Parasite density and impaired biochemical/hematological status are associated with severe clinical aspects of canine visceral leishmaniasis

We have performed a detailed investigation in 40 dogs naturally infected with Leishmania infantum (syn. chagasi), subdivided into three groups: asymptomatic (AD = 12), oligosymptomatic (OD = 12) and symptomatic (SD = 16), based on their clinical features. Twenty non-infected dogs (CD) were included as control group. Serological analysis, performed by IFAT and ELISA, demonstrated higher antibodies titers in SD in comparison to the AD. A positive correlation was found between parasite density in the spleen and skin smears as well as the bone marrow parasitism with clinical status of the infection. We observed that the progression of the disease from asymptomatic to symptomatic clinical form was accompanied by intense parasitism in the bone marrow. It is likely that this led to the impaired biochemical/hematological status observed. Finally, we believe that the follow-up of these parameters could be a relevant approach to be used as markers during therapeutic and vaccine evaluations.     

Canine visceral leishmaniasis: relationships between clinical status, humoral immune response, haematology and Lutzomyia (Lutzomyia) longipalpis infectivity

The main source of Leishmania infantum infection in humans is a naturally infected dog. This study reports on the infectivity to phlebotomine sandflies (Lutzomyia longipalpis) of serologically positive mongrel dogs that differed in clinical status, haematology and humoral responses to immunoglobulin (Ig) G(T) (total anti-Leishmania IgG), IgG(1) and IgG(2) subclasses of antibody to crude antigen of L. infantum. Forty-five female L. longipalpis were allowed to feed directly on the ears of dogs classified as asymptomatic, oligosymptomatic or symptomatic before being dissected five days later. Promastigotes were detected in 88% of the dissected sandflies. The highest rate of infectivity to sandflies was found in symptomatic dogs, followed by oligosymptomatic and asymptomatic animals. The results suggest that dogs naturally infected with L. infantum with higher total IgG and IgG(2) concentrations and lower haematocrit levels were able to infect the highest proportion of L. longipalpis. No correlation was observed between anaemia and the intensity of clinical signs. Symptomatic dogs presented the highest infection rate and intensity of infection.     

Cellular immunophenotypic profile in the splenic compartment during canine visceral leishmaniasis

To determine the role of the spleen in the pathogenesis of canine visceral leishmaniasis (CVL), we analyzed cellular immunophenotypic profiles of 52 dogs naturally infected with Leishmania infantum, clinically classified as follows: asymptomatic dogs-I (AD-I), seronegative/PCR+; asymptomatic dogs-II (AD-II), seropositive/PCR+; oligosymptomatic dogs (OD) and symptomatic dogs (SD). Seven non-infected dogs (CD) were included as a control group. AD-II presented higher levels of CD8+ T splenocytes and lower TCD4+/TCD8+ ratio in comparison with CD. OD and SD showed lower percentages of CD21+ as compared with AD-II. All seropositive dogs presented lower levels of CD45RA+ than CD. Regardless of the stimuli used, the proliferation index from splenocytes in vitro was inversely correlated with clinical status. After LSA stimulation, there was a higher percentage of specific CD8+ T in AD-II than CD and non-stimulated culture. In contrast, splenocytes from SD under in vitro LSA stimulation induced decreased MHC-II+ expression in comparison with all groups, and non-stimulated culture. In conclusion, the role of CD8+ T splenocytes seems to be important for an effective immunological response, a hallmark of asymptomatic CVL, whereas the pronounced loss of MHC-II expression upon LSA stimulation is a biomarker of symptomatic CVL.     

