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A 9-month-old mixed-breed dog developed generalised pustular dermatitis, accompanied by lethargy and hyperthermia, 7 days after oral fluralaner administration. Dermatopathological and microbiological evaluations were consistent with a pustular acantholytic dermatitis. A 4-month course of immunosuppressive therapy resulted in complete remission of lesions, which did not recur after therapy was withdrawn., (© 2024 ESVD and ACVD.)

The treatment of canine leishmaniosis commonly involves meglumine antimoniate with allopurinol or miltefosine with allopurinol. This study aims to compare the clinical and clinicopathological efficacy at 28-30 days of conventional dosing regimens for both treatments using the critically appraised topic methodology. A comprehensive search across three databases (PubMed, CAB Abstracts, and Web of Science) from March 2004 to September 2023 yielded 16 relevant articles, encompassing 325 ogs treated with meglumine antimoniate and allopurinol, and 273 dogs treated with miltefosine and allopurinol. The findings indicated a significantly higher rate of complete clinical cure in the group treated with meglumine antimoniate and allopurinol. Most dogs in both groups exhibited improvement in clinicopathological alterations after one month of treatment. No significant difference was observed in the number of dogs that showed a negative Leishmania qPCR between the two groups, one month post-treatment. However, quantitative serology results were not commonly reported in the available data and therefore this aspect could not be compared.

Papular dermatitis is a cutaneous manifestation of canine Leishmania infantum infection associated with mild disease. Although it is a typical presentation, nowadays, there is still no established treatment. This study evaluated the safety and clinical efficacy of local meglumine antimoniate, locally administered polyhexamethylene biguanide (PHMB) alone or PHMB in combination with a Toll-like receptor 4 agonist (TLR4a) for the treatment of papular dermatitis due to L. infantum and assessed parasitological and immunological markers in this disease. Twenty-eight dogs with papular dermatitis were divided randomly into four different groups; three of them were considered treatment groups: PHMB (n = 5), PHMB + TLR4a (n = 4), and meglumine antimoniate (n = 10)), and the remaining were considered the placebo group (n = 9), which was further subdivided into two sub-groups: diluent (n = 5) and TLR4a (n = 4). Dogs were treated locally every 12 h for four weeks. Compared to placebo, local administration of PHMB (alone or with TLR4a) showed a higher tendency towards resolution of papular dermatitis due to L. infantum infection at day 15 (χ 2 = 5.78; df = 2, p = 0.06) and day 30 (χ 2 = 4.; df = 2, p = 0.12), while local meglumine antimoniate administration demonstrated the fastest clinical resolution after 15 (χ 2 = 12.58; df = 2, p = 0.002) and 30 days post-treatment (χ 2 = 9.47; df = 2, p = 0.009). Meglumine antimoniate showed a higher tendency towards resolution at day 30 when compared with PHMB (alone or with TLR4a) (χ 2 = 4.74; df = 2, p = 0.09). In conclusion, the local administration of meglumine antimoniate appears to be safe and clinically efficient for the treatment of canine papular dermatitis due to L. infantum infection.

The effect of Leishmania infantum soluble antigen (LSA) and recombinant Kinetoplastid Membrane Protein 11 (rKMP11) on the induction of ex vivo specific IFN-γ (n = 69) and antibody responses (n = 108) was determined in dogs. All dogs were tested for serological response to both antigens and divided into Group 1: healthy (Asturias, Spain, n = 26), Group 2: sick (n = 46), Group 3: healthy Ibizan hounds (Mallorca, Spain, n = 22) and Group 4: healthy (Bari, Italy, n = 14). Antibody levels were higher for LSA when compared to rKMP11 (p = 0.001). Ibizan hounds were all seronegative to rKMP11 and 18% were low seropositive to LSA. Sick dogs presented higher antibody response to both antigens compared to the rest of the groups (p < 0.0001). All groups showed higher IFN-γ levels after LSA compared to rKMP11 responses (p < 0.05). The highest response to LSA was found in Ibizan hounds (p < 0.05). IFN-γ to LSA and rKMP11 stimulation was observed in 34% and in 2.8% of the sick dogs, respectively. Here, we demonstrated that anti-rKMP11 antibodies are mainly present in dogs with moderate to severe disease. Furthermore, cellular immune response measured by specific ex vivo IFN-γ production was more intense to LSA than stimulated to rKMP11.

