Baxarias et al. Parasites & Vectors (2022) 15:164 https://doi.org/10.1186/s13071-022-05246-2 Parasites & Vectors RESEARCH Open Access Detection of specific antibodies against Leishmania infantum in canine serum and oral transudate using an in‑house ELISA Marta Baxarias1, Júlia Viñals1, Alejandra Álvarez‑Fernández1, Mª Magdalena Alcover2 and Laia Solano‑Gallego1*    Abstract Background: Canine leishmaniosis caused by the protozoan Leishmania infantum is a complex infection due to its variable clinical signs and laboratory findings. Therefore, a broad range of techniques is available for diagnosis. Testing for specific antibodies in serum is the most commonly used technique, although the testing of other body fluids, such as oral transudate (OT), can be an alternative as its collection is non-invasive and testing can be performed by untrained personnel. The aim of this study was to assess and compare the detection of L. infantum-specific antibodies in paired samples of serum and OT collected from apparently healthy dogs and dogs with clinical leishmaniosis using an in-house enyzme-linked immunosorbent assay (ELISA). Methods: Serum and OT were collected from 407 dogs, which varied in breed, sex, age, lifestyle and clinical status, by many practicing veterinarians in Spain. The main geographical areas of sampling included Barcelona (n = 110), Mallorca (n = 94), Cadiz (n = 54) and Asturias (n = 47). The majority of infected dogs were apparently healthy (89.9%) while 41 presented clinical signs and/or clinicopathological abnormalities compatible with L. infantum infection and subsequently diagnosed with leishmaniosis (10.1%). An in-house ELISA was performed to quantify the anti-Leishmania antibodies in serum and OT. Results: The L. infantum infection rate determined by the in-house ELISA was 37.1% in serum samples and 32.7% in OT samples. Serum and OT ELISA results showed a positive correlation (Spearman’s correlation coefficient rs = 0.6687, P < 0.0001). The percent agreement between the serum and OT ELISA results was 84%, while agreement according to Cohen’s kappa statistic (κ) was substantial (0.66) when all samples were analyzed. The highest percent agreement (92.1%) between both tests was found in dogs from low endemicity regions and from sick dogs, with both groups presenting almost perfect agreement according to Cohen’s κ agreement test (0.84). Few seronegative dogs (n = 23) tested positive by the OT ELISA. The agreement between serum and OT went from almost perfect to moderate when the geographical distribution and clinical status were analyzed. Conclusions: The results of this study demonstrated an almost perfect to moderate agreement between OT and serum samples tested using the in-house ELISA. These results are particularly promising in sick dogs with high anti‑ body levels while the results seem less optimal in apparently healthy dogs with low antibody levels. Keywords: Leishmaniosis, Dog, Oral transudate, Serology, Diagnosis, Spain *Correspondence: laia.solano@uab.cat 1 Departament de Medicina i Cirurgia Animals, Facultat de Veterinària, Universitat Autònoma de Barcelona, Cerdanyola del Vallès, Barcelona, Spain Full list of author information is available at the end of the article © The Author(s) 2022. 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The Creative Commons Public Domain Dedication waiver (http://​creat​iveco​ mmons.​org/​publi​cdoma​in/​zero/1.​0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Baxarias et al. Parasites & Vectors (2022) 15:164 Page 2 of 12 Background several routine diagnostic tests, such as a complete blood Canine leishmaniosis (CanL), a zoonotic and endemic count, biochemical profile, urinalysis and serum electro- protozoan disease caused by Leishmania infantum, is phoresis, are necessary when there is a suspicion of CanL endemic in the Mediterranean basin [1, 2]. Transmission [15, 18]. In addition, several diagnostic techniques are is mostly through the bite of a female phlebotomine sand available that enable a definitive diagnosis of L. infantum fly following a digenetic life-cycle which consists of two infection, such as parasitological diagnosis (direct obser- different phases: an extracellular and mobile promastig- vation of the parasite), serological techniques (such as ote in the sand fly, and an intracellular and non-motile the enzyme-linked immunosorbent assay [ELISA] and amastigote in the mammalian host [3]. Other confirmed indirect fluorescent antibody test) and molecular studies transmission routes, such as venereal [4, 5] and transpla- (such as quantitative PCR) [1, 17, 18, 20]. Parasitological cental [5, 6] transmission and through blood transfusion, methods and molecular studies can detect the presence also occur [7, 8]. The dog is considered to be the main of the parasite, by direct observation or detection of domestic reservoir for L. infantum infection in the Medi- DNA, respectively, while serological techniques detect terranean basin [2, 9], while other mammals may be able serum anti-Leishmania antibodies. The diagnostic tech- to maintain a wild-life cycle [10–12]. niques must be used with full knowledge of the basis of The seroprevalence of L. infantum-infected healthy each test and its limitations, as well as how to correctly dogs in western Europe was 23% between 1971 and 2006 interpret the results [15, 17, 18]. [13]. In Spain, the seroprevalence has been reported Interestingly, these diagnostic