Detection of Leishmania infantum by PCR, serology and cellular immune response in a cohort study of Brazilian dogs

The sensitivity and specificity of PCR, serology (ELISA) and lymphoproliferative response to Leishmania antigen for the detection of Leishmania infantum infection were evaluated in a cohort of 126 dogs exposed to natural infection in Brazil. For PCR, Leishmania DNA from bone-marrow was amplified with both minicircle and ribosomal primers. The infection status and time of infection of each dog were estimated from longitudinal data. The sensitivity of PCR in parasite-positive samples was 98%. However, the overall sensitivity of PCR in post-infection samples, from dogs with confirmed infection, was only 68%. The sensitivity of PCR varied during the course of infection, being highest (78–88%) 0–135 days post-infection and declining to around 50% after 300 days. The sensitivity of PCR also varied between dogs, and was highest in sick dogs. The sensitivity of serology was similar in parasite-positive (84%), PCR-positive (86%) and post-infection (88%) samples. The sensitivity of serology varied during the course of infection, being lowest at the time of infection and high (93–100%) thereafter. Problems in determining the specificity of serology are discussed. The sensitivity and specificity of cellular responsiveness were low. These data suggest that PCR is most useful in detecting active or symptomatic infection, and that serology can be a more sensitive technique for the detection of all infected dogs

Antibody response to sand fly saliva is a marker of transmission intensity but not disease progression in dogs naturally infected with Leishmania infantum

BACKGROUND: Antibody responses to sand fly saliva have been suggested to be a useful marker of exposure to sand fly bites and Leishmania infection and a potential tool to monitor the effectiveness of entomological interventions. Exposure to sand fly bites before infection has also been suggested to modulate the severity of the infection. Here, we test these hypotheses by quantifying the anti-saliva IgG response in a cohort study of dogs exposed to natural infection with Leishmania infantum in Brazil. METHODS: IgG responses to crude salivary antigens of the sand fly Lutzomyia longipalpis were measured by ELISA in longitudinal serum samples from 47 previously unexposed sentinel dogs and 11 initially uninfected resident dogs for up to 2 years. Antibody responses were compared to the intensity of transmission, assessed by variation in the incidence of infection between seasons and between dogs. Antibody responses before patent infection were then compared with the severity of infection, assessed using tissue parasite loads and clinical symptoms. RESULTS: Previously unexposed dogs acquired anti-saliva antibody responses within 2 months, and the rate of acquisition increased with the intensity of seasonal transmission. Over the following 2 years, antibody responses varied with seasonal transmission and sand fly numbers, declining rapidly in periods of low transmission. Antibody responses varied greatly between dogs and correlated with the intensity of transmission experienced by individual dogs, measured by the number of days in the field before patent infection. After infection, anti-saliva antibody responses were positively correlated with anti-parasite antibody responses. However, there was no evidence that the degree of exposure to sand fly bites before infection affected the severity of the infection. CONCLUSIONS: Anti-saliva antibody responses are a marker of current transmission intensity in dogs exposed to natural infection with Leishmania infantum, but are not associated with the outcome of infection

Of cattle, sand flies and men : a systematic review of risk factor analyses for South Asian visceral leishmaniasis and implications for elimination

Background: Studies performed over the past decade have identified fairly consistent epidemiological patterns of risk factors for visceral leishmaniasis (VL) in the Indian subcontinent. Methods and Principal Findings: To inform the current regional VL elimination effort and identify key gaps in knowledge, we performed a systematic review of the literature, with a special emphasis on data regarding the role of cattle because primary risk factor studies have yielded apparently contradictory results. Because humans form the sole infection reservoir, clustering of kala-azar cases is a prominent epidemiological feature, both at the household level and on a larger scale. Subclinical infection also tends to show clustering around kala-azar cases. Within villages, areas become saturated over a period of several years; kala-azar incidence then decreases while neighboring areas see increases. More recently, post kalaazar dermal leishmaniasis (PKDL) cases have followed kala-azar peaks. Mud walls, palpable dampness in houses, and peridomestic vegetation may increase infection risk through enhanced density and prolonged survival of the sand fly vector. Bed net use, sleeping on a cot and indoor residual spraying are generally associated with decreased risk. Poor micronutrient status increases the risk of progression to kala-azar. The presence of cattle is associated with increased risk in some studies and decreased risk in others, reflecting the complexity of the effect of bovines on sand fly abundance, aggregation, feeding behavior and leishmanial infection rates. Poverty is an overarching theme, interacting with individual risk factors on multiple levels. Conclusions: Carefully designed demonstration projects, taking into account the complex web of interconnected risk factors, are needed to provide direct proof of principle for elimination and to identify the most effective maintenance activities to prevent a rapid resurgence when interventions are scaled back. More effective, short-course treatment regimens for PKDL are urgently needed to enable the elimination initiative to succeed

Measurement precision and evaluation of the diameter profiles of single wool fibers

