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Leishmaniasis caused by Leishmania infantum is an important zoonotic disease transmitted by sand flies with a high prevalence of infection in dogs and cats in regions whereby transmission occurs. Clinical disease is systemic with variable presenting signs and degrees of severity. It affects the skin, lymph nodes, eyes, bone marrow, kidneys, and other organs. The clinical findings in dogs and cats with L. infantum infection are generally similar. Subclinical infection of canines and felines in endemic areas is frequent. Long-term treatment of the disease with allopurinol, or combination of allopurinol with meglumine antimoniate or miltefosine, is needed, and clinical relapse is probable.

In Europe

The aim of the present study was to investigate the prevalence rates of the feline tick-borne pathogens (FTBPs)-Borrelia burgdorferi, Ehrlichia canis, and Anaplasma phagocytophilum in stray cats from Southern Bulgaria. Serum antibodies were used to estimate the prevalence of exposure to FTBPs from blood swabs. Of the 100 cat samples tested with in-clinic assay SNAP 4Dx Plus, the overall FTBP seroprevalence was 3% (3/100); with B. burgdorferi-1% (1/100) and E. canis-2% (2/100). This study provides the first evidence of exposure to B. burgdorferi and E. canis in cats from Bulgaria.

Prevention of canine Leishmania infantum infection is critical to management of visceral leishmaniasis in people living in endemic areas of Brazil. A bill (PL 1738/11), currently under consideration, proposes to establish a national vaccination policy against canine leishmaniasis in Brazil. However, there is no solid scientific evidence supporting the idea that this could reduce transmission from infected vaccinated dogs to sand flies to a level that would significantly reduce the risk of L. infantum infection or visceral leishmaniasis in humans. Thus, we advocate that insecticide-impregnated collars should the first line protective measure for public health purposes and that vaccines are applied on a case-by-case, optional basis for individual dog protection.

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

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 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 antibody levels while the results seem less optimal in apparently healthy dogs with low antibody levels. Graphical Abstract

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The role of the cat as a reservoir of Leishmania infantum and the characteristics of the immune response to this infection remains limited, contrary to the dog. This study aimed to compare the rate of L. infantum infection and parasite-specific humoral and cell-mediated immune responses in cats and dogs living in an area endemic to canine leishmaniosis (Córdoba, Spain). About one-third of the dogs and cats studied were positive for at least one molecular or serological diagnostic test. The immunopathogenesis of L. infantum infection in cats showed similarities when compared to dogs, although the parasite-specific immune response level in dogs was generally higher than in cats. This study shows that stray cats are exposed to L. infantum infection similarly to dogs in endemic areas, are able to mount a specific anti -Leishmania humoral and cell-mediated immune response as dogs, and can contribute to the endemicity of infection. Dogs are the main reservoir of Leishmania infantum and display different immunological patterns correlating with the progression of infection to disease. Data about feline L. infantum adaptive immune response are scant. This study aimed to compare the prevalence and immune response in cats and dogs from the same endemic area of canine leishmaniosis. Stray cats (109) and rescued dogs (59) from Córdoba (Spain) were enrolled. Data about their exposure to L. infantum were analyzed by detection of parasite DNA, measurements of Leishmania- specific interferon-γ (whole blood assay in 57 cats and 29 dogs), and antibodies (enzyme-linked immunosorbent assay and immunofluorescence antibody test). An overall L. infantum prevalence of 30.5% in dogs and 30% in cats were found according to serology and PCR tests. Prevalence was 44.8% in dogs and 35.1% in cats tested also for interferon-γ production. Dogs showed higher anti- L. infantum antibody levels compared to cats. More than one-third of cats had contact with or were infected by L. infantum and they may contribute to the endemicity of leishmaniosis in the investigated region. The immunopathogenesis of feline L. infantum infection has similarities with dogs but cats show a lower level of adaptive immune response compared to dogs.

Summary Previous studies have demonstrated the presence of Leishmania infantum amastigotes in the colonic mucosa of seropositive sick dogs. However, there are no studies that have investigated the presence of L. infantum infection in dogs diagnosed with inflammatory bowel disease (IBD). The aims of this study were: (1) to investigate retrospectively the presence of Leishmania spp. antigen by immunohistochemistry (IHC) in biopsy samples taken from the colon of dogs with IBD in an area endemic for leishmaniosis, and (2) to describe the main histopathological findings in these cases. Clinicopathological data and histopathological results were reviewed from 106 cases of canine colitis. IHC to detect Leishmania spp. antigen had been performed at the time of diagnosis in 13 cases and we performed IHC in 56 more cases. Five of the 69 cases (7.2%) were positive for Leishmania spp. antigen by IHC. Two positive biopsy samples had histiocytic inflammation and three had lymphoplasmacytic inflammation. The number of amastigotes was variable and independent of the type and grade of inflammatory infiltrate. The results suggest that Leishmania spp. infection is associated with chronic colitis in areas endemic for the infection. Therefore, Leishmania IHC should be used routinely as a diagnostic tool when evaluating colonic biopsy samples from dogs in endemic areas, to exclude or confirm an infection by this parasite in dogs with chronic colitis.

Altres ajuts: acords transformatius de la UAB 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.

Background Sand flies are the only biologically adapted vectors of Leishmania parasites, however, a possible role in the transmission of Leishmania has been proposed for other hematophagous ectoparasites such as ticks. In order to evaluate natural infection by Leishmania infantum in Rhipicephalus sanguineus ticks, taking into account its close association with dogs, 128 adult R. sanguineus ticks removed from 41 dogs living in endemic areas of canine leishmaniosis were studied. Methods Individual DNA extraction was performed from each tick and whole blood taken from dogs. Dog sera were tested for IgG antibodies to L. infantum antigen by ELISA and L. infantum real-time PCR was performed from canine whole blood samples and ticks. Results Leishmania infantum PCR was positive in 13 ticks (10.1%) including one female, (2.0%) and 12 males (15.2%), and in only five dogs (12.2%). Male ticks had a significantly higher infection rate when compared to female R. sanguineus. The percentage of L. infantum seroreactive dogs was 19.5%. All but two PCR positive dogs were seroreactive. Leishmania infantum PCR positive ticks were removed from seropositive and seronegative dogs with a variety of PCR results. Conclusions This study demonstrates high prevalence of L. infantum DNA in R. sanguineus ticks removed from L. infantum seropositive and seronegative dogs. The presence of L. infantum DNA was detected mainly in male ticks possibly due to their ability to move between canine hosts and feed on several canine hosts during the adult life stage. Additional studies are needed to further explore the role of R. sanguineus ticks and in particular, male adults, in both the epidemiology and immunology of L. infantum infection in dogs in endemic areas.