Canine babesiosis caused by Babesia canis vogeli in rural areas of the State of Minas Gerais, Brazil and factors associated with its seroprevalence

This epidemiological survey on canine babesiosis was carried out in three distinct rural regions (Lavras, Belo Horizonte and Nanuque) of the State of Minas Gerais, Brazil. Ticks and blood samples were collected during a dry season (Lavras, n = 92; Belo Horizonte, n = 50; Nanuque, n = 102) and the subsequent rainy season (Lavras, n = 71; Belo Horizonte, n = 28; Nanuque, n = 66) from dogs living on farms. Plasma samples were analyzed by the indirect fluorescent antibody test for detection of anti-Babesia canis vogeli antibodies. DNA was extracted from blood of serologically positive dogs and molecular characterization of Babesia species was performed. Rhipicephalus sanguineus, Amblyomma cajennense and Boophilus microplus were the tick species identified in all regions. In Lavras, in addition to those tick species, A. tigrinum and A. ovale were also identified. The most prevalent tick species was A. cajennense (35.3%), followed by R. sanguineus (19%) and B. microplus (4.0%). Dogs living in Nanuque region were more heavily infested with ticks than dogs living in Belo Horizonte and Lavras regions. The overall frequency of anti-B. c. vogeli antibodies in the canine population in rural areas of Minas Gerais was 28.7%, with prevalence rates of 49.0% in Nanuque, 34.0% in Belo Horizonte and 3.3% in Lavras. The age of the animals and tick infestation were associated with seroprevalence of B. c. vogeli. The sequence analysis showed that B. c. vogeli was the only Babesia species present in all three regions. This study showed different rates of prevalence and incidence of canine babesiosis among the three rural regions sampled in Minas Gerais State. The results point to the importance of canine babesiosis in rural areas and to the need for further studies related to its transmission and maintenance in nature.     

Parasitic load and histological aspects in different regions of the spleen of dogs with visceral leishmaniasis

Leishmania infantum causes from subclinical infection to severe disease in humans and dogs. The spleen is one of the organs most affected by the infection. Although evidence exists that the parasitic load distribution and histological alterations may not be homogeneous in the affected organs of naturally infected individuals, it has not been formally demonstrated using the current techniques used for studying the disease.In six dogs naturally infected with Leishmania, parasitic load and histological changes were compared in samples collected from the lower, middle and upper third of the spleen.Parasitic load in the spleen of the group of dogs was variable, revealing a difference of 61 times between animals with the lowest and the highest parasitism. The set of parasitic load values of each dog showed a cluster trend, when compared to the other animals. Nevertheless, the parasitic load values of each dog showed a variation ranging from 3.2 to 34.7 times between lowest and highest value. Histological changes showed recognizable variation in frequency (granulomas) or intensity (perisplenitis) in the spleen of 2 out of the 6 dogs. The agreement of histological findings between samples collected from the different thirds of the spleen was good (kappa coeficient, 0.61–0.80) very good (0.81–0.99) or perfect (1.00), for most of the parameters analyzed.Variability of parasitic load and, to a lesser extent, histological changes in spleen of dogs with visceral leishmaniasis is observed. Such variability may be taken in account in the design of studies on pathogenesis, vaccine and therapeutic drug development.     

Comparison of PCR methods for diagnosis of canine visceral leishmaniasis in conjunctival swab samples

Four PCR assays for detection of Leishmania DNA in conjunctival swab samples were compared. All methods had two steps: a first amplification followed by hybridization or by a new amplification (nested or seminested). Two methods (kDNA PCR-hybridization and kDNA snPCR) used primers targeted to the minicircles of kinetoplast DNA (kDNA) and the other two methods to the coding (LnPCR) and intergenic noncoding regions (ITS-1 nPCR) of ribosomal rRNA genes. kDNA PCR-hybridization was positive for 22/23 dogs (95.6%) and for 40/46 samples (86.9%), considering the right and the left conjunctivas. kDNA snPCR was positive for 21/23 dogs (91.3%) and for 40/46 samples (86.9%). The ITS-1 nPCR and LnPCR were both able to detect the parasites in 17/23 dogs (73.9%) and 29/46 (63%) and 30/46 (65.2%) samples, respectively. The positivities of the kDNA based methods were significantly higher; however the choice of the best method will depend on the kind of information required with the diagnosis.     