techniques can be per- to be around 10%, although it can vary from 0 to 57% formed using different types of samples, such as blood, depending on the region [14]. Moreover, the prevalence serum, urine and other infected tissues [15, 21–23]. The of dogs that develop the clinical disease is usually lower use of alternative samples, such as oral transudate (OT), than 10% [15, 16]. CanL is a complex infection due to hair or conjunctival swabs, has also been studied, with its variable clinical manifestations and wide spectrum interesting results [24–27]. Immunoglobin A (IgA) can of clinical signs and laboratory findings [9, 17, 18]. One be found in OT as it is secreted in the salivary glands by factor underlying this variability is the dog’s immune plasma cells, along with immunoglobin G (IgG) and response, which requires a balance between inflamma- immunoglobin M (IgM), both of which are derived from tory and regulatory responses to control L. infantum plasma [28]. Specific antibodies against L. infantum have infection [19]. For example, neutrophils and macrophages been previously detected in saliva samples of infected play distinctive roles in the dog’s initial immune ability sick dogs only by means of a time-resolved immuno- to control the infection or to allow progression towards fluorometric assay (TR-IFMA) [24, 29–31]. However, to disease. Both neutrophils and macrophages phagocytize the authors’ best knowledge, the detection of antibod- the parasite which can lead either to the elimination of ies against L. infantum by ELISA in OT from apparently the parasite through the production of reactive oxygen healthy dogs has not been previously documented. The species (ROS), or to the survival of parasites within mac- advantages of using OT instead of serum include a non- rophages, leading to parasite persistence and dissemi- invasive, cheap and painless collection of the sample, nation [19]. T lymphocytes also play an integral role in which can also be performed by untrained personnel. preventing parasite growth and disease development as The aim of this study was to assess and compare the these T cells produce interferon gamma (IFN-γ) among detection of L. infantum-specific antibodies in paired other cytokines, such as tumor necrosis factor alpha, samples of serum and OT from apparently healthy dogs interleukin-2 or chemokines, which results in the differ- and from dogs with clinical leishmaniosis, using an in- entiation, recruitment and activation of macrophages. house ELISA. However, as the infection progresses towards disease, there is a decrease in T cell proliferation and IFN-γ pro- Methods duction and a lack of macrophage activation, resulting in Dogs a reduction of parasite elimination [19]. Many other fac- A minimum sample size of 310 dogs was calculated tors can also affect the development of the disease, such [32] using an expected seroprevalence of L. infantum as age, sex, host genetics, among others. To date, how- infection of 10% [14] and a power of 80%. Both serum ever, the mechanisms responsible for the dog’s resistance and OT samples from 407 dogs varying in breed, sex, or susceptibility are still unknown [15, 17]. age, lifestyle and clinical status were collected between Due to this complexity, CanL diagnosis often requires January of 2018 and June of 2021 by several veterinar- an integrated approach, including a clinicopathological ians practicing in different areas of Spain (Fig. 1), a examination and specific laboratory tests [9, 15, 18]. A country endemic for CanL [14]. Dogs were chosen ran- full clinical history, thorough physical examination and domly from veterinary clinics, dog shelters and groups Baxarias et al. Parasites & Vectors (2022) 15:164 Page 3 of 12 Fig. 1 Geographical distribution of dogs sampled in Spain: 1 Pontevedra (n = 5), 2 Asturias (n = 47), 3 Álava (n = 3), 4 Navarra (n = 3), 5 La Rioja (n = 1), 6 Zaragoza (n = 10), 7 Huesca (n = 1), 8 Barcelona (n = 110), 9 Madrid (n = 8), 10 Teruel (n = 3), 11 Castellón (n = 19), 12 Cáceres (n = 3), 13 Toledo (n = 1), 14 Ciudad Real (n = 6), 15 Valencia (n = 15), 16 Mallorca (n = 94), 17 Córdoba (n = 6), 18 Jaén (n = 2), 19 Murcia (n = 10), 20 Cádiz (n = 54), 21 Málaga (n = 4), 22 Granada (n = 1), 22 Almería (n = 1) of hunting dogs. The clinical data recorded included Sampling the signalment and clinical status of all dogs (Table 1). Blood samples were obtained by jugular or cephalic None of the dogs were vaccinated against CanL. Dogs venepuncture and later centrifuged (Heraeus Labofuge were considered young if they were aged ≤ 1.5 years, 400R Centrifuge; Thermo Fisher Scientific, Waltham, while dogs aged > 1.5 years were considered to be MA, USA) at 789 g for 10 min to obtain serum. adult. Dog characteristics, such as sex, age, breed and OTs were collected by foam swabs (Ecouvillon PP; clinical status, and the significant differences between Dominique Dutscher, Bernolsheim, France) impregnated dogs are shown in Table 1. with hypertonic saline (NaCl 7.5%; B. Braun Melsungen The main sampling areas included Barcelona AG, Melsungen, Germany) mainly as described previ- (n = 110 dogs), Mallorca (n = 94), Cádiz (n = 54) and ously [34] but with some modifications. The swabs were Asturias (n = 47) (Table 1). In the additional sampling kept in the dog’s mouth between the gum and the inner areas, fewer than 20 dogs were sampled per area, with mucosa of the upper or lower lip for around 2 min and a total