Cutaneous immune mechanisms in canine leishmaniosis due to Leishmania infantum

Canine leishmaniosis (CanL) caused by the parasite Leishmania infantum is a systemic disease with variable clinical signs. The disease is endemic in the Mediterranean countries and dogs are the main domestic reservoir of the parasite. The quite complicated immune response against the parasite is crucial for the evolution of CanL infection with the skin playing a major role in its immunopathogenesis.After the inoculation of Leishmania promastigotes into the dermis by sand fly bites, complement factors, Langerhan's cells, neutrophils, fibroblasts and keratinocytes are involved in the activation of the innate arm of the skin immune system, with the macrophages and dendritic cells to play a major key role.The effective activation of cellular immunity is the cornerstone of dog's resistance against the parasite. Promastigotes reaching the dermis are engulfed, processed and transferred by APCs to draining lymph nodes to stimulate naïve T-cells for proliferation and differentiation into armed effector T-cells. Th1 cells activate the infected macrophages to kill Leishmania, whereas Th2 cells divert the immune response to humoral immunity and down regulation of cellular immunity with Th1 cell anergy. Inhibition of co-stimulatory molecules expression by infected macrophages contributes to T-cell anergy. In canine subclinical infections cutaneous lymphocytic infiltrate and parasites are absent, as opposed to dogs with clinical leishmaniosis. CD8+ cells constitute a significant population of cellular immunity in CanL since they outnumber CD4+ cells in the dermis, producing IFN-γ in sub clinically infected dogs and high levels of IL-4 in dogs with clinical leishmaniosis.Numerous B-lymphocytes have been shown to heavily infiltrate the dermis at least in exfoliative dermatitis in CanL. A mixed Th1/Th2 cytokine profile has been found in the dermis of naturally infected with L. infantum dogs. In the skin of dogs with clinical leishmaniosis, where plasma cells outnumber T lymphocytes in the dermal infiltrate, there is an overproduction of IL-4, IL-13 and TNF-α leading to Th2-biased humoral immune response. The issue of humoral immunity polarization in CanL remains controversial. Much still needs to be learned about other mechanisms underlying the complex interaction between the skin immune system and the parasite.     

A minimally invasive approach to spleen histopathology in dogs: A new method for follow-up studies of spleen changes in the course of Leishmania infantum infection

Severe forms of zoonotic visceral leishmaniosis (ZVL) are associated with disruption of the spleen structure. However, the study of spleen histology requires splenectomy or necropsy. In this work, we present a minimally invasive cell-block technique for studying spleen tissue histology in dogs with ZVL. We examined 13 dogs with and seven dogs without Leishmania infantum infection. The dogs with Leishmania infection had a lower frequency of lymphoid follicles (2/13, Fisher’s test, P < 0.02) and a higher density of plasma cells (score 3, Fisher’s test, P < 0.02) than uninfected dogs (5/7 exhibiting lymphoid follicles and a plasma cell score of 1). The dogs with Leishmania infection also presented with granulomas (8/13) and infected macrophages (5/13). These differences in the histological presentations of spleen tissue from infected and uninfected dogs corresponded to changes observed in conventional histology. Hence, the cell-block technique described here may be used in the follow-up care and study of dogs with ZVL and other diseases in both clinical practice and research.     

Canine vector-borne disease in travelled dogs in Germany--a retrospective evaluation of laboratory data from the years 2004-2008

When importing dogs from various Mediterranean countries into Western Europe canine vector-borne infections are often considered as a major issue. Several diseases including babesiosis, leishmaniosis, hepatozoonosis, canine heartworm disease or ehrlichiosis can potentially be endemic in this region and pose a potential health risk for travelling dogs. Information on such infections in travelled dogs is scarce and therefore this study has been undertaken to examine the frequency of vector-borne infections in travelled dogs from the years 2004-2008. A total of 997 samples were screened by direct and/or indirect methods. Total seroprevalence was 7.5% with individual seroprevalence for the 3 species Leishmania spp., Ehrlichia canis and Babesia canis spp. ranging from 3.1 to 4.9%. Total detection rate for pathogens by direct methods was 3.5%. Ninteen Giemsa-stained blood smears were positive for large Babesia. None of the samples screened for microfilariae by Knott's test or for Dirofilaria immitis antigen by DiroChek^® were positive. Using PCR methods Leishmania-DNA was detected in 1/42 samples but none of 59 animals screened for E. canis-DNA was positive. The prevalence values as established by indirect and direct pathogen detection are considered as rather low.     

