Emerging Leishmania donovani Lineages Associated with Cutaneous Leishmaniasis, Himachal Pradesh, India, 2023.

The clinical manifestation of leishmaniasis has historically been determined by the Leishmania species involved. However, recent emergence of novel Leishmania lineages has caused atypical pathologies. We isolated and characterized 2 new Leishmania donovani parasites causing cutaneous leishmaniasis in Himachal Pradesh, India.

Vibrio cholerae O1 experiences mild bottlenecks through the gastrointestinal tract in some but not all cholera patients.

Vibrio cholerae O1 causes the diarrheal disease cholera, and the small intestine is the site of active infection. During cholera, cholera toxin is secreted from V. cholerae and induces a massive fluid influx into the small intestine, which causes vomiting and diarrhea. Typically, V. cholerae genomes are sequenced from bacteria passed in stool, but rarely from vomit, a fluid that may more closely represents the site of active infection. We hypothesized that V. cholerae O1 population bottlenecks along the gastrointestinal tract would result in reduced genetic variation in stool compared to vomit. To test this, we sequenced V. cholerae genomes from 10 cholera patients with paired vomit and stool samples. Genetic diversity was low in both vomit and stool, consistent with a single infecting population rather than coinfection with divergent V. cholerae O1 lineages. The amount of single-nucleotide variation decreased from vomit to stool in four patients, increased in two, and remained unchanged in four. The variation in gene presence/absence decreased between vomit and stool in eight patients and increased in two. Pangenome analysis of assembled short-read sequencing demonstrated that the toxin-coregulated pilus operon more frequently contained deletions in genomes from vomit compared to stool. However, these deletions were not detected by PCR or long-read sequencing, indicating that interpreting gene presence or absence patterns from short-read data alone may be incomplete. Overall, we found that V. cholerae O1 isolated from stool is genetically similar to V. cholerae recovered from the upper intestinal tract. IMPORTANCE: Vibrio cholerae O1, the bacterium that causes cholera, is ingested in contaminated food or water and then colonizes the upper small intestine and is excreted in stool. Shed V. cholerae genomes from stool are usually studied, but V. cholerae isolated from vomit may be more representative of where V. cholerae colonizes in the upper intestinal epithelium. V. cholerae may experience bottlenecks, or large reductions in bacterial population sizes and genetic diversity, as it passes through the gut. Passage through the gut may select for distinct V. cholerae mutants that are adapted for survival and gut colonization. We did not find strong evidence for such adaptive mutations, and instead observed that passage through the gut results in modest reductions in V. cholerae genetic diversity, and only in some patients. These results fill a gap in our understanding of the V. cholerae life cycle, transmission, and evolution.

Diversity of Vibrio cholerae O1 through the human gastrointestinal tract during cholera.

Vibrio cholerae O1 causes the diarrheal disease cholera, and the small intestine is the site of active infection. During cholera, cholera toxin is secreted from V. cholerae and induces a massive fluid influx into the small intestine, which causes vomiting and diarrhea. Typically, V. cholerae genomes are sequenced from bacteria passed in stool, but rarely from vomit, a fluid that may more closely represents the site of active infection. We hypothesized that the V. cholerae O1 population bottlenecks along the gastrointestinal tract would result in reduced genetic variation in stool compared to vomit. To test this, we sequenced V. cholerae genomes from ten cholera patients with paired vomit and stool samples. Genetic diversity was low in both vomit and stool, consistent with a single infecting population rather than co-infection with divergent V. cholerae O1 lineages. The number of single nucleotide variants decreased between vomit and stool in four patients, increased in two, and remained unchanged in four. The number of genes encoded in the V. cholerae genome decreased between vomit and stool in eight patients and increased in two. Pangenome analysis of assembled short-read sequencing demonstrated that the toxin-coregulated pilus operon more frequently contained deletions in genomes from vomit compared to stool. However, these deletions were not detected by PCR or long-read sequencing, indicating that interpreting gene presence or absence patterns from short-read data alone may be incomplete. Overall, we found that V. cholerae O1 isolated from stool is genetically similar to V. cholerae recovered from the upper intestinal tract.

Production of leishmanin skin test antigen from Leishmania donovani for future reintroduction in the field.