of 102 dogs (Fig. 1). Dogs were also classified later centrifuged (Eppendorf Centrifuge 5418; Merck according to their clinical status. The majority of dogs KGaA, Darmstadt, Germany) at 16,000 g for 1 min. After were apparently healthy (89.9%) while 41 presented that, OTs were collected. clinical signs and/or clinicopathological abnormali- All samples, including both serum samples and OTs, ties compatible with L. infantum infection and were were identified and stored at – 80 °C until further use. diagnosed with leishmaniosis (10.1%) [9] (Table 1). Most dogs were sampled at the time of diagnosis and Quantitative in‑house ELISA for the detection of L. had not previously been treated with anti-Leishmania infantum‑specific antibodies drugs, with the exception of three dogs that had been Serum ELISA recently treated with allopurinol. Dogs from Asturias, The in-house ELISA was performed on serum samples an area with very low endemicity [14, 33], were classi- of all dogs studied as previously described [21]. Briefly, fied as negative controls, while samples from sick dogs samples were diluted to 1:800 in phosphate buffered that were diagnosed with leishmaniosis were classi- saline (PBS) Tween with 1% dry milk and incubated at fied as positive controls. 37 °C for 1 h, following which they were washed three Baxarias et al. Parasites & Vectors Table 1 Signalment and geographical distribution of dogs enrolled in the study Geographical Sex (%, number of Breed (%, number of Most common breeds (%, number of dogs) Age (%, number of Age, median Clinical status (%, distribution dogs) dogs) dogs)a (years, min– number of dogs) (2022) 15:164 (number of max)a, e dogs) Femaleb Maleb Purebredc Crossbreedc Youngd Adultd Healthyf Sickf Asturias (47) 51.1%, 24 48.9%, 23 89.4%, 42 10.6%, 5 English setter (17%, 8) and Mastiff (10.6%, 5) 8.9%, 4 91.1%, 41 5.5%, 0.5–12 100%, 47 0%, 0 Barcelona (110) 46.4%, 51 53.6%, 59 20.9%, 23 79.1%, 87 German Shepherd (4.5%, 5) and Labrador Retriever (3.6%, 4) 25.5%, 24 74.5%, 70 4%, 0.3–12 87.3%, 96 12.7%, 14 Cádiz (54) 44.4%, 24 55.6%, 30 29.6%, 16 70.4%, 38 Spanish sighthound (11.1%, 6) 17.5%, 7 82.5%, 33 3.5%, 0.5–16 100%, 54 0%, 0 Mallorca (94) 68.1%, 64 31.9%, 30 80.9%, 18 19.1%, 76 Ibizan Hound (54.3%, 51), Mallorca Shepherd dog (5.3%, 5) and 38%, 35 62%, 57 3%, 0.5–14 92.6%, 87 7.4%, 7 Andalusian wine-cellar rat-hunting dog (5.3%, 5) Total of prov‑ 51.4%, 209 48.6%, 198 46.7%, 190 53.3%, 217 Ibizan Hound (12.8%, 52), German Shepherd (3.9%, 16) and 22.8%, 79 77.2%, 267 4%, 0.3–16 89.9%, 366 10.1%, 41 inces of origin Mastiff (3.4%, 14) (407) max maximum, min minimum a Age was not recorded in 2 dogs from Asturias, 16 dogs from Barcelona, 14 dogs from Cádiz, 2 dogs from Mallorca and 27 dogs from other Spanish regions b Mallorca had a higher rate of female dogs (Chi-square test: X2 = 11.7, df = 3, P = 0.008) c Asturias and Mallorca had a higher rate of purebred dogs (Chi-square test: X2 = 110.9, df = 3, P < 0.001) d Mallorca had significantly more young dogs than Asturias (Fisher’s exact test: P < 0.0001) and Cádiz (Fisher’s exact test: P = 0.025), while Asturias had significantly more adult dogs than Barcelona (Fisher’s exact test: P = 0.024) e Dogs from Mallorca were significantly younger than those from Asturias (Mann–Whitney test: U = 2740, n1 = 45, n2 = 92, P = 0.002) and Barcelona (Mann–Whitney test: U = 5106, n1 = 94, n2 = 92, P = 0.032) f Barcelona and Mallorca had some sick dogs (Chi-square test: X2 = 13.4, df = 3, P = 0.004) while all dogs in Asturias and Cádiz were apparently healthy Page 4 of 12 Baxarias et al. Parasites & Vectors (2022) 15:164 Page 5 of 12 times (3 min each wash) with PBS-Tween and once (1 Statistical analysis min) with PBS. The samples were then incubated for A descriptive analysis of all collected data was per- 1 h at 37 °C with peroxidase-conjugated Protein A (Per- formed. Qualitative variables (sex [female/male], oxidase Conjugate Protein A; Merck KGaA) at a con- breed [purebred/mixed breed], age [young/adult] and centration of 0.16 ng/µl. After incubation, the plates ELISA results [positive/negative]) were assessed with were washed three times with PBS-Tween followed a Fisher’s exact test when only two groups were com- by an additional wash with PBS. Then, o-phenylenedi- pared and with a Chi-square test when there were amine and substrate buffer (SIGMAFAST OPD; Merck more than two groups. Quantitative variables (age, EU) KGaA) were added to the plates and the reaction was were assessed using a non-parametric Mann–Whitney finally stopped with 5 M ­H2SO4. The results were read U-test when two groups were compared (clinical status: at 492 nm in a spectrophotometer (MB-580 HEALES; apparently healthy/sick), and the Kruskal–Wallis H-test Shenzhen Huisong Technology Development Co., Ltd, was used when more than two groups were compared Shenzhen, China) and were defined as ELISA units (geographical distribution). Spearman’s correlation test (EU) in relation with a positive canine serum sample was carried out to detect a relationship between ELISA used as a calibrator set at 100 EU. The cut-off of the quantitative results of the serum and OT. serum in-house ELISA was already determined to be The agreement between the interpretation of the 35 EU using the ELISA results of 80 dogs from a non- results of serum and OT ELISAs was calculated by per- endemic area, as previously described [35]. Cut-off was cent agreement and by Cohen’s kappa