The first report of autochthonous non-vector-borne transmission of canine leishmaniosis in the Nordic countries

Background

Leishmania spp. are zoonotic protozoans that infect humans and other mammals such as dogs. The most significant causative species in dogs is L. infantum. In dogs, leishmaniosis is a potentially progressive, chronic disease with varying clinical outcomes. Autochthonous cases of canine leishmaniosis have not previously been reported in the Nordic countries.

Results

In this report we describe the first diagnosed autochthonous cases of canine leishmaniosis in Finland, in which transmission via a suitable arthropod vector was absent. Two Finnish boxers that had never been in endemic areas of Leishmania spp., had never received blood transfusions, nor were infested by ectoparasites were diagnosed with leishmaniosis. Another dog was found with elevated Leishmania antibodies. A fourth boxer dog that had been in Spain was considered to be the source of these infections. Transmission occurred through biting wounds and semen, however, transplacental infection in one of the dogs could not be ruled out.Two of the infected dogs developed a serious disease and were euthanized and sent for necropsy. The first one suffered from membranoproliferative glomerulonephritis and the second one had a chronic systemic disease. Leishmania sp. was detected from tissues by PCR and/or IHC in both dogs. The third infected dog was serologically positive for Leishmania sp. but remained free of clinical signs.

Conclusions

This case report shows that imported Leishmania-infected dogs may pose a risk for domestic dogs, even without suitable local arthropod vectors.     

A survey on canine leishmaniasis and phlebotomine sand flies in central Italy

Zoonotic visceral leishmaniasis (ZVL) is a vector-transmitted zoonosis caused by the parasitic protozoan Leishmania infantum. Bloodsucking sand flies of the subfamily Phlebotominae are the obligatory insect hosts, and the dog is the only domestic reservoir.This study reports data from a survey of canine infection and sand fly phlebotomine monitoring in the province of Perugia in central Italy. The overall seroprevalence in a total of 100 dogs tested was 8% (95% confidence interval: 3.8-15.6%). Data analysis revealed that serological positivity was statistically associated with age (p-value = 0.03) and the area where dogs lived. Standard blacklight traps employed for sampling Culicoides midges in bluetongue disease surveillance were used in phlebotomine monitoring. A total of 5698 sand flies were collected and the two species, Leishmania competent vectors, were identified, Phlebotomus perfiliewi (50%) and Phlebotomus perniciosus (30%).     

A systematic review of the efficacy of prophylactic control measures for naturally-occurring canine leishmaniosis,part I: Vaccinations

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.     

Detection of anti-Leishmania infantum antibodies in sylvatic lagomorphs from an epidemic area of Madrid using the indirect immunofluorescence antibody test

An outbreak of human leishmaniosis was confirmed in the southwest of the province of Madrid, Spain, between July 2009 and December 2012. Incidence of Leishmania infection in dogs was unchanged in this period, prompting a search for alternative sylvatic infection reservoirs. We evaluated exposure to Leishmania in serum samples from animals in the area with an indirect immunofluorescence test (IFAT). Using promastigotes from six culture passages and a 1/25 threshold titer, we found anti-Leishmania infantum seroreactivity in 9.3% of cats (4 of 43), 45.7% of rabbits (16/35) and 74.1% of hares (63/85). Use of promastigotes from >10 in vitro passages resulted in a notably IFAT lower titer, suggesting antigenic changes during extended culture. Postmortem inspection of seropositive animals showed no clinical signs of infection. The results clearly suggest that asymptomatic hares were the main reservoir in the outbreak, and corroborate IFAT as a sensitive serological surveillance method to detect such cryptic Leishmania infections.     