Background: Canine leishmaniosis (CanL) is a common infectious disease. Age, sex and breed might influence the type of clinical and pathological manifestations that dogs develop. The main objective of this retrospective cross-sectional study was to determine if an association between age, sex, breed and size and the clinical findings of CanL exists., Material and Methods: Dogs with a diagnosis of leishmaniosis were enrolled (n = 123). Clinical information, including signalment, clinical signs and laboratory abnormalities, was retrieved from medical records from different veterinary facilities from Catalonia., Results: Young dogs developed less frequently systemic signs (p = 0.0046), renal (p = 0.0019) and haematologic (p = 0.0275) abnormalities, while dermatologic signs were more common in young and adult dogs compared with old ones (p = 0.0451). Young dogs showed proteinuria less often than adult and old dogs (p = 0.0029). Young dogs did not present renal azotemia, while old dogs showed occasionally renal azotemia (p = 0.0478). Young dogs were mainly classified as mild-moderate LeishVet clinical stages of the disease, and very rarely as severe-very severe LeishVet clinical stages, compared with adult and old dogs (p = 0.0457). Purebred dogs significantly developed ulcerative dermatitis more frequently than crossbred dogs (p = 0.0460)., Conclusion: This study describes that age is associated with differences in clinicopathological findings of CanL., (© 2021 The Authors. Veterinary Record published by John Wiley & Sons Ltd on behalf of British Veterinary Association.)

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

The leishmanin skin test (LST) is an in vivo technique commonly used to evaluate the Leishmania -specific cellular immune response in dogs. However, information regarding the local immune response in LST-positive reactions is scarce. We examined the pattern of toll-like receptor 2 (TLR2), TLR4, TLR7, interleukin- (IL-) 10, interferon gamma (IFN- γ ), and (program death ligand) PD-L1 gene expression in LST-positive reactions and paired normal-looking skin of nine infected Ibizan hound dogs. Healthy skin from ten seronegative dogs from a nonendemic area was analysed as a negative control. Immune gene expressions were examined by quantitative PCR (qPCR) analysis. LST-positive reactions presented significant upregulation of TLR2, TLR4, IL-10, IFN- γ , and PD-L1 and downregulation of TLR7 when compared with healthy skin of seronegative control dogs from a nonendemic area. All transcripts but TLR7 were significantly higher in LST-positive reaction than in paired normal-looking skin of Ibizan hound. The expression profile of immune genes in LST-positive reactions was similar to that previously observed in clinically lesioned skin of mildly diseased dogs with papular dermatitis due to Leishmania infantum infection. This data provide additional support for the important role of TLRs in canine leishmaniosis., Competing Interests: The authors declare that there is no conflict of interest regarding the publication of this paper., (Copyright © 2020 Laura Ordeix et al.)

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 < 0.0001) and IL-10 (P = 0.021) and downregulation of TLR7 (P = 0.004) when compared with more severely affected dogs. Normal-looking skin of mildly affected dogs was characterized by a significant lower expression of TLR7 (P = 0.003), IFN-γ (P < 0.0001) and PD-L1 (P = 0.001) when compared with more severely affected dogs. TLR2, TLR4, IL-10 and IFN-γ upregulation in clinically-lesioned skin was correlated with lower disease severity while TLR7 upregulation was correlated with markers of disease severity. Upregulation of TLR7, IL-10, IFN-γ and PD-L1 in normal-looking skin was correlated with disease severity., Conclusions: This study demonstrated different expression profiles of immune genes in clinically-lesioned and normal-looking skin among mildly and more severely affected dogs. These immunological conditions might favor the maintenance and replication of the parasite in the skin of more severely affected dogs.

Background: Detection of Leishmania in cutaneous lesions is possible by visualization of amastigotes. Detection of Leishmania DNA by PCR presents greater sensitivity, and PCR has been used to diagnose cutaneous leishmaniosis in humans using noninvasive clinical specimens., Objectives: Study I: to determine if Leishmania DNA could be efficiently extracted and amplified from archived Diff-Quik ® -stained slides of cytological specimens from canine cutaneous lesions. Study II: to evaluate the diagnostic performance of a Leishmania-quantitative (q)PCR on stained cytological specimens and on filter paper impressions (FPI) obtained from cutaneous lesions suggestive of canine leishmaniosis (CanL)., Animals: Samples from cutaneous lesions of 54 dogs., Methods and Materials: Study I: Leishmania-qPCR was performed on 19 glass slides (from nine dogs) with cytologically visible amastigotes. Fifteen slides with no visible amastigotes, obtained from 12 dogs seronegative for Leishmania by ELISA, served as controls. Study II: Leishmania-qPCR was performed on glass slides and FPI from cutaneous lesions compatible with clinical leishmaniosis in 33 dogs., Results: Study I: all slides with visible amastigotes had positive qPCR, whereas all control slides yielded negative results. Study II: of 13 dogs definitively diagnosed with clinical leishmaniosis, eight had visible amastigotes on cytology, whereas Leishmania-qPCR was positive on 11 glass slides and 13 FPI. Leishmaniosis was ruled out by standard methods in 20 dogs, four of which yielded positive qPCR on FPI and/or glass slides., Conclusions and Clinical Importance: Leishmania-DNA can be detected efficiently by qPCR from cutaneous cytological specimens and FPI to diagnose Leishmania infection in dogs with cutaneous lesions suggestive of CanL., (© 2019 ESVD and ACVD.)

