Glomerular lesions associated with proteinuria in clinically healthy dogs.

Spontaneous proteinuria in otherwise clinically normal adult Beagles 4-6 years old was studied for 2 years. Eighteen dogs, representing a population of 218 Beagles, were placed into three groups: group I, nonproteinuric; group II, intermittently proteinuric; group III, persistently proteinuric. The groups were alike on the basis of laboratory tests, except urinary protein loss. Proteinuria was persistent in most affected dogs but not progressive during the 2 years. The loss of proteins with high molecular weight, including alpha-, beta-, and gamma-globulins, suggested the proteinuria was of glomerular origin. There were glomerular lesions but no other significant change in the kidneys and urogenital system. Lesions were generalized and characterized by prominent, local or diffuse mesangial proliferation and by thickening, wrinkling, and splitting of the glomerular basement membrane. The subendothelial space was often widened and contained electron-dense deposits. Similar electron-dense deposits, as well as lipid and mineral, were in the mesangium. Alterations in visceral epithelial cells and endothelium were prominent. Periglomerular sclerosis was present but tended not to correlate with the severity of mesangial change in any given renal corpuscle. The severity of both mesangial and periglomerular changes increased with increasing proteinuria. Immunofluoescence studies demonstrated granular discontinuous localization of IgG and betaIC-globulins in the glomerular capillaries and mesangium. Similar localization was seen but to a lesser extent in nonproteinuric dogs. The glomerular lesions seen in these clinically healthy, proteinuric dogs are similar to those described in various canind diseases associated with terminal renal failure.     

Antileishmanial antibody profile in dogs naturally infected with Leishmania chagasi

Visceral leishmaniasis (VL) presents vigorous Th2 immune response, which is mainly characterized in human by augmented expression of Il-4, polyclonal B cell activation, intense hypergammaglobulinemia and production of antileishmanial IgE antibodies. However, few aspects of this type of immune response have been demonstrated in studies of canine visceral leishmaniasis (CVL). This work investigated by ELISA and western immunoblotting the production of antileishmanial IgE antibodies (IgE Ab) in symptomatic and asymptomatic dogs naturally infected by Leishmania chagasi, and also compared this IgE immune response with those of IgG, IgG1 and IgG2 antibodies. Three groups of dogs were evaluated: 12 VL dogs with positive Leishmania biopsies (GI), 44 dogs with a positive leishmanial indirect fluorescent antibody test (IFAT), 30 of them presenting clinical signs of VL and 14 asymptomatic (GII) and 21 healthy dogs living in kennels located in leishmaniasis endemic areas (GIII), which were seronegative in the IFAT. Eighteen dogs from an area free of CVL were used as controls (GIV). Antileishmanial IgE antibodies were detected in 4 of 12 VL dogs from group I (33%) and 14 of 30 symptomatic dogs from group II (47%). While all asymptomatic dogs from group II (100%) were seronegative for antileishmanial IgE Ab, 7 of 21 healthy animals from group III (33%) had these immunoglobulins. A strong correlation was verified between antileishmanial IgG and IgG2 antibody titers in all symptomatic dogs, but only 15 of these 42 animals (36%) produced simultaneously IgE, IgG, IgG1 and IgG2 antibodies to Leishmania. IgE antibodies recognized leishmanial antigens of 12, 36, 61, 81 and 118 KDD, while a more complex pattern of immunoblotting was verified mainly for IgG and IgG2 antibodies from symptomatic animals. IgG1 and IgG2 antibodies shared the recognition of L. chagasi polypeptides of 118, 81, 61, 36, 18, 14 and 12 KDD, being more intense the immune reactions between IgG1 Ab and the leishmanial polypeptides of 61 and 36 KDD, and also between IgG2 antibodies and the antigens of 26, 21, 18, 14 and 12 KDD. Our results suggest that the polyclonal production of antileishmanial antibodies that includes IgE Ab could characterize a Th2 immune response in CVL and can help the laboratory diagnosis of this disease.     