Rapid detection of Leishmania infantum infection in dogs: comparative study using an immunochromatographic dipstick rk39 test and direct agglutination

A rapid, sensitive and specific tool for detection of Leishmania infantum infection in dogs, would be highly desirable, because it would allow control interventions in endemic areas of Zoonotic visceral leishmaniosis (ZVL). In this study, we compared an immunochromatographic dipstick test with direct agglutination test (DAT) for detecting L. infantum infections in dogs from areas of ZVL endemic in Iran. The validity of the dipstick rk39 (Cypress Diagnostic Company, Belgium) for canine visceral leishmaniosis (CVL) was compared with a standard direct agglutination test on 116 clinically suspected dogs and 152 healthy controls from endemic areas of Ardabil and East Azerbaijan provinces, north-western of Iran for 1 year. A sensitivity of 70.9% and specificity of 84.9% were found at a 1:320 cut off titer when DAT confirmed cases were compared with healthy control. As the dipstick rk39 test is rapid, noninvasive and does not require much expertise or elaborate equipment, it can be used for screening and diagnosis of canine visceral leishmaniosis in remote endemic areas.     

Methods for diagnosis of canine leishmaniasis and immune response to infection

Canine leishmaniasis (CanL) caused by Leishmania infantum (syn. L. chagasi, in Latin America), which is transmitted by the bite of phlebotomine sand flies, is endemic and affects millions of dogs in Europe, Asia, North Africa and South America. It is an emergent disease in North America. Early detection and treatment of infected animals may be critical in controlling the spread of the disease and is an essential part of human zoonotic visceral leishmaniasis control. The laboratory diagnosis of CanL still poses a challenge, despite progress made in the development of several direct and indirect methods. An effective diagnosis test, apart of being able to confirm a clinical suspicion in a single patient as well as to detect infection in asymptomatic dogs, should have high sensitivity, specificity and reproducibility; it must be simple, easy to perform, non-expensive, feasible in regional laboratories or adaptable for field conditions. Ideally, it should detect all Leishmania-infected dogs, preferentially using non-invasive collection of biological samples. In this paper we review the advantages and shortcomings of the available procedures for CanL diagnosis in the different phases, e.g. pre-patent and patent period of the infection and methods to determine the related immune response.     

Leishmania infantum in wild animals in endemic areas of southern Italy

Leishmania infantum infection in wildlife is increasingly reported in Europe, but scant data are available in Italy so far. This study aimed to investigate the circulation of L. infantum among sylvatic hosts in Sicily (southern Italy), a highly endemic area for canine leishmaniosis, through serological and molecular tools. Target tissues (skin, spleen, lymph nodes) collected from 71 European rabbits, 2 European hares, 7 red foxes, 11 European wildcats and 1 pine marten, were qPCR analysed for the detection of L. infantum DNA. Additionally, 40 rabbits, older than one year, were serologically screened for specific anti-Leishmania antibodies. Leishmania infantum was molecularly diagnosed in 5.4% (n = 5) of the examined animals (3/71 European rabbits, 2/7 red foxes). In many of the qPCR positive animals (4/5), the parasite DNA was more prevalent in visceral than cutaneous tissues. None of the positive animal showed signs of disease and/or macroscopic alterations of organs; low parasitic burden in all positive tissue samples was also recorded. Only one rabbit serum (i.e., 2.5%) tested positive for anti-Leishmania antibodies. The seropositive rabbit was in good health status and no amastigotes were observed in lymph-node aspirate and blood smears.This study provides first evidence of L. infantum infection in wild animals from Sicily (southern Italy). Despite the low prevalence of infection here reported, the circulation of the Leishmania in wild reservoirs in Sicily remains worthy of future investigations for a better understanding of their role in the epidemiology of the disease as well as to fine-tune control strategies in the area.     