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.

Background: Autosomal recessive congenital ichthyosis (ARCI) in golden retrievers is due to a PNPLA1 gene mutation, which plays a role in epidermal lipid organization and metabolism. Topical therapies are used to reduce scaling; however, there are few published efficacy studies., Objectives: To examine the efficacy of topical treatment based on gluconolactone, a polyhydroxy acid with known beneficial effects on stratum corneum structure., Animals: Sixteen golden retriever dogs with clinical signs of ARCI and PCR-confirmed PNPLA1 gene mutation., Methods: This was a prospective, multicentre, noncontrolled study. Dogs were treated with a shampoo and lotion containing gluconolactone and other hydroxyl acids. Treatments were administered initially twice weekly for two weeks, then once weekly for two weeks and finally once monthly. Examinations were performed prior to and at 14 and 30 days of treatment to assess scaling, presence of other skin lesions and pruritus. In two dogs, pre- and 30 day post-treatment, skin biopsies were obtained., Results: The extent and size of the scales were reduced by 60% and 75% after 14 and 30 days of treatment, respectively (P < 0.001). In 20% of the dogs, scaling was no longer observed after the first 30 days of treatment. No other skin lesions or pruritus were observed in any dog. Post-treatment biopsies showed normalization of the stratum corneum morphology and reduced hyperpigmentation., Conclusion and Clinical Importance: The frequent use of a shampoo and lotion containing gluconolactone may be an effective measure to improve skin scaling in golden retrievers with ARCI., (© 2018 ESVD and ACVD.)

The aims of the study were to determine whether symmetric dimethylarginine (SDMA) was increased in dogs with leishmaniosis and to assess its relationship with creatinine concentration and urinary protein : creatinine ratio (UPC) to determine its utility as a marker of early excretory dysfunction. Fifty-three dogs with leishmaniosis classified according to the LeishVet clinical staging (stage I, n = 5, stage II, n = 30; stage III, n = 12; stage IV, n = 6) were selected and compared with 41 clinically healthy dogs. Thirty-nine dogs with leishmaniosis were also followed up for six months. SDMA concentrations on the day of diagnosis were significantly higher in dogs with leishmaniosis with respect to control dogs and in dogs from LeishVet stage IV when compared with the other stages. Increased UPC (>0.5), SDMA (>19  μ g/dL), and creatinine concentrations (≥1.4 mg/dL) were found in 47.1%, 15.1%, and 9.4% of dogs with leishmaniosis, respectively. SDMA concentration was increased in 24% of proteinuric dogs, in 7% of nonproteinuric dogs, and in four of five dogs with increased creatinine. SDMA concentration ≥ 25  μ g/dL was associated with clinical chronic kidney disease (CKD) after six months. Our results did not demonstrate advantages in using SDMA concentration as an early marker of CKD when compared to creatinine and UPC in canine leishmaniosis.

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.

Canine leishmaniosis has a wide range of disease severity from mild (stage I), to severe (stages II-III), or very severe disease (stage IV). The objective of the study was to evaluate and compare serum antibody levels, Leishmania infantum specific IFN-γ production and TLR2 and TLR4 transcripts in non-stimulated blood from dogs with different clinical stages at the time of diagnosis as well as blood parasitemia. Enzyme-Linked ImmunoSorbent Assay (ELISAs) were performed to determine serum antibody levels and IFN-γ production and quantitative polymerase chain reaction ( q PCRs) in order to determine blood parasite load and TLR2 and TLR4 transcripts. Older dogs were significantly affected by more severe disease with higher antibody levels and blood parasitemia than dogs with mild disease. IFN-γ production was significantly higher in dogs with stage I disease when compared to dogs with more severe disease. Relative quantification of TLR2 in dogs with mild disease was similar to that of control dogs. On the other hand, TLR2 transcripts were significantly higher in dogs with severe disease as compared with that from control healthy dogs. No differences were found in TLR4 relative quantification between groups. This study demonstrates that dogs with different clinical stages of leishmaniosis present different levels of biological markers indicative of different immune responses., Competing Interests: The authors declare no conflict of interest.