statistic (κ) for established by the standard deviation (SD) method, agreement (kappa agreement test). When evaluating consisting of multiplying the SD of the results by four kappa agreement, the agreement was considered to and adding up the mean of the results obtained by the be slight when it ranged from 0.00 to 0.20, fair when ELISA (mean + 4 SD). Serum was classified as high at range 0.21–0.40, moderate at range 0.41–0.60, sub- positive when the result was ≥ 300 EU, medium posi- stantial at range 0.61–0.80 and almost perfect at range tive when the result was ≥ 150 EU and < 300 EU, low 0.81–1.00 [36]. positive when the result was ≥ 35 EU and < 150 EU and A P-value of < 0.05 was considered to be statistically negative when the result was < 35 EU [35]. significant. The Shapiro–Wilk test was performed to detect normal distribution of quantitative variables. Areas where < 20 dogs were sampled were excluded Oral transudate ELISA from the geographical distribution analysis. The statis- The in-house ELISA was performed on OTs of all dogs tical analysis was performed using the package Stats for studied as previously described [21] with some modifi- R software version i386 3.6.1 for Windows. Cohen’s κ cations. OT samples were diluted to 1:5 in PBS-Tween statistic for agreement was calculated using free on-line with 1% dry milk and incubated at 37 °C for 1 h. Washes GraphPad software (https://​w ww.​graph​pad.​com/​quick​ were performed as described for the serum samples, and calcs/​kappa1/). Graphs were plotted using Graphad peroxidase conjugated Protein A (Peroxidase Conjugate Prism version 5.00 for Windows (GraphPad Software, Protein A; Merck KGaA) at a concentration of 0.5 ng/µl San Diego, CA, USA). was added and then incubated at 37 °C for 1 h. Washes were repeated and o-phenylenediamine and substrate buffer (SIGMAFAST OPD; Merck KGaA) were added to Results the samples. The reaction was stopped with 5 M ­H2SO4. Serum ELISA results As described for the serum samples, the results were read The rate of L. infantum infection determined by serum in a spectrophotometer (MB-580 HEALES; Shenzhen ELISA and the serological status of dogs (negative, low Huisong Technology Development Co., Ltd.) at 492 nm positive, medium positive or high positive) are shown and were quantified as EU relative to a positive canine in Table 2. The infection rate was significantly higher in OT sample used as a calibrator set at 100 EU. The cut- adult dogs than in young dogs (42.7 vs 21.5%; Fisher’s off of the OT in-house ELISA was established using the exact test: P = 0.001), and lower in apparently healthy ELISA results of 30 non-infected healthy Beagles. With dogs than in sick dogs (29.5 vs 100%; Fisher’s exact the values of these 30 dogs, the SD was calculated and test: P < 0.0001) (Table 2). No significant differences multiplied by 4, and then added up to the mean of all were observed between dogs of different sex and breed the results (mean + 4 SD), resulting in a cut-off value of (Table 2). When dogs from different geographical loca- 28 EU. The OTs were then classified as positive when the tions were compared, a significantly lower rate of infec- result was ≥ than 28 EU and negative when it was < 28 tion was found in Asturias when compared to the other EU. Baxarias et al. Parasites & Vectors (2022) 15:164 Page 6 of 12 Table 2 Rate of L. infantum infection, percent agreement and Cohen’s kappa agreement between enzyme-linked immunosorbent assay results for serum and oral transudate samples Classification (number of dogs) Number of positive dogs (%) Percent Cohen’s κ agreement (interpretation) 95% CI of agreement (%) Cohen’s κ Serum ELISA OT ELISA agreement Total of dogs (407) 149 (36.6) 133 (32.7) 345 (84.8) 0.66 (substantial agreement) 0.59–0.74 Sex Female (209) 78 (37.3) 71 (34) 174 (83.3) 0.64 (substantial agreement) 0.53–0.75 Male (198) 71 (35.9) 62 (31.3) 171 (86.4) 0.7 (substantial agreement) 0.59–0.8 Agea Young (79) 17 (21.5)b 15 (19)c 69 (87.3) 0.61 (substantial agreement) 0.39–0.83 b Adult (267) 114 (42.7) 103 (38.6)c 224 (83.9) 0.67 (substantial agreement) 0.58–0.76 Breed Purebred (190) 63 (33.2) 63 (33.2) 158 (83.2) 0.62 (substantial agreement) 0.5–0.74 Mixed breed (217) 86 (39.6) 70 (32.3) 187 (86.2) 0.7 (substantial agreement) 0.6–0.8 Geographical distribution Asturias (47) 0 (0)d 3 (6.4)e 44 (93.6) -h -h Barcelona (110) 30 (27.3)d 23 (20.9)e 99 (90) 0.73 (substantial agreement) 0.58–0.88 Cádiz (54) 9 (16.7)d 7 (13)e 48 (88.9) 0.56 (moderate agreement) 0.25–0.87 Mallorca (94) 33 (35.1)d 28 (29.8)e 74 (79.6) 0.54 (moderate agreement) 0.36–0.72 Clinical status Sick (41) 41 (100)f 37 (90.2)g 37 (90.2) -h -h f g Apparently healthy (366) 108 (29.5) 96 (26.2) 308 (84.2) 0.61 (substantial agreement) 0.52–0.7 Negative control (Asturias) and 41 (46.6) 40 (45.5) 81 (92.1) 0.84 (almost perfect agreement) 0.73–0.95 positive control dogs (Sick) (88) Serological status High positive (26) 26 (100) 26 (100) 26 (100) -h -h h Medium positive (40) 40 (100) 34 (85) 34 (85) - -h h Low positive (83) 83 (100) 50 (60.2) 50 (60.2) - -h h Negative (258) 0 (0) 23 (8.9) 235 (91.1) - -h Negative control (Asturias) and 66 (58.4) 63 (55.8) 104 (92) 0.84 (almost perfect agreement) 0.74–0.94 high and medium positive dogs (113) CI confidence interval, ELISA enzyme-linked immunosorbent assay, OT oral transudate a Age was not recorded in 61 dogs b Fisher’s Exact test: P = 0.001 c Fisher’s Exact test: P = 0.001 d