Human cellular and humoral immune responses to Phlebotomus papatasi salivary gland antigens in endemic areas differing in prevalence of Leishmania major infection.

BACKGROUND: Sand fly saliva compounds are able to elicit specific immune responses that have a significant role in Leishmania parasite establishment and disease outcome. Characterizing anti-saliva immune responses in individuals living in well defined leishmaniasis endemic areas would provide valuable insights regarding their effect on parasite transmission and establishment in humans. METHODOLOGY/PRINCIPAL FINDINGS: We explored the cellular and humoral immune responses to Phlebotomus (P.) papatasi salivary gland extracts (SGE) in individuals living in cutaneous leishmaniasis (CL) old or emerging foci (OF, EF). OF was characterized by a higher infection prevalence as assessed by higher proportions of leishmanin skin test (LST) positive individuals compared to EF. Subjects were further subdivided into healed, asymptomatic or naïve groups. We showed anti-SGE proliferation in less than 30% of the individuals, regardless of the immune status, in both foci. IFN-γ production was higher in OF and only observed in immune individuals from OF and naïve subjects from EF. Although IL-10 was not detected, addition of anti-human IL-10 antibodies revealed an increase in proliferation and IFN-γ production only in individuals from OF. The percentage of seropositive individuals was similar in immune and naïves groups but was significantly higher in OF. No correlation was observed between anti-saliva immune responses and LST response. High anti-SGE-IgG responses were associated with an increased risk of developing ZCL. No differences were observed for anti-SGE humoral or cellular responses among naïve individuals who converted or not their LST response or developed or not ZCL after the transmission season. CONCLUSIONS/SIGNIFICANCE: These data suggest that individuals living in an old focus characterized by a frequent exposure to sand fly bites and a high prevalence of infection, develop higher anti-saliva IgG responses and IFN-γ levels and a skew towards a Th2-type cellular response, probably in favor of parasite establishment, compared to those living in an emerging focus.

Leishmania major large RAB GTPase is highly immunogenic in individuals immune to cutaneous and visceral leishmaniasis.

BACKGROUND: We previously identified a Leishmania (L.) major large RAB GTPase (LmlRAB), a new atypical RAB GTPase protein. It is highly conserved in Leishmania species while displaying low level of homology with mammalian homologues. Leishmania small RAB GTPases proteins have been involved in regulation of exocytic and endocytic pathways whereas the role of large RAB GTPases proteins has not been characterized yet. We report here the immunogenicity of both recombinant rLmlRAB and rLmlRABC, in individuals with immunity against L. major or L. infantum. METHODS: PBMC were isolated from individuals cured of L. major (CCLm) or from healthy individuals. The latter were subdivided into high or low IFN-γ responders. Healthy high IFN-γ responders, considered as asymptomatics, were living in an endemic area for L. major (HHRLm) or L. infantum (HHRLi). Healthy low IFN-γ responders (HLR) were considered as naïve controls. Cells from all volunteers were stimulated with rLmlRAB or rLmlRABC. Cytokines were analysed by CBA and ELISA and phenotypes of IFN-γ-producing cells were analysed by flow cytometry. RESULTS: Both rLmlRAB and rLmlRABC induced high significant levels of IFN-γ in CCLm, HHRLm and HHRLi groups. Phenotype analysis of rLmlRAB and rLmlRABC-stimulated T cells in CCLm individuals showed a significant increase in the percentage of specific IFN-γ-producing CD4+ and CD8+ T cells. rLmlRAB induced significant granzyme B levels in CCLm and HHRLm. Low but significant granzyme B levels were detected in naïve group. IL-10 was detected in immune and naïve individuals. CONCLUSION: We showed that rLmlRAB protein and its divergent carboxy-terminal part induced a predominant Th1 response in individuals immune to L. major or L. infantum. Our results suggest that rLmlRAB and rLmlRABC proteins are potential cross-species vaccine candidates against cutaneous and visceral leishmaniasis.

Characterisation of Early-Life Fecal Microbiota in Susceptible and Healthy Pigs to Post-Weaning Diarrhoea.

