Detection of Leishmania infantum DNA in the non-parasitized lung of dogs with visceral leishmaniasis

Background

Despite the very low or absent parasitism in the lungs, the interstitial pneumonitis is a common lesion found in humans and dogs with visceral leishmaniasis. The lung is a neglected organ in the study of dogs and humans with visceral leishmaniasis, but interstitial pneumonitis represents an important lesion characterized by thickening of the alveolar septum due to fibrosis and inflammatory exudate, and its pathogenesis is still uncertain. In this study, the polymerase chain reaction (PCR) was used to detect Leishmania infantum in paraffin-embedded lung biopsies from naturally infected dogs from an endemic area in Minas Gerais State, Brazil; PCR was compared to histological and immunohistochemical techniques for detecting Leishmania.

Results

Eighteen dogs in which leishmaniasis had been diagnosed by serological tests - indirect immunofluorescence assay (IFAT), enzyme-linked immunosorbent assay (ELISA) and complement fixation tests (CFT) - were classified as asymptomatic, oligosymptomatic or symptomatic. Nine of the 18 dogs studied had a positive PCR (50%) but parasites were not detected by histopathological and immunocytochemistry methods.

Conclusions

These data indicate that PCR on DNA extracted from paraffin-embedded tissue is a valuable method for detecting Leishmania infantum parasites in lungs of naturally infected dogs, despite the apparent absence of parasites from standard HE (hematoxylin and eosin) stained slides and of labeled parasites from immunocytochemical preparations.

     

Apoptosis of Leishmania (Leishmania) chagasi amastigotes in hamsters infected with visceral leishmaniasis

Apoptosis in amastigotes from hamsters infected with visceral leishmaniasis was absent 30-day post-infection but appeared 90-day post-infection in the liver and spleen, as analysed using the TUNEL method. Necrosis was not present in these tissues and the nuclei of macrophages harbouring apoptotic amastigotes were preserved. Amastigote DNA fragmentation was demonstrated using agarose gel electrophoresis. DNA fragmentation was evident 90-day post-infection, coinciding with the occurrence of apoptosis of amastigotes in the tissues. Apoptosis of Leishmania amastigotes in vivo may constitute a mechanism that regulates growth of the parasite population during infection.     

Heterogeneity among zymodemes of Leishmania infantum from HIV-positive patients with visceral leishmaniasis in south Italy

Abstract Eleven zymodemes of Leishmania infantum were identified among 38 parasite stocks isolated from Italian HIV-positive patients with visceral leishmaniasis (VL). Only one zymodeme is a common agent of Mediterranean VL in HIV-negative individuals, five zymodemes usually cause simple, self-resolving cutaneous leishmaniasis (CL), and five belong to unique genotypes which have not been previously reported from either VL or CL cases in immunocompetent individuals. This last group of parasites showed reassortaient patterns within electromorphs frequently observed in dermotropic L. infantum zymodemes. The highest zymodeme heterogeneity was found in south Italy (Sicily), with six zymodemes identified among 12 HIV-positive patients surveyed.     

Leishmania chagasi/infantum: further investigations on Leishmania tropisms in atypical cutaneous and visceral leishmaniasis foci in Central America

In Central America, apparently genetically identical Leishmania chagasi/infantum parasites cause cutaneous (CL) and visceral leishmaniasis (VL), the latter being more frequent in young children. The present study investigated if there were pathology-related differences in virulence between Honduran CL and VL strains using Mediterranean L. infantum strains as a reference. Macrophage infectivity and serum sensitivity, properties thought to be associated with virulence, were similar between CL and VL strains from both regions. Attention focused on the genome organisation of genes for two candidate virulence factors: Leishmania mitogen activated protein kinase (LMPK) and cysteine proteinase b (Cpb). Interestingly, the Mediterranean strains exhibited restriction enzyme polymorphisms associated with tropism for both LMPK and Cpb genes whereas no differences were observed for the Honduran strains. We also report relative genetic homogeneity of the Honduran strains as compared to the Mediterranean strains and discuss it in terms of the probable origin for the Central American L. chagasi/infantum.     

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

Background

A nationwide survey on the microbial etiology of cases of subclinical mastitis in dairy cows was carried out on dairy farms in Sweden. The aim was to investigate the microbial panorama and the occurrence of antimicrobial resistance. Moreover, differences between newly infected cows and chronically infected cows were investigated.