There is limited data regarding Leishmania infantum specific T cell mediated immunity in naturally infected sick dogs at the time of diagnosis and during anti-Leishmania treatment. Our aim was to investigate the kinetics of L. infantum specific IFN-γ production in dogs with leishmaniosis at the time of diagnosis and during treatment and to correlate it with specific L. infantum antibodies, blood parasitemia and clinicopathological findings. Thirty-four dogs were diagnosed with leishmaniosis based on physical examination, routine laboratory tests and L. infantum-specific antibody levels by quantitative ELISA. Heparinized whole blood was stimulated with L. infantum soluble antigen (LSA) and concanavalin A (ConA) and incubated for 5days. IFN-γ concentration was evaluated in supernatants of stimulated blood using a commercial sandwich ELISA. Leishmania real-time PCR was also performed for assessing blood parasitemia. Dogs were treated with meglumine antimoniate and allopurinol. Sixteen dogs were classified as IFN-γ non-producers after LSA stimulation (mean±SD: 0±0pg/mL) and 18 dogs as IFN-γ producers (mean±SD: 2885.3±4436.1pg/mL) at the time of diagnosis (P<0.0001). IFN-γ non-producers were classified in a more severe clinical staging than IFN-γ producers that presented a mild to moderate clinical staging (P=0.03). In the IFN-γ non-producer group, production of IFN-γ after LSA stimulation was significantly increased during treatment especially at day 365 (P=0.018) together with clinical improvement when compared with day 0. In contrast, IFN-γ producers maintained their IFN-γ production after LSA stimulation and no statistically significant changes were found during treatment follow-up. At diagnosis, IFN-γ non-producers showed a significantly higher blood parasitemia versus IFN-γ -producers (P=0.005). IFN-γ non-producers drastically reduced blood parasitemia to minimum values at day 365 when compared with day 0 (P=0.017). No significant differences were found at day 365 in blood parasitemia of IFN-γ producers compared to pre-treatment. At diagnosis, L. infantum specific antibodies were higher in IFN-γ non-producers than IFN-γ producers (P=0.014). A marked reduction of antibody levels was found at day 365 when compared with day 0 in IFN-γ non-producers (P=0.005) and producers (P=0.001). These results demonstrate that IFN-γ concentration increases with long-term anti-Leishmania treatment together with clinical improvement in dogs that do not produce IFN-γ at diagnosis. Together with clinical recovery, reduction in blood parasitemia and L. infantum specific antibodies, tracking IFN-γ concentration could constitute an important prognostic tool for immune monitoring in CanL., (Copyright © 2017 Elsevier B.V. All rights reserved.)

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

Background: A wide spectrum of clinical manifestations and immune responses exist in canine L. infantum infection. Ibizan hounds are more "resistant" to disease than other dog breeds. Recognition of pathogen-associated molecule patterns by toll like receptors (TLRs) rapidly triggers a variety of anti-microbial immune responses through the induction of pro-inflammatory cytokines such as TNF-α and IL-6 which may play an important role in controlling Leishmania infection. The main objective of this study was to investigate and compare the effect of a TLR2 agonist (TLR2a) alone or in combination with L. infantum antigen (LSA) on ex vivo whole blood cytokine production from healthy seronegative IFN-γ non-producer dogs from an area of low in canine leishmaniosis endemicity (n = 11); sick seropositive dogs with low production of IFN-γ (n = 17) and healthy seronegative or low positive Ibizan hounds with a predominant IFN-γ production (n = 21) from a highly endemic area. Whole blood was stimulated with medium alone (Ø), LSA, concanavalin A, TLR2 (Pam3CSK4) receptor agonist (Ø + TLR2a) and TLR2a and LSA (LSA + TLR2a) for 48 h. Supernatants were harvested for measurement of canine TNF-α and IL-6 cytokines by ELISA., Results: A significant increase of TNF-α was found in the supernatants of stimulated blood from all groups (Ø + TLR2a and LSA + TLR2a) when compared with medium alone. A similar pattern was observed for IL-6. Interestingly, a significant increase of TNF-α production was only observed when stimulation with LSA + TLR2a was compared with TLR2a alone in Ibizan hounds. A significant increase of TNF-α production was observed with stimulation of LSA + TLR2a when compared with LSA in all groups. Significantly higher concentrations of TNF-α and IL-6 were detected in Ibizan hounds, especially for the Ø + TLR2a and LSA + TLR2a treatments compared with other groups., Conclusions: This study demonstrated that TLR2a alone enhances the production of the inflammatory cytokines TNF-α and IL-6 in sick, "resistant" and healthy non-infected dogs. In addition, a combination of LSA+TLR2a promoted a synergistic pro-inflammatory effect with TNF-α in Ibizan hounds but not in seropositive sick dogs and seronegative healthy dogs. These findings might suggest the importance of Pam3CSK4 as a possible immunomodulator for CanL.