Chemical composition of Eucalyptus spp. essential oils and their insecticidal effects on Lutzomyia longipalpis

The chemical composition of essential oils from three species of plants belonging to the Eucalyptus genus was determined and, their insecticidal effects on egg, larva and adult phases of Lutzomyia longipalpis were assessed. The insects were collected in the municipality of Sobral in the State of Ceará, Brazil. Five treatments with different concentrations were performed along with two negative controls, distilled water and Tween 80 (3%), and a positive control, cypermethrin (0.196 mg/ml). The tests were carried out in plastic pots internally coated with sterile plaster and filled with a substrate made of rabbit feces and crushed cassava leaves. The eggs, larvae and adults were sprayed with the oils. The hatched larvae were counted for 10 consecutive days and observed until pupation. Insect mortality was observed after 24, 48 and 72 h. E. staigeriana oil was the most effective on all three phases of the insect, followed by E. citriodora and E. globulus oils, respectively. The major constituents of the oils were Z-citral and alpha-citral (E. staigeriana), citronellal (E. citriodora) and 1,8-cineole (E. globulus). The Eucalyptus essential oils constitute alternative natural products for the control of L. longipalpis since the median effective concentration (EC₅₀) values revealed relevant action as compared with other natural products, some of their chemical constituents are already known for their insecticidal activity and these oils are produced in commercial scale in Brazil.     

Distinct antigen recognition pattern during zoonotic visceral leishmaniasis in humans and dogs

Leishmania infantum is a causative agent of endemic zoonotic visceral leishmaniasis (VL) in regions of South America and the Mediterranean. Dogs are the major reservoirs for L. infantum in these regions, and control of disease in dogs could have a significant impact on human disease. Although dogs share many symptoms of VL with humans as a result of L. infantum infection, they also show some unique clinical manifestations, which are often a combination of visceral and cutaneous leishmaniasis, suggesting different mechanisms of disease development in dogs and humans. Here, we compare antibody responses of dogs and humans with VL to various defined leishmanial antigens. Parasite lysate and K39, the two most commonly used antigens for serodiagnosis of VL, detected the highest levels of antibodies in both humans and dogs with VL, whereas the recognition patterns of these antigens were distinct between the hosts. Among other defined antigens tested, LmSTI1 and CPB detected higher levels of antibodies in dogs and humans, respectively. These results indicate there is a difference between humans and dogs in antigen recognition patterns during VL. We infer that different strategies may need to be used in development of vaccines and diagnostics for humans and for dogs. In addition, we show a correlation between antibody titers to several antigens and severity of clinical symptoms during canine VL.     

Evaluation of renal and hepatic functions in dogs naturally infected by visceral leishmaniasis submitted to treatment with meglumine antimoniate

The present study aimed to evaluate the renal and hepatic responses in eight dogs with visceral leishmaniasis submitted to treatment with meglumine antimoniate and to verify the occurrence of possible side effects. Urinalysis, hepatic and renal function tests were carried out in all animals at up to seven moments. After the end of a six-month observation period, all dogs were euthanized. Before the beginning of the experiment urinary and biochemical alterations were observed in four dogs due to the changes caused by the parasite itself. These alterations included the presence of renal cells, cylindruria, proteinuria, azotemia, hyperproteinemia and hypoalbuminemia. One dog died on the third day after treatment because an aggravation of the clinical picture, probably due to the medication. During the course of the study, an increase in hepatic enzymes was verified in two animals. Sixty days after the beginning of the treatment four dogs showed remission of clinical signs. The other three were asymptomatic with persistent biochemical alterations. From these, two presented recurrence of clinical signs about 150 days after the beginning of the treatment while in the other, hyperproteinemia persisted. Meglumine antimoniate was not efficient to treat dogs with severe renal dysfunction and the side effects observed were pain at the site of injection and the probable transient hepatotoxicity, evidenced by biochemical examinations, but without the presence of clinical signs.     