Methods

In total, 583 quarter milk samples were collected from 583 dairy cows at 226 dairy farms from February 2008 to February 2009. The quarter milk samples were bacteriological investigated and scored using the California Mastitis Test. Staphylococci were tested for betalactamase production and presence of resistance was evaluated in all specific udder pathogens. Differences between newly infected cows and chronically infected cows were statistically investigated using logistic regression analysis.

Results

The most common isolates of 590 bacteriological diagnoses were Staphylococcus (S) aureus (19%) and coagulase-negative staphylococci (CNS; 16%) followed by Streptococcus (Str) dysgalactiae (9%), Str. uberis (8%), Escherichia (E.) coli (2.9%), and Streptococcus spp. (1.9%). Samples with no growth or contamination constituted 22% and 18% of the diagnoses, respectively. The distribution of the most commonly isolated bacteria considering only bacteriological positive samples were: S. aureus - 31%, CNS - 27%, Str. dysgalactiae - 15%, Str. uberis - 14%, E. coli - 4.8%, and Streptococcus spp. - 3.1%. There was an increased risk of finding S. aureus, Str. uberis or Str. dysgalactiae in milk samples from chronically infected cows compared to findings in milk samples from newly infected cows. Four percent of the S. aureus isolates and 35% of the CNS isolates were resistant to penicillin G. Overall, resistance to other antimicrobials than penicillin G was uncommon.

Conclusions

Staphylococcus aureus and CNS were the most frequently isolated pathogens and resistance to antimicrobials was rare.     

Development and evaluation of Leishmania infantum rK26 ELISA for serodiagnosis of visceral leishmaniasis in Iran

The purpose of this study was to prepare recombinant K26 antigen from Leishmania infantum and evaluate its performance by enzyme-linked immunosorbent assay (ELISA) test for serodiagnosis of visceral leishmaniasis (VL) in endemic regions of Iran. The results were compared with those obtained by direct agglutination test (DAT) and whole cell ELISA using crude parasite antigen. Of 93 sera from patients with confirmed VL, 90 sera were positive with rK26 ELISA (sensitivity=96.8%), whereas 85 sera were positive with DAT (sensitivity=91.4%) and 89 sera were positive with whole cell ELISA (sensitivity=95.7%). Of 130 subjects who either had other infectious diseases (n=30) or were healthy (n=100), rK26 ELISA were negative in all cases (specificity=100%), whereas DAT were negative in 116 cases (specificity=89.2%) and whole cell ELISA was negative in 114 cases (specificity=87.7%). The results of this study indicate that the rK26 ELISA is more sensitive and specific than conventional methods and could be used for reliable diagnosis of VL caused by Leishmania infantum.     

Leishmania chagasi naturally resistant to nitric oxide isolated from humans and dogs with visceral leishmaniasis in Brazil

Nitric oxide (NO) plays an important role as a leishmanicidal agent in murine macrophages. NO resistant Escherichia coli and Mycobacterium tuberculosis have been associated with poor outcomes of their resulting diseases. NO resistant Leishmania braziliensis has also been identified and exacerbates the clinical course of human leishmaniasis. We report, for the first time, natural resistance of Leishmania chagasi promastigotes to NO. These parasites were isolated from humans and dogs with visceral leishmaniasis. We also demonstrate that this resistance profile was associated with a greater survival capacity and a greater parasite burden in murine macrophages, independent of activation and after activation by IFN-γ and LPS.     

The genetic structure of Leishmania infantum populations in Brazil and its possible association with the transmission cycle of visceral leishmaniasis