Early-life microbial exposure is of particular importance to growth, immune system development and long-lasting health. Hence, early microbiota composition is a promising predictive biomarker for health and disease but still remains poorly characterized in regards to susceptibility to diarrhoea. In the present study, we aimed to assess if gut bacterial community diversity and composition during the suckling period were associated with differences in susceptibility of pigs to post-weaning diarrhoea. Twenty piglets from 5 sows (4 piglets / litter) were weaned in poor housing conditions to challenge their susceptibility to post-weaning diarrhoea. Two weeks after weaning, 13 pigs exhibited liquid faeces during 2 or 3 days and were defined as diarrhoeic (D) pigs. The other 7 pigs did not have diarrhea during the whole post-weaning experimental periodand were defined as healthy (H) pigs. Using a molecular characterisation of fecal microbiota with CE-SSCP fingerprint, Next Generation Sequencing and qPCR, we show that D and H pigs were mainly discriminated as early as postnatal day (PND) 7, i.e. 4 weeks before post-weaning diarrhoea occurence. At PND 7 H pigs displayed a lower evenness and a higher abundance of Prevotellaceae, Lachnospiraceae, Ruminocacaceae and Lactobacillaceae compared to D pigs. The sPLS regression method indicates that these bacterial families were strongly correlated to a higher Bacteroidetes abundance observed in PND 30 H pigs one week before diarrhoea. These results emphasize the potential of early microbiota diversity and composition as being an indicator of susceptibility to post-weaning diarrhoea. Furthermore, they support the health promoting strategies of pig herds through gut microbiota engineering.

Inulin Supplementation Lowered the Metabolic Defects of Prolonged Exposure to Chlorpyrifos from Gestation to Young Adult Stage in Offspring Rats.

Increasing evidence indicates that chlorpyrifos (CPF), an organophosphorus insecticide, is involved in metabolic disorders. We assess the hypothesis whether supplementation with prebiotics from gestation to adulthood, through a modulation of microbiota composition and fermentative activity, alleviates CPF induced metabolic disorders of 60 days old offspring. 5 groups of Wistar rats, from gestation until weaning, received two doses of CPF pesticide: 1 mg/kg/day (CPF1) or 3.5 mg/kg/day (CPF3.5) with free access to inulin (10g/L in drinking water). Then male pups received the same treatment as dams. Metabolic profile, leptin sensitivity, insulin receptor (IR) expression in liver, gut microbiota composition and short chain fatty acid composition (SCFAs) in the colon, were analyzed at postnatal day 60 in the offspring (PND 60). CPF3.5 increased offspring's birth body weight (BW) but decreased BW at PND60. Inulin supplementation restored the BW at PND 60 to control levels. Hyperinsulinemia and decrease in insulin receptor ß in liver were seen in CPF1 exposed rats. In contrast, hyperglycemia and decrease in insulin level were found in CPF3.5 rats. Inulin restored the levels of some metabolic parameters in CPF groups to ranges comparable with the controls. The total bacterial population, short chain fatty acid (SCFA) production and butyrate levels were enhanced in CPF groups receiving inulin. Our data indicate that developmental exposure to CPF interferes with metabolism with dose related effects evident at adulthood. By modulating microbiota population and fermentative activity, inulin corrected adult metabolic disorders of rats exposed to CPF during development. Prebiotics supply may be thus considered as a novel nutritional strategy to counteract insulin resistance and diabetes induced by a continuous pesticide exposure.

Antioxidant properties of formula derived Maillard reaction products in colons of intrauterine growth restricted pigs.

OBJECTIVE: the present study has been conducted to evaluate the impact of the consumption of high MRP formula on changes in the microbiota and the oxidative status, during development, in the colons of intrauterine growth restricted (IUGR) juvenile pigs. METHODS: over a 3-week period, fifteen-day old piglets received formula with two different heat treatments. A formula heated at high temperature (HHF, n = 8) and another one heated at a low temperature (LHF, n = 8). After weaning, animals were fed, ad libitum, a solid diet until postnatal day 54 (PND54). The diversity and composition of the major microbiota were analyzed by CE SSCP and qPCR at postnatal day 36 (PND36) and PND54. Protein oxidation levels, glutathione peroxidase (GPX) activity, catalase (CAT), manganese dependent superoxide dismutase (Mn SOD), NFκB and inducible nitric oxide synthase (iNOS) gene expression were measured in the colon at the juvenile stage (PND54). RESULTS: HHF resulted in a significant decrease in bacterial diversity in the colon at PND36. An increase in the total count of Bifidobacteria, Lactobacillus, Bacteroidetes and Enterobacteria, without major changes in total microbiota was evidenced by qPCR, suggesting qualitative changes in the bacterial population of the HHF group. The imbalance of microbiota observed at PND36 was significantly modified at PND54, when animals received a solid diet. Colon GPX activity (p < 0.05) and gene expression of CAT and iNOS were significantly (p < 0.05) upregulated in the HHF group. No differences in the total protein oxidation and carbonyl score were found in the HHF group. Colon redox enzyme gene expression and pro-inflammatory factor NFκB negatively correlated (p < 0.05) with the bacterial population, suggesting the involvement of certain phyla in controlling the oxidative status of the IUGR piglets, fed on the high AGE formula. CONCLUSION: during development, consuming a high load MRP formula was associated with a major modification in the diversity and composition of the microbiota. The onset of an IUGR adaptive oxidant defense mechanism was found to counteract the oxidative stress induced by the presence of MRPs in formula.

