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Abstract Background The accuracy of blood cell ratios (BCRs) as cost-effective and easily accessible diagnostic and prognostic markers of inflammatory conditions has been investigated in veterinary medicine in recent years. Methods Neutrophil-to-lymphocyte (NLR), monocyte-to-lymphocyte (MLR), and platelet-to-lymphocyte (PLR) ratios were studied in 195 dogs clinically evaluated and tested for anti-Leishmania infantum (Li) antibodies (Li-seronegative (Li −), n = 10; Li-seropositive clinically healthy (Li + healthy), n = 100; Li-seropositive with clinical and/or clinicopathological abnormalities (Li + sick), n = 85). The Li + sick dogs were classified in LeishVet stages IIa/IIb (Li + IIa/IIb) (n = 66) and III/IV (Li + III/IV) (n = 19). BCR relationships with LeishVet clinical stage, antibody levels, and serum protein electrophoretic fraction concentrations were investigated. Results Higher NLR values were found in Li +, Li + healthy, and Li + IIa/IIb sick dogs compared to Li − dogs (P

Abstract Background Domperidone (Leisguard®) is an immunomodulatory drug used as a preventive measure in healthy dogs. However, no studies have been published in healthy Leishmania infantum-seropositive dogs. The aim of this study was to evaluate the clinical efficacy and safety of domperidone as immunotherapy in Leishmania-seropositive healthy dogs. Methods Sixty-seven dogs were treated with domperidone at 0.5 mg/kg and 44 dogs received placebo, once daily for 4 consecutive weeks. Monthly treatments were repeated every 4 months until the end of the 1-year follow-up period. Veterinary examinations were performed on days 0, 30, 120, 150, 240, 270 and 360. Samples of blood and urine were collected on days 0, 120, 240 and 360 for routine laboratory tests and quantitative in-house ELISA for the detection of L. infantum-specific antibodies. Furthermore, Leishmania real-time PCR and IFN-γ ELISA were performed at day 0 and the end of the study. Dogs that developed disease were withdrawn from the study and classified as sick dogs. Adverse drug reactions were reported. Results Thirty dogs developed disease during the follow-up period: 13/67 (19.4%) in the group treated with domperidone and 17/44 (38.6%) in the placebo-treated group (P = 0.03). Low-seropositive dogs treated with domperidone (4/40, 9.1%) were significantly less likely to develop disease compared to low-seropositive dogs treated with placebo (7/24, 29.2%; P = 0.04), while no differences were found between domperidone (9/23, 39.1%) and placebo (10/20, 50%) in medium- to high-seropositive dogs. At the end of the study, a higher proportion of Leishmania PCR-positive dogs was observed in the placebo-treated group (16/33, 48.5%) compared to the domperidone group (13/51, 25.5%; P = 0.04). Furthermore, low-seropositive dogs treated with domperidone with an increase of IFN-γ concentration presented a higher increase than those treated with placebo at the end of the study. Four dogs treated with domperidone presented self-limiting diarrhea. Conclusions Healthy dogs with low L. infantum antibody levels treated with domperidone were less likely to develop disease compared to placebo-treated dogs. Furthermore, domperidone presented a good safety profile. Graphical abstract

Abstract Background Canine leishmaniosis caused by Leishmania infantum is an endemic disease in Spain. The dog is considered the main reservoir, and the detection of specific serum antibodies against L. infantum antigen is the most used technique for diagnosing this infection. The LEISCAN LEISHMANIA ELISA test is a commercialized enzyme‐linked immunosorbent assay for the detection and measurement of canine anti‐Leishmania serum antibodies. Objectives The aim of this study was to assess seroprevalence results of apparently healthy dogs in different areas of Spain using LEISCAN. Methods Collection of sera from 5451 apparently healthy dogs was performed between 2020 and 2021 in different areas of Spain. Dogs were of adult age (≥12 months), were not previously diagnosed with clinical leishmaniosis or vaccinated against Leishmania and did not present clinical signs compatible with L. infantum infection. LEISCAN was performed following the manufacturer's protocol. Results The overall seroprevalence was 5.5%. The highest seroprevalences were found in the Southeast of Spain: Comunidad Valenciana (14%) and Región de Murcia (14%), whereas the lowest seroprevalences were found in Northern Spain: Galicia (1%), Navarra (2%) and Castilla y León (2%) (p‐value