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.

SUMMARYCanine leishmaniosis (CanL) is caused by the parasiteLeishmania infantumand is a systemic disease, which can present with variable clinical signs, and clinicopathological abnormalities. Clinical manifestations can range from subclinical infection to very severe systemic disease. Leishmaniosis is categorized as a neglected tropical disease and the complex immune responses associated withLeishmaniaspecies makes therapeutic treatments and vaccine development challenging for both dogs and humans. In this review, we summarize innate and adaptive immune responses associated withL. infantuminfection in dogs, and we discuss the problems associated with the disease as well as potential solutions and the future direction of required research to help control the parasite.

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.

A wide spectrum of clinical and clinicopathological findings in dogs with canine leishmaniosis (CanL) due to Leishmania infantum exists. However, the majority of clinical descriptions have been published a long time ago and recent studies in Europe are almost lacking. In addition, clinical classification of sick dogs is not well-standardized, with different classification systems used by clinical and epidemiological studies, making comparison of studies a difficult task. The aims of the study were to describe the clinicopathological findings of dogs naturally infected with L. infantum at the time of diagnosis and to review and compare the various clinical classification systems for CanL available in the literature. Eighty-one healthy dogs and fifty-one dogs with CanL were studied and clinical and clinicopathological data were recorded. The most common clinical findings at diagnosis were skin lesions (78.4%), lymphadenomegaly (64.7%) and weight loss (47.1%). The most frequent clinicopathological abnormalities included mild to moderate non-regenerative anemia (62.7%), lymphopenia (25.5%), hyperproteinemia (52.9%) dysproteinemia (78.4%). and proteinuria (47.8%). Renal azotemia was rare (5.9%). Only 5.9% of the patients studied were classified in similar categories (mild, moderate and severe disease) when five clinical classifications systems were compared, while 11.8% of cases were classified in similar categories when only two clinical classification systems were considered based on the fact that they included therapeutic and prognosis recommendations. In conclusion, anemia and protein-related alterations are common in dogs with CanL. In contrast, renal azotemia is infrequent despite the high percentage of diseased dogs with proteinuria, indicating kidney involvement. Adequate clinical staging system is desirable in order to establish proper management, treatment and prognosis in dogs with CanL and to facilitate the comparison of clinical and epidemiological studies.

A French bulldog with a previous history of leishmaniosis was presented due to respiratory distress associated with a laryngeal mass. The mass was excised and cytological and histopathological examination revealed pyogranulomatous inflammation with Leishmania spp. amastigotes. After surgery, the respiratory condition resolved; however, 3 months later the dog developed clinicopathological signs of leishmaniosis, which improved with systemic treatment. This case shows an atypical presentation of leishmaniosis with a focal tumour-like mass in the vocal folds as the only clinical sign.

This study presents the results of a national survey in Spain on the distribution of ticks on owned dogs, their phenology and the associated pathogens over one year. In the study, 1628 adult ticks were collected on 660 dogs presented to 26 veterinary practices, of which 507 dogs (76.8%) carried at least one adult tick. The primary species of ticks were Rhipicephalus sanguineus s.l. (53%), Dermacentor reticulatus (9%), Ixodes ricinus (9%), and I. hexagonus (4%). Parasitism by two species of these ticks was rare. The four species showed a clear association with the biogeographical features of the country, with I. ricinus associated with the wettest northern regions, I. hexagonus and D. reticulatus associated with the north of the territory, and R. sanguineus s.l. prevalent throughout the entire country. Dogs living in rural areas had a higher prevalence of all species, but R. sanguineus s.l. was the most prevalent; however, this tick was also more common on dogs living indoors. R. sanguineus s.l. adults were active throughout the year, with a maximum peak from March to July. The other tick species were collected throughout the year, with an autumn-winter peak of D. reticulatus, but without clear seasonality for either I. ricinus or I. hexagonus. Combined real-time PCR and conventional PCR of the feeding ticks recorded Piroplasmida (Hepatozoon canis, Babesia canis, B. gibsoni, and several sequences compatible with Theileria spp.), Rickettsia spp. (R. massiliae, R. sibirica mongolitimonae, R. monacensis), Ehrlichia canis, Anaplasma platys, A. phagocytophilum and Borrelia spp. (B. afzelii, B. garinii, B. valaisiana). Hepatozoon canis and B. canis were the most frequently detected pathogens, with variable rates of infection according to the region. Other than a close association of Borrelia spp. with I. ricinus (and therefore to the wet northern areas of the territory), the other tick-borne pathogens were recorded throughout the country. Although a potential transmission role for ticks carrying unusual pathogens cannot be attributed to these results, these findings introduce a change of paradigm on the tick-borne pathogen distribution in Spain and emphasize the importance of performing active surveys to understand the complex patterns of tick-borne pathogen distributions and their vectors.