Serum cobalamin concentrations in dogs with leishmaniosis before and during treatment

Hypocobalaminemia in dogs is most commonly associated with gastrointestinal disorders leading to impaired absorption and utilization of cobalamin. The objectives of this study were to compare serum cobalamin concentrations between dogs with leishmaniosis and clinically healthy dogs, and to assess possible alterations of serum cobalamin concentrations in dogs with leishmaniosis at different timepoints during treatment. Fifty-five dogs with leishmaniosis and 129 clinically healthy dogs were prospectively enrolled. Diagnosis of leishmaniosis was based on clinical presentation, positive serology and microscopic detection of Leishmania amastigotes in lymph node aspiration smears. Twenty of the dogs with leishmaniosis were treated with a combination of meglumine antimonate and allopurinol for 28 days and serum cobalamin concentrations were measured in blood samples that were collected before initiation of treatment (timepoint 0) and on days 14 and 28. In order to estimate alterations of serum cobalamin concentrations during treatment, cobalamin concentrations were measured in blood samples from 20 out of 55 dogs with leishmaniosis at all timepoints. Serum cobalamin concentrations were significantly lower in dogs with leishmaniosis before treatment (median: 362 ng/L; IQR: 277−477 ng/L) compared to clinically healthy dogs (median: 470 ng/L; IQR: 367−632 ng/L; P = 0.0035). Serum cobalamin concentrations increased significantly in dogs with leishmaniosis on day 14 of treatment compared to timepoint 0 (P = 0.02).In the present study, serum cobalamin concentrations were significantly lower in dogs with leishmaniosis compared to clinically healthy dogs. In addition, there was an increase in serum cobalamin concentrations during treatment. The clinical significance of hypocobalaminemia in dogs with leishmaniosis remains to be determined.     

Perspectives on canine and feline hepatozoonosis

Two species of Hepatozoon are currently known to infect dogs and cause distinct diseases. Hepatozoon canis prevalent in Africa, Asia, southern Europe, South America and recently shown to be present also in the USA causes infection mainly of hemolymphoid organs, whereas Hepatozoon americanum prevalent in the southeastern USA causes myositis and severe lameness. H. americanum is transmitted by ingestion of the Gulf Coast tick Amblyomma maculatum and also by predation on infected prey. H. canis is transmitted by Rhipicephalus sanguineus, in South America also by Amblyomma ovale, and has also been shown to be transmitted transplacentally. Hepatozoonosis of domestic cats has been described mostly from the same areas where canine infection is present and the exact identity of the species which infect cats, their pathogenicity and vectors have not been elucidated. The diagnosis of hepatozoonosis is made by observation of gamonts in blood smears, histopathology, PCR or serology. The main treatment for H. canis is with imidocarb dipropionate whereas H. americanum infection is treated with an initial combination of trimethoprim-sulfadiazine, pyrimethamine and clindamycin followed by maintenance with decoquinate. Treatment for both diseases has not been reported to facilitate complete parasite elimination and new effective drugs are needed for the management of these infections. Prevention of hepatozoonosis should be based on avoidance of oral ingestion of infected tick vectors and infected prey.     

Evaluation of transformation growth factor beta1, interleukin-10, and interferon-gamma in male symptomatic and asymptomatic dogs naturally infected by Leishmania (Leishmania) chagasi

The aims of this study were to evaluate the immunomodulatory role of TGF-β₁, IL-10, and INF-γ in spleen and liver extracts and supernatant cultures of white spleen cells from male symptomatic and asymptomatic dogs, naturally infected by Leishmania (Leishmania) chagasi. Thirty dogs from Araçatuba, São Paulo, Brazil, an endemic leishmaniosis area, were selected by positive ELISA serological reaction for Leishmania sp. and divided into two groups: asymptomatic (n = 15) and symptomatic (n = 15) consisting of animals with at least three characteristic signs (fever, dermatitis, lymphoadenopathy, onychogryphosis, weight loss, cachexia, locomotion problems, conjunctivitis, epistaxis, hepatosplenomegaly, edema, and apathy). After euthanasia, spleen and liver fragments were collected for ex vivo quantification of TGF-β₁, IL-10, and INF-γ. Naturally active in vitro produced TGF-β₁ was also evaluated in spleen cell culture supernatant. Spleen and liver extract of asymptomatic dogs had higher mean TGF-β₁ levels than symptomatic dogs. High concentrations of IL-10 were found in spleen, and mainly in liver extract of both groups. Higher INF-γ concentrations were found in spleen extracts of symptomatic dogs, and in liver extracts of asymptomatic dogs. Extract of this cytokine was lower in spleen extract. Although INF-γ is being produced in canine infection, mean levels of TGF-β₁ and IL-10 from spleen and liver extracts were quantitatively much higher; suggesting that immune response in both asymptomatic and symptomatic dogs was predominantly type Th2.     