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 r s = 0.6687, P

Abstract Background There are several screening tools for detecting Leishmania infantum infection in dogs and various preventive measures to protect against it. Some studies have investigated them, but not many have described their current use. The aim of this study was to investigate which preventive measures and serological screening tools for L. infantum infection were employed from 2012 to 2018 in dogs from different endemic European countries. Methods A set of electronic datasheets was completed for each dog from several veterinary centres. Classification of preventive measures included: (1) repellents, (2) vaccines and (3) immunomodulators. Classification of serological tests included the: (1) direct agglutination test (DAT), (2) enzyme-linked immunosorbent assay (ELISA), (3) indirect immunofluorescence (IFI), (4) rapid tests and (5) other assays. Dogs were also classified depending on their risk of exposure and living area. Results Information from 3762 dogs was gathered. Preventive measures were applied in 91.5% of dogs and the most frequently used were repellents (86.2%) followed by vaccines (39.8%) and Leisguard® (15.3%). The different types of repellents (collar and spot-on) were used similarly. A combination of a vaccine and repellents was preferred in the high-risk group while the low-risk preferred a combination of Leisguard® and a repellent (Chi-square test: X 2 = 88.41, df = 10, P

This study aimed to investigate the role of neutrophils in canine leishmaniosis by assessing neutrophil activation and its relationship with different states of L. infantum infection and antibody and IFN-γ production. Dogs were categorized into five groups: healthy-seronegative (n = 25), healthy-seropositive (n = 21), LeishVet-stage I (n = 25), Leishvet-stage II (n = 41), and LeishVet-stage III–IV (n = 16). Results of the nitroblue tetrazolium reduction test (NBT) showed significantly higher neutrophil activation in stage I (median:17.17, range: [7.33–31.50]%) compared to in healthy-seronegative (4.10 [1.20–18.00]%), healthy-seropositive (7.65 [3.98–21.74]%), stage II (6.50 [1.50–28.70]%), and stage III–IV (7.50 [3.00–16.75]%) groups (p < 0.0001). Healthy-seropositive dogs also displayed higher values than all groups except stage I. Stages II and III–IV did not show significant differences compared to healthy-seronegative. Regarding IFN-γ, stage I dogs had higher concentrations (median:127.90, range: [0–3998.00] pg/mL) than healthy-seronegative (0 [0–109.50] pg/mL) (p = 0.0002), stage II (9.00 [0–5086.00] pg/mL) (p = 0.045), and stage III–IV (3.50 [80.00–548.80] pg/mL) (p = 0.02) dogs. Stage II dogs showed increased IFN-γ compared to healthy-seronegative dogs (p = 0.015), while stage III–IV dogs had no significant differences compared to healthy-seronegative dogs (p = 0.12). Healthy-seropositive dogs had elevated IFN-γ concentrations compared to healthy-seronegative dogs (p = 0.001) and dogs in stage III–IV (p = 0.03). In conclusion, neutrophil activation was higher in dogs with mild disease and healthy-seropositive dogs, and a relationship between neutrophil activation and the production of IFN-γ was found.