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.Study I: to determine if Leishmania DNA could be efficiently extracted and amplified from archived Diff-QuikSamples from cutaneous lesions of 54 dogs.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.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.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.Détection de Leishmania dans les lésions cutanées est possible par visualisation des amastigotes. La détection de l’ADN de Leishmania par PCR présente une meilleure sensibilité et la PCR a été utilisée pour diagnostiquer la leishmaniose cutanée de l'homme à l'aide de techniques cliniques non invasives.Etude I : déterminer si l’ADN de Leishmania peut être efficacement extraite et amplifiée de lames colorées au Diff-QuikEchantillons de lésions cutanées de 54 chiens. MATÉRIEL ET MÉTHODE: Etude I : la PCRq a été réalisée sur 19 lames (de 9 chiens) avec des amastigotes visibles à la cytologie. Quinze lames sans amastigote visible, obtenu de 12 chiens séronégatifs pour Leishmania par ELISA, ont servi de contrôle. Etude II : Une PCRq Leishmania a été réalisée sur des lames et FPI de lésions cutanées compatibles avec une leishmaniose clinique chez 33 chiens. RÉSULTATS: Etude I : toutes les lames avec amastigotes visibles avaient une PCRq positive tandis que les lames contrôles étaient négatives. Etude II : sur les 13 chiens diagnostiqués définitivement comme leishmaniose clinique, huit avaient des amastigotes visibles à la cytologie tandis que la PCRq Leishmania était positive sur 11 des lames et 13 FPI. LA leishmaniose a été exclue par méthodes standard chez 20 chiens, dont quatre présentaient une PCRq positive sur FPI et/ou lames.L’ADN de Leishmania peut être détectée efficacement par PCRq de cytologies cutanées et FPI pour le diagnostic de leishmaniose chez les chiens présentant des lésions cutanées évocatrices de CanL.INTRODUCCIÓN: la visualización de amastigotes permite la detección de Leishmania en lesiones cutáneas. La detección del DNA de Leishmania por PCR es más sensible, y la PCR se ha utilizado para diagnosticar la leishmaniasis cutánea en humanos utilizando muestras clínicas no invasivas. OBJETIVOS: Estudio I: determinar si el DNA de Leishmania se podría extraer y amplificar de manera eficiente a partir de preparaciones teñidas con Diff-Quik® archivadas de muestras citológicas de lesiones cutáneas caninas. Estudio II: evaluar el valor diagnóstico de una PCR cuantitativa (qPCR) de Leishmania en muestras citológicas teñidas y en impresiones de papel de filtro (FPI) obtenidas de lesiones cutáneas sugestivas de leishmaniasis canina (CanL). ANIMALES: muestras de lesiones cutáneas de 54 perros. MÉTODOS Y MATERIALES: Estudio I: se realizó qPCR para Leishmania en 19 portaobjetos de vidrio (de nueve perros) con amastigotes citológicamente visibles. Quince preparaciones sin amastigotes visibles, obtenidas de 12 perros seronegativos para Leishmania por ELISA, sirvieron como controles. Estudio II: se realizó qPCR para Leishmania en portaobjetos de vidrio y FPI de lesiones cutáneas compatibles con leishmaniasis clínica en 33 perros. RESULTADOS: Estudio I: todas las preparaciones con amastigotes visibles tuvieron qPCR positiva, mientras que todas las preparaciones de control dieron resultados negativos. Estudio II: de 13 perros diagnosticados definitivamente con leishmaniasis clínica, ocho tuvieron amastigotes visibles en la citología, mientras que qPCR para Leishmania fue positiva en 11 portaobjetos de vidrio y 13 FPI. La leishmaniasis se descartó mediante métodos estándar en 20 perros, cuatro de los cuales produjeron qPCR positiva en FPI y/o portaobjetos de vidrio. CONCLUSIONES E IMPORTANCIA CLÍNICA: la qPCR para DNA de Leishmania puede realizarse de forma efectiva a partir de muestras citológicas cutáneas y FPI para diagnosticar la infección por Leishmania en perros con lesiones cutáneas sugestivas de CanL.Leishmanien können in Hautveränderungen durch eine Sichtbarmachung der Amastigoten gefunden werde. Die Feststellung von Leishmania DNA mittels PCR bedeutet eine größere Sensibilität. Außerdem wurde die PCR verwendet, um kutane Leishmaniose bei Menschen mittels nichtinvasiver klinischer Proben zu diagnostizieren.Studie I: festzustellen, ob ausreichend Leishmania DNA aus archivierten Diff-Quick® gefärbten zytologischen Präparaten aus kutanen Veränderungen extrahiert und amplifiziert werden konnte. Studie II: eine Evaluierung der diagnostischen Leistung einer quantitativen Leishmania (q) PCR aus gefärbten Zytologiepräparaten sowie aus Abdrücken auf Filterpapier (FPI), die aus Hautveränderungen stammten, die verdächtig für eine canine Leishmaniose (CanL) waren.Es wurden Proben von Hautveränderungen von 54 Hunden verwendet.Studie I: Es wurde ein Leishmania-qPCR von 19 Objektträgern (von neun Hunden) mit zytologisch sichtbaren Amastigoten durchgeführt. Fünfzehn Objektträger ohne sichtbare Amastigoten, die von 12 Hunden stammten, die mittels ELISA seronegativ auf Leishmania waren, dienten als Kontrollen. Studie II: Ein Leishmania-qPCR wurde von Objektträgern und FPI kutaner Läsionen von 33 Hunden, die mit klinischer Leishmaniose kompatibel waren, durchgeführt.Studie I: alle Objektträger mit sichtbaren Amastigoten zeigten eine positive qPCR, während die Kontrollen ein negatives Ergebnis lieferten. Studie II: von den 13 Hunden, die definitiv mit einer Leishmaniose diagnostiziert worden waren, zeigten acht sichtbare Amastigoten in der Zytologie, während der Leishmania-PCR von 11 Objektträgern und 13 FPI positiv war. Eine Leishmaniose war bei 20 Hunden mit Standardmethoden ausgeschlossen worden, von denen vier eine positive qPCR von FPI und/oder Objektträgern lieferten.Leishmania-DNA kann effektiv mittels qPCR aus kutanen zytologischen Hautproben und aus FPI festgestellt werden, um eine Leishmania Infektion bei Hunden mit Hautveränderungen, die auf CanL hinweisen zu diagnostizieren.背景: 皮膚病変におけるリーシュマニアの検出は、無鞭毛期虫体の視覚化によって可能となる。 PCRによるリーシュマニアDNAの検出はより高い感度を示し、PCRは非侵襲性臨床標本を用いて人の皮膚リーシュマニア症を診断するために使用されてきた。 目的: 研究I:研究Iの目的は、犬の皮膚病変から得られた細胞学的標本のアーカイブされたDiff-Quik®染色スライドからLeishmania DNAを効率的に抽出および増幅できるかどうかを決定することである。研究II:研究IIの目的は、染色した細胞学的標本および犬リーシュマニア症(CanL)を示唆する皮膚病変から得られたfilter paper impressions (FPI)に対するリーシュマニア定量的(q)PCRの診断性能を評価することである。 被験動物: 54頭の犬の皮膚病変からのサンプル。 材料と方法: 研究I:Leishmania-qPCRを、細胞学的に観察可能な無鞭毛期虫体を含む19枚のスライドガラス(9頭の犬から)によって実施した。 ELISAによってリーシュマニアに対して血清陰性の12頭の犬から得られた、目に見える無鞭毛期虫体のない15枚のスライドを対照として用いた。研究II:33頭の犬において、リーシュマニアqPCRを、臨床的にリーシュマニア症と適合性のある皮膚病変からのスライドガラスおよびFPIに対して実施した。 結果: 研究I:目に見える無鞭毛期虫体を含む全てのスライドがqPCR陽性であったのに対し、全対照スライドは陰性結果をもたらした。研究II:臨床リーシュマニア症と確定診断された13頭の犬のうち、8頭が細胞診で目に見える斑点があったのに対し、Leishmania-qPCRは11枚のスライドガラスと13枚のFPIで陽性を示した。リーシュマニア症は、20頭の犬において標準的な方法で除外され、そのうちの4頭はFPIおよび/またはスライドガラス上でqPCR陽性を示した。 結論と臨床的重要性: リーシュマニアDNAは、CanLを示唆する皮膚病変を有する犬におけるリーシュマニア感染を診断するために、皮膚細胞診標本およびFPIからqPCRによって効率的に検出することができる。.背景: 通过发现无鞭毛体可以检测皮肤病变中的利什曼原虫。 PCR检测利什曼原虫DNA具有更高的灵敏度,并且无创临床样本的PCR检测已被用于诊断人类皮肤利什曼病。 目的: 研究I:确认是否能从已被Diff-Quik®染色的犬皮肤病变细胞学载玻片中,有效提取和扩增利什曼原虫DNA。研究II:染色细胞学和滤纸印迹(FPI)均采自疑似犬利什曼病(CanL)的皮肤病变,评估利什曼原虫定量(q)PCR方法对这些样本的诊断效果。 动物: 54只犬的皮肤病变样本。 方法和材料: 研究I:细胞学检查可见无鞭毛体的19张载玻片(来自9只犬),对其进行利什曼原虫-qPCR检测;从 12只犬身上获得的15张载玻片样本作为对照,细胞学检查未见无鞭毛体,经ELISA检测利什曼原虫血清阴性。研究II:33只症状符合利什曼病的犬,用利什曼原虫-qPCR法检测其皮肤病变载玻片和FPI。 结果: 研究I:所有可见无鞭毛体的载玻片,其qPCR检测均为阳性,而所有对照载玻片的检测结果均为阴性。 研究II: 13只确诊利什曼病的患犬中,8只细胞学上可见无鞭毛体,而利什曼原虫-qPCR检测中11张载玻片和13份FPI呈阳性。用标准方法排除利什曼病的20只犬中,经qPCR检测FPI和/或载玻片,有4只呈阳性。 结论和临床价值: 用qPCR技术检测皮肤细胞学和FPI中的利什曼原虫DNA,可有效诊断皮肤病变疑似CanL的利什曼原虫感染。.A detecção de Leishmania em lesões cutâneas é possível pela visualização de amastigotas. A detecção de DNA de Leishmania por PCR possui maior sensibilidade, e a PCR tem sido utilizada para diagnosticar leishmaniose cutânea em humanos utilizando espécimens clínicos não invasivos.Estudo I: determinar se o DNA de Leishmania pode ser eficientemente extraído e amplificado de lâminas coradas em Diff-QuikAmostras cutâneas de lesões de 54 cães. MÉTODOS E MATERIAIS: Estudo I: Leishmania-qPCR foi realizado em 19 lâminas de vidro (de nove cães) com amastigotas citologicamente visíveis. Quinze lâminas sem amastigotas visíveis, obtidas de 12 cães soronegativos para Leishmania por ELISA serviram como controle. Estudo II: Leishmania-qPCR foi realizado em lâminas de vidro e em FPI de lesões cutâneas compatíveis com leishmaniose clínica em 33 cães.Estudo I: todas as amostras com amastigotas visíveis apresentaram qPCR positivo, enquanto todas as lâminas controle tiveram resultados negativos. Estudo II: dos 13 cães diagnosticados definitivamente com leishmaniose clínica, oito apresentaram amastigotas visíveis na citologia, enquanto Leishmania-qPCR foi positivo em 11 lâminas de vidro e 13 FPI. Descartou-se a leishmaniose pelos métodos convencionais em 20 cães, dos quais quatro apresentaram qPCR positiva na FPI e/ou nas lâminas de vidro. CONCLUSÕES E IMPORTÂNCIA CLÍNICA: O DNA de Leishmania pode ser eficientemente detectado por qPCR de espécimens citológicos cutâneos e FPI para diagnosticar uma infecção por Leishmania em cães com lesões cutâneas sugestivas de CanL.