Innate immunity, in particular, the role of toll-like receptors (TLRs), has not been extensively studied in canine L. infantum infection. The main aim of this study was to determine the transcription of TLR2 and TLR4 in the blood of dogs with natural clinical leishmaniosis at the time of diagnosis and during treatment follow-up and subsequently correlate these findings with clinical, serological and parasitological data. Forty-six Leishmania-seropositive sick dogs with a high antibody level at the time of diagnosis were studied and compared with 34 healthy seronegative dogs. Twenty-two of these sick dogs were treated with meglumine antimoniate and allopurinol and followed-up at 30, 180 and 365days following the start of treatment. Clinical status was defined by a thorough physical examination, complete blood count, biochemistry profile, electrophoresis of serum proteins, and urinary protein/creatinine ratio (UPC). EDTA blood was stored in RNAlater ® solution before RNA extraction and cDNA production were performed. TLR2, TLR4 and three reference genes (HPRT-1, CG14980 and SDHA) were studied in each blood sample by real time PCR. The relative quantification of TLR2 was higher (mean 3.5) in sick dogs when compared with seronegative healthy dogs (mean 1.3; P=0.0001) while the relative quantification of TLR4 was similar in both groups. In addition, the relative quantification of TLR2 significantly decreased during follow-up at all time points compared with day 0 whereas no changes were observed with TLR4 transcription. A significant positive correlation was noted between TLR2 and UPC, total protein, beta and gamma globulins, specific L. infantum antibodies and blood parasite load while a negative correlation was observed with albumin, albumin/globulin ratio, hematocrit and hemoglobin. TLR4 transcript did not correlate with any parameter. These findings indicate an up-regulation of TLR2 transcription in unstimulated blood in naturally infected sick dogs as compared to healthy dogs suggesting active innate immune and proinflammatory responses. In addition, TLR2 transcription is reduced with clinical improvement during treatment. In contrast, TLR4 transcription appears to be similar among groups at the time of diagnosis with no changes during treatment follow-up suggesting a less important role for this TLR in clinical canine leishmaniosis., (Copyright © 2016 Elsevier B.V. All rights reserved.)

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

Background: Leishmania infantum-specific antibodies are used extensively for the diagnosis and monitoring of treatment in canine leishmaniosis. Different views have been described for the measurement of L. infantum antibody levels for the monitoring of anti-leishmanial treatment. In addition, molecular techniques using blood are frequently employed in the clinical setting. However, there are not enough studies to prove the usefulness of PCR in diagnosis, treatment monitoring and in assessing the prognosis of the disease. The objectives of this study were to evaluate L. infantum-specific antibodies and blood parasitemia at the time of diagnosis and during treatment and to correlate these with the dog's clinical status., Methods: Thirty-seven dogs were diagnosed and followed-up during treatment (days 30, 180 and 365). The treatment protocol consisted of a combination of meglumine antimoniate for one month and allopurinol for at least one year. Leishmania infantum-specific antibodies and blood parasitemia were assessed by an end point sera dilution ELISA and by real-time PCR, respectively., Results: The majority of dogs were classified as LeishVet stage II (moderate disease) at the time of diagnosis (86 %) and the rest as stage III. Results showed variable levels of specific antibodies at the time of diagnosis [median ± interquartile range (IQR): 1372 ± 8803 ELISA units (EU)]. Twenty-three seropositive dogs (64 %) were detected as PCR-positive at the time of diagnosis. Interestingly, a rapid significant antibody level reduction was observed by day 30 of treatment (median ± IQR: 604 ± 2168 EU). A continuing significant decrease of specific antibodies was also found at days 180 (median ± IQR: 201 ± 676 EU) and 365 (median ± IQR: 133 ± 329 EU) in association with clinical improvement. A significant blood parasitemia reduction was also observed at all time points studied. Mean parasites/ml ± SD were 19.4 ± 79.1 on day 0, 2.2 ± 11.7 on day 30, 0.9 ± 2.9 on day 180, and 0.3 ± 0.7 on day 365., Conclusions: This study reports a significant reduction of L. infantum antibodies measured by an end point sera dilution ELISA method after 30 days of treatment associated with clinical improvement. A low proportion of sick dogs with moderate disease were negative by blood real-time PCR at the time of diagnosis.

