PBST-M) as a blocking agent, was added to each well.The plates were incubated for 1 h at 37 °C in a moist chamber.After washing the plates for 3 min 3 times with PBST followed by 1 wash with PBS for 1 min, 100 µl of Protein A/G conjugated to horseradish peroxidase (Thermo Fisher Scientific, Waltham, Massachusetts, USA) diluted 1:10000 in PBST-M was added per well.The standardisation of the ideal concentration of serum dilution and conjugated dilution was based on a previous case report of leishmaniosis in a small ruminant (Ruiz et al. 2023).The plates were incubated for 1 h at 37 °C in the moist chamber and were washed again with PBST and PBS as described above.The substrate solution (ortho-phenylene-diamine) and stable substrate buffer (Thermo Fisher Scientific, Waltham, Massachusetts, USA) was added at 100 µl per well and developed for 20 ± 5 min at room temperature in the dark.The reaction was stopped by adding 100 µl of 2.5 M H2SO4 to each well.Absorbance values were read at 492 nm in an automatic microELISA reader (Microplate Photometer Biosan Hipo MPP-96, Riga, Latvia).As a positive control, each plate included three serum samples, including a sick seropositive dog, a sick seropositive cat and a sick seropositive goat diagnosed with clinical leishmaniosis (Ruiz et al. 2023).Optical density (OD) units of the positive controls were > 1.1 OD units.By contrast, serum from healthy, non-infected sheep was used as a negative control with a value < 0.10 OD units.The same positive and negative sera were used for all assays, and the plates with an inter-assay variation greater than 10% were discarded.All samples were run in duplicate.The cut-off was set to 0.38 OD units (mean + 3 standard deviations (SD) of values from 90 sheep from non-endemic areas such as northern Spain), and results above this value were considered positive.These 90 sheep were classified as healthy according to Leishvet guidelines (Solano-Gallego et al. 2011;Pennisi et al. 2015).This classification was based on a complete physical examination and the absence of clinicopathological abnormalities detected by routine red blood cell count (Idexx Procyte Dx, Westbrook, ME, USA), clinical chemistry (AmiShield, Protect Life International Biomedical Inc. Taiwan), urinalysis and serum protein electrophoresis by agarose gel electrophoresis system (Hydragel Kit 1-2, Sebia, Evry, France).Laboratory parameters of these animals were not considered altered because they were within the reference intervals.

Statistical analysis
Data collected for the entire population were analysed using descriptive statistics.Univariate analysis of categorical data was performed to determine possible associations between L. infantum seropositivity and the following variables: age, gender, the elevation of farm location, season, and collection year.The significance of this difference was assessed using the Fisher's exact test or Chi-square.A p ≤ 0.05 was considered significant.The SPSS v.22 software (SPSS Inc., Chicago, USA) was used.
No significant association (p > 0.05) was detected between Leishmania seropositivity and the year of collection, season, and elevation of farm location.However, a significant association was found between age and seropositivity (p = 0.006) and gender and seropositivity (p = 0.02).