Prevalence of human visceral leishmaniasis in Iran: A systematic review and meta-analysis

Leishmania infantum is the main cause of human visceral leishmaniasis (HVL; also known as kala-azar) in the Middle East and may be fatal if left untreated. This disease was first reported in 1949 in Iran. Despite marked improvements in hygiene and sanitation conditions, the disease is still endemic in some parts of Iran. It is difficult to determine the current prevalence of HVL in Iran due to the scarcity of comprehensive studies in this regard. In response to this gap, a systematic review and meta-analysis was conducted to gain better understanding of HVL epidemiology in the general population of Iran. English and Persian databases were searched for studies reporting the prevalence and risk factors associated with HVL in the Iranian people from January 1995 to December 2019. The reported data were selected according to inclusion and exclusion criteria. The pooled prevalence of HVL infection and its 95 % confidence intervals were calculated. Quality assessment, heterogeneity testing and publication bias assessment were also done. Literature search revealed 3634 studies, of which 35 studies met our eligibility criteria, with a total of 50,716 individuals. The meta-analysis was performed on 31 out of 35 included studies. The estimated pooled prevalence of HVL infection according to seropositivity was 2% (95 % CI: 1–2%) in the general population of Iran in which western and northern provinces had the lowest and the highest prevalence, 0.5 % (95 %CI, 0.2−0.7%) and 3% (95 %CI, 1–5 %), respectively. The seroprevalence of HVL among females (2%; 95 %CI, 1–2 %) was more than males (1%; 95 %CI, 1–2 %). The ≤10 and >10 years age groups had similar seroprevalence rates (1%, 95 %CI, 1–2 % versus 1%, 95 %CI, 0–1 %, respectively). There was no significant difference in terms of geographic area, age and sex. Of 31 studies included in the meta-analysis, the most common diagnostic test was the direct agglutination test (96.77 %). To the best of our knowledge, this is the first systematic review of the prevalence of HVL in Iran. The results showed a low seroprevalence of HVL infection. However, the lack of published reports of HVL in an area does not exclusively mean the absence of the disease or carrier. We therefore recommend further studies in this regard.     

PD-1 and PD-L1 regulate cellular immunity in canine visceral leishmaniasis

PD-1 is a negative costimulator of chronic infectious diseases In this study, we investigated the expression of PD-1 and its ligands in the spleen of dogs with visceral leishmaniasis and lymphoproliferative response to soluble antigen, in lymph node cells in the presence or absence of antibodies blocking PD-1 and its ligands. Our results showed expression of PD-1 and its ligands is higher after L. infantum infection and in the spleen of infected dogs, PD-1 blockage was able to restore the antigen-dependent lymphoproliferative response and regulated production of the cytokines IL-4 and IL-10 and NO production. We concluded that L. infantum infection modulates PD-1 and its ligands expression in canine VL and that blockage of PD-1 restores the immune response. Thus, blockage of PD-1 is a target for therapeutic drug development.     