Abstract Background There is limited clinical or epidemiological knowledge regarding Bartonella infection in cats, and no serological studies have compared the presence of antibodies against different Bartonella species. Moreover, there are limited feline Bartonella studies investigating co-infections with other vector-borne pathogens and the associated risk factors. Therefore, the objective of this study was to investigate Bartonella spp. infections and co-infections with other pathogens in cats from Barcelona (Spain) based on serological and/or molecular techniques and to determine associated risk factors. Methods We studied colony and owned cats (n = 135). Sera were tested for Bartonella henselae-, Bartonella quintana-, and Bartonella koehlerae-specific antibodies using endpoint in-house immunofluorescence antibody assays. Bartonella real-time PCR (qPCR) and conventional PCR (cPCR) were performed. In addition, cPCR followed by DNA sequencing was performed for other pathogenic organisms (Anaplasma, Babesia, Cytauxzoon, Ehrlichia, Hepatozoon, hemotropic Mycoplasma, and Theileria spp.). Results From 135 cats studied, 80.7% were seroreactive against at least one Bartonella species. Bartonella quintana, B. koehlerae, and B. henselae seroreactivity was 67.4, 77.0, and 80.7%, respectively. Substantial to almost perfect serological agreement was found between the three Bartonella species. Colony cats were more likely to be Bartonella spp.-seroreactive than owned cats. Moreover, cats aged ≤ 2 years were more likely to be Bartonella spp.-seroreactive. Bartonella spp. DNA was detected in the blood of 11.9% (n = 16) of cats. Cats were infected with B. henselae (n = 12), B. clarridgeiae (n = 3), and B. koehlerae (n = 1). Mycoplasma spp. DNA was amplified from 14% (n = 19) of cat blood specimens. Cats were infected with Mycoplasma haemofelis (n = 8), Candidatus M. haemominutum (n = 6), Candidatus Mycoplasma turicensis (n = 4), and Mycoplasma wenyonii (n = 1). Anaplasma, Babesia, Cytauxzoon, Ehrlichia spp., Hepatozoon, and Theileria spp. DNA was not amplified from any blood sample. Of the 16 Bartonella spp.-infected cats based on PCR results, six (37%) were co-infected with Mycoplasma spp. Conclusions Bartonella spp. and hemoplasma infections are prevalent in cats from the Barcelona area, whereas infection with Anaplasma spp., Babesia, Cytauxzoon, Ehrlichia spp., Hepatozoon, and Theileria infections were not detected. Co-infection with hemotropic Mycoplasma appears to be common in Bartonella-infected cats. To our knowledge, this study is the first to document M. wenyonii is infection in cats. Graphical Abstract

Canine leishmaniosis caused by Leishmania infantum is a disease with a wide range of clinical manifestations. Epidemiological serosurveys performed in Europe often lack a thorough assessment of clinical health status of studied dogs. The aim of this study was to evaluate signalment, immunological and parasitological status and clinicopathological findings of L. infantum-seropositive apparently healthy dogs (n = 212) living in endemic areas. Routine laboratory tests, endpoint in-house ELISA to quantify the anti-Leishmania antibodies, blood Leishmania qPCR and IFN-γ ELISA were performed. All dogs enrolled were L. infantum-seropositive and were classified as healthy (n = 105) or sick (n = 107) according to LeishVet guidelines. The sick group presented a higher proportion of medium to high antibody levels and positive qPCR and lower IFN-γ concentration compared to the healthy group. Sick dogs were mostly classified in LeishVet stage IIa. Biochemical alterations (98%) were the most common clinicopathological findings, with fewer urinary tract (46%) and hematological (40%) alterations. Apparently healthy L. infantum-seropositive dogs can be classified between truly healthy dogs and sick dogs with clinicopathological findings. Sick dogs presented medium to high seropositivity and parasitemia and low IFN-γ concentrations, and their most common clinicopathological abnormalities were serum protein alterations followed by proteinuria and lymphopenia.

Abstract Background Canine leishmaniosis (CanL) caused by Leishmania infantum can have several dermatological manifestations. The type of immune response elicited against the parasite appears to be at the basis for such clinical variability. Much of the work in CanL has focused on adaptive immune response and there are scarce data on the importance of the innate immune responses. Moreover, few studies have evaluated the immunological response in the cutaneous lesions in dogs naturally infected with L. infantum and with different degrees of disease severity, and no study has compared clinically-lesioned with normal-looking skin. Methods We determined and compared the transcription of toll like receptors (TLRs) 2, 4 and 7, interferon gamma (IFN-γ), interleukin (IL) 10 and programmed cell death protein ligand (PD-L) 1 by real-time PCR in paired clinically-lesioned and normal-looking skin from 25 diseased dogs (mild disease-stage I (n = 11) and moderate to severe disease-stages II and III (n = 14) as well as in normal-looking skin from healthy dogs (n = 10) from a non-endemic area. We also assessed the association between the transcripts in clinically-lesioned and normal-looking skin of dogs with leishmaniosis with clinicopathological, immunological and parasitological findings. Results Clinically-lesioned skin from mildly affected dogs was characterized by a significant upregulation of TLR2 (P