Canine leishmaniosis in the United Kingdom: A zoonotic disease waiting for a vector?

Leishmaniosis is an important sand fly transmitted protozoan disease of dogs and humans. In northern Europe, infection is mainly restricted to dogs that have travelled to and/or from endemic areas of the Mediterranean region during periods when there is high sand fly exposure, mostly between March and November. Infected dogs in these areas in northern latitudes are a potential reservoir should incursion of a competent vector occur. However, information on the scale of the potential reservoir in the UK is lacking. Confirmed cases of canine leishmaniosis entering the United Kingdom between 2005 and 2007 were identified using diagnostic samples submitted to the Department of Clinical Veterinary Science, University of Bristol and from collaborating laboratories (n = 257). All study dogs had clinico-pathological signs compatible with leishmaniosis, as typically reported in endemic countries and were leishmania positive in real time or conventional PCR tests, IFA serology and/or tissue microscopic examination for amastigote identification. Information obtained from each case included travel history, habitat, clinico-pathological findings and geographical location once located in the UK. The majority of dogs with complete travel history (n = 183) had spent at least 6 months in Spain (105/183), 28/183 were rescued from re-homing centres in the country of origin and 26/183 entered the UK with confirmed leishmaniosis. Once located in the UK, the majority of positive cases were resident in south and central England. The spectrum of clinico-pathological signs for this group of dogs is similar to that reported in endemic countries. These data confirm that a potentially significant reservoir of infected dogs is resident in areas where future climatic conditions may support introduction of competent vectors.     

Detection of Leishmania infantum DNA by real-time PCR in canine oral and conjunctival swabs and comparison with other diagnostic techniques

The use of non invasive sampling, such as collection of conjunctival swabs, as a diagnostic tool for the detection of Leishmania DNA is of interest. The purpose of this study was to evaluate the diagnostic utility of detecting Leishmania infection with the use of conjunctival swab samples in dogs living in a highly endemic area for leishmaniosis and to investigate, for the first time, the presence of Leishmania DNA in oral swabs in the same population. One hundred sixty-three dogs living outdoor and recruited in various provinces of Sicily were studied. Leishmania infantum indirect fluorescent antibody test (IFAT), delayed-type hypersensitivity reaction to leishmanin (DTH) and real-time PCR of blood (BL), lymph node (LN), conjunctival (CS) and oral swab (OS) samples were performed. The positive PCR percentages in LN, CS, OS and BL samples were: 24.5%, 22.1%, 8.7% and 5.5%, respectively. Serological and DTH positive percentages were 27.0% and 73.8%, respectively. Seropositive and LN-PCR positive dogs had a high likelihood to be positive by CS-PCR. The similar positive PCR percentages found in CS and LN samples suggest the use of CS-PCR as non-invasive alternative technique to LN-PCR for the detection of Leishmania infection in dogs. In addition, this study demonstrated, for the first time, the presence of Leishmania DNA in oral swabs in dogs.     

Feline leishmaniosis due to Leishmania infantum in Italy

A case of leishmaniosis in domestic cats (Felis catus domesticus) is described. The subject showed a nodular lesion on the eyelid. The diagnosis was achieved by serological, parasitological, and light and electron microscopic investigations. By molecular techniques the aetiological agent was identified as belonging to Leishmania infantum, the species implicated in human and canine leishmaniosis in southern Europe. A preliminary study on the prevalence of asymptomatic feline leishmaniosis, performed in the areas where the infected cat was identified, revealed a low seroprevalence of infection: only 1 (0.9%) of the 110 cat sera examined by indirect fluorescent antibody test was positive for anti-Leishmania antibodies. Because clinical signs in feline leishmaniosis are unspecific and similar to those observed in other diseases commonly found in this species, leishmaniosis must be added to the differential diagnosis by feline veterinary practitioners and adequate serologic and histopathologic investigations must be performed in endemic areas.     