Anti-Leishmania IgA in urine samples from dogs with clinical leishmaniasis

Recently, anti-Leishmania IgG has been detected in urine samples from Leishmania-infected dogs and its concentrations have been correlated with impairment of renal function. The presence and relationship with other anti-Leishmania Ig isotypes in urine have not yet been investigated. The current study analyzed the concentrations of anti-Leishmania IgA and IgG in sera (Ig-S) and urine (Ig-U) samples by ELISA in 64 untreated dogs with clinical leishmaniasis. All 64 serum samples tested were positive for anti-Leishmania IgG. Fifty of them (78.1%) were also positive for anti-Leishmania IgA. The results showed the presence of anti-Leishmania IgA-U in 38% of the 50 dogs that were positive for specific IgA-S. Thirty-eight of the 64 dogs positive for Leishmania-specific IgG-S (59.4%) were also positive for Leishmania-specific IgG in urine (IgG-U). The concentrations of anti-Leishmania IgA-U were significantly correlated with urine protein/creatinine (uP/C) ratio (ρ = 0.542; P < 0.001) and with serum biochemical parameters, such as γ-globulins, urea and creatinine. Goldmann–Witmer coefficient (C value) indicated that detection of specific IgA in urine samples from dogs with leishmaniasis might not only be due to impairment of filtration of the glomerular barrier but also be due to local production of this isotype, which might reflect a local immunological response to the presence of the parasite in the genitourinary tract. Anti-Leishmania IgG-U concentrations were highly correlated with uP/C ratio (ρ = 0.779; P < 0.001) and C value did not support in any case local production of this isotype. IgG isotype might be a more suitable and specific tool to evaluate renal damage due to the lower IgA-U sensitivity and correlation coefficients and evidence of IgA local production. However, dogs found positive for both Ig isotypes in urine presented significantly higher specific IgG-U concentrations and higher uP/C ratios than dogs found positive only for IgG-U, thus suggesting that the first group suffered more severe renal damage. This fact makes it necessary to evaluate the prognosis of dogs showing both anti-Leishmania IgA-U and IgG-U in future studies.     

Expression of inducible nitric oxide synthase in macrophages inversely correlates with parasitism of lymphoid tissues in dogs with visceral leishmaniasis

Background

There are only a few studies reporting the role of nitric oxide metabolites for controlling macrophage intracellular parasitism, and these are controversial. Therefore, the present study aimed to evaluate the expression of inducible nitric oxide synthase (iNOS) in the lymph nodes and spleen of dogs affected by visceral leishmaniasis through immunohistochemistry and to determine its correlation with tissue parasite burden and serum interferon (IFN)-γ levels. Twenty-eight dogs were selected and assigned to one of two groups, symptomatic (n = 18) and asymptomatic (n = 10), according to clinical status and laboratory evaluation. A negative control group (n = 6) from a non-endemic region for visceral leishmaniasis was included as well.

Results

Parasite density (amastigotes/mm²) was similar between clinical groups in the lymph nodes (P = 0.2401) and spleen (P = 0.8869). The density of iNOS⁺ cells was higher in infected dogs compared to controls (P < 0.05), without a significant difference in lymph node (P = 0.3257) and spleen (P = 0.5940) densities between symptomatic and asymptomatic dogs. A positive correlation was found between the number of iNOS⁺ cells in lymph nodes and interferon-γ levels (r = 0.3776; P = 0.0303), and there was a negative correlation between parasites and iNOS⁺ cell densities both in lymph nodes (r = −0.5341; P = 0.0034) and spleen (r = −0.4669; P = 0.0329).

Conclusion

The negative correlation observed between tissue parasitism and the expression of iNOS may be a reflection of NO acting on the control of parasites.     

Sero-prevalence and risk indicators for canine ehrlichiosis in three rural areas of Brazil

Ehrlichia canis has a worldwide geographic distribution, occurring particularly in tropical and subtropical areas. In Brazil, the main vector in urban areas is believed to be the brown dog tick Rhipicephalus sanguineus, but little is known about the occurrence, transmission and other epidemiological aspects of canine ehrlichiosis in rural areas, where Amblyomma ticks are found more frequently than R. sanguineus. A sero-prevalence study of canine ehrlichiosis was carried out in three distinct rural regions of the State of Minas Gerais, Brazil. Serum samples were collected from 226 dogs living on farms in Lavras (n=85), Belo Horizonte (n=45), and Nanuque (n=96) and were analyzed by an indirect fluorescent antibody test for the detection of anti-Ehrlichia canis antibodies. Age, breed, sex, presence of ticks and packed cell volume were also recorded. There were 65.6% positive dogs in Nanuque, 37.8% in Belo Horizonte, and 24.7% in Lavras. Animals living in Nanuque were 4.6 times more likely to be serologically positive than dogs living in the other two regions and antibody titres were considerable higher in this area. Male dogs, dogs >5 years of age, those infested with ticks, and mongrels all showed higher rates of positivity. The results point to the importance of canine ehrlichiosis in rural areas and indicate the need for further studies on natural transmission and maintenance of the disease.     

