Comparative analyses of whole genome sequences of Leishmania infantum isolates from humans and dogs in northeastern Brazil.

The genomic sequences of 20 Leishmania infantum isolates collected in northeastern Brazil were compared with each other and with the available genomic sequences of 29 L. infantum/donovani isolates from Nepal and Turkey. The Brazilian isolates were obtained in the early 1990s or since 2009 from patients with visceral or non-ulcerating cutaneous leishmaniasis, asymptomatic humans, or dogs with visceral leishmaniasis. Two isolates were from the blood and bone marrow of the same visceral leishmaniasis patient. All 20 genomic sequences display 99.95% identity with each other and slightly less identity with a reference L. infantum genome from a Spanish isolate. Despite the high identity, analysis of individual differences among the 32 million base pair genomes showed sufficient variation to allow the isolates to be clustered based on the primary sequence. A major source of variation detected was in chromosome somy, with only four of the 36 chromosomes being predominantly disomic in all 49 isolates examined. In contrast, chromosome 31 was predominantly tetrasomic/pentasomic, consistent with its regions of synteny on two different disomic chromosomes of Trypanosoma brucei. In the Brazilian isolates, evidence for recombination was detected in 27 of the 36 chromosomes. Clustering analyses suggested two populations, in which two of the five older isolates from the 1990s clustered with a majority of recent isolates. Overall the analyses do not suggest individual sequence variants account for differences in clinical outcome or adaptation to different hosts. For the first known time, DNA of isolates from asymptomatic subjects were sequenced. Of interest, these displayed lower diversity than isolates from symptomatic subjects, an observation that deserves further investigation with additional isolates from asymptomatic subjects.

Protective immunity against the protozoan Leishmania chagasi is induced by subclinical cutaneous infection with virulent but not avirulent organisms.

Protective immunity against Leishmania major is provided by s.c. immunization with a low dose of L. major promastigotes or with dihydrofolate-thymidylate synthase gene locus (DHFR-TS) gene knockout L. major organisms. Whether these vaccine strategies will protect against infection with other Leishmania species that elicit distinct immune responses and clinical syndromes is not known. Therefore, we investigated protective immunity to Leishmania chagasi, a cause of visceral leishmaniasis. In contrast to L. major, a high dose s.c. inoculum of L. chagasi promastigotes was required to elicit protective immunity. Splenocytes from mice immunized with a high dose produced significantly greater amounts of IFN-gamma and lower TGF-beta than mice immunized with a low dose of promastigotes. The development of protective immunity did not require the presence of NK cells. Protection was not afforded by s.c. immunization with either attenuated L. chagasi or with L. major promastigotes, and s.c. L. chagasi did not protect against infection with L. major. Subcutaneous immunization with DHFR-TS gene knockouts derived from L. chagasi, L. donovani, or L. major did not protect against L. chagasi infection. We conclude that s.c. inoculation of high doses of live L. chagasi causes a subclinical infection that elicits protective immune responses in susceptible mice. However, L. chagasi that have been attenuated either by long-term passage or during the raising of recombinant gene knockout organisms do not elicit protective immunity, either because they fail to establish a subclinical infection or because they no longer express critical antigenic epitopes.

BCG expressing LCR1 of Leishmania chagasi induces protective immunity in susceptible mice.

Cellular immune responses are required for protective immunity against Leishmania chagasi. Immunization strategies using live intracellular bacteria (e.g., bacille-Calmette Guerin strain of Mycobacterium bovis) expressing recombinant antigens can induce cellular immune responses to these antigens. Previous studies demonstrated that the L. chagasi antigen LCR1 stimulates IFN-gamma production from T cells of infected BALB/c mice, and immunization with recombinant LCR1 partially protects against L. chagasi infection. To determine whether live bacteria could enhance the immunization potential of LCR1, we engineered BCG expressing LCR1 (BCG-LCR1). Subcutaneous immunization with BCG-LCR1, but not with BCG containing plasmid only (BCG-pMV261), elicited better protective immunity against L. chagasi infection than LCR1 protein alone. BCG-LCR1 administered intraperitoneally did not protect. Splenocytes from mice immunized s.c. with either BCG-LCR1 or BCG-pMV261 and then infected with L. chagasi promastigotes had increased antigen-induced IFN-gamma and reduced IL-10 production compared to splenocytes of control mice. We propose that BCG-LCR1 promotes a Th1-type protective immune response, and it may be a useful component of a Leishmania vaccine.

Visceral leishmaniasis.

Visceral leishmaniasis presents a serious problem in endemic regions that is difficult to treat or prevent. Several epidemiologic problems make the disease particularly troublesome to manage. These include the facts that classic visceral leishmaniasis is fatal if untreated and there is not reliable access to medical care in many endemic regions. When available, treatment has associated toxicity and requires the use of intravenous medications with careful monitoring for toxicity, which are complex to administer in underdeveloped nations. There is an increasing incidence of the disease in HIV-infected individuals in southern Europe, in part because of the fact that eradication of the organism from infected persons using currently available drugs appears to be difficult if not impossible. Furthermore, chronic cutaneous forms of the disease allow humans and animals to maintain the organism long-term in a bodily site that is easily accessible to the sandfly vector. More effective and less toxic treatment modalities as well as a protective vaccine are badly needed to manage this disease.

Developmental changes in the expression of Leishmania chagasi gp63 and heat shock protein in a human macrophage cell line.

The ability of the protozoan Leishmania chagasi to infect a vertebrate host depends on its ability to survive intracellularly in a mammalian macrophage. Novel patterns of gene expression are probably important for conversion from the extracellular promastigote to the obligate intracellular amastigote parasite form. We found that the human macrophage-like cell line U937 provided an in vitro model of phagocytosis of L. chagasi promastigotes and intracellular conversion to amastigotes, allowing examination of parasite protein and RNA expression. The Leishmania surface protease gp63 assumed three isoforms during stage conversion, and a 64-kDa form of gp63 not present in promastigotes became the most prominent form in amastigotes. gp63 RNAs derived from the three different classes of msp genes (mspS, mspL, and mspC) were also differentially expressed. Infectious promastigotes contained mRNAs from mspS and mspC genes, whereas converting parasites expressed only mspL and mspC mRNAs. Sequence analysis of clones from an amastigote cDNA library confirmed the presence of gp63 mRNAs only from mspL and mspC class genes in tissue-derived amastigotes. Finally, 24 h after phagocytosis, there was a transient increase in the level of hsp70 and hsp90 proteins that subsequently decreased to baseline; this increase was not due to heat shock alone. We conclude that a unique pattern of selected L. chagasi proteins and RNAs is induced following phagocytosis by macrophages.

Prolonged elevation of alphafetoprotein and detectable acetylcholinesterase after death of an anomalous twin fetus.

Persistence of elevated alphafetoprotein (AFP) levels and the presence of an acetylcholinesterase (AChE) band in amniotic fluid have been reported to occur up to 11 weeks following intrauterine fetal demise (IUFD) of one twin (Bass et al., 1986). We now report a case where such prolongation of these findings was observed in a case of unrecognized monochorionic, monoamniotic twinning, in which case cord entanglement resulted in IUFD at an estimated 10-12 weeks and 25-26 weeks. The fetus suffering early demise (Fetus B) had multiple congenital anomalies, including a neural tube defect. The presence of this defect and/or fetal demise and bleeding into the amniotic sac is entertained as continuing sources of documented elevated AChE and AFP 9-11 weeks after the initial fetal death. We re-emphasize the possibility of unrecognized twinning as a cause of abnormal maternal serum and amniotic fluid study results in the face of one apparently normal fetus.