Serological and parasitological follow-up in dogs experimentally infected with Leishmania infantum and treated with meglumine antimoniate.

Six healthy beagle dogs were infected with Leishmania infantum (MCAN/ES/92/BCN-83/MON-1) by intravenous inoculation of 5 x 10(7) promastigotes and two others were used as controls. When animals showed clinical signs of disease at 29, 37, 41 and 45 weeks post-infection (p.i.), they were treated with meglumine antimoniate (20.4 mg Sb/kg/12 h) subcutaneously for two periods of 10 days each. Sera were tested periodically for Leishmania antibodies by Dot-ELISA, ELISA and Western blot (WB). Aspirates of popliteal lymph node (PLN), peripheral blood sample (PB) and healthy skin were cultured in NNN and Schneider's medium. PLNs were positive between 8 and 20 weeks p.i. and in one animal PB was positive 6 weeks p.i. Samples of healthy skin, obtained before treatment, were also positive. Dot-ELISA and ELISA detected specific antibodies at an early stage between 4 and 12 weeks p.i and surpassed the cut-off between 16-24 weeks p.i., while the WB was positive between 10-19 weeks p.i. The pattern of bands revealed during the first stages of infection was variable and only in two cases did the positivity start with bands of low molecular weight (12-14 kD); the number of bands increased until 15-24 weeks p.i., after which sera revealed a complete pattern of bands, from 12 to 85 kD, in the antigen of Leishmania. After treatment the clinical improvement of the animals was accompanied by a decrease in antibody titers (Dot-ELISA and ELISA) although the parasites remained in the PLN. This was reflected in the WB by a decrease in the intensity of bands, especially those in the region of 12-30 kD. A new increase in the antibody levels between 3 and 5 months after terminating the therapy was detected in the WB by a restoration of the initial complete pattern of bands.     

Idiotype expression of IgG1 and IgG2 in dogs naturally infected with Leishmania infantum

Here we analyzed, by Western blot analysis, the idiotype expression of IgG1 and IgG2 in 109 canine sera corresponding to 50 dogs from endemic areas of leishmaniosis in order to detect markers related to Leishmania infantum infection and clinical condition (asymptomatic or symptomatic). Twenty-four dogs from an area free of leishmaniosis were used as controls. IgG1 and IgG2 responses in symptomatic and asymptomatic L. infantum infections differed mainly in subclass production (ELISA values), with higher IgG2 production occurring particularly in symptomatic dogs. Nevertheless, we observed little difference in the idiotype expression of these IgG subclasses, which, in general, recognized the same antigenic fractions. While early L. infantum infection was characterized by recognition of polypeptide fractions of low molecular weight, mainly fractions of 14, 16 and 18 kDa by IgG1 and 14 and 16 kDa by IgG2, symptomatology was associated with recognition by both IgG subclasses of a 24 kDa fraction and other antigens belonging to the AG24 family.     

Presence of anti-platelet IgM and IgG antibodies in dogs naturally infected by Leishmania infantum

Thirty-three dogs, naturally infected by Leishmania infantum, were enrolled in the study and were classified as oligo-symptomatic (n. 15) and symptomatic or markedly symptomatic (n. 18). A control group was 10 healthy dogs. A haematological profile was obtained and the dogs serum was employed to assess the presence of platelet binding IgM and IgG antibodies (PBIgM, PBIgG) using flow cytometry. FITC labelled goat anti-dog IgM or IgG were used to detect PBIgM and PBIgG. Samples with a mean fluorescence intensity (MFI) that was 100 channels higher on a log scale for more than 30% of the platelets than seen in negative control platelets from a healthy dog were considered positive for the presence of anti-platelet antibodies (PBIg). Twenty-one (63.3%) dogs revealed the presence of PBIg. Six of them were oligo-symptomatic while 15 showed moderate or severe clinical signs of illness. All the dogs with PBIg showed the presence of PBIgM, with nine animals showing both PBIgM and PBIgG. Nine of 18 symptomatic or markedly symptomatic dogs showed thrombocytopenia, while normal platelet counts were observed in all oligo-symptomatic animals. Eight of 9 thrombocytopenic animals showed the presence of PBIgM, while six of them showed PBIgG. One thrombocytopenic dog was negative for PBIg. This study is the first report documenting the presence of PBIg in natural canine leishmaniasis implying a pathogenic association between thrombocytopenia and the presence of antibody against platelet membrane.     