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.     

Canine visceral leishmaniasis: comparison of in vitro leishmanicidal activity of marbofloxacin, meglumine antimoniate and sodium stibogluconate

The control of canine leishmaniasis largely depends on the success of treatment. Drugs currently available to treat this disease are toxic and partially effective. The curative effect of marbofloxacin, a third-generation fluoroquinolone developed for veterinarian individual treatment, was evaluated in vitro in the presence of Leishmania infantum promastigotes and dog-monocyte-derived macrophages; meglumine antimoniate and sodium stibogluconate were used as comparative treatments. We observed that the killing of Leishmania promastigotes and intracellular amastigotes by marbofloxacin was dose-dependent. We demonstrated that successful treatment of canine infected macrophages for 48 h was possible with 500 microg/ml of marbofloxacin. Leishmanicidal activity acted through a TNF-alpha and nitric oxide pathway and correlated with the generation of nitric oxide (NO(2)) production by monocytes derived macrophages from infected (23+/-5 microM) or healthy (21+/-6 microM) dogs, in comparison with NO(2) concentration in infected/non-treated macrophages (< 3 microM, P<0.01). This significant induced parasiticidal effect correlated with extensive elimination of amastigotes by macrophages derived from infected (11+/-5) and healthy dogs (6+/-2), when compared to infected/non-treated macrophages (530+/-105 and 472+/-86 amastigotes, respectively, P< 0.01). Marbofloxacin was shown to be non-toxic at 500 microg/ml in vitro and no cell apoptosis was observed. The molecule was able to induce a parasitic process after significant elimination of amastigotes in leishmania-infected dog macrophages. We propose that marbofloxacin, compared to standard chemotherapeutic agents (meglumine antimoniate and sodium stibogluconate), could be an effective and pragmatic oral route alternative to treat canine leishmaniasis.     

A systematic review of the efficacy of prophylactic control measures for naturally occurring canine leishmaniosis. Part II: Topically applied insecticide treatments and prophylactic medications

The objective of this study was to systematically review the efficacy of topically applied insecticide treatments of dogs (impregnated collars, spot-ons), and prophylactic medications to prevent natural Leishmania infantum (L. infantum) infection in dogs.     

Therapy of ocular and visceral leishmaniasis in a cat

An 8-year-old, spayed female Domestic Short-haired cat was referred for further evaluation of chronic lymphocytic-plasmacytic stomatitis and bilateral ocular disease. The cat had been treated with systemic glucocorticoids for several months. Initial ophthalmic examination revealed bilateral deep stromal corneal ulcers, exudative panuveitis and secondary glaucoma. Mature mild neutrophilia and monocytosis were detected on complete blood cell count. Abnormalities in the serum profile were hyperglycemia, mild azotemia, hyperglobulinemia and moderate polyclonal gammapathy. Urinalysis revealed glucosuria without ketonuria. Diabetes mellitus was diagnosed and treatment with long-acting insulin was started. An enzyme-linked immunosorbent assay was highly positive for leishmaniasis, and treatment with allopurinol was started. Although specific topical treatment was applied, melting ulcers progressed to corneal perforation and both eyes were enucleated. Ocular histology showed large numbers of intracellular organisms compatible with amastigotes of the genus Leishmania located in the uveal tract, cornea, sclera and retina. Results of inmunohistochemistry staining on ocular samples were positive for Leishmania. Bone marrow cytology demonstrated numerous macrophages with intracytoplasmatic Leishmania. Polymerase chain reaction results on bone marrow for Leishmania were positive. Three weeks later, hypoglycemic episodes permitted withdrawal of the insulin therapy. To the authors' knowledge this is the first case of ocular and visceral leishmaniasis diagnosed in vivo and under systemic treatment in a cat.