Chi-square test: χ2 = 23.7, df = 3, P < 0.001 e Chi-square test: χ2 = 12.8, df = 3, P = 0.004 f Fisher’s Exact test: P < 0.0001 g Fisher’s Exact test: P < 0.0001 h Cohen’s kappa (κ) agreement could not be calculated in the Asturias, the seropositive sick dogs and the serological status groups because of the lack of positivity to both tests or the lack of negativity to both tests locations (Chi-square test: χ2 = 23.7, df = 3, P < 0.001) No significant differences were observed when differ- (Table 2). ent sexes and breeds were compared (Table 3). When Regarding the quantitative ELISA results shown in groups from different geographical locations were com- Table 3, adult and sick dogs presented significantly pared (Table 3; Fig. 3a), Asturias (3.7 EU) presented a higher median EU values than young and apparently significantly lower median value than Barcelona (11.4 healthy dogs, respectively (Fig. 2; Mann–Whitney EU), Cádiz (6.3 EU) and Mallorca (25.3 EU) (Kruskal– test: U = 12,389, n1 = 267, n2 = 79, P = 0.018; Mann– Wallis H-test: χ2 = 99.2, df = 3, P < 0.0001) while Bar- Whitney test: U = 829, n1 = 366, n2 = 41, P < 0.0001). celona and Mallorca had significantly higher median values than Cádiz. Baxarias et al. Parasites & Vectors (2022) 15:164 Page 7 of 12 Table 3 Median values of serum and OT EU according to the degree of reactivity to sera ELISA Classification of dogs (number of dogs) Median of serum EU (min–max)a Median of OT EU (min–max)a Total of dogs (407) 17.7 (0–300) 14.9 (0–300) Sex Female (209) 22.3 (0–300) 13.8 (0–300) Male (198) 15.9 (0–300) 15.8 (0–300) Ageb Young (79) 11.0 (1.8–300)c 9.9 (0–250.5)f c Adult (267) 22.3 (0–300) 18.1 (0–300)f Breed Purebred (190) 16.9 (0–300) 16.0 (0–300) Mixed breed (217) 18.2 (0–300) 13.6 (0–300) Geographical location Asturias (47) 3.7 (0–7.4)d 8.6 (0.2–39.9)g d Barcelona (110) 11.4 (2.7–300) 12.0 (0.2–300)g d Cádiz (54) 6.3 (0–300) 4.1 (0–300)g d Mallorca (94) 25.3 (3.2–300) 14.7 (2.2–166.5)g Clinical status Sick (41) 300.0 (39.3–300)e 111.7 (11.6–300)h e Apparently healthy (366) 12.8 (0–300) 12.9 (0–300)h Serological status Negative (258) 7.0 (0–34.7) 9.7 (0–76.4) Low positive (83) 59.2 (35–142.9) 38.1 (0–166.5) Medium positive (40) 210.4 (150.4–291.8) 80.4 (0–300) High positive (26) 300.0 (300) 160.9 (28.5–300) Total positives (149) 132.8 (35–300) 59.2 (0–300) EU ELISA units, OT oral transudate, max maximum, min minimum a Samples with a value of 300 EU may actually be higher as the spectrophotometer is only able to read up to 3 units of optical density b Age was not recorded in 61 dogs c Mann–Whitney test: U = 12,389, n1 = 267, n2 = 79, P = 0.018 d Kruskal–Wallis H-test: χ2 = 99.2, df = 3, P < 0.0001 e Mann–Whitney test: U = 829, n1 = 366, n2 = 41, P < 0.0001 f Mann–Whitney test: U = 12,863, n1 = 267, n2 = 79, P = 0.003 g Kruskal–Wallis H-test: χ2 = 38.7, df = 3, P < 0.0001 h Mann–Whitney test: U = 1461, n1 = 366, n2 = 41, P < 0.0001 Oral transudate ELISA results Regarding the quantitative ELISA results shown in The rate of L. infantum infection determined on OT Table 3, as found in the serum results, adult and sick ELISA is shown in Table 2. Similar to the results for the dogs presented a significantly higher mean EU value serum samples, the rate of OT sample positivity was than young and apparently healthy dogs, respectively also significantly higher in adult (Fisher’s exact test: (Mann–Whitney test: U = 12,863, n1 = 267, n2 = 79, P = 0.001) and sick dogs (Fisher’s exact test: P < 0.0001) P = 0.003; Mann–Whitney test: U = 1461, n1 = 366, (38.6%) when compared to young dogs (19%) while it n2 = 41, P < 0.0001) (Fig. 2b). No significant differ- was lower in apparently healthy dogs than in sick dogs ences were observed between different sex and breed (26.2% vs 90.2%) (Table 2). No significant differences (Table 3). When groups of dogs from different geo- were observed in terms of sex and breed (Table 2). graphical location were compared (Table 3; Fig. 3b), When comparisons were made between groups of dogs Asturias (8.6 EU) and Cádiz (4.1 EU) presented a sig- from different geographic locations, a significantly nificantly lower mean EU value than Barcelona (12 lower rate of infection was still found for dogs from EU) and Mallorca (14.7 EU) (Kruskal–Wallis H-test: Asturias compared to those from other locations (Chi- χ2 = 38.7, df = 3, P < 0.0001). square test: χ2 = 12.8, df = 3, P = 0.004) (Table 2). Baxarias et al. Parasites & Vectors (2022) 15:164 Page 8 of 12 Fig. 2 Antibody levels against L. infantum (EU) as determined by the in-house ELISA performed on serum (a) and OT (b) samples collected from dogs classified according to clinical status (apparently healthy vs sick). Horizontal solid black lines indicate the median. Horizontal black dashed lines indicate the cut-off: 35 EU in serum ELISA and 28 EU in OT ELISA. ELISA enzyme-linked immunosorbent assay, EU ELISA units, OT oral transudate Fig. 3 Antibody levels against L. infantum (EU) by the in-house ELISA performed on serum (a) and OT (b) samples collected from dogs classified according to geographical distribution. Horizontal solid black lines indicate the median. Horizontal black dashed lines indicate the cut-off: 35 EU in serum ELISA and 28 EU in OT ELISA. ELISA enzyme-linked immunosorbent assay, EU ELISA units, OT oral transudate Correlation and comparison between ELISA results for correlation was lower (Spearman’s correlation coeffi- serum and OT samples cient ­rs = 