Infection of sandflies by a cat naturally infected with Leishmania infantum

Despite the recent reports of feline leishmaniosis from Southern Europe, cats are still regarded as unusual Leishmania hosts. A cat found chronically infected with Leishmania was submitted to xenodiagnosis. After being sedated, the animal was exposed to the bite of 100 laboratory-reared Phlebotomus perniciosus in a fine net cage for 90 min. Four out of 19 blood-fed sandflies (21%) showed motile promastigotes at the dissection. Parasites cultured from cat's lymph node and an infected fly were identical at PCR-RFLP genotyping and identified as Leishmania infantum MON-1, the main zymodeme responsible for human and canine leishmaniosis in Southern Europe. This is the first evidence of transmissibility of feline parasites to a proven vector, suggesting that cats may represent an additional domestic reservoir for L. infantum.     

Cervical, mandibular, and parotid lymph nodes of dogs naturally infected with Leishmania infantum: a histopathologic and immunohistochemistry study and its correlation with facial skin lesions

The parasite load in cervical, mandibular, and parotid lymph nodes and in the skin of the nose and the pinna from dogs infected with Leishmania infantum were investigated by histologic and immunohistochemical studies. Twenty-two infected dogs with and without signs of infection were examined to demonstrate correlation of signs with parasite load and the correlation of facial skin lesions with parasites in regional lymph nodes. Chronic inflammation of the skin was demonstrated in infected dogs that had no gross skin lesions, confirming that normal-appearing skin can harbor the parasite, likely playing a role in transmission. Dogs with facial skin lesions showed a higher parasite load in parotid lymph nodes than dogs without lesions of the facial skin, based on Leishman-Donovan unit analysis. Based on immunohistochemical analysis, parasite load in parotid and cervical nodes was correlated with that of skin of the nose and pinna, as was the parasite load in mandibular lymph nodes and skin of the external nose. We demonstrated a logical involvement of the lymphatic vessels and their specific anatomic draining sites.     

Changes in the plasma concentrations of lipids and lipoprotein fractions in dogs infected with Leishmania infantum.

A study was made of serum concentrations of cholesterol, phospholipids, triglycerides, cholesterol bound to high-density lipoproteins (HDL), HDL1-cholesterol, HDL2-cholesterol and cholesterol bound to low-density lipoproteins (LDL) in 16 dogs naturally infected with Leishmania infantum (ZMON-1) taken from an endemic focus. Results were compared with those of a control group of ten healthy dogs. Statistically significant increases in cholesterol, triglyceride and LDL-cholesterol levels were observed. There was, however, a statistically significant decrease in HDL-cholesterol level, mainly at the expense of the HDL2-cholesterol subfraction. Cholesterol transport is therefore shown to undergo changes which may be attributed to the consumptive evolution of the disease, immunocomplex deposits in cells, hepatic disorders and interactions between the parasite and the normal cholesterol metabolism of the host.     

Platelet aggregation and haemostatic response in dogs naturally co-infected by Leishmania infantum and Ehrlichia canis

Haemostatic alterations in dogs naturally infected by ehrlichiosis and/or leishmaniasis were studied. Platelet count, ADP and collagen-induced platelet aggregation, prothrombin time, activated partial thromboplastin time (APTT) and plasma fibrinogen concentration were measured. An evident reduction of platelet aggregation response was shown for Leishmania-Ehrlichia co-infected dogs where platelet aggregation was lower in comparison with control and leishmaniotic dogs (ADP and collagen, P < or = 0.01) and ehrlichiotic dogs (ADP 10 and 7.5 microm, P < or = 0.05). Moreover, a significant increase in APTT as well as a reduction of the albumin/globulin rate (A/G) for leishmaniotic and co-infected dogs versus control and ehrlichiotic dogs was detected. The hypothesis of a synergism between leishmaniosis and ehrlichiosis in altering platelet function by different pathways is discussed.     

A Leishmania infantum multi-component antigenic protein mixed with live BCG confers protection to dogs experimentally infected with L. infantum

The capacity of a quimeric protein, formed by the genetic fusion of five antigenic determinants from four Leishmania proteins, formulated with BCG, to protect dogs against Leishmania infantum infection is described. The data showed that after i.v. administration of 500,000 parasites of the L. infantum M/CAN/ES/96/BCN150 strain, zymodeme MON-1, the animals became infected as suggested by the humoral response against the parasite antigens. All control unvaccinated dogs had parasites in the lymph nodes at day 150 post-infection. One of these unvaccinated infected dog was parasite negative at day 634 behaving, thus, as resistant. In contrast, only 50% of the immunized dogs had parasites in the lymph nodes at day 150 post-infection. Four of these dogs became parasite negative by day 634 post-infection. The control animals developed at various times during the follow-up period clinical symptoms associated with Leishmaniasis. The control diseased dogs developed also in the liver and spleen some of the abnormal histological features associated with natural visceral Leishmaniasis. The immunized dogs, however, were not only normal at the clinical but also at the anatomo-pathological level. A positive delayed type hypersensitivity (DTH) response was observed in nine of the immunized protected dogs. The data indicated that Q+BCG confers 90% protection against infection and at least 90% protection at the clinical level.     