Cytologic patterns of lymphadenopathy in dogs infected with Leishmania infantum

BACKGROUND: Lymphadenopathy in canine leishmaniosis has been reported as reactive lymphoid hyperplasia or granulomatous (histiocytic) lymphadenitis. However, we are unaware of information on the effect of latent Leishmania infection on lymph node cytology compared with clinically affected dogs. OBJECTIVES: The aim of the present study was to investigate cytologic patterns of lymphadenopathy in dogs with clinical and subclinical forms of leishmaniosis and to correlate cytologic findings with the density of Leishmania amastigotes in fine needle aspiration (FNA) smears. METHODS: FNA cytology of prescapular or popliteal lymph nodes was evaluated on 32 dogs with clinical evidence of leishmaniosis (group A), 24 subclinically infected dogs (group B), and 17 clinically healthy noninfected dogs (group C); groups were based on the results of serologic and PCR tests for Leishmania sp. Differential nucleated cell counts (based on 300 cells) and amastigote density were determined microscopically. Cytologic findings were categorized and compared among groups. RESULTS: Cytologic abnormalities were found in 19 of 32 (59.4%) dogs in group A, 1 of 24 (4.2%) dogs in group B, and 2 of 17 (11.8%) dogs in group C and were significantly more frequent in group A than group B (P <.001) or C (P = .001). In group A, 68.7% of the dogs had lymphoid hyperplasia, 12.5% had lymphoid hyperplasia and histiocytic lymphadenitis, 6.3% had histiocytic lymphadenitis, and 3.1% had lymphoid hyperplasia and neutrophilic lymphadenitis. Lymphoid hyperplasia was also noted in 1 dog in group B, and lymphoid hyperplasia and eosinophilic lymphadenitis were each found in 1 dog in group C. Lymph node smears from 31 (96.9%) dogs in group A and 6 (25%) dogs in group B were positive for Leishmania amastigotes; however, no correlation was found between the density of amastigotes and cytopathologic patterns of lymphadenopathy. CONCLUSION: Abnormal lymph node cytology is much more common in dogs with clinical leishmaniosis than in dogs with subclinical infection, and primarily involves lymphoid hyperplasia. Despite finding no association between the density of amastigotes and type of lymphadenopathy, lymph node cytology still is a valuable diagnostic tool for diagnosing canine leishmaniosis.     

Immune response against Leishmania antigens in dogs naturally and experimentally infected with Leishmania infantum

Cell-mediated and humoral immune response in naturally and experimentally infected dogs was studied using crude and pure antigens. Both types of infections induced severe signs of visceral disease, but the symptoms observed in natural infections were more pronounced than in experimental infections. In addition, asymptomatic infections were not observed in experimentally infected animals. Disease evolution in laboratory infections was rapid and an increase in antibody titer to crude parasite antigen was correlated with the appearance and aggravation of clinical symptoms. Peripheral blood lymphocyte proliferation to crude antigen and pure gp63 was observed early following experimental infection, but was abolished once the infected dogs began to exhibit clinical signs. A similar pattern was observed in naturally infected dogs. Serum from all patent dogs showed high antibody titers to rK39 in enzyme-linked immunosorbent assays (ELISA), and reacted by western blotting with several antigens, 12 to 120 KDa, including gp63 and gp70. In the case of asymptomatic dogs. antibody titers to crude antigen were low and only a few antigens were identified by western blotting. None of the pure proteins examined, gp63, gp70, and rK39 were recognized by western blotting or ELISA. However, asymptomatic dogs exhibited specific lymphocyte proliferation to both crude antigen and the potential vaccine candidate gp63.     

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.     

Genital lesions and distribution of amastigotes in bitches naturally infected with Leishmania chagasi

Recent reports indicate that Leishmania chagasi has tropism to the male canine genital system, which is associated with shedding of the organism in the semen, supporting the hypothesis of venereal transmission. The aim of this study was to describe the lesions and assess parasite load in the genital system of bitches with canine visceral leishmaniasis (CanL). Symptomatic (n=5) and asymptomatic (n=5) bitches seropositive for CanL were randomly selected at the Center for Zoonosis Control (Belo Horizonte, State of Minas Gerais, Brazil). Five serologically negative, healthy, adult bitches also from the CZC were used as controls. Samples from genital organs (vulva, vagina, cervix, uterine body, uterine horns, uterine tubes, and ovaries), liver, and spleen were histologically evaluated and processed for immunodetection of Leishmania sp., and PCR. The most significant histological change was a mild to moderate vulvar dermatitis, characterized by a histio-plasma-lymphocytic infiltrate. This change was detected in all asymptomatic, four symptomatic, and three uninfected control bitches. In one symptomatic and one asymptomatic bitch intracytoplasmic amastigotes were observed within macrophages in the inflammatory infiltrate. Samples from all the segments of the genital tract were positive in at least one infected animal, in the absence of detectable amastigotes in the tissue. These findings support the notion that L. chagasi does not have genital tropism in the bitch, which is in contrast to our previous findings in naturally infected male intact dogs.     