Leishmania infantum is the etiologic agent of visceral leishmaniasis (VL) in the Americas, Mediterranean basin and West and Central Asia. Although the geographic structure of L. infantum populations from the Old World have been described, few studies have addressed the population structure of this parasite in the Neotropical region. We employed 14 microsatellites to analyze the population structure of the L. infantum strains isolated from humans and dogs from most of the Brazilian states endemic for VL and from Paraguay. The results indicate a low genetic diversity, high inbreeding estimates and a depletion of heterozygotes, which together indicate a predominantly clonal breeding system, but signs of sexual events are also present. Three populations were identified from the clustering analysis, and they were well supported by F statistics inferences and partially corroborated by distance-based. POP1 (111 strains) was observed in all but one endemic area. POP2 (31 strains) is also well-dispersed, but it was the predominant population in Mato Grosso (MT). POP3 (31 strains) was less dispersed, and it was observed primarily in Mato Grosso do Sul (MS). Strains originated from an outbreak of canine VL in Southern Brazil were grouped in POP1 with those from Paraguay, which corroborates the hypothesis of dispersal from Northeastern Argentina and Paraguay. The distribution of VL in MS seems to follow the west-east construction of the Bolivia-Brazil pipeline from Corumbá municipality. This may have resulted in a strong association of POP3 and Lutzomyia cruzi, which is the main VL vector in Corumbá, and a dispersion of this population in this region that was shaped by human interference. This vector also occurs in MT and may influence the structure of POP2. This paper presents significant advances in the understanding of the population structure of L. infantum in Brazil and its association with eco-epidemiological aspects of VL.     

Mark-release-recapture of sand flies fed on leishmanial dogs: the natural life-cycle of Leishmania infantum in Phlebotomus ariasi

Wild-caught Phlebotomus ariasi Tonnoir permitted to feed on dogs infected with Leishmania infantum Nicolle were marked with fluorescent powder and released into their natural habitat in an uninhabited area of the Cévennes in southern France. Over a period of 29 days after release, 253 females were recaptured with CDC miniature light traps or by active search at night with portable UV lamps. The ovaries and infections in the alimentary tract were then examined. The females oviposited 6 nights after in infecting blood meal. Second blood meals were never taken during the maturation of eggs. During the first ovarian cycle, midgut infections with promastigotes were only moderately heavy. The intensity of infection increased markedly during the second ovarian cycle and, in the third ovarian cycle, the first pharynx infected with paramastigotes was seen (on day 19). From day 19 to day 29, 76% of the flies had pharyngeal infections. Three out of 19 sand flies with pharyngeal infections recaptured during this period had metacyclic promastigotes in their mouthparts. The long time required for parasites to reach the proboscis in completely natural conditions suggests that their presence in the mouthparts is not a prerequisite for transmission by bite. It is more likely that transmission is most commonly by the regurgitation of metacyclic promastigotes from the thoracic midgut following damage to the stomodaeal valve by chitinase produced by the parasite during its development in the gut of the fly. Nevertheless, it is reasonable to assume that the bite of a fly with metacyclic promastigotes in the proboscis (or salivary glands) would also be infective.     

Amyloidosis in adrenal glands of hamsters experimentally infected with Leishmania infantum

Thirty 10-week-old hamsters were inoculated intraperitoneally with leishmania infantum amastigotes and were serially killed after 15, 30, 45, 60, 75 and 89-90 days. The adrenal glands of each of them were examined by means of light and electron microscopy. The cortex and medulla of the adrenal gland showed a progressive deposition of amyloid, selectively identified by both optical and ultrastructural techniques. It has been suggested that adrenal amyloidosis during visceral leishmaniasis is directly related to a stimulation of the phagocitary mononuclear system due to the persistence of the antigenic stimulation of the parasite. In addition to these deposits, the presence of inflammatory infiltrates containing lymphocytes, plasmocytes and macrophages with leishmanias confirmed the establishment of leishmaniasis. The deposition of the amyloid fibrils finally leads to the partial destruction of the adrenal parenchyma.     

Serological markers of sand fly exposure to evaluate insecticidal nets against visceral leishmaniasis in India and Nepal: a cluster-randomized trial

Background

Visceral leishmaniasis is the world'' second largest vector-borne parasitic killer and a neglected tropical disease, prevalent in poor communities. Long-lasting insecticidal nets (LNs) are a low cost proven vector intervention method for malaria control; however, their effectiveness against visceral leishmaniasis (VL) is unknown. This study quantified the effect of LNs on exposure to the sand fly vector of VL in India and Nepal during a two year community intervention trial.

Methods

As part of a paired-cluster randomized controlled clinical trial in VL-endemic regions of India and Nepal we tested the effect of LNs on sand fly biting by measuring the antibody response of subjects to the saliva of Leishmania donovani vector Phlebotomus argentipes and the sympatric (non-vector) Phlebotomus papatasi. Fifteen to 20 individuals above 15 years of age from 26 VL endemic clusters were asked to provide a blood sample at baseline, 12 and 24 months post-intervention.