0.3079, P = 0.005). A positive correlation was established between the Of the total of 407 dogs, 235 (57.7%) were negative by results of the in-house ELISA for the serum and OT both serum and OT ELISA while 110 (27%) were posi- samples (Spearman’s correlation coefficient rs = 0.6687, tive to both tests. In contrast, there was disagreement P < 0.0001) when all samples were studied (Fig. 4). The regarding the remaining 62 dogs (15.3%). Six medium positive correlation improved when only Asturias dogs seropositive and 33 low seropositive dogs (9.6%) with (negative control) and sick dogs (positive control) a median of 55.3 EU (ranging from 35 to 288.9 EU) were investigated (Spearman’s correlation coefficient were negative by OT ELISA with a median of 12.4 EU ­rs = 0.7479, P < 0.0001) and also when only Asturias (ranging from 0 to 27.2 EU) while 23 seronegative dogs seronegative dogs and high and medium seropositive (5.7%) with a median of 16.7 EU (ranging from 0.9 to dogs were studied (Spearman’s correlation coefficient 30.8 EU) were positive by OT ELISA with a median of ­rs = 0.7585, P < 0.0001). On the other hand, when only 43.4 EU (ranging from 29.4 to 76.4 EU) (Fig. 5). The low seropositive dogs were investigated, the positive percentage agreement and Cohen’s kappa agreement between serum and OT ELISA results was substantial Baxarias et al. Parasites & Vectors (2022) 15:164 Page 9 of 12 Discussion A quantitative in-house ELISA technique [21] was adapted in the present study to detect specific anti-Leish- mania antibodies in OT canine samples and to assess the diagnostic performance of this ELISA. This ELISA is currently performed on serum samples to detect specific immunoglobulins as it has been proven that most dogs infected with an active disease show high levels of dif- ferent isotypes of antibodies [9, 18, 37]. The presence of several types of immunoglobulins has also been studied in saliva [28]. IgA has been proven to be present in saliva as it is secreted in the salivary glands by plasma cells, and plasma-derived antibodies have been found, such as IgG Fig. 4 Spearman’s correlation coefficient (rs) for the serum and OT and IgM [28]. Specific canine anti-Leishmania antibod- ELISA results (rs [407] = 0.6687, P < 0.0001). Red filled circles indicate the individual results for each sampled dog. The horizontal solid black ies have also been documented in oral fluid samples by line indicates the cut-off: 35 EU in serum ELISA (Y-axis) and 28 EU using a TR-IFMA [24, 29–31], which is a technique that in OT ELISA (X-axis). ELISA enzyme-linked immunosorbent assay, EU has shown a broader range of detection of antibodies ELISA units, OT oral transudate in serum than ELISA. These studies showed great suc- cess at discriminating between seropositive and seron- egative dogs with no overlapping in terms of evaluating IgG2 [24, 29–31]. However, the authors of these studies were not successful at correctly differentiating seroposi- tive dogs from seronegative based on IgA evaluation [24, 29–31]. These studies provided the first evidence of the potential of oral fluid for the quantification of anti-Leish- mania IgG2 to diagnose CanL [24, 29–31]. Nonetheless, no studies have evaluated the ability to detect anti-Leish- mania antibodies by using a quantitative in-house ELISA technique in OT samples until now. Additionally, the first study performed on oral fluid samples for the diagnosis of CanL was carried out on a very homogeneous group of dogs, using dogs with advanced clinical leishmaniosis and high antibody levels [24], while in the present study, dogs with subclinical infection and low antibody titers were also included. In the present study, the agreement between the quali- tative interpretation of serum and OT ELISA results was Fig. 5 Proportion of positive and negative samples based on the evaluated using two methods: (1) percent agreement and results of both the serum and OT ELISAs. Neg Negative, Pos positive (2) agreement according to the kappa agreement statis- tic. The percent agreement is easy to calculate and can be interpreted directly, but it does not take into account (0.66) when studying the whole group while it went the agreements made by chance [38]. On the other hand, from almost perfect to moderate depending on the Cohen’s kappa agreement statistic is a statistical value classification studied (Table 2). useful for assessing inter-rater or intra-rater reliability Comparison of the EU values for the serum and OT and takes into consideration the possibility of chance samples according to degrees of reactivity is shown in [38]. A Cohen’s kappa agreement of > 0.80 is needed to Table 3. When comparing the OT EU, antibody levels be able to validate a new test [38]. When Cohen’s kappa were found to be significantly higher in OT samples agreement was interpreted for the 407 dogs, a substan- with a high or medium positive EU value for the serum tial agreement of 0.66 was found. As stated earlier in this ELISA than in those with a low positive serum ELISA text, this agreement is not sufficient to affirm that OT can (Kruskal–Wallis H-test: χ2 = 43.2, df = 2, P < 0.0001). be used to correctly differentiate between seropositive and seronegative dogs by means of an in-house ELISA. However, a high number of dogs in this study presented Baxarias et al. Parasites & Vectors (2022) 15:164 Page 10 of 12 subclinical infection and low seropositive antibody lev- unexpected. One possible