A recent model of the Single Fiber Analyzer 3001 (SIFAN3001) was firstly employed to obtain the single wool fiber diameter profiles (SfFDPs) at multiple orientations. The results showed that using SIFAN3001 to measure fiber diameter at four orientations for 50 single fibers randomly sub-sampled from each mid-side sample can produce average fiber diameter profiles (AS fFDPs) of fibers within staples. Within the testing regime used, the precision estimates for the total samples were &plusmn;1.3 &micro;m for the mean fiber diameter of staples and 1.4 &micro;m for the average fiber diameter of the AS fFDPs at each scanned step in the diameter profile. The mean diameter ratio (ellipticity) obtained from the four orientations was 1.08&plusmn;0.01, confirming that the Merino wool fibers under review were elliptical rather than circular. The elliptical morphology of wool fibers and the precision of the fiber diameter measurement at each point along a fiber will be considered in the development of a mechanical model of Staple Strength testing.<br /

Spatial and Genetic Epidemiology of Hookworm in a Rural Community in Uganda

There are remarkably few contemporary, population-based studies of intestinal nematode infection for sub-Saharan Africa. This paper presents a comprehensive epidemiological analysis of hookworm infection intensity in a rural Ugandan community. Demographic, kinship, socioeconomic and environmental data were collected for 1,803 individuals aged six months to 85 years in 341 households in a cross-sectional community survey. Hookworm infection was assessed by faecal egg count. Spatial variation in the intensity of infection was assessed using a Bayesian negative binomial spatial regression model and the proportion of variation explained by host additive genetics (heritability) and common domestic environment was estimated using genetic variance component analysis. Overall, the prevalence of hookworm was 39.3%, with the majority of infections (87.7%) of light intensity (≤1000 eggs per gram faeces). Intensity was higher among older individuals and was associated with treatment history with anthelmintics, walking barefoot outside the home, living in a household with a mud floor and education level of the household head. Infection intensity also exhibited significant household and spatial clustering: the range of spatial correlation was estimated to be 82 m and was reduced by a half over a distance of 19 m. Heritability of hookworm egg count was 11.2%, whilst the percentage of variance explained by unidentified domestic effects was 17.8%. In conclusion, we suggest that host genetic relatedness is not a major determinant of infection intensity in this community, with exposure-related factors playing a greater role

The Hookworm Tissue Inhibitor of Metalloproteases (Ac-TMP-1) Modifies Dendritic Cell Function and Induces Generation of CD4 and CD8 Suppressor T Cells

Hookworm infection is a major cause of disease burden for humans. Recent studies have described hookworm-related immunosuppression in endemic populations and animal models. A Tissue Inhibitor of Metalloproteases (Ac-TMP-1) has been identified as one of the most abundant proteins released by the adult parasite. We investigated the effect of recombinant Ac-TMP-1 on dendritic cell (DC) and T cell function. Splenic T cells from C57BL/6 mice injected with Ac-TMP-1 showed reduced proliferation to restimulation with anti CD3 or bystander antigens such as OVA. Incubation of bone marrow-derived DCs with Ac-TMP-1 decreased MHC Class I and, especially, Class II expression but increased CD86 and IL-10 expression. Co-incubation of splenic T cells with DCs pulsed with Ac-TMP-1 induced their differentiation into CD4+ and, particularly, CD8+ CD25+Foxp3+ T cells that expressed IL-10. These cells were able to suppress proliferation of naïve and activated CD4+ T cells by TGF-Β-dependent (CD4+ suppressors) or independent (CD8+ suppressors) mechanisms. Priming of DCs with non-hookworm antigens, such as OVA, did not result in the generation of suppressor T cells. These data indicate that Ac-TMP-1 initiates the development of a regulatory response through modifications in DC function and generation of suppressor T cells. This is the first report to propose a role of suppressor CD8+ T cells in gastrointestinal helminthic infections

Immuno-epidemiology of human Schistosoma haematobium infection: preferential IgG3 antibody responsiveness to a recombinant antigen dependent on age and parasite burden

