Comparison of Mucosal,Subcutaneous and Intraperitoneal Routes of Rat Leptospira Infection

Leptospirosis is a zoonosis found worldwide that is caused by a spirochete. The main reservoirs of Leptospira, which presents an asymptomatic infection, are wild rodents, including the brown rat (Rattus norvegicus). Experimental studies of the mechanisms of its renal colonization in rats have previously used an intraperitoneal inoculation route. However, knowledge of rat-rat transmission requires the use of a natural route of inoculation, such as a mucosal or subcutaneous route. We investigated for the first time the effects of subcutaneous and mucosal inoculation routes compared to the reference intraperitoneal route during Leptospira infection in adult rats. Infection characteristics were studied using Leptospira renal isolation, serology, and molecular and histological analyses. Leptospira infection was asymptomatic using each inoculation route, and caused similar antibody production regardless of renal colonization. The observed renal colonization rates were 8 out of 8 rats, 5 out of 8 rats and 1 out of 8 rats for the intraperitoneal, mucosal and subcutaneous inoculation routes, respectively. Thus, among the natural infection routes studied, mucosal inoculation was more efficient for renal colonization associated with urinary excretion than the subcutaneous route and induced a slower-progressing infection than the intraperitoneal route. These results can facilitate understanding of the infection modalities in rats, unlike the epidemiological studies conducted in wild rats. Future studies of other natural inoculation routes in rat models will increase our knowledge of rat-rat disease transmission and allow the investigation of infection kinetics.     

Kinetics of Leptospira interrogans Infection in Hamsters after Intradermal and Subcutaneous Challenge

Background

Leptospirosis is a zoonosis caused by highly motile, helically shaped bacteria that penetrate the skin and mucous membranes through lesions or abrasions, and rapidly disseminate throughout the body. Although the intraperitoneal route of infection is widely used to experimentally inoculate hamsters, this challenge route does not represent a natural route of infection.

Methodology/Principal Findings

Here we describe the kinetics of disease and infection in hamster model of leptospirosis after subcutaneous and intradermal inoculation of Leptospira interrogans serovar Copenhageni, strain Fiocruz L1-130. Histopathologic changes in and around the kidney, including glomerular and tubular damage and interstitial inflammatory changes, began on day 5, and preceded deterioration in renal function as measured by serum creatinine. Weight loss, hemoconcentration, increased absolute neutrophil counts (ANC) in the blood and hepatic dysfunction were first noted on day 6. Vascular endothelial growth factor, a serum marker of sepsis severity, became elevated during the later stages of infection. The burden of infection, as measured by quantitative PCR, was highest in the kidney and peaked on day 5 after intradermal challenge and on day 6 after subcutaneous challenge. Compared to subcutaneous challenge, intradermal challenge resulted in a lower burden of infection in both the kidney and liver on day 6, lower ANC and less weight loss on day 7.

Conclusions/Significance

The intradermal and subcutaneous challenge routes result in significant differences in the kinetics of dissemination and disease after challenge with L. interrogans serovar Copenhageni strain Fiocruz L1-130 at an experimental dose of 2×10⁶ leptospires. These results provide new information regarding infection kinetics in the hamster model of leptospirosis.     

In vivo gene expression and immunoreactivity of Leptospira collagenase

Pulmonary hemorrhage is an increasing cause of death of leptospirosis patients. Bacterial collagenase has been shown to be involved in lung hemorrhage induced by various infectious agents. According to Leptospira whole genome study, colA, a gene suggested to code for bacterial collagenase has been identified. We investigated colA gene expression in lung tissues of Leptospira infected hamsters. Golden Syrian Hamsters were injected intraperitoneally with Leptospira interrogans serovar Pyrogenes. The hamsters were sacrificed on days 3, 5 and 7 post-infection and lung tissues were collected for histological examination and RNA extraction. Lung pathologies including atelectasis and hemorrhage were observed. Expression of colA gene in lung tissues was demonstrated by both RT-PCR and real time PCR. In addition, ColA protein was cloned and the purified protein could react with sera from leptospirosis patients. Leptospira ColA protein may play a role in Leptospira survival or pathogenesis in vivo. Its reaction with leptospirosis sera suggests that this protein is immunogenic and could be another candidate for vaccine development.     

Leptospira Serovars for Diagnosis of Leptospirosis in Humans and Animals in Africa: Common Leptospira Isolates and Reservoir Hosts

The burden of leptospirosis in humans and animals in Africa is higher than that reported from other parts of the world. However, the disease is not routinely diagnosed in the continent. One of major factors limiting diagnosis is the poor availability of live isolates of locally circulating Leptospira serovars for inclusion in the antigen panel of the gold standard microscopic agglutination test (MAT) for detecting antibodies against leptospirosis. To gain insight in Leptospira serovars and their natural hosts occurring in Tanzania, concomitantly enabling the improvement of the MAT by inclusion of fresh local isolates, a total of 52 Leptospira isolates were obtained from fresh urine and kidney homogenates, collected between 1996 and 2006 from small mammals, cattle and pigs. Isolates were identified by serogrouping, cross agglutination absorption test (CAAT), and molecular typing. Common Leptospira serovars with their respective animal hosts were: Sokoine (cattle and rodents); Kenya (rodents and shrews); Mwogolo (rodents); Lora (rodents); Qunjian (rodent); serogroup Grippotyphosa (cattle); and an unknown serogroup from pigs. Inclusion of local serovars particularly serovar Sokoine in MAT revealed a 10-fold increase in leptospirosis prevalence in Tanzania from 1.9% to 16.9% in rodents and 0.26% to 10.75% in humans. This indicates that local serovars are useful for diagnosis of human and animal leptospirosis in Tanzania and other African countries.     