Results

A total of 305 individuals were included in the study, 68 participants provided two blood samples and 237 gave three samples. A random effect linear regression model showed that cluster-wide distribution of LNs reduced exposure to P. argentipes by 12% at 12 months (effect 0.88; 95% CI 0.83–0.94) and 9% at 24 months (effect 0.91; 95% CI 0.80–1.02) in the intervention group compared to control adjusting for baseline values and pair. Similar results were obtained for P. papatasi.

Conclusions

This trial provides evidence that LNs have a limited effect on sand fly exposure in VL endemic communities in India and Nepal and supports the use of sand fly saliva antibodies as a marker to evaluate vector control interventions.     

Non-sand fly transmission of a North American isolate of Leishmania infantum in experimentally infected BALB/c mice

Leishmania infantum, an etiologic agent of zoonotic visceral leishmaniasis, is endemic in the foxhound population in the United States and Canada. Leishmaniasis is usually transmitted by blood-feeding sand flies; however, epidemiological data do not support a significant role for sand flies in the maintenance of foxhound infections in North America, and an alternate mode of transmission may exist. The present study was conducted to determine if transplacental or direct transmission occurs in pregnant BALB/c mice experimentally infected with L. infantum isolated from a naturally infected foxhound from Virginia as well as to determine if the parasite was directly transmitted to the males used to breed the mice. Female BALB/c mice were intravenously inoculated with 1 x 10(6) promastigotes of the LIVT-1 strain of L. infantum. Mice were bred to uninfected male BALB/c mice 2 mo postinoculation. Pregnant mice were killed between days 13 and 18 of gestation. Pups and placentas were collected at necropsy, divided, and used for parasite culture and polymerase chain reaction (PCR) analyses. Culture and PCR analyses were performed on spleens from the male mice to determine the possibility of sexual transmission. Leishmania sp. DNA was detected in 4 of 88 pups and 3 of 16 placentas from LIVT-1-inoculated mice. One male mouse used to breed infected females was PCR positive. This work provides evidence for a low level of nonvector transmission of North American L. infantum in a mouse model.     

Isolation of Leishmania infantum, zymodeme MON-1 from canine and human visceral leishmaniasis on Margarita Island, Venezuela.

An increase in the incidence of human visceral leishmaniasis (HVL) has been detected in recent years on Margarita Island, located off the NE coast of Venezuela. Recent studies have revealed reactivity to rK39 antigen (Leishmania chagasi) in 20% of 541 sera from domestic dogs in endemic communities; PCR reactions were positive using primers for the L. donovani complex. Here we report that isolates from human and canine infection, identified by isoenzyme analysis, correspond to L. infantum, zymodeme MON-1. This appears to be the first isolation and identification of an isolate from HVL on Margarita Island and demonstrates the presence of this zymodeme in the canine population.     

Immunodominant antigens of Leishmania chagasi associated with protection against human visceral leishmaniasis

Background

Protection and recovery from visceral leishmaniasis (VL) have been associated with cell-mediated immune (CMI) responses, whereas no protective role has been attributed to humoral responses against specific parasitic antigens. In this report, we compared carefully selected groups of individuals with distinct responses to Leishmania chagasi to explore antigen-recognizing IgG present in resistant individuals.

Methodology and Principal Findings

VL patients with negative delayed-type hypersensitivity (DTH) were classified into the susceptible group. Individuals who had recovered from VL and converted to a DTH+ response, as well as asymptomatic infected individuals (DTH+), were categorized into the resistant group. Sera from these groups were used to detect antigens from L. chagasi by conventional and 2D Western blot assays. Despite an overall reduction in the reactivity of several proteins after DTH conversion, a specific group of proteins (approximately 110–130 kDa) consistently reacted with sera from DTH converters. Other antigens that specifically reacted with sera from DTH+ individuals were isolated and tandem mass spectrometry followed by database query with the protein search engine MASCO were used to identify antigens. The serological properties of recombinant version of the selected antigens were tested by ELISA. Sera from asymptomatic infected people (DTH+) reacted more strongly with a mixture of selected recombinant antigens than with total soluble Leishmania antigen (SLA), with less cross-reactivity against Chagas disease patients'' sera.