Topical treatment with cyclosporine A (CsA) has recently become possible with the development of novel nanotechnology pharmaceutical formulations of CsA able to penetrate through the epidermis providing good absorption and dermal action. The aim of this multicentre, blinded, parallel, randomized, placebo controlled trial was to evaluate the efficacy of a new topical CsA formulation in dogs with atopic dermatitis (AD). Dogs (n=32) with severe and moderate clinical signs of non-seasonal AD, but few localized lesions, were randomly allocated to receive topical CsA (17 dogs) or placebo (15 dogs) and were treated twice a day for 6 weeks. Before and 21 and 45 days after starting the treatment, the severity of a previously selected skin lesion was evaluated according to a dermatological scoring system. Owners using a visual analogue scale also assessed pruritus weekly and effectiveness of the treatment was defined as a reduction of at least 50% in these variables after 45 days. After 21 and 45 days the lesion severity score in animals treated with CsA was significantly lower than at baseline (P<0.01, both times). In contrast, the animals on placebo showed no significant improvement at days 21 or 45. The percentage of dogs with an effective reduction in pruritus at the end of the trial was 87.5% and 28.6% in the CsA and placebo groups, respectively. These results suggest that topical administration of CsA is effective in reducing the severity of skin lesions and pruritus in dogs with moderate to severe AD as soon as 3 weeks after starting treatment., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Purpose: Canine lymphoma (CL) is one of the most common forms of spontaneous canine neoplasia. Improved understanding of the genetic and environmental factors associated with CL may provide benefits for the study of non-Hodgkin's and Hodgkin's lymphoma in humans. Uveodermatologic lymphoma is reported in a single household of Portuguese water dog, and the etiology is discussed., Methods: A 1-year-old female intact Portuguese water dog was referred to the Ophthalmology Service of the Veterinary Teaching Hospital of the Autonomous University of Barcelona because of acute blepharospasm of the right eye in association with a multinodular dermatosis. Physical and ophthalmological examination and a complete diagnostic work-up, including skin and ocular histopathology and immunohistochemistry, were performed. Three months prior, in Galicia (Spain), 1200 km away from Barcelona, a male dog of the same litter showed very similar oculodermatological clinical signs and skin histopathology, and immunohistochemistry were obtained., Results: The clinical diagnoses were anterior exudative uveitis, iridal masses, and secondary glaucoma. Histopathology and immunohistochemistry revealed a nonepitheliotropic lymphoma rich in B cell in dog 1 and rich in T cell in dog 2., Conclusion: It is proposed that an underlying genetic predisposition could have played a role in the development of lymphoma in these two young related dogs., (© 2012 American College of Veterinary Ophthalmologists.)

Unlabelled: PRESENTING SIGNS: Three Devon Rex cats were presented with multiple erythematous papules, occasionally associated with crusting and hyperpigmentation, with a linear distribution on the head, neck, chest and abdomen. One cat also had multifocal alopecia with hyperpigmentation on the dorsum., Diagnosis and Treatment: Clinical and histopathological features were suggestive of papular eosinophilic/mastocytic dermatitis (urticaria pigmentosa-like dermatitis). In all cases, dermatophytosis was diagnosed: in cases 1 and 2 there was histopathological evidence of dermatophytosis, while fungal culture was positive for Microsporum canis in cases 2 and 3. In all cats, lesions disappeared following antifungal treatment., Clinical Significance: Papular eosinophilic/mastocytic dermatitis in Devon Rex cats may represent either an atypical presentation of dermatophytosis or a clinical and histological reaction pattern to various diseases, including dermatophytosis and allergic diseases. Clinical differentiation is crucial as there are important implications regarding treatment and, in particular, the use of glucocorticoids, which are contraindicated in cases of dermatophytosis.

The aim of this case-control study was to evaluate and compare the bacterial microflora from the conjunctival sac of dogs with atopic dermatitis and healthy dogs. Twenty-one atopic dogs without clinical and/or cytopathological signs of bacterial blepharoconjunctivitis and 21 breed-matched healthy dogs were enrolled. Under topical anaesthesia, the inferior conjunctival sac of one eye was scraped twice. Material was collected with a Kimura spatula, spread over a slide and stained with a Diff Quick(®) -type stain (Medion Diagnostics GmbH, Düdingen, Switzerland) for cytological examination. An area of 0.5 cm(2) was examined at ×1000 magnification, and the types and numbers of cells and bacteria were recorded. A bacterial swab was collected and inoculated into culture media for the growth of aerobic bacteria. Before sampling, each atopic dog was evaluated for severity of cutaneous lesions, pruritus and conjunctival inflammation. Significant differences were observed between atopic and healthy dogs for the presence of bacteria on cytology (P = 0.015), keratinized (P = 0.001) and nonkeratinized epithelial cells (P = 0.013), eosinophils (P = 0.019) and lymphocytes (P = 0.008). Bacteria were recovered from 12 atopic dogs and three healthy dogs (P = 0.004). Staphylococcus pseudintermedius was the most commonly isolated species in atopic dogs (seven of 12). In atopic dogs, no significant relation was found between conjunctival bacterial colonization (on cytology and culture) and the severity of any of the clinical parameters. This study suggests differences in conjunctival bacterial colonization and cytological features between atopic and healthy dogs., (© 2011 The Authors. Veterinary Dermatology. © 2011 ESVD and ACVD.)