Leptospira Seroprevalence and Risk Factors in Health Centre Patients in Hoima District,Western Uganda

BackgroundThe burden of human leptospirosis in Uganda is unknown. We estimated the seroprevalence of Leptospira antibodies, probable acute/recent leptospirosis, and risk factors for seropositivity in humans in rural Western Uganda.Conclusions/SignificanceThe 35% prevalence of Leptospira antibodies suggests that exposure to leptospirosis is common in rural Uganda, in particular the Nigeria serovar (Pyrogenes serogroup). Leptospirosis should be a diagnostic consideration in febrile illness and “smear-negative malaria” in rural East Africa.     

Differential Cytokine Gene Expression According to Outcome in a Hamster Model of Leptospirosis

Background

Parameters predicting the evolution of leptospirosis would be useful for clinicians, as well as to better understand severe leptospirosis, but are scarce and rarely validated. Because severe leptospirosis includes septic shock, similarities with predictors evidenced for sepsis and septic shock were studied in a hamster model.

Methodology/Principal Findings

Using an LD50 model of leptospirosis in hamsters, we first determined that 3 days post-infection was a time-point that allowed studying the regulation of immune gene expression and represented the onset of the clinical signs of the disease. In the absence of tools to assess serum concentrations of immune effectors in hamsters, we determined mRNA levels of various immune genes, especially cytokines, together with leptospiraemia at this particular time-point. We found differential expression of both pro- and anti-inflammatory mediators, with significantly higher expression levels of tumor necrosis factor α, interleukin 1α, cyclo-oxygenase 2 and interleukin 10 genes in nonsurvivors compared to survivors. Higher leptospiraemia was also observed in nonsurvivors. Lastly, we demonstrated the relevance of these results by comparing their respective expression levels using a LD100 model or an isogenic high-passage nonvirulent variant.

Conclusions/Significance

Up-regulated gene expression of both pro- and anti-inflammatory immune effectors in hamsters with fatal outcome in an LD50 model of leptospirosis, together with a higher Leptospira burden, suggest that these gene expression levels could be predictors of adverse outcome in leptospirosis.     

Pulmonary haemorrhage as the earliest sign of severe leptospirosis in hamster model challenged with Leptospira interrogans strain HP358

BackgroundSevere leptospirosis is challenging as it could evolve rapidly and potentially fatal if appropriate management is not performed. An understanding of the progression and pathophysiology of Leptospira infection is important to determine the early changes that could be potentially used to predict the severe occurrence of leptospirosis. This study aimed to understand the kinetics pathogenesis of Leptospira interrogans strain HP358 in the hamster model and identify the early parameters that could be used as biomarkers to predict severe leptospirosis.Methodology/Principal findingsMale Syrian hamsters were infected with Leptospira interrogans strain HP358 and euthanized after 24 hours, 3, 4, 5, 6 and 7 days post-infection. Blood, lungs, liver and kidneys were collected for leptospiral detection, haematology, serum biochemistry and differential expression of pro- and anti-inflammatory markers. Macroscopic and microscopic organ damages were investigated. Leptospira interrogans strain HP358 was highly pathogenic and killed hamsters within 6–7 days post-infection. Pulmonary haemorrhage and blood vessel congestion in organs were noticed as the earliest pathological changes. The damages in organs and changes in biochemistry value were preceded by changes in haematology and immune gene expression.Conclusion/SignificanceThis study deciphered haemorrhage as the earliest manifestation of severe leptospirosis and high levels of IL-1β, CXCL10/IP-10, CCL3/MIP-α, neutrophils and low levels of lymphocytes and platelets serve as a cumulative panel of biomarkers in severe leptospirosis.     

Household transmission of leptospira infection in urban slum communities

Background

Leptospirosis, a spirochaetal zoonotic disease, is the cause of epidemics associated with high mortality in urban slum communities. Infection with pathogenic Leptospira occurs during environmental exposures and is traditionally associated with occupational risk activities. However, slum inhabitants reside in close proximity to environmental sources of contamination, suggesting that transmission during urban epidemics occurs in the household environment.