The leishmanin skin test was used for almost a century to detect exposure and immunity to Leishmania, the causative agent of leishmaniasis, a major neglected tropical disease. Due to a lack of antigen used for the intradermal injection, the leishmanin skin test is no longer available. As leishmaniasis control programs are advancing and new vaccines are entering clinical trials, it is essential to re-introduce the leishmanin skin test. Here we establish a Leishmania donovani strain and describe the production, under Good Laboratory Practice conditions, of leishmanin soluble antigen used to induce the leishmanin skin test in animal models of infection and vaccination. Using a mouse model of cutaneous leishmaniasis and a hamster model of visceral leishmaniasis, soluble antigen induces a leishmanin skin test response following infection and vaccination with live attenuated Leishmania major (LmCen-/-). Both the CD4+ and CD8+ T-cells are necessary for the leishmanin skin test response. This study demonstrates the feasibility of large-scale production of leishmanin antigen addressing a major bottleneck for performing the leishmanin skin test in future surveillance and vaccine clinical trials.

Centrin-deficient Leishmania mexicana confers protection against Old World visceral leishmaniasis.

Leishmaniasis is one of the top neglected tropical diseases with significant morbidity and mortality in low and middle-income countries (LMIC). However, this disease is also spreading in the developed world. Currently, there is a lack of effective strategies to control this disease. Vaccination can be an effective measure to control leishmaniasis and has the potential to achieve disease elimination. Recently, we have generated centrin gene-deleted new world L. mexicana (LmexCen-/-) parasites using CRISPR/Cas9 and showed that they protect mice against a homologous L. mexicana infection that causes cutaneous disease. In this study, we tested whether LmexCen-/- parasites can also protect against visceral leishmaniasis caused by L. donovani in a hamster model. We showed that immunization with LmexCen-/- parasites is safe and does not cause lesions. Furthermore, such immunization conferred protection against visceral leishmaniasis caused by a needle-initiated L. donovani challenge, as indicated by a significant reduction in the parasite burdens in the spleen and liver as well as reduced mortality. Similar control of parasite burden was also observed against a sand fly mediated L. donovani challenge. Importantly, immunization with LmexCen-/- down-regulated the disease promoting cytokines IL-10 and IL-4 and increased pro-inflammatory cytokine IFN-γ resulting in higher IFN-γ/IL-10 and IFN-γ/IL4 ratios compared to non-immunized animals. LmexCen-/- immunization also resulted in long-lasting protection against L. donovani infection. Taken together, our study demonstrates that immunization with LmexCen-/- parasites is safe and efficacious against the Old World visceral leishmaniasis.

Investigating the Leishmania donovani sacp Gene and Its Role in Macrophage Infection and Survival in Mice.

The protozoan parasite Leishmania donovani is a causative agent of the neglected tropical disease known as visceral leishmaniasis, which can be lethal when untreated. Studying Leishmania viru-lence factors is crucial in determining how the parasite causes disease and identifying new targets for treatment. One potential virulence factor is L. donovani's abundantly secreted protein: secreted acid phosphatase (SAcP). Whole-genome analysis revealed that the sacp gene was present in three copies in wild type L. donovani. Using CRISPR-Cas9 gene editing; we generated a sacp gene knockout termed LdΔSAcP, which demonstrated a loss of both the SAcP protein and an associated reduction in secreted acid phosphatase activity. Genome sequencing confirmed the precise dele-tion of the sacp gene in LdΔSAcP and identified several changes in the genome. LdΔSAcP demonstrated no significant changes in promastigote proliferation or its ability to infect and survive in macrophages compared to the wildtype strain. LdΔSAcP also demonstrated no change in murine liver infection; however, survival was impaired in the spleen. Taken together these results show that SAcP is not necessary for the survival of promastigotes in culture but may support long-term survival in the spleen. These observations also show that the use of CRISPR gene editing and WGS together are effective to investigate the function and phenotype of complex potential drug targets such as multicopy genes.

Centrin-deficient Leishmania mexicana confers protection against New World cutaneous leishmaniasis.

Leishmaniasis is a neglected protozoan disease affecting over 12 million people globally with no approved vaccines for human use. New World cutaneous leishmaniasis (CL) caused by L. mexicana is characterized by the development of chronic non-healing skin lesions. Using the CRISPR/Cas9 technique, we have generated live attenuated centrin knockout L. mexicana (LmexCen-/-) parasites. Centrin is a cytoskeletal protein important for cellular division in eukaryotes and, in Leishmania, is required only for intracellular amastigote replication. We have investigated the safety and immunogenicity characteristics of LmexCen-/- parasites by evaluating their survival and the cytokine production in bone-marrow-derived macrophages (BMDMs) and dendritic cells (BMDCs) in vitro. Our data shows that LmexCen-/- amastigotes present a growth defect, which results in significantly lower parasitic burdens and increased protective cytokine production in infected BMDMs and BMDCs, compared to the wild type (WT) parasites. We have also determined the safety and efficacy of LmexCen-/- in vivo using experimental murine models of L. mexicana. We demonstrate that LmexCen-/- parasites are safe and do not cause lesions in susceptible mouse models. Immunization with LmexCen-/- is also efficacious against challenge with WT L. mexicana parasites in genetically different BALB/c and C57BL/6 mouse models. Vaccinated mice did not develop cutaneous lesions, displayed protective immunity, and showed significantly lower parasitic burdens at the infection site and draining lymph nodes compared to the control group. Overall, we demonstrate that LmexCen-/- parasites are safe and efficacious against New World cutaneous leishmaniasis in pre-clinical models.