explanation is that sand flies els, which is a likely explanation of why the agreement mainly feed on skin areas with very little hair, such as the was lower than found in previous studies where the dog face [15], which could lead to a local expression of par- populations studied were mostly sick dogs with advanced asite-specific immunoglobulins before the parasite dis- clinical leishmaniosis [24, 31]. When Cohen’s kappa seminates systemically. A second possibility is that there agreement was obtained only for seronegative dogs from may be an as-yet unknown cross-reactivity with another Asturias (a low endemicity area) and for sick dogs with pathogen, such as oral bacteria, in some dogs with poor clinical signs and/or clinicopathological abnormalities dental hygiene and dental disease, such as gingivitis, sto- compatible with L. infantum infection, an almost per- matitis and periodontal disease. Further studies on the fect agreement of 0.84 was obtained. The same result diagnostic performance of the OT ELISA are needed to (0.84) was found when Cohen’s kappa agreement was evaluate this hypothesis. obtained for seronegative dogs from Asturias and sero- When taking locations of origin into consideration, the positive dogs with high or medium levels of antibody lev- percent agreement was higher in Asturias (93.6%), fol- els. These findings agree with those reported in previous lowed by Barcelona (90%), Cádiz (88.9%) and Mallorca studies [24, 31] and highlight the usefulness of detecting (79.6%). In comparison, Cohen’s kappa agreement was antibodies against L. infantum in OT in dogs with clinical substantial in Barcelona (0.73), followed by Cádiz with leishmaniosis or progressing towards disease. a moderate agreement (0.56) and Mallorca, also with a When the percent agreement was evaluated, an agree- moderate agreement (0.54). ment of 84.8% was found. The remaining samples from Despite the OT showing a lower diagnostic value than 15.2% (62) dogs showed disagreements between the serum according to the quantitative in-house ELISA used serum and OT ELISA. Included in these samples that in this study, a good percentage of success was obtained disagreed, 39 were from seropositive dogs (39/62 dogs; for the OT samples. In addition, OT sample collection is 62.9%) that were negative by the OT ELISA. There are easy, cheap, non-invasive and painless; consequently, OT several reasons that could explain this disagreement in could be of use in specific cases, such as dogs who do not results from the OT and serum ELISA. First, there may have easy access to veterinary clinics, dogs that need a be a lesser ability to detect seropositive dogs with a low continued follow-up or aggressive dogs that can only be serum antibody, as detected when comparing the Cohen’s touched by its owner. κ agreement statistic described above. This seems to be Further studies are needed to increase the reliability of the most plausible reason as when only seronegative the results of the present study. First, an investigation of dogs from Asturias and sick dogs with clinical signs and/ the OT quality must be performed to confirm the correct or clinicopathological abnormalities compatible with collection of the samples before performing OT ELISA. L. infantum infection were studied, the percent agree- In addition, a group of dogs with poor dental hygiene and ment increased to 92.1%. A similar result, i.e. 92%, presenting dental diseases could be added to the study was obtained when the results from only seronegative population to assess the possibility of poor dental health dogs from Asturias and seropositive dogs with high or being a factor of false positivity by OT ELISA. Also, it medium levels of antibody levels were considered. This would be also of interest to perform a longitudinal study result was to be expected as the sick group presented a of those dogs that were seronegative yet tested positive higher proportion of high serum antibody levels com- by OT ELISA, as well as those dogs that tested negative pared to the apparently healthy group which had a higher for the OT ELISA yet tested positive by the serum ELISA, proportion of low antibody levels. Another explanation to describe antibody kinetics. Finally, other techniques could be a lack of homogenous OT sample collection, as using OT could also be developed and improved. Even even if untrained personnel can perform this procedure, ELISA as a serological test has some limitations in terms it is difficult to perform correctly if the standardized of the detection of infection as it can detect antibodies protocol is not followed as described [39]. For example, elicited by Leishmania vaccines in dogs [17]. if the impregnated swabs were not kept in the mouth The seroprevalence of canine L. infantum infection was of the dog for at least 2 min, insufficient OT could have around 10% [14, 33, 40] between 2011 and 2020 in Spain, been absorbed. As the samples in this study were col- which is lower than the seroprevalence detected in the lected by several veterinarians, even though a standard- present study (36.6%). In terms of specific Spanish areas, ized protocol was recommended and agreed to, we could Asturias has always presented one of the lowest sero- not confirm that all samples were always collected in a prevalence rates [14, 33, 40], usually around 1%, while similar manner. On the other hand, of these 62 disagree- the rates from Cádiz and Mallorca are