BACKGROUND: Schistosomiasis is a major parasitic disease affecting over 200 million people in the developing world with a further 400 million people at risk of infection. The aim of this study was to identify a single antigen from adult Schistosoma haematobium worms and subsequently use this antigen to study the development of schistosome-acquired immunity in a human population. METHODS: The full-length cDNA sequence of a S. haematobium protein, a putative orthologue of the S. mansoni tegumental antigen Sm13, was obtained from a cDNA library of adult S. haematobium worms and named Sh13 following a small-scale expressed sequence tags (EST) project. The recombinant Sh13 protein expressed in E. coli, was used to investigate immuno-epidemiological patterns in 147 Zimbabweans (7–18 years old) exposed to S. haematobium. RESULTS: Sequence analysis of the full-length cDNA sequence of the S. haematobium protein Sh13, indicated that the protein has an N-terminal signal peptide and encodes an 85-amino acid mature protein with a highly conserved predicted transmembrane domain (86 % identity with the S. mansoni tegumental antigen Sm13). The recombinant Sh13 protein was used in ELISA assays to determine the reactivity of sera from the study participants. Antibody responses against Sh13 were predominantly IgG3 isotype compared to responses against crude worm antigens which were predominantly IgG1 and IgG4. The relationship between anti-Sh13 IgG3 levels and infection intensity varied significantly with host age. The youngest children (7–10 years old) had relatively low levels of both infection and anti-Sh13 IgG3. In older children (11–12 years old) rising infection levels were accompanied by a significant increase in anti-Sh13 IgG3 levels. Subsequently, infection intensity declined significantly in 13–18 year olds but levels of the antibody continued to rise. The changing relationship between infection intensity and anti-Sh13 IgG3 levels with host age is consistent with the profile of a protective immune response predicted from theoretical work. CONCLUSION: We have identified and characterised a novel S. haematobium antigen Sh13, a putative tegumental protein, and shown that it is recognised predominantly by IgG3 antibodies from people infected with/exposed to S. haematobium parasites. We have also shown that, the anti-Sh13 IgG3 response is maximal in older individuals with the lowest infection intensity, and that the age profile of the relationship between anti-Sh13 IgG3 and infection intensity is consistent with that predicted by theoretical work for a protective response stimulated by and directed against adult worms

High-Throughput Analysis of Synthetic Peptides for the Immunodiagnosis of Canine Visceral Leishmaniasis

Globally, the number of new human cases of visceral leishmaniasis (VL) is estimated to be approximately 500,000 per year. This is the most severe of all forms of leishmaniasis, and the zoonotic form of VL, caused by Leishmania infantum (also known as Leishmania chagasi), represents 20% of human visceral leishmaniasis worldwide; additionally, its prevalence is increasing in urban and peri-urban areas of the tropics. In Brazil, the identification and elimination of infected dogs, which act as a reservoir for Leishmania parasites, is a control measure employed in addition to the use of insecticides against the vectors and the identification and treatment of infected humans. Currently, the diagnostic methods employed to identify infected animals are not able to detect all of these dogs, which compromises the effectiveness of control measures. Moreover, one of the most important issues in controlling VL is the difficulty of diagnosing asymptomatic dogs, which act as parasite reservoirs. Therefore, to contribute to the improvement of the diagnostic methods for CVL, we aimed to identify and characterize new antigens that were more sensitive and specific and could be applied in epidemiologic surveys

IDENTIFICATION OF SANDFLIES (Diptera: Psychodidae: Phlebotominae) BLOOD MEALS IN AN ENDEMIC LEISHMANIASIS AREA IN BRAZIL

SUMMARY The aim of this study was to identify blood meals of female sandflies captured in the municipality of Governador Valadares, an endemic area of visceral and cutaneous leishmaniasis, in the State of Minas Gerais, Brazil. From May 2011 to January 2012, captures were performed using HP light traps in four districts. There were 2,614 specimens (2,090 males and 524 females) captured; 97 engorged females were identified belonging to the species Lutzomyia longipalpis (82.1%) and Lutzomyia cortelezzii (17.9%). Considering simple and mixed feeding, the enzyme-linked immunosorbent assay revealed a predominance of chicken blood (43.6%) in Lutzomyia longipalpis, showing the important role that chickens exert around the residential areas of Governador Valadares. This finding increases the chances of sandflies contact with other vertebrates and consequently the risk of leishmaniasis transmission

A randomised controlled trial of the effects of albendazole in pregnancy on maternal responses to mycobacterial antigens and infant responses to bacille Calmette-Guérin (BCG) immunisation [ISRCTN32849447]

BACKGROUND: Maternal schistosomiasis and filariasis have been shown to influence infant responses to neonatal bacille Calmette-Guérin (BCG) immunisation but the effects of maternal hookworm, and of de-worming in pregnancy, are unknown. METHODS: In Entebbe, Uganda, we conducted a randomised, double-blind, placebo-controlled trial of a single dose of 400 mg of albendazole in the second trimester of pregnancy. Neonates received BCG. Interferon-gamma (IFN-γ) and interleukin (IL)-5 responses to a mycobacterial antigen (crude culture filtrate proteins (CFP) of Mycobacterium tuberculosis) were measured in a whole blood assay. We analysed results for binary variables using χ(2 )tests and logistic regression. We analysed continuous variables using Wilcoxon's tests. RESULTS: Maternal hookworm was associated with reduced maternal IFN-γ responses to CFP (adjusted odds ratio for IFN-γ > median response: 0.14 (95% confidence interval 0.02–0.83, p = 0.021). Conversely, maternal hookworm was associated with subsequent increased IFN-γ responses in their one-year-old infants (adjusted OR 17.65 (1.20–258.66; p = 0.013)). Maternal albendazole tended to reduce these effects. CONCLUSION: Untreated hookworm infection in pregnancy was associated with reduced maternal IFN-γ responses to mycobacterial antigens, but increased responses in their infants one year after BCG immunisation. The mechanisms of these effects, and their implications for protective immunity remain, to be determined