The outcome of infection with Leishmania infantum in dogs is variable, which is thought to be due to the nature of the immune response mounted by the host. As a consequence, the clinical signs and severity of canine leishmaniosis vary between individual dogs. Host immunogenetic factors might play an important role in determining the outcome of infection. The aim of this study was to examine polymorphisms in innate and adaptive immune response genes, to determine whether any of these were associated with susceptibility or resistance to L. infantum infection. Genomic DNA was obtained from two groups: pet dogs in endemic regions of Europe and a group of Beagles exposed to sand fly infection as part of a vaccine study. Genotyping was performed using a SNP (single nucleotide polymorphism) array for selected immune response genes. The first part of the study compared 62 clinical cases with 101 clinically unaffected dogs that were seronegative for Leishmania antibodies. One SNP in the CIITA gene demonstrated a significantly higher minor allele frequency in the case group, compared with the control group at the individual SNP level after permutation, but was not significant after correction for multiple testing. The second part of the study examined 48 Beagle dogs exposed to L. infantum over two transmission seasons. Twenty-seven dogs with a resistant phenotype (no evidence of clinical disease, seronegative at the end of the study period, negative on lymph node culture and only transiently PCR positive in bone marrow) were compared with 21 dogs demonstrating a susceptible phenotype (clinical disease, seropositive, positive lymph node culture and consistently PCR positive in bone marrow). Three SNPs in TLR3, two SNPs in PTPN22 and one SNP in TLR4 and IL1A were associated with the susceptible phenotype in the Beagle group at the individual SNP level after permutation analysis, but were not significant after correction for multiple testing. Further validation of these SNPs is required in a larger cohort of dogs, ideally with extreme phenotypes to confirm an association with the outcome of L. infantum infection.