Prediction of T Cell Epitopes from Leishmania major Potentially Excreted/Secreted Proteins Inducing Granzyme B Production.

Leishmania-specific cytotoxic T cell response is part of the acquired immune response developed against the parasite and contributes to resistance to reinfection. Herein, we have used an immune-informatic approach for the identification, among Leishmania major potentially excreted/secreted proteins previously described, those generating peptides that could be targeted by the cytotoxic immune response. Seventy-eight nonameric peptides that are predicted to be loaded by HLA-A*0201 molecule were generated and their binding capacity to HLA-A2 was evaluated. These peptides were grouped into 20 pools and their immunogenicity was evaluated by in vitro stimulation of peripheral blood mononuclear cells from HLA-A2+-immune individuals with a history of zoonotic cutaneous leishmaniasis. Six peptides were identified according to their ability to elicit production of granzyme B. Furthermore, among these peptides 3 showed highest affinity to HLA-A*0201, one derived from an elongation factor 1-alpha and two from an unknown protein. These proteins could constitute potential vaccine candidates against leishmaniasis.

Immunomodulatory Effects of Four Leishmania infantum Potentially Excreted/Secreted Proteins on Human Dendritic Cells Differentiation and Maturation.

Leishmania parasites and some molecules they secrete are known to modulate innate immune responses through effects on dendritic cells (DCs) and macrophages. Here, we characterized four Leishmania infantum potentially excreted/secreted recombinant proteins (LipESP) identified in our laboratory: Elongation Factor 1 alpha (LiEF-1α), a proteasome regulatory ATPase (LiAAA-ATPase) and two novel proteins with unknown functions, which we termed LiP15 and LiP23, by investigating their effect on in vitro differentiation and maturation of human DCs and on cytokine production by DCs and monocytes. During DCs differentiation, LipESP led to a significant decrease in CD1a. LiP23 and LiEF-1α, induced a decrease of HLA-DR and an increase of CD86 surface expression, respectively. During maturation, an up-regulation of HLA-DR and CD80 was found in response to LiP15, LiP23 and LiAAA-ATPase, while an increase of CD40 expression was only observed in response to LiP15. All LipESP induced an over-expression of CD86 with significant differences between proteins. These proteins also induced significant IL-12p70 levels in immature DCs but not in monocytes. The LipESP-induced IL-12p70 production was significantly enhanced by a co-treatment with IFN-γ in both cell populations. TNF-α and IL-10 were induced in DCs and monocytes with higher levels observed for LiP15 and LiAAA-ATPase. However, LPS-induced cytokine production during DC maturation or in monocyte cultures was significantly down regulated by LipESP co-treatment. Our findings suggest that LipESP strongly interfere with DCs differentiation suggesting a possible involvement in mechanisms established by the parasite for its survival. These proteins also induce DCs maturation by up-regulating several costimulatory molecules and by inducing the production of proinflammatory cytokines, which is a prerequisite for T cell activation. However, the reduced ability of LipESP-stimulated DCs and monocytes to respond to lipopolysaccharide (LPS) that can be observed during human leishmaniasis, suggests that under certain circumstances LipESP may play a role in disease progression.

In vitro evaluation of a soluble Leishmania promastigote surface antigen as a potential vaccine candidate against human leishmaniasis.

