A panel of recombinant Leishmania donovani cell surface and secreted proteins identifies LdBPK_323600.1 as a serological marker of symptomatic infection

Visceral leishmaniasis is a deadly infectious disease and is one of the world’s major neglected health problems. Because the symptoms of infection are similar to other endemic diseases, accurate diagnosis is crucial for appropriate treatment. Definitive diagnosis using splenic or bone marrow aspirates is highly invasive, and so, serological assays are preferred, including the direct agglutination test (DAT) or rK39 strip test. These tests, however, are either difficult to perform in the field (DAT) or lack specificity in some endemic regions (rK39), making the development of new tests a research priority. The availability of Leishmania spp. genomes presents an opportunity to identify new diagnostic targets. Here, we use genome data and a mammalian protein expression system to create a panel of 93 proteins consisting of the extracellular ectodomains of the Leishmania donovani cell surface and secreted proteins. We use these panel and sera from murine experimental infection models and natural human and canine infections to identify new candidates for serological diagnosis. We observed a concordance between the most immunoreactive antigens in different host species and transmission settings. The antigen encoded by the LdBPK_323600.1 gene can diagnose Leishmania infections with high sensitivity and specificity in patient cohorts from different endemic regions including Bangladesh and Ethiopia. In longitudinal sampling of treated patients, we observed reductions in immunoreactivity to LdBPK_323600.1 suggesting it could be used to diagnose treatment success. In summary, we have identified new antigens that could contribute to improved serological diagnostic tests to help control the impact of this deadly tropical infectious disease. IMPORTANCE Visceral leishmaniasis is fatal if left untreated with patients often displaying mild and non-specific symptoms during the early stages of infection making accurate diagnosis important. Current methods for diagnosis require highly trained medical staff to perform highly invasive biopsies of the liver or bone marrow which pose risks to the patient. Less invasive molecular tests are available but can suffer from regional variations in their ability to accurately diagnose an infection. To identify new diagnostic markers of visceral leishmaniasis, we produced and tested a panel of 93 proteins identified from the genome of the parasite responsible for this disease. We found that the pattern of host antibody reactivity to these proteins was broadly consistent across naturally acquired infections in both human patients and dogs, as well as experimental rodent infections. We identified a new protein called LdBPK_323600.1 that could accurately diagnose visceral leishmaniasis infections in humans

Detection and identification of blood-borne infections in dogs in Nigeria using light microscopy and the polymerase chain reaction

Many sick dogs brought to the University of Ibadan Veterinary Teaching Hospital (UIVTH) are infested with ticks and are anemic. Up until recently, light microscopy (LM) has been the only available means used for detection of blood-borne infections. In other parts of the world, PCR-based assays been used as a gold standard for accurate diagnosis of blood-borne infections. In this study, we used LM and broad-spectrum rRNA gene PCR-based assays on 116 blood samples from dogs brought to the UIVTH for detection of the 18S rRNA gene of Babesia and the 16S rRNA genes of Ehrlichia and hemotropic mycoplasmas. The relationship between clinicopathological findings and PCR results was evaluated. Age, sex, presence of ticks, anemia, co-infection status, and fever were also assessed in relation to PCR positivity to determine the risk factors using stepwise logistic regression analyses. Light microscopic examination revealed an overall prevalence of infection of 14.7% (17/116). Organisms detected were Babesia canis (3.5%), Ehrlichia canis (10.3%) and Trypanosoma congolense (0.9%) and a single co-infection with Babesia canis and Ehrlichia canis (0.9%). PCR analysis revealed 89/116 (76.7%) positive samples. Infections with 1, 2 and 3 infectious agents occurred in 49 (55.1%), 36 (40.4%) and 4 (4.5%) samples, respectively. Specifically, among the 89 PCR positive samples, Babesia spp. (85.4%) was the most abundant infection followed by Ehrlichia spp. (46.1%) and hemoplasmas (13.5%). Sequencing of PCR products identified two samples (1.7%) that contained Hepatozoon canis DNA. Sequencing of hemoplasma positive samples identified 'Candidatus Mycoplasma haemobos' in 0.8% of dogs. Using PCR, a 5-fold higher prevalence of blood-borne infections was found in the dogs (76.7%, 89/116) than with LM (14.7%, 17/116) alone" Dogs between 1 and 12months were the most frequently infected with multiple agents (47.2% double and 50.0% triple infections). Male dogs had the highest prevalence of infection (80.4%) and more triple infections (75.0%). A total of 57.3% of infected dogs were anemic. Anemic dogs were 2.77 times more likely to test positive for Ehrlichia spp. (OR: 2.77 95% CI: 1.25-6.16) and dogs with ticks were 3.6 times more likely to test positive for hemoplasmas (OR=3.60 95% CI: 1.05-12.38). This study underscores the abundance of blood-borne infections in dogs in Ibadan, Nigeria, which is underestimated using light microscopy. This is also the first evidence of existence of 'Candidatus Mycoplasma haemobos' in a dog in Nigeria and in Africa. Consequently there is a need for molecular diagnostic facilities for routine screening of sick animals, as multiple infections were not found by light microscopy

Role of NK-like CD8+ T cells during asymptomatic Borrelia burgdorferi infection

Lyme disease (LD) due to Borrelia burgdorferi is the most prevalent vector-borne disease in the United States. There is a poor understanding of how immunity contributes to bacterial control, pathology, or both during LD. Dogs in an area of endemicity were screened for B. burgdorferi an

Leishmania-Derived Trimannose Modulates the Inflammatory Response To Significantly Reduce Leishmania major-Induced Lesions

Leishmania lipophosphoglycan (LPG) is a key virulence factor, initiating inflammation resulting in cutaneous lesions. LPG is capped by various oligosaccharides. How these glycans are recognized and how they alter the course of Leishmania infection are poorly understood. Previous studies synthesized α-1,2-trimannose cap sugars on latex beads and demonstrated that C57BL/6 mice coinoculated with Leishmania major and trimannose-coated beads produced significantly higher levels of interleukin-12p40 (IL-12p40) and other proinflammatory, type 1 cytokines than mice inoculated with L. major alone within the first 48 h of infection. However, as L. major infection typically progress over weeks to months, the role of trimannose in altering disease progression over the course of infection was unknown. Wild-type mice were inoculated with either trimannose-coated or carrier (uncoated) beads, infected with L. major alone, coinoculated with carrier beads and L. major, or coinoculated with trimannose-coated beads and L. major. Trimannose treatment of L. major-infected mice decreased the parasite load and significantly decreased the lesion size at 14 days postinfection (p.i.) compared to results for nontreated, infected mice. Infected, trimannose-treated mice had decreased IL-12p40 and IL-10 secretion and increased interferon gamma secretion at 14 days p.i. Mannose receptor knockout (MR$^{−/−}$) mice lack the ability to detect trimannose. When MR$^{−/−}$ mice were infected with L. major and treated with trimannose beads, they did not have decreased lesion size. Leishmania-derived trimannose represents a novel immunomodulator that provides early type 1-skewed cytokine production to control the parasite load and alter the course of cutaneous leishmaniasis