Background Feline leishmaniosis caused by Leishmania infantum is considered a rare disease in endemic areas, whereas subclinical infections are common. Immune response plays a key role in driving the course of L. infantum infection in other host species; however, the feline cell-mediated immune response to L. infantum infection has not yet been investigated. The aim of this study was to determine the cell-mediated immune response specific to L. infantum by means of interferon (IFN)-γ release in whole blood assay from cats living in endemic areas (66 in Sicily and 113 in Catalonia) and to compare with antibody levels to L. infantum [enzyme-linked immunosorbent assay (ELISA) and immunofluorescence antibody test (IFAT)], blood parasite load and retroviral infections. Results Most cats (n = 140) were L. infantum antibody negative and only 22% (n = 39) were positive. Only 9 and 2% of tested cats had a feline immunodeficency virus (FIV) infection or a feline leukemia virus (FeLV) infection, respectively. Thirty-two cats out of 179 (18%) produced IFN-γ after stimulation with L. infantum soluble antigen (LSA) while the majority of cats (93%) produced IFN-γ after stimulation with concanavalin A (ConA). Six LSA-IFN-γ-producer cats were seropositive (three to ELISA and five to IFAT) but they were polymerase chain reaction (PCR) negative, while only one cat was antibody- and PCR-positive. Significant positive correlations were found between IFN-γ concentrations after stimulation with LSA and ConA, and between serology and PCR testing. No association was found between FIV status and LSA or ConA-IFN-γ production. Combining PCR, serology and specific IFN-γ concentration results, we found that 36% of cats studied were exposed to L. infantum. Conclusions As expected, cats from endemic areas produce IFN-γ after ex vivo blood stimulation with LSA and therefore are able to activate a cell-mediated adaptive immune response against the parasite that is variably associated with antibody or blood PCR positivity. The association of this assay to serological and molecular tests provides a better estimate of cat exposure to L. infantum.

Cutaneous mast cell tumours (cMCTs) are one of the most common cutaneous tumours in ferrets (Mustela putorius furo). However, limited information is available regarding cytological and histological features of these tumours and studies evaluating KIT expression are lacking in this species. The aims of this prospective study were to describe the most common clinical, cytological and histological features of cMCTs in ferrets and to compare the usefulness of different staining techniques in the diagnosis of these tumours in ferrets as well as evaluating KIT expression in neoplastic mast cells (MCs) by immunohistochemistry. Macroscopically, the tumours were small, round to plaque-like and frequently associated with surface crusting. The most common locations were the extremities and the trunk. MC granules were stained in all cases using toluidine blue (TB) and Wright-Giemsa stains in cytological specimens, but none stained with modified Wright's stain. Haematoxylin and eosin and TB on histological sections failed to stain MC granules in all the cases. Cytological and histological examination revealed low to moderate anisocytosis and anisokaryosis. An infiltrative rather than a delineated or encapsulated growth pattern was noted histologically in all cases. Eosinophilic infiltration was not uncommon and 'collagenolysis' was detected on cytological and histological examination. KIT expression was detected in all cases evaluated. In approximately one third of the cases the MCs exhibited KIT labelling pattern I and in the remaining ferrets, KIT pattern III. No correlation was found between KIT expression pattern and biological behaviour.

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.

Venezuela is a country where human and canine leishmaniosis due to Leishmania infantum, Leishmania braziliensis and other Leishmania spp. is endemic. However, only limited data is available on canine Leishmania infection in Venezuela. The aim of this cross-sectional study was to evaluate the prevalence of Leishmania infection in dogs (n = 152) from the states of Lara (n = 91) and Yaracuy (n = 61) in Venezuela by means of serological and molecular methods. Physical examination was performed and blood samples were collected from all dogs. Serology for antibodies reactive with L. infantum and L. braziliensis antigens was assessed by the enzyme-linked immunosorbent assay (ELISA) and detection of Leishmania DNA from blood samples was evaluated by kinetoplast Leishmania real-time polymerase chain reaction (RT-PCR). In addition, Leishmania internal transcribed spacer (ITS-1) RT-PCR was performed on the samples positive by kinetoplast RT-PCR. The prevalence of Leishmania infection based on serological and/or molecular techniques was 11.8%. The seroprevalence for L. infantum and L. braziliensis antigens were 2.1% (3/144) and 8.3% (12/144), respectively. All dogs from the state of Yaracuy were serologically negative to L. infantum while 4.6% (4/86) of the dogs were reactive to L. braziliensis antigen. Fourteen percent (8/58) of the dogs from the state of Lara were positive to L. infantum and 5.2% (3/58) to L. braziliensis antigen. Three dogs were positive to both Leishmania spp. antigens. By RT-PCR, 6.5% (4/61) and 4.4% (4/91) of the dogs were positive for infection in the states of Lara and Yaracuy, respectively. The RT-PCR product of one dog from the state of Yaracuy was sequenced revealing a 100% identity with L. infantum. However, all RT-PCR positive dogs were seronegative to both Leishmania spp. antigens. In conclusion, the positivity for Leishmania spp. infections observed indicates that dogs are frequently infected by L. infantum, L. braziliensis or related Leishmania spp. in Venezuela.

Leishmaniosis due to Leishmania infantum is a complex infection that can affect both humans and dogs, and present a wide range of clinical signs and clinicopathological abnormalities. The conventional treatment of this disease is challenging due to the fact that complete parasitological cure commonly does not occur. Furthermore, treatment of the disease with the conventionally used drugs has several shortcomings. These include the need for long-term treatment, side effects and the formation of drug resistance. Moreover, it is important to highlight that the host immune responses play a crucial role in the outcome of this infection. For this reason, the use of immunotherapy in clinical leishmaniosis to improve the result of treatment with the conventional anti-leishmanial drugs by enhancing the immune response is imperative. The aim of this review is to provide a comparative overview of the wide range of immunotherapeutical approaches and strategies for the treatment of L. infantum infection in animals focusing on dogs.