PSA (Promastigote Surface Antigen) belongs to a family of membrane-bound and secreted proteins present in several Leishmania (L.) species. PSA is recognized by human Th1 cells and provides a high degree of protection in vaccinated mice. We evaluated humoral and cellular immune responses induced by a L. amazonensis PSA protein (LaPSA-38S) produced in a L. tarentolae expression system. This was done in individuals cured of cutaneous leishmaniasis due to L. major (CCLm) or L. braziliensis (CCLb) or visceral leishmaniasis due to L. donovani (CVLd) and in healthy individuals. Healthy individuals were subdivided into immune (HHR-Lm and HHR-Li: Healthy High Responders living in an endemic area for L. major or L. infantum infection) or non immune/naive individuals (HLR: Healthy Low Responders), depending on whether they produce high or low levels of IFN-γ in response to Leishmania soluble antigen. Low levels of total IgG antibodies to LaPSA-38S were detected in sera from the studied groups. Interestingly, LaPSA-38S induced specific and significant levels of IFN-γ, granzyme B and IL-10 in CCLm, HHR-Lm and HHR-Li groups, with HHR-Li group producing TNF-α in more. No significant cytokine response was observed in individuals immune to L. braziliensis or L. donovani infection. Phenotypic analysis showed a significant increase in CD4+ T cells producing IFN-γ after LaPSA-38S stimulation, in CCLm. A high positive correlation was observed between the percentage of IFN-γ-producing CD4+ T cells and the released IFN-γ. We showed that the LaPSA-38S protein was able to induce a mixed Th1 and Th2/Treg cytokine response in individuals with immunity to L. major or L. infantum infection indicating that it may be exploited as a vaccine candidate. We also showed, to our knowledge for the first time, the capacity of Leishmania PSA protein to induce granzyme B production in humans with immunity to L. major and L. infantum infection.

Pathocenosis: a holistic approach to disease ecology.

The history of medicine describes the emergence and recognition of infectious diseases, and human attempts to stem them. It also throws light on the role of changing environmental conditions on disease emergence/re-emergence, establishment and, sometimes, disappearance. However, the dynamics of infectious diseases is also influenced by the relationships between the community of interacting infectious agents present at a given time in a given territory, a concept that Mirko Grmek, an historian of medicine, conceptualized with the word "pathocenosis". The spatial and temporal evolution of diseases, when observed at the appropriate scales, illustrates how a change in the pathocenosis, whether of "natural" or anthropic origin, can lead to the emergence and spread of diseases.

When domestic cat (Felis silvestris catus) population structures interact with their viruses.

Many theoretical studies have proposed different causal mechanisms by which the structure of a host population could have important implications for life history traits of pathogens. However, little information is available from real systems to test these hypotheses. The domestic cat, Felis silvestris catus, whose populations exhibit a great variability in social and spatial structure, represent an ideal case study to assess this question. In the present article, we show how cat population structure may have influenced the evolution of feline viruses and, in return, how these viruses may have modified the genetic structure of cat populations.

Stochastic extinction and the selection of the transmission mode in microparasites.

Stochastic fluctuations in the transmission process of microparasites generate a risk of parasite extinction that cannot be assessed by deterministic models, especially in host populations of small size. While this risk of extinction represents a strong selection pressure for microparasites, it is usually not clearly separated from the deterministic ones. We suggest here that this stochastic selection pressure can affect the selection of the transmission mode of microparasites. To avoid extinction, parasites should maximize their inter-population transmission to ensure frequent reintroductions. Since the types of contacts may differ if congeners belong to the same or distinct populations, strains that are mainly transmitted through inter-population contacts might be selected. To examine this assumption, we analyse the issue of the competition between two strains differing in their transmission mode using a stochastic metapopulation model in which hosts may display different behaviours inside and outside their populations. We show that stochastic selection pressures may drive parasite evolution towards a transmission mode that maximizes the persistence of the parasite. We study the conditions under which stochastic selection pressures may surpass the deterministic ones. Our results are illustrated by the cases of feline immunodeficiency virus in cats and of sexually transmitted diseases in mammals.

The canine distemper epidemic in Serengeti: are lions victims of a new highly virulent canine distemper virus strain, or is pathogen circulation stochasticity to blame?