Clinical and serological aspects of visceral leishmaniasis in northeast Brazilian dogs naturally infected with Leishmania chagasi

Human visceral leishmaniasis is endemic in the northeast of Brazil, where the domestic dog is an important parasite reservoir in the infectious cycle of Leishmania chagasi. In this study, we evaluated the clinical signs of canine visceral leishmaniasis (CVL), serum protein profile and the antileishmanial IgG antibody production in 86 dogs living in northeast endemic areas of leishmaniasis. Thirty dogs from a leishmaniasis-free area were used as a control group. The major clinical signs of CVL seen were emaciation and skin ulcers (80%), followed by onychogryphosis and conjunctivitis (73%). Depilation was observed in 60% of animals while lymphadenomegaly, splenomegaly, liver enlargement or kidney involvement was less frequent (< or =20%). VL seropositive dogs presented with serum hyperproteinemia, hypoalbuminemia, hypergammaglobulinemia and decreased albumin/globulin ratio. A lower sensitivity and higher specificity was observed for promastigote indirect fluorescent antibody test (IFAT) (83 and 100%, respectively) compared with enzyme-linked immunosorbent assay (ELISA) (94 and 90%), which uses a crude extract of Leishmania. There was a positive correlation between IFAT and ELISA titers of antileishmanial IgG antibodies (Spearman test, P < 0.05), which was augmented in CVL dogs. This study found that the determination of serum protein, A/G ratio and the use of two different leishmanial serological tests like IFAT and ELISA are essential in CVL screening.     

Identification of highly specific and cross-reactive antigens of Leishmania species by antibodies from Leishmania (Leishmania) chagasi naturally infected dogs

The Leishmania species present a genetic homology that ranges from 69 to 90%. Because of this homology, heterologous antigens have been used in the immunodiagnosis and vaccine development against Leishmania infections. In the current work, we describe the identification of species-specific and cross-reactive antigens among several New World Leishmania species, using symptomatic and asymptomatic naturally Leishmania chagasi-infected dog sera. Soluble antigens from five strains of New World Leishmania were separated by electrophoresis in SDS-PAGE and immunoblotted. Different proteins were uniquely recognized in the L. chagasi panel by either symptomatic or asymptomatic dog sera suggesting their use as markers for the progression of disease and diagnosis of the initial (sub-clinical) phase of the infection. Cross-reactive antigens were identified using heterologous antigenic panels (L. amazonensis strains PH8 and BH6, L. guyanensis and L. braziliensis). L. guyanensis panel showed the highest cross-reactivity against L. chagasi specific antibodies, suggesting that proteins from this extract might be suitable for the diagnosis of visceral canine leishmaniasis. Interestingly, the 51 and 97 kDa proteins of Leishmania were widely recognized (77.8% to 100%) among all antigenic panels tested, supporting their potential use for immunodiagnosis. Finally, we identified several leishmanial antigens that might be useful for routine diagnosis and seroepidemiological studies of the visceral canine leishmaniasis.     

Longitudinal analysis of cytokine gene expression and parasite load in PBMC in Leishmania infantum experimentally infected dogs

Canine visceral leishmaniasis (CVL) is caused by Leishmania infantum, an intracellular protozoan parasite that causes a severe infectious disease. To evaluate the gene expression profile associated to CVL in vivo, we have measured monthly by real-time PCR over one year the IL-4, IL-10, IL-12, IL-13, IFN-gamma, TGF-beta and TNF-alpha mRNA levels in peripheral blood mononuclear cells in 6 experimentally infected dogs that exhibited different progressions of the illness. While in two dogs no parasite, or a very low number of parasites, was detected and the two dogs did not show any clinico-pathological abnormalities at the end of the study (L dogs), for the remaining dogs high parasite loads were detected and they developed clinical leishmaniasis (H dogs). The L dogs have null expression of both IL-4 and IL-13 for the first 4 months after the infection, whereas an early IL-4 and IL-13 expression occurs in this period of infection in most of the dogs that developed clinical leishmaniasis (H dogs). Furthermore, a higher IFN-gamma expression was associated with the increase of parasite load and clinical status in these dogs. Moreover, the high variability of expression at the pre-infection stage causes us to reject the possibility that the basal levels of these cytokines indicate the prognosis of the subsequent response against infection.     