Background Dogs with canine leishmaniosis (CanL) due to Leishmania infantum can show a wide spectrum of clinical and clinicopathological findings at the time of diagnosis. The aim of this paper is to describe the possible application of acute phase proteins (APPs) for the characterization and management of this disease, based on previously published information on the utility of APPs in CanL and the experience of the authors in using APPs as analytes in the profiling of canine diseases. Main body Dogs diagnosed with L. infantum infection by serology, polymerase chain reaction, cytological or histopathological identification, can be divided into three groups based on their clinical condition at physical examination and their APPs concentrations: Group 1: dogs with no clinical signs on physical examination and APPs in reference range; Group 2: dogs with changes in APPs but no clinical signs on physical examination; Group 3: dogs with clinical signs and changes in APPs. This report describes the main characteristics of each group as well as its association with the clinical classification schemes of CanL. Conclusion APPs concentration can be a useful clinical tool to characterize and manage CanL.

Background: In the Mediterranean basin, Leishmania infantum is a major cause of disease in dogs, which are frequently co-infected with other vector-borne pathogens (VBP). However, the associations between dogs with clinical leishmaniosis (ClinL) and VBP co-infections have not been studied. We assessed the risk of VBP infections in dogs with ClinL and healthy controls. Methods: We conducted a prospective case-control study of dogs with ClinL (positive qPCR and ELISA antibody for L. infantum on peripheral blood) and clinically healthy, ideally breed-, sex- and age-matched, control dogs (negative qPCR and ELISA antibody for L. infantum on peripheral blood) from Paphos, Cyprus. We obtained demographic data and all dogs underwent PCR on EDTA-blood extracted DNA for haemoplasma species, Ehrlichia/Anaplasma spp., Babesia spp., and Hepatozoon spp., with DNA sequencing to identify infecting species. We used logistic regression analysis and structural equation modelling (SEM) to evaluate the risk of VBP infections between ClinL cases and controls.Results: From the 50 enrolled dogs with ClinL, DNA was detected in 24 (48%) for Hepatozoon spp., 14 (28%) for Mycoplasma haemocanis, 6 (12%) for Ehrlichia canis and 2 (4%) for Anaplasma platys. In the 92 enrolled control dogs, DNA was detected in 41 (45%) for Hepatozoon spp., 18 (20%) for M. haemocanis, 1 (1%) for E. canis and 3 (3%) for A. platys. No Babesia spp. or “Candidatus Mycoplasma haematoparvum” DNA was detected in any dog. No statistical differences were found between the ClinL and controls regarding age, sex, breed, lifestyle and use of ectoparasitic prevention. A significant association between ClinL and E. canis infection (OR = 12.4, 95% CI: 1.5–106.0, P = 0.022) was found compared to controls by multivariate logistic regression. This association was confirmed using SEM, which further identified that younger dogs were more likely to be infected with each of Hepatozoon spp. and M. haemocanis, and dogs with Hepatozoon spp. were more likely to be co-infected with M. haemocanis.Conclusions: Dogs with ClinL are at a higher risk of co-infection with E. canis than clinically healthy dogs. We recommend that dogs diagnosed with ClinL should be tested for E. canis co-infection using PCR.

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 (qPCRs) 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.

The aim of this study was to investigate the presence of canine immunoglobulins (Ig) G against Demodex proteins in the sera of healthy dogs and of dogs with juvenile generalized demodicosis (CanJGD) with or without secondary pyoderma. Demodex mites were collected from dogs with CanJGD. Protein concentration was measured and a western blot technique was performed. Pooled sera from healthy dogs reacted mainly with antigen bands ranging from 55 to 72 kDa. Pooled sera from dogs with CanJGD without secondary pyoderma reacted either with 10 kDa antigen band or 55 to 72 kDa bands. Pooled sera from dogs with CanJGD with secondary pyoderma reacted only with a 10 kDa antigen band. The results of this study suggest that both healthy dogs and dogs with CanJGD develop a humoral response against different proteins of Demodex canis.

A broad spectrum of clinical manifestations ranging from a chronic subclinical infection to a non-self-limiting illness has been described for canine leishmaniosis (CanL). This clinical variation is determined by a variable immune response, presumably genetically determined, against the infection. Although different types of adaptive immune response in dogs with CanL have been investigated in several studies, the mechanisms that underlie and determine this variability are still poorly understood. It is currently thought that innate immune response, and particularly the role of specific mediators of the innate immune system, such as toll-like receptors (TLRs), plays a central role in this polarization. However, there is limited data available concerning the role that TLRs play in canine Leishmania infantum infection. The objective of this descriptive study was to characterize and compare the inflammatory pattern, the Leishmania burden and expression of TLR2 in skin lesions derived from dogs with different clinical stages of leishmaniosis and cutaneous lesions. Routine histology, Leishmania and TLR2 immunohistochemistry assays were performed in 11 patients with papular dermatitis (stage I - mild disease) and 10 patients with other cutaneous lesions (stage II-III - moderate to severe disease). A significantly higher frequency of granuloma formation was demonstrated in skin samples of dogs with stage I when compared with dogs of stage II-III. Although not statistically significant, a trend for a lower parasite burden was observed for skin lesions of dogs with stage I when compared with dogs of stage II-III. A lower expression of TLR2 in skin biopsies from dogs with stage I was statistically significant compared with stage II-III. The results obtained in this study indicated an association with TLR2 in the pathogenesis of canine cutaneous leishmaniosis. Further studies are required to fully elucidate these findings.

Cell cannibalism refers to the engulfment of cells by nonprofessional phagocytic cells. Studies in human medicine have demonstrated a relationship between the presence of cell cannibalism by neoplastic cells and a poor outcome, and have shown a positive correlation with the presence of metastasis at the time of diagnosis. The biologic significance of cell cannibalism is unknown, but it is proposed that it may represent a novel mechanism of tumor immune evasion as a survival strategy in cases of unfavorable microenvironmental conditions. This report describes clinical and morphologic features of 3 cases of dogs with malignant neoplasia in which the presence of cellular cannibalism was observed in cytologic and histologic specimens. In the 1(st) case, a dog with a primary tonsillar squamous cell carcinoma with metastasis to retropharyngeal lymph nodes had neoplastic epithelial cells engulfing neutrophils noted in cytologic examination of the lymph nodes. In the 2(nd) case, neoplastic epithelial cells were seen engulfing each other in fine-needle aspirates from a primary mammary carcinoma with lung metastasis. In the 3(rd) case, poorly differentiated neoplastic mast cells from a recurrent, metastatic grade III mast cell tumor were observed cannibalizing eosinophils. A brief review of the literature describing known cell-into-cell relationships and the possible biologic significance and mechanisms involved in this phenomenon is provided. The relationship between cell cannibalism and distant metastasis should be explored in further studies, as it may prove to be a criterion of malignancy, as it is proposed in human medicine.