Discussion
The dog has been implicated as the domestic reservoir of L. infantum infection in European Mediterranean countries including Spain as an endemic vector-borne disease.Therefore, most of the epidemiological information is focused on dogs and cats.However, new evidence supports the potential importance of other animal species, such as ferrets and goats, where the first description of clinical cases of leishmaniosis has been published (Giner et al. 2020;Ruiz et al. 2023).The seroprevalence of L. infantum infection in dogs in Spain was around 10% between 2011 and 2020 (Baxarias et al. 2023).Little information is available concerning L. infantum and livestock in Europe.In the case of sheep, different pieces of evidence suggest that the proliferation of promastigotes of different Leishmania species such as L. infantum, L. donovani, Leishmania tropica and Leishmania major in the biphasic Novy-MacNeal-Nicolle (NNN) 1 3 agriculture productivity, and these sheep are more exposed to sandflies bites.However, the lambs are kept indoor, in livestock facilities, with less exposure to the vector.In this sense, the association of canine leishmaniosis with age has been explained by a longer exposure time of the animals with sandflies vectors (Martín-Sánchez et al. 2009).Finally, a possible explanation of the association of seropositivity with the females can be related to the high number of ewes in the study, especially in the Mediterranean breed (Ortiz 1983).
Differences to the study performed in Greece (Kantzoura et al. 2013) and the studies performed in Spain include geographic location, age of animals, type of serological technique, serum dilution and type of antigen.In the case of Kantzoura et al. 2013, samples were in a double dilution (1:200) to that of the present work (1:100) in the ELISA.No information related to the antigen is available in this serological study, however in the Spanish studies, the antigen was prepared from promastigotes of L. infantum (strain MHOM/ FR/78/LEM 75 belonging to zymodeme MON-1).Different antigenic sources are employed in serological techniques, including soluble leishmania antigen and different parasite antigenic fractions such as individual recombinant proteins medium prepared using sheep blood is possible.The sheep blood is able to produce cultures in lower numbers requiring longer than 7 days to appear (Ladopoulus et al. 2015).
The epidemiological role of sheep in L. infantum infection has been investigated in an endemic area as it is Greece, with the absence of L. infantum antibodies detected by ELISA in the natural environment in 361 sheep from 34 different farms from Thessaly (Kantzoura et al. 2013).By contrast, low antibody titres were detected by Dot-ELISA in sheep in Tarragona with a seroprevalence of 11.90% (Portus et al. 2002).
In the present study, the age and gender of the sheep tested showed significant associations with Leishmania seropositivity.This association has not been previously described in other European studies related to L. infantum infection in sheep.Nevertheless, age association has been described in canine leishmaniosis, with the presence of a bimodal distribution with a peak of sick dogs with less than three years and a second peak between eight and ten years (Alvar et al. 2004).In our study, the highest number of seropositive was detected in the adult group (16/101), followed by lambs (7/159), and the senior group (5/41).In general, adult and senior sheep raised in an extensive system in areas with low .The presence of anti-Leishmania spp.antibodies was detected by DAT in 40.1% of sheep in Iran.In this sense, due to the coexistence of different Leishmania species in this country, it was necessary to perform a specific quantitative molecular technique and sequencing for the identification and detection L. infantum and L. major species (Rezaei et al. 2022).
One advisable situation in the validation of serological techniques would be the inclusion of seropositive samples for other pathogens to evaluate cross-reaction phenomenon with importance in the specificity value of the analysed technique.Little is known about which sheep pathogens could be potentially cross-reacted with anti-Leishmania antibodies detected by ELISA.In this sense, we have included a panel of seropositive samples of the most common vector borne disease and bacterial disease with importance in Spain.The inclusion of positive serum samples to another type of Leishmania species phylogenetically similar would be desirable in areas where different Leishmania species are present and we cannot rule out the potential of or small peptides containing defined antigenic determinants (Ramírez et al. 2019).The use of soluble leishmanial total extract preparation as antigen appears to be more sensitive than recombinant protein antigen for the detection of anti-Leishmania antibodies in dogs (Miró et al. 2008) and humans (Kühne et al. 2019).
The presence of seropositive sheep to Leishmania infection has been detected in other countries outside of Europe.An investigation of the zoonotic leishmaniasis outbreak in China detected the presence of Leishmania DNA in different livestock species, including sheep, goats, cattle, and donkeys (Gao et al. 2015).In Africa, the presence of anti-Leishmania antibodies against L. donovani was detected in donkeys, cows, and goats in Sudan.However, specific antibodies against L. donovani were not detected in the sheep from eastern Sudan (Mukhtar et al. 2000).The presence of antibodies against L. donovani by modified agglutination test (DAT) has been detected in different domestic animals in Ethiopia, such as cows, dog, donkeys, goats and finally in sheep with the lowest seropositivity rate in comparison to the remaining species included in the study (Kenubih et al.

Fig. 1
Fig. 1 Distribution of the location of the seropositive sheep per municipalities in Spain from autumn 2020 to winter 2022.The coloured areas indicate the location of the animals

Table 1
The twenty-eight seropositive samples from sheep evaluated in detail: by province of origin, altitude of farm location, breed, season of sampling collection, year of sampling collection, age group, age, sex, and optical density units detected by ELISA technique