Leishmania DNA load and cytokine expression levels in asymptomatic naturally infected dogs

The factors responsible for the clinical progress of visceral leishmaniasis (VL) in dogs have not been yet established. The starting hypothesis was the possibility of associating the changing level of a specific type of cytokines with the evolution of the infection towards infection-manifested disease or resistant behaviour. For this purpose the authors have established a connection between Leishmania load, cytokine mRNA accumulation, and the progression of the disease in naturally infected asymptomatic dogs. We made use of real-time (RT) PCR system to detect the expression of cytokine mRNA levels during all the phases of the infection. In particular, we measured the amount of parasites in samples such as blood, lymph nodes and skin, and the expression levels of IFN-gamma, IL-2, IL-4, IL-10, IL-12 and IL-18 cytokines in the blood. We employed different targeted real-time PCR assay on 40 naturally infected dogs, initially asymptomatic; 20 of these progressed to overt disease, and the 20 remaining dogs remained asymptomatic throughout the period of study (2 years). Two other groups included: 20 naturally infected dogs with clinical signs of VL, and 20 healthy dogs living in a non-endemic area. All these animals were employed as positive and negative controls, respectively. The overall results obtained demonstrate that the simultaneous evaluation of parasites and cytokine levels represents a reliable tool for predicting disease development, and thus for choosing the best treatment for the asymptomatic form of the disease.     

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.     

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.     

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.     

Clinical and parasitological evaluation of dogs naturally infected by Leishmania (Leishmania) chagasi submitted to treatment with meglumine antimoniate

Aiming to evaluate the efficacy of the treatment of canine visceral leishmaniasis, to verify the occurrence of a possible disease relapse, and to search for the presence of the parasites after the end of the treatment, seven dogs naturally infected by Leishmania (Leishmania) chagasi were used. The dogs were subjected to a treatment with 75 mg/kg meglumine antimoniate subcutaneously every 12 h for 21 days, and followed-up for a period of 6 months. During the whole experimental period the animals wore deltamethrin collars and were kept in a screened kennel to avoid reinfection. Lymph node and bone marrow aspiration biopsy was carried out to search for the parasite at seven moments: before the treatment, 30, 60, 90, 120, 150 and 180 days after the start of the treatment. After the end of the experiment all dogs were humanely euthanized. Then, spleen and liver "imprints" and in vitro cultures were carried out to search for amastigote forms of the parasite. During the treatment all animals presented remission of symptoms. However, two dogs were observed to present new symptoms in the course of the experiment. At the end of the experiment, the presence of amastigote forms of the parasite was evidenced in five of the seven dogs. This enabled us to conclude that the treatment promoted clinical cure but did not eliminate the parasites completely.     

Histopathological and parasitological study of the gastrointestinal tract of dogs naturally infected with Leishmania infantum

Background

The aim of this study was to provide a systematic pathological and parasitological overview of the gastrointestinal tract (GIT), including the stomach, duodenum, jejunum, ileum, caecum and colon, of dogs naturally infected with Leishmania.

Methods

Twenty mongrel dogs naturally infected with Leishmania (Leishmania) infantum and obtained from the Control Zoonosis Center of the Municipality of Ribeirão das Neves, Belo Horizonte Metropolitan area, Minas Gerais (MG) state, Brazil, were analyzed. The dogs were divided into two groups: Group 1 comprised nine clinically normal dogs and group 2 comprised 11 clinically affected dogs. After necropsy, one sample was collected from each GIT segment, namely the stomach, duodenum, jejunum, ileum, caecum and colon. Furthermore, paraffin-embedded samples were used for histological and parasitological (immunohistochemistry) evaluation and a morphometrical study were carried out to determine the parasite load (immunolabeled amastigote forms of Leishmania). The Friedman and the Mann Whitney tests were used for statistical analysis. The Friedman test was used to analyze each segment of the GIT within each group of dogs and the Mann Whitney test was used to compare the GIT segments between clinically unaffected and affected dogs.

Results

The infected dogs had an increased number of macrophages, plasma cells and lymphocytes, but lesions were generally mild. Parasite distribution in the GIT was evident in all intestinal segments and layers of the intestinal wall (mucosal, muscular and submucosal) irrespective of the clinical status of the dogs. However, the parasite load was statistically higher in the caecum and colon than in other segments of the GIT.

Conclusion

The high parasite burden evident throughout the GIT mucosa with only mild pathological alterations led us to consider whether Leishmania gains an advantage from the intestinal immunoregulatory response (immunological tolerance).     

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.     

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.     

Studies on the pathogenesis of heart lesions in dogs infected with pseudorabies virus.

Pseudorabies virus was inoculated by various routes into dogs to determine the relationship of pseudorabies virus to the development of heart lesions. Electrocardiograms and serum samples for lactate dehydrogenase isoenzymes were taken twice daily. Transitory and persistent arrhythmias were a consistent finding. Heart lesions were noted within each of the inoculated groups. These changes varied from severe multifocal areas of hemorrhage and myocardial degeneration to small zones of myolysis. A ganglioneuritis of the stellate ganglia and autonomic ganglia within the heart were also consistent findings. Herpes-like viral particles were found by electron microscopy in various autonomic ganglia and in myocardial endothelial cells. No viral particles were found in myocardial cells. Significant increases in lactate dehydrogenase-1 were noted. It was concluded that fatal arrhythmias resulted from pseudorabies virus infections in the dog after the occurrence of myocardial and ganglionic lesions.