Zinc deficiency causes skin diseases both in humans and in animals. The underlying pathogenic mechanisms remain unclear, but a growing body of evidence indicates a role for zinc in skin protection against free radical-induced oxidative damage. The immunohistochemical expression of heat shock proteins (HSPs; Hsp27, Hsp72, Hsp73 and Hsp90), Cu/Zn superoxide dismutase (SOD), metallothionein (MT), Ki-67 antigen and active caspase-3 were evaluated in normal canine skin and in samples from eight dogs with zinc-responsive dermatosis. All investigated HSPs showed intense cytoplasmic immunostaining in the affected epidermis. Focal nuclear positivity of Hsp72 was also detected in keratinocytes. Although Cu/Zn SOD expression was similar to that observed in normal skin, MT immunoreactivity occurred in both the cytoplasm and the nucleus of basal cells in normal skin but was absent from the affected epidermis. Caspase-3 activation was also absent in the involved epidermis, which revealed a high Ki-67 index (a 3.5- to 9-fold increase compared with normal skin). These results support the hypothesis that cellular response to stress, particularly oxidative stress, is involved in the pathogenesis of skin lesions in canine zinc-responsive dermatosis. The lack of MT immunoreactivity in the affected epidermis may be indicative of low zinc levels, thus resulting in vulnerability to oxidative damage. In contrast, high expression levels of HSPs in skin during zinc deficiency may confer protection against a variety of dangerous stimuli, contributing to inhibition of apoptosis and to cell cycle regulation of proliferating keratinocytes., (© 2010 The Authors. Journal compilation © 2010 ESVD and ACVD.)

Inverted papillomas are uncommon papillomavirus (PV)-induced canine skin lesions. They consist of cup- to dome-shaped dermal nodules with a central pore filled with keratin. Histologically they are characterized by endophytic projections of the epidermis extending into dermis. Cytopathic effects of PVs infection include the presence of clumped keratohyalin granules, koilocytes and intranuclear inclusion bodies. Different DNA hybridization studies carried out with a canine oral papillomavirus (COPV) probe suggested that a different PV than COPV might cause these lesions. Canine papillomavirus 2 (CPV2) was discovered a few years ago in inverted papillomas of immunosuppressed beagles. Two other cases, presenting with distinct clinical and histological features have also been described. This study was carried out on four dogs with clinical and histological signs of inverted papillomas. Molecular biological analyses confirmed that PV DNA was present in all four lesions but demonstrated that the sequences in each case were different. One corresponded to COPV, the second to CPV2, and the third and fourth to unknown PVs. These findings suggest that inverted papillomas are not caused by one single PV type. Similar observations have also been made in human medicine.

Demodex canis is thought to be present in small numbers in the skin of most healthy dogs; however, available data on the prevalence of normal dogs harbouring D. canis are scarce. The purpose of this study was to investigate, using microscopic examination of plucked hairs, the prevalence of healthy dogs harbouring D. canis. Seventy-eight clinically healthy dogs with no history of dermatological problems and clinically normal skin and hair coat were included in the study. Five areas (perioral skin 2-3mm from both labial commissures, periungual skin of the third digit of both anterior paws and chin) were examined in each dog. Fifty to sixty hairs were plucked from each skin site and microscopically examined. No D. canis mites were observed and only one adult form of Demodex injai was found in the labial commissure of one dog. Based on these results, the estimated prevalence of healthy dogs harbouring D. canis in clinically normal skin should not exceed the threshold of 5.4%, with 95% confidence level. Considering our and previous findings, we propose that, although small numbers of D. canis might inhabit the skin of normal dogs, the probability of finding these mites in normal dogs is low. Consequently, in most cases, the presence of a D. canis mite in the skin should not be considered as indicative of normality.

Demodex injai mites were detected on trichoscopic examinations and/or deep skin scrapings in eight wirehaired fox terrier dogs with dorsal greasy skin and hair. Histological examination performed in five dogs revealed marked sebaceous gland hyperplasia with lympho-plasmacytic periadnexal dermatitis in all of them. One mite section was observed in one patient. Seven dogs were parasitologically cured after 2 to 7 months of oral ivermectin treatment. Greasy skin and hair resolved in four dogs, was partially reduced in two dogs and persisted in the remaining dog. Skin biopsies were repeated after parasitological cure in two dogs and revealed the persistence of sebaceous gland hyperplasia with mild lympho-plasmacytic periadnexal dermatitis and no parasites. Based on the findings in this case series, the terrier dog breed might be at increased risk for the development of D. injai mite infestation associated with dorsal greasy skin and hair, and microscopically with sebaceous gland hyperplasia. Persistence of sebaceous gland hyperplasia after parasitological cure in some patients suggested that this histological finding may not always be resulting from Demodex infestation. Moreover, low numbers of adult mites and variable clinical responses to acaricidal therapy suggested a contributory rather than a major role of D. injai in this skin condition. Dermatopathological diagnosis of sebaceous gland hyperplasia, particularly in case of dorsal trunk specimens from terrier dog breeds, warrants the search for D. injai mites on trichoscopic examinations and/or deep skin scrapings.