Abstract Background Canine leishmaniosis (CanL) due to Leishmania infantum is characterized by the development of both cellular and humoral immune responses. The dysfunction of T cell-mediated immunity leads to a lack of proliferation of T cells in response to Leishmania antigens with the consequence of parasite dissemination that seems to be related to a T cell exhaustion mediated by regulatory B cells expressing immunoglobulin D (IgD). The aim of this study was to determine and compare the total serum IgD in dogs with clinical leishmaniosis and in clinically healthy dogs. Results A total of 147 dog sera were studied. All dogs were tested for L. infantum-specific antibodies by quantitative ELISA. Interferon-gamma (IFN-γ) production was also determined by sandwich ELISA after blood stimulation with L. infantum soluble antigen (LSA) or concanavalin A (ConA). The quantification of total IgD was performed using a human IgD sandwich ELISA quantification set. Dogs were classified in three different groups. Group 1 included 40 clinically healthy non-infected dogs, all serologically negative to L. infantum-specific antibodies and non-producers of IFN-γ upon LSA stimulation. Group 2 included 63 clinically healthy infected dogs that were LSA IFN-γ producers (n = 61) and/or IFN-γ non-producers (n = 2) as well as negative to medium seropositive to L. infantum antigen. Finally, Group 3 included 44 dogs with clinical leishmaniosis (IFN-γ producers, n = 23; and IFN-γ non-producers, n = 21) that were negative to highly positive to L. infantum-specific antibodies. No significant differences were observed when the total IgD concentration was compared within groups. Additionally, total IgD of sick IFN-γ producers and IFN-γ non-producers was not significantly different. Finally, total IgD concentration was not statistically related to demographic parameters such as age, sex and breed. Conclusions The results of this study demonstrated that there were no differences between groups in total serum IgD. Total serum IgD does not appear to be a marker of disease in CanL.

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

Cats are the primary reservoir host for Bartonella henselae(B. henselae), an etiological agent of human bartonellosis, including cat scratch disease. Although Bartonella DNA has been amplified from salivary swabs from cats, dogs and humans, we are not aware of studies investigating Bartonella antibodies in oral fluid (OF). Using inhouse and commercial immunofluorescence antibody assays (IFA), the objective of this study was to detect and compare antibodies against B. henselae in paired OF and serum specimens from cats. Specimens were collected from shelter and client-owned cats. For serum specimens, B. henselae seroreactivity was 78% for both the inhouse and commercial IFA assays and 56.8% for OF specimens. Comparing serum and OF specimens, there was moderate Kappa agreement (Cohen’s k = 0.434) for detection of B. henselae antibodies. Oral fluid antibodies were more likely measurable in cats with high B. henselae serum antibody titers when compared with low antibody titers. In conclusion, B. henselae OF IFA antibody measurements were less sensitive compared to serum IFA measurements of ≥1:64. Oral fluid antibodies were detected more often in cats with high B. henselae serum antibody titers. Therefore, OF antibodies, detectable by IFA, is of limited utility for epidemiological or diagnostic testing in cats.

Dogs are the main reservoir for Leishmania infantum, manifesting from a subclinical to a fatal disease. Limited treatments are available, although new antiparasitics and immunomodulators are pursued. Polyhexamethylene biguanide (PHMB) has a broad antimicrobial spectrum, including antiparasitic activity. Here, we evaluated the potential for Toll-like receptor agonists (TLRa) and PHMB alone, and as polyplex nanoparticles containing PHMB and TLR4 or TLR9 agonists, to selectively kill L. infantum. Susceptibility of L. infantum promastigotes to PHMB, miltefosine, and allopurinol was performed, and the half-maximum inhibitory concentrations (IC50) were determined. Then, DH-82 cells were infected and treated with PHMB alone or combined with TLR4a (MPLA-SM) or TLR9a (CpG ODNs) and allopurinol alone. The IC50 values of L. infantum promastigotes were PHMB (1.495 µM), miltefosine (9.455 µM), and allopurinol (0.124 µM). After infection, treated DH-82 cells displayed a lower percentage (p = 0.0316), intensity (p = 0.0002), and index of infection (p = 0.0022) when compared to non-treated cells. PHMB induced lower percentage of infection alone (p = 0.043), in combination with TLR9a (p = 0.043), and with TLR4a (p = 0.0213). Supernatants were collected and used to measure TNF-α and IL-6 levels. Increased TNF-α was observed after PHMB plus TLR4a, relative to uninfected and infected untreated macrophages (p = 0.043). PHMB combined with TLR4a shows promise as a potential anti-L. infantum drug combination, as well as inducer of proinflammatory response, as demonstrated by decreased infection and increased TNF-α production.