家养野猪自然感染猪圆环病毒2型的病理学观察

经PCR检测南京某养殖场送检的表现为多系统衰竭综合征的家养野猪病原为猪圆环病毒2型(PCV2)。采用常规石蜡切片法和免疫组织化学法对患病仔猪的病理组织学变化特点及PCV2抗原在淋巴结内的分布进行了研究。结果显示,剖检病死仔猪病变主要表现为全身淋巴结肿大和间质性肺炎。组织病理学变化主要表现为淋巴结、脾和扁桃体等免疫器官淋巴滤泡萎缩,淋巴细胞数量减少,有大量巨噬细胞浸润及多核巨细胞;非淋巴组织病变主要为肺脏呈典型的间质性肺炎,肝脏局灶性坏死及淋巴细胞浸润;肺脏和肝脏病灶内也可见少量多核巨细胞。PCV2主要分布于淋巴组织内单核巨噬细胞、支气管黏膜上皮、肺泡壁Ⅱ型上皮细胞内,肾小球内也可见少量分布。     

Genital lesions associated with visceral leishmaniasis and shedding of Leishmania sp. in the semen of naturally infected dogs

Although visceral leishmaniasis is primarily transmitted by a biological invertebrate vector, transmission in the absence of the vector has been reported, including venereal transmission in humans. Considering the possibility of venereal transmission, we studied genital lesions in dogs naturally infected with visceral leishmaniasis and shedding of Leishmania sp. in the semen. Approximately 200 dogs were serologically tested for anti-Leishmania antibodies and divided into three groups: 1) serologically negative dogs (n = 20), 2) asymptomatic serologically positive dogs (n = 20), and 3) symptomatic serologically positive dogs (n = 20). Samples from both testes, all segments of both epididymes, prostate gland, glans penis, and prepuce were histologically evaluated and processed for immunodetection of Leishmania sp. Semen samples were obtained from 22 symptomatic serologically positive dogs and processed for detecting Leishmania DNA by polymerase chain reaction. A significantly higher frequency of inflammation was observed in the epididymes, glans penis, and prepuce of dogs with visceral leishmaniasis, which was associated with a high frequency of immunohistochemically positive tissues (up to 95% of tissues from symptomatic dogs were positive by immunohistochemistry). Leishmania DNA was detected in eight of 22 semen samples from symptomatic dogs. Together these findings indicate that genital lesions and shedding of Leishmania sp. (donovani complex) in the semen are associated with visceral leishmaniasis. Additional studies should address the possibility of venereal transmission of the disease in the dog.     

Humoral and cellular immune responses against Type I cysteine proteinase of Leishmania infantum are higher in asymptomatic than symptomatic dogs selected from a naturally infected population

Canids are natural reservoirs of Leishmania infantum and have been promoted as experimental hosts to decipher the pathogenesis of human visceral leishmaniasis (VL). In this study, the presence of IgG antibodies as well as the presence of mononuclear leukocytes reactive to different cysteine proteinases (CPs) were examined in 13 L. infantum-infected dogs (six with symptoms, seven asymptomatic). Cysteine proteinases which belong to papain-like enzymes known as clan CA are the most studied CPs of parasite protozoa. These molecules are expressed by the intracellular stages of the parasite and could be immunogenic. We studied Type II CP (CPA) and Type I CP (CPB) with its long C-terminal extension (CTE) which could be highly immunogenic. We showed that the level of antibodies reactive to rCPA is low in both symptomatic and asymptomatic dogs. In contrast, when CPB and CTE were used as antigens, the level of total IgG (with IgG2 superior to IgG1) reached higher values in asymptomatic dogs than in dogs with VL. While the peripheral blood mononuclear cell (PBMC) reactivity was significant when cultured in the presence of freezed/thawed (F/T) lysate, it remained low in presence of CP although always higher for PBMC recovered from asymptomatic dogs. We showed the importance of CPB and CTE in particular as a target of immune response and their potential use for serodiagnosis in asymptomatic dogs.     