In the year 1994, the Serengeti lion population was decimated by a canine distemper disease outbreak. Retrospective investigations showed that this host population had already been in contact with the pathogen in 1981 without any detected sign of disease. As an alternative to the virus mutation hypothesis to explain this difference in virulences observed in 1981 and 1994, we propose a novel mechanism of disease emergence based on variation in population immunity. We use a stochastic model to show that stochastic fluctuations in pathogen circulation, owing to a low probability of virus transmission from its reservoir to the target host and thereby resulting in variations in the global immunity level of the target host population, can explain the observations made in Serengeti. This mechanism may also be involved in other infectious disease emergences or re-emergences.

Selection of endogenous reference genes for gene expression analysis in Leishmania major developmental stages.

At the era of post-genomics, gene expression analysis constitutes an important step for understanding the biological functions of genes. For this, reverse transcription and real-time polymerase chain reaction (RT-PCR) is one of the most accurate techniques available to date. Normalization with a proper internal control is critical for the generation of reliable results with biological significance. This is particularly true for pathogens, like Leishmania (L.) parasites, that alternate between different stages during their life cycle. In this study, we evaluate six different sequences for their potential as suitable internal control for the study of gene expression in three different developmental stages (procyclic and metacyclic promastigotes and amastigotes) of the parasite Leishmania major. Experiments were performed on RNA purified from three L. major isolates using the RT-PCR technique. Data analysis was performed using GeNorm and NormFinder programs. We could determine that a sequence encoding rRNA45 is the most stable in the three developmental stages of the parasite and can thus be used as a reference gene in gene expression studies in L. major.

The role of maternal antibodies in the emergence of severe disease as a result of fragmentation.

Population fragmentation is a major problem for the conservation of mammalian species. Since the spread of an infectious disease is related to the intensity of contacts between individuals, fragmentation destabilizes the way the parasites circulate in their host population. Recently, Zinkernagel has proposed that a reduction in the frequency of infections by a parasite could lead to the emergence of severe forms of the disease, previously avoided because the disease was contracted early in life and attenuated by maternal antibodies. However, it is still unclear whether this change in disease expression increases the global mortality it induces because the disease becomes more severe and also less frequent. Here, we use a mathematical model to link population fragmentation with the hypothesis of Zinkernagel. Firstly, we show that there is a change in the severity of the disease during the fragmentation process, especially at a local scale, suggesting that host population fragmentation could be a widespread mechanism of disease emergence. Secondly, we show that the emergence of the severe form of the disease can lead to a significant increase in its induced mortality. Finally, we determine the types of interactions for which the fragmentation of the host population could be the most dangerous.

Modeling transmission of directly transmitted infectious diseases using colored stochastic Petri nets.

In order to improve our understanding of directly transmitted pathogens within host populations, epidemic models should take into account individual heterogeneities as well as stochastic fluctuations in individual parameters. The associated cost results in an increasing level of complexity of the mathematical models which generally lack consistent formalisms. In this paper, we demonstrate that complex epidemic models could be expressed as colored stochastic Petri nets (CSPN). CSPN is a mathematical tool developed in computer science. The concept is based on the Markov Chain theory and on a standard well codified graphical formalism. This approach presents an alternative to other computer simulation methods since it offers both a theoretical formalism and a graphical representation that facilitate the implementation, the understanding and thus the replication or modification of the model. We explain how common concepts of epidemic models--such as the incidence function--can be easily translated into an individual based point of view in the CSPN formalism. We then illustrate this approach by using the well documented susceptible-infected model with recruitment and death.

Behavioral plasticity and virus propagation: the FIV-cat population example.

A new theoretical approach is proposed to investigate the effect of intra-individual variability in behavior on the spread of directly transmitted diseases within host populations. The classical hawk-dove game is used to describe interactions between individuals on a fast time scale (the day). Individuals may exhibit both tactics according to their own experience, to environment conditions, and to the opponent. They are not able to recognize the epidemiological state of their opponents. This fast-time part of the model is coupled to a classical compartmental epidemic model describing the demography of the population and the transmission of the disease from an infected individual to a healthy one on a slow time scale (the year). The model is applied to the case of feline immunodeficiency virus (FIV)-domestic cat population system. Our model gives rise to different predictions according to values of cost and gain due to fights: extinction of the epidemic, FIV endemicity at low, intermediate and high prevalence. These predictions are in good agreement with results from domestic cat populations living in different environmental conditions.