usually higher ments, 23 seronegative dogs (23/62 dogs; 37.1%) turned than 15% [14]. These results resemble those found in the out to be positive in the OT ELISA. These results were present study, with low rates in Asturias (0%) and high Baxarias et al. Parasites & Vectors (2022) 15:164 Page 11 of 12 rates in Cádiz (16.7%) and Mallorca (35.1%). Regarding (CV) Albaitari (Álava, Spain); CV Alta Paterna (Valencia, Spain); CV Bocairent (Valencia, Spain); CV El Acebo (Madrid, Spain); CV Faunivet (Castellón, Spain); the results found in Barcelona (27.3%), a previous study CV Germanias (Valencia, Spain); CV Jara (Córdoba, Spain); CV La Corraliza performed in 27 sick and 20 clinically healthy dogs in (Navarra, Spain); CV Malaka (Málaga, Spain); CV Nutrizoo (Murcia, Spain); CV 2006 [41] documented a 65% seroprevalence of L. infan- Pawspatas (Murcia, Spain); CV Sevilla (Jaén, Spain); Hospital Pratdesaba veteri‑ naris (Barcelona, Spain); Hospital Veteralia Movet (Barcelona, Spain); Hospital tum in Barcelona, but no other studies in this area have Veterinari (HV) de Catalunya (Barcelona, Spain); HV Desvern (Barcelona, Spain); been carried out in the last decade. However, seropreva- HV Garbí (Barcelona, Spain); HV San Bernardo (Castellón, Spain); Infantes vet‑ lence rates of around 13% were detected in other areas of erinarios (Ciudad Caminando con ellos (Murcia, Spain); Protectora Modepran (Valencia, Spain); Provença Centre veterinari (Barcelona, Spain); Refugio canino Catalonia [33, 40]. Interestingly, the seroprevalence rates La Reserva (Almeria, Spain); and Vilazoo veterinaris (Islas Baleares, Spain). detected in this study seem to be slightly higher than the 16th CVBD® World Forum Symposium. The paper has been sponsored by Elanco Animal Health in the framework of those described in previous studies [14, 33, 40, 41]. This could be related to the number of sick dogs included in Authors’ contributions the Barcelona (12.7%) and Mallorca (7.4%) groups. The MB, AAF, MMA and LSG collected serum and OT samples. MB and JV incidence rate of human leishmaniosis in Spain was 0.62 performed laboratory work (ELISAs). MB performed statistical analysis and prepared the figures. MB and LSG contributed with data analysis and interpre‑ cases per 100,000 inhabitants between 2005 and 2017, tation. JV wrote the first draft of the manuscript. MB and LSG wrote the final with cases mainly distributed throughout the Mediter- manuscript. All authors read and approved the final manuscript. ranean region [42]. However, asymptomatic infections Funding are also common in humans in Spain and Mediterranean This work was founded by Spanish ministry grant, Ministerio de Economía basin countries as recently reviewed elsewhere [43]. y competitividad y Fondo Europeo de Desarrollo Regional (FEDER, EU) We also detected higher serological rates of L. infantum (AGL2015-68477). This work was also partially supported by Ecuphar veteri‑ naria SLU. infection in both adult and sick dogs. A high rate should be expected in sick dogs that have been already diag- Availability of data and materials nosed with leishmaniosis and still present clinical signs The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. and/or clinicopathological abnormalities [9, 15]. In terms of age of dogs, previous studies have found that puppies (< 1 year old) have a lower rate of L. infantum infection Declarations than dogs aged > 1 year old [33, 40] and that the risk of Ethics approval and consent to participate Leishmania infection increases with increasing age [40]. Ethical review and approval were waived for the study, due to non-invasive procedures (OT sampling) and use of residual samples (serum). A signed con‑ sent form was obtained from the owner or the person in charge of the dog(s). Conclusions Consent for publication Not applicable. In conclusion, the present study demonstrates an almost perfect to moderate agreement between OT and serum Competing interests samples using a quantitative in-house ELISA for Leish- The authors declare that they have no competing interests. mania antibodies. These results are promising for the Author details detection of infection in sick dogs with high antibody 1 Departament de Medicina i Cirurgia Animals, Facultat de Veterinària, Universi‑ levels while they seem to be less optimal in apparently tat Autònoma de Barcelona, Cerdanyola del Vallès, Barcelona, Spain. 2 Departa‑ ment de Biologia, Sanitat i Medi Ambient, Facultat de Farmàcia i Ciències de healthy dogs with low antibody levels. Further studies l’Alimentació, Universitat de Barcelona, Barcelona, Barcelona, Spain. could improve OT serology and its reliability and value as a future diagnostic technique for L. infantum infection Received: 24 January 2022 Accepted: 18 March 2022 when compared with other diagnostic methods for CanL. Abbreviations References CanL: Canine leishmaniosis; ELISA: Enzyme-linked immunosorbent assay; EU: 1. Ribeiro RR, Suzan M, Michalick M, Eduardo M, Cheim C, Jean F, et al. ELISA units; IFN-γ: Interferon gamma; IgA: Immunoglobin A; IgG: Immuno‑ Canine leishmaniasis: an overview of the current status and strategies for globin G; IgM: Immunoglobin M; OT: Oral transudate; PBS: Phosphate buffered control. Biomed Res Int. 2018;2018:1–12. saline; ROS: Reactive oxygen species; SD: Standard deviation; TR-IFMA: Time- 2. Baneth G, Koutinas AF, Solano-Gallego L, Bourdeau P, Ferrer L. 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