An intraspecies Leishmania donovani hybrid from the Indian subcontinent is associated with an atypical phenotype of cutaneous disease.

Leishmaniasis is a neglected tropical disease endemic in over 90 countries. The disease has two main pathologies; cutaneous leishmaniasis (CL) that generally self-heals, and visceral leishmaniasis (VL) that is fatal if untreated. The majority of VL cases, concentrated on the Indian subcontinent (ISC) and East Africa, are caused by Leishmania donovani. However, recent foci of CL on the ISC have been attributed as an atypical phenotype of L. donovani including a recent outbreak in Himachal Pradesh, India. Whole genome sequencing and phylogenetic analysis was undertaken to investigate the origins and genetic factors leading to this pathology atypical of L. donovani. Here we demonstrate the isolate from Himachal Pradesh is derived from a genetic hybridization between two independent L. donovani parents from the 'Yeti' ISC1 divergent clade of parasites, identified in the Nepalese highlands. This reveals that intraspecies L. donovani hybrids can give rise to a novel strain associated with CL.

A review of the leishmanin skin test: A neglected test for a neglected disease.

The leishmanin skin test (LST) has been used for decades to detect exposure and immunity to the parasite Leishmania, the causative agent of the neglected tropical disease leishmaniasis. In the LST, Leishmania antigen (leishmanin) is intradermally injected into the forearm. In an individual who has been previously infected, a delayed-type hypersensitivity (DTH) reaction results in a measurable induration at the site of the injection, indicating that previous exposure to Leishmania has resulted in the development of cell-mediated immunity. LST positivity is associated with long-lasting protective immunity against reinfection, most notably as reported for visceral leishmaniasis (VL). Despite efforts over the past few decades, leishmanin antigen is no longer produced under good manufacturing practice (GMP) conditions anywhere in the world. Consequently, the use of the LST in epidemiological studies has declined in favor of serological and molecular tests. In this review, we provide a historical overview of the LST and justification for the reintroduction of leishmanin. A GMP-grade leishmanin can be used to detect immunity in vivo by the LST and can be investigated for use in an interferon-γ release assay (IGRA), which may serve as an in vitro version of the LST. The LST will be a valuable tool for surveillance and epidemiological studies in support of the VL elimination programs and as a surrogate marker of immunity in vaccine clinical trials. METHODS: A review of the literature was conducted using PubMed as the primary database, with MeSH terms "leishmanin skin test" OR "Montenegro test" OR "Montenegro skin test." Articles written in English that describe the history or standardization of leishmanin, the use of leishmanin in an IGRA, or the use of the LST in epidemiological studies or vaccine trials were prioritized in our appraisal of the literature.

Evidence that a naturally occurring single nucleotide polymorphism in the RagC gene of Leishmania donovani contributes to reduced virulence.

Leishmaniasis is a widespread neglected tropical disease transmitted by infected sand flies resulting in either benign cutaneous infection or fatal visceral disease. Leishmania donovani is the principal species responsible for visceral leishmaniasis, yet an atypical L. donovani has become attenuated in several countries including Sri Lanka and causes cutaneous leishmaniasis. Previous studies have identified 91 genes altered in the atypical cutaneous L. donovani compared to typical visceral disease associated L. donovani including mutations in the RagC and Raptor genes that are part of the eukaryotic conserved TOR pathway and its upstream sensing pathway. In the present study, we investigate whether the RagC R231C mutation present in atypical cutaneous L. donovani introduced into the virulent L. donovani 1S2D chromosome by CRISPR gene editing could affect virulence for survival in visceral organs. Through bioinformatic analysis, we further investigated the presence of sensing pathway components upstream of TOR in L. donovani including RagC complexing proteins, RagA and Raptor. L. donovani 1S2D edited to express mutant RagC R231C were viable in promastigote but had reduced visceral parasitemia in infected BALB/c mice. The RagC R231C mutant retained the ability to interact with RagA and gene knockout experiments revealed that although the RagA gene was essential, the RagC gene was not essential under promastigote culture conditions but was essential for survival in the liver of experimentally infected mice. These results provide evidence that the TOR associated sensing pathway plays a prominent role in L. donovani visceral disease and the RagC R231C mutation contributed to the atypical pathology of cutaneous L. donovani in Sri Lanka.