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. 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). 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. 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.

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 5 days. 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 ± 0 pg/mL) and 18 dogs as IFN-γ producers (mean ± SD: 2885.3 ± 4436.1 pg/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.

Summary The leishmanin skin test (LST), a delayed-type hypersensitivity (DTH) reaction to Leishmania infantum, can specifically identify dogs that have made a cell-mediated immune response to L. infantum infection. The Ibizan hound appears to be more resistant to L. infantum infection than other breeds of dog. The aim of this study was to describe the histological and immunohistochemical changes induced by the LST in Ibizan hounds living in an area highly endemic for leishmaniosis. The majority of dogs were apparently healthy, lacked serum antibody to L. infantum and blood parasitaemia, but had marked specific interferon gamma production after in-vitro blood stimulation with L. infantum. Leishmanin (3 × 108 killed promastigotes of L. infantum/ml) was injected intradermally and biopsy samples were obtained from a positive reaction at 72 h from nine Ibizan hounds. A moderate to intense, perivascular to interstitial dermatitis and panniculitis characterized the inflammatory response at the injection site. In addition, three samples had diffuse inflammation in the deep dermis and panniculus. Oedema and necrosis were present in the deep dermis and panniculus. Congestion and haemorrhage were observed in five biopsies. T lymphocytes (CD3+) and large mononuclear cells (lysozyme−) were the most prevalent cells. CD3+ cells were significantly more numerous than CD20+ B cells and lysozyme+ cells. B cells were sparsely distributed, especially in the deep dermis and panniculus. Rare neutrophils and macrophages (lysozyme+) were observed with few eosinophils. Toll-like receptor (TLR)-2 protein was expressed in large mononuclear cells mainly located in the superficial dermis. Leishmania immunohistochemistry was negative and quantitative polymerase chain reaction was positive in all cases. The intradermal injection of killed L. infantum promastigotes in Ibizan hounds causes similar histological and immunohistochemical findings to those described for human subjects and are indicative of a DTH response. Moreover, TLR2 protein is expressed in inflammatory cells similar to findings in clinically affected skin biopsy samples.

The clinical outcome of Leishmania infantum infection in dogs varies from subclinical infection to severe disease. Researchers attribute this variability in clinical manifestations to the ability of the immune response to limit pathogen multiplication and dissemination, which is, in part, likely determined by the immune response genes. The aim of this study was to test the hypothesis that MHC class II genes are associated with disease outcome of experimental L. infantum infection in Beagles. Dog leukocyte antigen (DLA) class II haplotypes were characterised by sequence-based typing of Beagle dogs experimentally infected with L. infantum during vaccine challenge studies. Variability of response to infection was determined by clinical score, serology and quantification of L. infantum DNA in the bone marrow over the study period. Dogs showed limited DLA diversity and the DLA profiles of dogs recruited for the different vaccine challenge studies differed. There were variable responses to infection, despite the apparent restriction in genetic diversity. One haplotype DLA-DRB1*001:02–DQA1*001:01–DQB1*002:01 was associated with increased anti-Leishmania antibodies in one infection model, but no DLA associations were found in other groups or with parasite load or clinical score. Examination of this particular DLA haplotype in a larger number of dogs is required to confirm whether an association exists with the immune or clinical responses to L. infantum infection.

Background Feline infectious agent studies are lacking in Cyprus. The aims of this study were to determine the prevalence and risk factors for various feline infectious agents, including feline vector-borne pathogens (FVBP), in cats from Cyprus. Methods A cross-sectional, descriptive, multicentre study was performed on 174 feline samples [138 owned and 36 shelter-feral, including both healthy (43) and non-healthy (131), cats] from private veterinary clinics from all six districts of Cyprus. Real-time quantitative polymerase chain reaction (qPCR) assays were used to detect Mycoplasma haemofelis (Mhf), “Candidatus Mycoplasma haemominutum” (CMhm) and “Candidatus Mycoplasma turicensis” (CMt). The population was tested for four FVBP including Bartonella henselae and Leishmania spp. using qPCR, while conventional PCR assays were used to detect Ehrlichia/Anaplasma spp. and Hepatozoon spp. Serological assays were performed to detect Leishmania infantum antibodies, feline leukaemia virus (FeLV) antigen and feline immunodeficiency virus (FIV) antibodies. Statistical analysis was performed to test associations and possible risk factors between variables and infectious agents. Results Ninety-six (55.2%) of the 174 cats were PCR-positive for at least one infectious agent. Forty-six cats (26.4%) were haemoplasma positive, including 13 (7.5%) for Mhf, 36 (20.7%) for CMhm and 12 (6.9%) for CMt. Sixty-six cats (37.9%) were positive for Hepatozoon spp., while 19 (10.9%) were positive for B. henselae, four (2.3%) for Leishmania spp. and one (0.6%) for Ehrlichia/Anaplasma spp. Sequencing revealed the presence of Hepatozoon felis, L. infantum and Anaplasma platys. Of the 164 cats that underwent retroviral serology, 10 (6.1%) were FeLV-positive and 31 (18.9%) were FIV-positive, while L. infantum serology was positive in 7 (4.4%) of the 160 cats tested. Multivariable logistic regression revealed significant associations for various infectious agents including L. infantum with each of Hepatozoon spp. and CMt infection. Conclusions A high prevalence of infectious agents was found in cats from Cyprus with Mhf, CMhm, CMt, L. infantum, B. henselae, H. felis, A. platys, FeLV and FIV infections reported for the first time. The significant associations between different pathogens provide a better understanding of similarities in the epidemiology of these pathogens and interactions between them. Electronic supplementary material The online version of this article (doi:10.1186/s13071-017-2063-2) contains supplementary material, which is available to authorized users.