Significance

Our results are the first evidence of leishmania proteins that are specifically recognized by sera from individuals who are putatively resistant to VL. In addition, these data highlight the possibility of using specific proteins in serological tests for the identification of asymptomatic infected individuals.     

Studies on the attachment of Leishmania flagella to sand fly midgut epithelium

An in vitro assay was developed to study the recognition mechanism for attachment of Leishmania flagella to sand fly midgut epithelium. Frozen sections of sand fly guts were incubated with flagella preparations, and probed with a flagella-specific monoclonal antibody. Tissue-specific adhesion of flagella to midgut epithelium was demonstrated by indirect immunofluorescence. None of the 13 sugars, screened to test for possible lectin-mediation, appeared to significantly inhibit the adhesion of flagella to gut sections. Similarly no inhibition was achieved by incubating flagella with pep 63 which inhibits the promastigote-macrophage recognition mechanism. Significant inhibition was attained by incubating flagella preparations with a monoclonal antibody which binds to a flagellar membrane-component. The possible relevance of the described mechanism for the biology of Leishmania in their sand fly hosts, is discussed.     

Canine cutaneous leishmaniasis caused by neotropical Leishmania infantum despite of systemic disease: A case report

Visceral leishmaniasis is an anthropozoonosis caused by a protozoan Leishmania infantum (syn. Leishmania chagasi). Here, we report a typical case of canine cutaneous leishmaniasis due to L. infantum infection without any other systemic symptom in one dog in the city of Rio de Janeiro, Brazil. A mongrel female dog was admitted in a veterinary clinic with reports of chronic wounds in the body. Physical examination revealed erosive lesions in the limbs, nasal ulcers, presence of ectoparasites and seborrheic dermatitis. Blood samples and fragments of healthy and injured skin were collected. The complete hemogram revealed aregenerative normocytic normochromic anemia and erythrocyte rouleaux, and biochemical analysis revealed normal renal and hepatic functions. Cytology of the muzzle and skin lesions suggested pyogranulomatous inflammatory process. The histopathology of a skin fragment was performed and revealed suspicion of protozoa accompanied by necrotizing dermatitis. The diagnosis of leishmaniasis was accomplished by positive serology, isolation of Leishmania from the skin lesion, and also by molecular test (PCR targeting the conserved region of Leishmania kDNA). Culture was positive for damaged skin samples. PCR targeting a fragment of Leishmania hsp70 gene was performed employing DNA extracted from damaged skin. RFLP of the amplified hsp70 fragment identified the parasite as L. infantum, instead of Leishmania braziliensis, the main agent of cutaneous leishmaniasis in Rio de Janeiro. Characterization of isolated promastigotes by five different enzymatic systems confirmed the species identification of the etiological agent. Serology was positive by ELISA and rapid test. This case warns to the suspicion of viscerotropic Leishmania in cases of chronic skin lesions and brings the discussion of the mechanisms involved in the parasite tissue tropism.     

The molecular detection of different Leishmania species within sand flies from a cutaneous and visceral leishmaniasis sympatric area in Southeastern Brazil

Over the last 20 years, there has been an increase in the number of leishmaniasis cases in Brazil. Belo Horizonte (BH) is one of the most highly populated Brazilian cities that is affected by visceral leishmaniasis (VL). The health services in BH are coordinated by a central nucleus that is subdivided into nine sanitary districts. Historically, the highest level of human VL cases was found in the northeast sanitary district (NSD). The objective of our study was to detect Leishmania infection in the phlebotomine sand flies collected in the NSD by dissection and molecular approaches. Following the occurrence of human VL cases in 2005, entomological captures were performed from July 2006-June 2007. Out of the 245 sand flies dissected, only three Lutzomyia longipalpis spp contained flagellates. The female sand flies were grouped into 120 pools according to date, collection site and species, with approximately 10 individual sand flies in each pool. Subsquently, the DNA was extracted and Leishmania spp and other parasites were detected and identified by polymerase chain reaction (PCR) and PCR-restriction fragment length polymorfism. Leishmania infantum was present in at least 19% of the Lu. longipalpis collected, in 3.8% of the Nyssomiya whitmani collected, in 33.3% of the Evandromiya termitophila collected and in 14.3% of the Nyssomiya intermedia collected. When the females of the cortelezzii complex were compared with each other, 3.2% of the females were infected with Leishmania braziliensis, whereas 3.2% of the females were infected with trypanosomatids.