IL-6 and TNF-alpha production during active canine visceral leishmaniasis

We investigated the production of interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) during canine visceral leishmaniasis (VL) to gain a better understanding of the role of such multi-functional cytokines in parasite resistance. IL-6 and TNF-α levels were measured by capture ELISA in sera from 8 healthy dogs from a non-endemic area (control group) and in sera from 16 dogs from Araçatuba, SP, Brazil, an area endemic for leishmaniosis. The dogs from the endemic area were selected by positive ELISA serology against total Leishmania chagasi antigen, positive spleen imprints for Leishmania, and the presence of at least three clinical signs associated with active visceral leishmaniasis (fever, dermatitis, lymphoadenopathy, onychogryphosis, weight loss, cachexia, locomotory difficulty, conjunctivitis, epistaxis, hepatosplenomegaly, edema, and apathy).Enhanced systemic IL-6 production was found in sera from dogs with the active disease compared to healthy dogs (t-test, P < 0.05). In contrast, TNF-α did not differ between the two groups studied. There was no correlation between IL-6 production and anti-leishmanial antibody titers in the sera. Our findings suggest that IL-6 is a good marker of active disease during leishmaniasis, and that other cytokines may be involved in the hypergammaglobulinemia characteristic of canine visceral leishmaniasis.     

Efficacy of a combination of 10% imidacloprid/50% permethrin for the prevention of leishmaniasis in kennelled dogs in an endemic area

The efficacy of imidacloprid 10%and permethrin 50% (Advantix; Bayer AG, Germany) in a spot-on formulation was evaluated in the field as a control measure to prevent canine leishmaniasis (CanL) in dogs in an endemic area of southern Italy. In February 2005, out of 845 dogs initially tested for CanL, 631 dogs which tested negative (315 from a kennel in Bari (KB) and 316 from a kennel in Ginosa (KG)) in a serological and a parasitological examination were allocated to one of three groups: Group A-treated with imidacloprid 10% and permethrin 50% once a month; Group B-treated every 2 weeks; and Group C-untreated control animals. All the dogs were examined serologically and parasitologically for CanL prior to the start of the study, in November 2005 (end of the sandfly season) and in March 2006 (end of the study). An initial CanL seroprevalence of 24.7% (209 dogs) was detected in KB and KG. In KB Leishmania infection, inferred by positivity in at least one of the three tests performed at the interim or final follow-up, was found in one animal from Group A and in nine from Group C. No positive animals were detected in Group B, thus giving a final protection efficacy of 88.9% in Group A and 100% in Group B. In KG Leishmania infection was identified in one animal from Groups A and B, respectively, and 11 from Group C (protection efficacy of 90.36% in Group A and 90.73% in Group B). The incidence density rates (IDRs) of infection in both Groups A and B at each kennel were statistically significantly lower than that registered in Group C (KB p<0.05 and KG p<0.01). The results clearly show that a combination of imidacloprid 10% and permethrin 50%, by virtue of its repellent activity against sandflies, is effective under both application regimes in preventing CanL in the field in endemic areas.     

Equine ehrlichiosis: A comparison between E. equi and other pathogenic species of Ehrlichia

Ehrlichia equi, etiologic agent of equine ehrlichiosis, is a rickettsia which morphologically closely resembles the agents of bovine petechial fever, tick-borne fever, and canine ehrlichiosis (tropical canine pancytopenia). Natural infections of E. equi have been reported only in horses; however, the experimental host range of E. equi has been broadened to include burros, sheep, goats, dogs, cats, monkeys and baboons. Infection of primates indicates that E. equi may be a zoonotic agent. An indirect fluorescent antibody test employing E. equi-infected granulocytes as antigen has been developed and used to show that infected horses develop a prolonged antibody response to E. equi. Cell-mediated immune responses measured by the leukocyte migration inhibition test were also detected in horses recovered from acute illness. Protective immunity in horses, monkeys and baboons to reinfection is long-lasting. In contrast to the blood of dogs recovered from clinical E. canis infection, the blood of horses and dogs recovered from clinical infection with E. equi is not infectious for susceptible animals. Infection of dogs with E. equi does not provide protection against subsequent infection with E. canis.