Canine leishmaniosis (CanL) is an important zoonotic disease; however, the efficacy of available vaccines for the prevention of naturally-occurring Leishmania infantum (L. infantum) infection in dogs remains unclear. The objective of this review was to determine the efficacy of currently available vaccines to prevent naturally-occurring L. infantum infection in dogs. Four bibliographic databases (CAB Direct 2011, Web of Science 2011, U.S. National Library of Medicine 2011 and Literatura Latino Americana e do Caribe em Ciências da Saúde) were searched along with eight sets of conference proceedings and the International Veterinary Information Service (IVIS) database, from 1980 to November 2012. Randomised controlled trials (RCTs), non-randomised clinical trials (NRCTs), cohort studies and case-control studies that investigated vaccine efficacy for natural L. infantum infection in dogs were eligible for inclusion. Two review authors independently assessed each study against the inclusion criteria, independently extracted relevant data from all included studies and assessed the risk of methodological shortcomings in each individual study. The odds ratio (OR) and absolute risk reduction (ARR) for dichotomous outcomes and mean difference for continuous outcomes were calculated. Meta-analysis was not performed due to heterogeneity of the studies identified. The search was conducted for all mitigations for CanL and yielded the title and abstract of 937 articles, from which 84 articles were screened based on full text. Twelve studies on vaccinations (five RCTs, seven NRCTs) were identified. Ten studies were at a high risk of methodological shortcomings, whilst two were at an unclear risk. The use of 200 μg ALM protein, Leishmune(®), CaniLeish(®), LiESAp with MDP, and ALM with BCG tended to significantly reduce the proportion of dogs infected with L. infantum based on either parasitological or serological evidence. The use of lyophilized protein vaccine significantly increased the proportion of dogs infected with L. infantum based on either parasitological or serological evidence. There is peer-reviewed evidence that control measures are effective in preventing CanL with the results suggesting that between 6 and 54% of infections could be prevented with vaccination. However, this evidence is based on a small number of RCTs, all of which are either at high or unclear risk of methodological shortcomings. Well-designed, adequately powered and properly reported randomised clinical trials are needed to clearly establish efficacy of vaccines as CanL control measures.

This study aimed to evaluate the frequency and spatial distribution of canine leishmaniosis (CanL) in France, Greece, Italy, Portugal and Spain. An online questionnaire investigated the location and frequency of CanL cases diagnosed by veterinary practitioners. Further data from the practice management systems of veterinary clinics in France were provided by a financial benchmarking company in relation to all treatment and test invoice data from participating practices. The geographical and temporal web interest in leishmaniosis was explored using Google Trends. Veterinary practitioners from France, Greece, Italy, Portugal and Spain completed 1231 questionnaires. The percentage of practice-attending dogs with a veterinary diagnosis of CanL ranged from 0.71% in France to 7.80% in Greece. However, due to regional differences in response rates, particularly in France, the mean regional estimates may better reflect the disease burden. Benchmarking data relating to approximately 180,000 dogs estimated that 0.05% of dogs attending veterinary clinics were treated for CanL or euthanased with suspected CanL in France. The regional frequency of Google web queries for leishmaniosis generally reflected the spatial patterns of disease identified from the other data sources. In conclusion, CanL was a relatively common diagnosis in veterinary clinics in many regions of the countries studied. Knowledge of CanL in endemic areas can direct the use of preventative measures and help estimate the likelihood of infection in dogs visiting or inhabiting these countries.

An increasing number of non-endemic vector-borne pathogens have been described in dogs imported to the UK in the past two decades. Recently, an outbreak of canine babesiosis in south-east England has raised veterinary awareness with regard to the impact of such diseases on the UK canine population. Canine hepatozoonosis, caused by Hepatozoon canis and transmitted by the ingestion of Rhipicephalus sanguineus ticks, is widespread in the Mediterranean basin. Herein we describe the first three molecularly confirmed clinical cases of canine hepatozoonosis in dogs imported into the UK. Veterinarians in the UK should be aware of H. canis as a potential infection in imported dogs, especially in the face of the expanding distribution of R. sanguineus ticks in Europe.

Prevalence of Babesia microti-like infection in red foxes (Vulpes vulpes) from Portugal Abstract The prevalence of piroplasm (order Piroplasmida) infection was assessed in blood and bone marrow samples from 91 red foxes (Vulpes vulpes) from northern, central and southern Portugal by means of molecular methods. PCR for the 18S rRNA gene of Babesia spp. followed by sequencing revealed 63 foxes positive for the Babesia microti-like piroplasm (syn. Theileria annae) (69.2%; 95% confidence interval [CI]: 58.7–78.5%) and one fox positive for Babesia canis (1.1%; 95% CI: 0.0–6.0%). Positivity to the B. microti-like piroplasm or B. canis in 43 blood samples (83.7%) was significantly higher (p < 0.001) than in 43 paired bone marrow samples (20.9%). There were no statistically significant differences in the prevalence of infection between genders (p = 0.219) or age groups (

Indexación: Web of Science. Piroplasmids are tick-borne protozoan parasites that infect blood cells (erythrocytes, lymphocytes or other leukocytes) or endothelial cells of numerous wild and domestic vertebrates worldwide. They cause severe disease in livestock, dogs, cats, wild mammals and, occasionally, in humans. Piroplasmid infections are prevalent in wild carnivores worldwide although there is limited information about their clinical and epidemiological importance. There are currently nine recognized species of Babesia, two of Theileria, two of Cytauxzoon and one of Rangelia infecting captive and wild carnivores, including members of Canidae, Felidae, Mustelidae, Procyonidae, Ursidae, Viverridae, Hyaenidae and Herpestidae in the Americas, Eurasia and Africa. However, the number of piroplasmid species is likely higher than currently accepted due to the reported existence of DNA sequences that may correspond to new species and the lack of studies on many host species and biogeographical areas. Indeed, many species have been recognized in the last few years with the advancement of molecular analyses. Disease and mortality have been documented in some wild carnivores, whereas other species appear to act as natural, subclinical reservoirs. Various factors (e.g. unnatural hosts, stress due to captivity, habitat degradation, climate fluctuation or immunosuppression) have been associated with disease susceptibility to piroplasmid infections in some species in captivity. We aimed to review the current knowledge on the epidemiology of piroplasmid infections in wild carnivores and associated tick vectors. Emphasis is given to the role of wild carnivores as reservoirs of clinical piroplasmosis for domestic dogs and cats, and to the importance of piroplasmids as disease agents for endangered carnivores. http://parasitesandvectors.biomedcentral.com/articles/10.1186/s13071-016-1808-7

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.