A series of 18 allergic cats with multifocal Malassezia spp. overgrowth is reported: atopic dermatitis was diagnosed in 16, an adverse food reaction in another and one was euthanized 2 months after diagnosis of Malassezia overgrowth. All the cats were otherwise healthy and those tested (16 out of 18) for feline leukaemia or feline immunodeficiency virus infections were all negative. At dermatological examination, multifocal alopecia, erythema, crusting and greasy adherent brownish scales were variably distributed on all cats. Cytological examination revealed Malassezia spp. overgrowth with/without bacterial infection in facial skin (n = 11), ventral neck (n = 6), abdomen (n = 6), ear canal (n = 4), chin (n = 2), ear pinnae (n = 2), interdigital (n = 1) and claw folds skin (n = 1). Moreover, in two cats Malassezia pachydermatis was isolated in fungal cultures from lesional skin. Azoles therapy alone was prescribed in seven, azoles and antibacterial therapy in eight and azoles with both antibacterial and anti-inflammatory therapy in three of the cats. After 3-4 weeks of treatment, substantial reduction of pruritus and skin lesions was observed in all 11 cats treated with a combined therapy and in five of seven treated solely with azoles. Malassezia spp. overgrowth may represent a secondary cutaneous problem in allergic cats particularly in those presented for dermatological examination displaying greasy adherent brownish scales. The favourable response to treatment with antifungal treatments alone suggests that, as in dogs, Malassezia spp. may be partly responsible for both pruritus and cutaneous lesions in allergic cats.

Papular dermatitis due to Leishmania spp. infection was diagnosed in three boxers and two Rottweilers with Leishmania-specific cellular immunity. Diagnosis was based on histological and immunohistochemical examination of papules in four dogs and on cytological examination in one dog. Serum protein electrophoresis was within reference ranges and low antibody levels to Leishmania infantum were detected. Delayed-type hypersensitivity (DTH) reaction to leishmanin was evaluated before treatment in three dogs with positive results. After meglumine antimoniate therapy for 3 to 4 weeks and allopurinol treatment for 6 to 10 months, all dogs were clinically normal, had positive DTH reactions to leishmanin and reduced antibody titres. In conclusion, we suggest that this previously unreported cutaneous presentation of canine leishmaniosis appears to be associated with specific immunocompetence and, consequently, with a favourable prognosis.

This study determined the frequency of occurrence and the mean number of 'flame follicles' per skin section and assessed their diagnostic significance in cutaneous biopsies of Shar-pei dogs. The number of 'flame follicles' per section was recorded in skin sections from 42 Shar-pei dogs, of which 40 had non-neoplastic skin disease and non-atrophic dermatoses and 2 had healthy skin. Forty-two skin sections from dogs of different breeds served as control specimens, 28 of which were examples of non-neoplastic and non-atrophic dermatoses and 14 were from dogs with healthy skin. Differences among groups were analysed by means of the unpaired Student's t-test. It was concluded that 'flame follicles' were more frequent and found in significantly higher numbers in the Shar-pei group when compared with the control group suggesting that 'flame follicles' in skin sections from Shar-pei dogs do not have the same diagnostic significance as in other breeds.

A 4-year-old, intact male rottweiler was presented with a 10-day history of papulonodular dermatitis. At the time of presentation, the dog was receiving prednisone and azathioprine to treat pemphigus foliaceus. Cutaneous neosporosis was diagnosed by immunohistochemistry on skin biopsy specimens and a high serum antibody titer to Neospora caninum by Neospora agglutination test. Electron microscopy examination of skin specimens further supported the diagnosis. Clindamycin therapy, together with withdrawal of immunosuppressive medication, resulted in prolonged clinical remission. This report documents cutaneous neosporosis in an adult dog and suggests that immunosuppressive therapy might be a predisposing factor.

Cutaneous mast cells from skin biopsies of three healthy dogs and three dogs with atopic dermatitis were assessed for their proliferative potential using bromodeoxyuridine and proliferating cell nuclear antigen labelling. Mast cells isolated from the skin of two healthy dogs were also studied using bromodeoxyuridine labelling. Mast cells in skin biopsy specimens and mast cells isolated from the skin of healthy dogs did not incorporate bromodeoxyuridine. Two mast cells expressing proliferating cell nuclear antigen were seen around two superficial vessels in the dermis of one atopic dog. Epidermal cells, glandular epithelial cells, fibroblasts and endothelial cells incorporated bromodeoxyuridine and showed positive staining for proliferating cell nuclear antigen. These results suggest that canine mature mast cells do not proliferate in the dermis.