Lymphocytes of dogs immunised with purified excreted-secreted antigens of Leishmania infantum co-incubated with Leishmania infected macrophages produce IFN gamma resulting in nitric oxide-mediated amastigote apoptosis

The role of nitric oxide (NO) in the anti-leishmanial activity has been confirmed both in vitro and in vivo. Recently, we demonstrated that NO-mediated apoptosis-like amastigote death pathway is an important and highly regulated mechanism used for the clearance of Leishmania within infected murine macrophages stimulated to produce NO endogenously. To further characterize these important effector mechanisms in dog, a natural host-reservoir of L. infantum/L. chagasi, we have developed an ex vivo infection model of canine macrophages. Exposure of L. infantum-infected macrophages to autologous peripheral lymphocytes derived from dogs immunised with purified excreted-secreted antigens of L. infantum promastigotes (LiESAp) formulated with muramyl dipeptide (MDP) as adjuvant resulted in a significant leishmanicidal effect due to interferon (IFN)-gamma dependent macrophage activation. Concomitant accumulation of NO(3)(-)/NO(2)(-) in supernatants of co-cultured cells and in situ staining of parasites with terminal deoxynucleotidyltransferase-mediated dUTP nick-end labelling (TUNEL) and YOPRO-1 showed that NO-mediated apoptosis of intracellular L. infantum amastigotes is occurring in canine macrophages as previously observed in mouse models. Monitoring these parameters in dogs after immunisation and before experimental challenge can represent a useful and easy way to rapidly evaluate vaccine candidates against canine visceral leishmaniasis.     

Evaluation of miltefosine for the treatment of dogs naturally infected with L. infantum (=L. chagasi) in Brazil

Dogs naturally infected with Leishmania Infantum (=L. chagasi) were treated with miltefosine using different therapeutic regimens. The animals were evaluated for clinical evolution, biochemical parameters, parasite load (by real-time PCR), cytokine levels and humoral response. After treatment and during the following 24 months, there was progressive clinical improvement and complete recovery in 50% (7/14) of the treated animals. There was a decrease in the smear positivity of the bone marrow after treatment, and there was also a gradual and constant decrease in positive cultures at the end of the follow-up period. However, the PCR detection of parasite DNA remained positive. In general, all animals presented a significant increase in parasite load 6 months after treatment. The IFN-γ levels in all the groups tended to increase during follow-up period, regardless of the miltefosine dose administered. The IL-4 and IL-10 levels of the animals tended to decrease during follow-up, except after 300 days when only IL-10 increased. The serum antibodies identified antigens that ranged from 116 kDa to less than 29 kDa in the Western blot assay. Furthermore, 300 days after treatment, qualitative and quantitative differences in the antigen profiles were observed. Antigens of 97 and 46 kDa were the most intensely recognized. Higher levels of antigen-specific Leishmania IgG were detected before and 300 days after treatment in all groups. Taking together, the improvement in the clinical symptoms was not followed by parasitological clearance, suggesting that treatment with miltefosine is not recommended, especially in endemic areas like Brazil, where children are the major victims and dogs are involved in the maintenance of the parasite cycle.     

Glomerular lesions in dogs infected with Leishmania organisms

OBJECTIVE: To histologically identify glomerular lesions in dogs infected with Leishmania organisms. ANIMALS: 41 dogs (17 sexually intact males and 14 sexually intact and 10 ovariohysterectomized females) that had positive results when tested for leishmaniosis as determined by use of serologic evaluation (indirect fluorescent antibody test, titers of 1:80 to 1:640) and direct microscopic identification of the protozoal organisms. PROCEDURE: Urine samples were collected by use of cystocentesis and examined by qualitative SDS-agarose gel electrophoresis (AGE). All dogs had non-selective (glomerular) or mixed (glomerular and tubular) proteinemia. Specimens were obtained from each dog during ultrasound-assisted renal biopsy and used for histologic examination. Each specimen was stained with H&E, periodic acid-Schiff, Goldner's trichrome, methenamine silver, and Congo Red stains. Specimens were adequate for evaluation when they contained at least 5 glomeruli/section, except for specimens stained with Congo Red in which 1 glomerulus/section was adequate. RESULTS: Examination of renal biopsy specimens revealed various glomerular lesions in all dogs and interstitial or tubular (or both) lesions in 23 of 41 (55%) dogs. CONCLUSIONS AND CLINICAL RELEVANCE: Glomerular lesions that develop in dogs during infection with Leishmania organisms can be classified histologically as mesangial glomerulonephritis, membranous glomerulonephritis, membranoproliferative glomerulonephritis, and focal segmental glomerulonephritis. Tubulointerstitial histopathologic conditions were not observed as the primary lesion, despite being evident in 23 of 41 (55%) dogs. Use of SDS-AGE for qualitative evaluation of proteinuria and successive collection of specimens during renal biopsies following diagnosis of nonselective glomerular proteinuria provides the possibility for early identification of renal lesions.