Methods and Findings

A survey was performed to determine whether Leptospira infection clustered within households located in slum communities in the city of Salvador, Brazil. Hospital-based surveillance identified 89 confirmed cases of leptospirosis during an outbreak. Serum samples were obtained from members of 22 households with index cases of leptospirosis and 52 control households located in the same slum communities. The presence of anti-Leptospira agglutinating antibodies was used as a marker for previous infection. In households with index cases, 22 (30%) of 74 members had anti-Leptospira antibodies, whereas 16 (8%) of 195 members from control households had anti-Leptospira antibodies. Highest titres were directed against L. interrogans serovars of the Icterohaemorrhagiae serogroup in 95% and 100% of the subjects with agglutinating antibodies from case and control households, respectively. Residence in a household with an index case of leptospirosis was associated with increased risk (OR 5.29, 95% CI 2.13–13.12) of having had a Leptospira infection. Increased infection risk was found for all age groups who resided in a household with an index case, including children <15 years of age (P = 0.008).

Conclusions

This study identified significant household clustering of Leptospira infection in slum communities where recurrent epidemics of leptospirosis occur. The findings support the hypothesis that the household environment is an important transmission determinant in the urban slum setting. Prevention therefore needs to target sources of contamination and risk activities which occur in the places where slum inhabitants reside.     

Epidemiology of Leptospirosis in Africa: A Systematic Review of a Neglected Zoonosis and a Paradigm for ‘One Health’ in Africa

BackgroundLeptospirosis is an important but neglected bacterial zoonosis that has been largely overlooked in Africa. In this systematic review, we aimed to summarise and compare current knowledge of: (1) the geographic distribution, prevalence, incidence and diversity of acute human leptospirosis in Africa; and (2) the geographic distribution, host range, prevalence and diversity of Leptospira spp. infection in animal hosts in Africa.MethodsFollowing Preferred Reporting Items for Systematic Reviews and Meta-analyses (PRISMA) guidelines, we searched for studies that described (1) acute human leptospirosis and (2) pathogenic Leptospira spp. infection in animals. We performed a literature search using eight international and regional databases for English and non-English articles published between January 1930 to October 2014 that met out pre-defined inclusion criteria and strict case definitions.

Results and Discussion

We identified 97 studies that described acute human leptospirosis (n = 46) or animal Leptospira infection (n = 51) in 26 African countries. The prevalence of acute human leptospirosis ranged from 2 3% to 19 8% (n = 11) in hospital patients with febrile illness. Incidence estimates were largely restricted to the Indian Ocean islands (3 to 101 cases per 100,000 per year (n = 6)). Data from Tanzania indicate that human disease incidence is also high in mainland Africa (75 to 102 cases per 100,000 per year). Three major species (Leptospira borgpetersenii, L. interrogans and L. kirschneri) are predominant in reports from Africa and isolates from a diverse range of serogroups have been reported in human and animal infections. Cattle appear to be important hosts of a large number of Leptospira serogroups in Africa, but few data are available to allow comparison of Leptospira infection in linked human and animal populations. We advocate a ‘One Health’ approach to promote multidisciplinary research efforts to improve understanding of the animal to human transmission of leptospirosis on the African continent.     

Correlation between renal distribution of leptospires during the acute phase and chronic renal dysfunction in a hamster model of infection with Leptospira interrogans

BackgroundLeptospirosis has been described as a biphasic disease consisting of hematogenous dissemination to major organs in the acute phase and asymptomatic renal colonization in the chronic phase. Several observational studies have suggested an association between leptospirosis and chronic kidney disease (CKD). We investigated the dynamics of leptospires and histopathological changes in the kidney to understand the relationship between them, and also investigated the extent of renal dysfunction in the acute and chronic phases of leptospirosis using a hamster model.FindingsHamsters (n = 68) were subcutaneously infected with 1 × 10⁴ cells of the Leptospira interrogans serovar Manilae strain UP-MMC-SM. A total of 53 infected hamsters developed fatal acute leptospirosis, and the remaining 15 hamsters recovered from the acute phase, 13 of which showed Leptospira colonization in the kidneys in the chronic phase. Five asymptomatic hamsters also had renal colonization in the chronic phase. Immunofluorescence staining showed that leptospires were locally distributed in the renal interstitium in the early acute phase and then spread continuously into the surrounding interstitium. The kidneys of the surviving hamsters in the chronic phase showed patchy lesions of atrophic tubules, a finding of chronic tubulointerstitial nephritis, which were substantially consistent with the distribution of leptospires in the renal interstitium. The degree of atrophic tubules in kidney sections correlated statistically with the serum creatinine level in the chronic phase (rs = 0.78, p = 0.01).ConclusionSubcutaneous infection with pathogenic leptospires could cause acute death or chronic leptospirosis in hamsters after surviving the acute phase. We suggest that the renal distribution of leptospires during the acute phase probably affected the extent of tubular atrophy, leading to CKD.     

Leptospirosis as a cause of fever associated with jaundice in the Democratic Republic of the Congo

BackgroundFever with jaundice is a common symptom of some infectious diseases. In public health surveillance within the Democratic Republic of the Congo (DRC), yellow fever is the only recognized cause of fever with jaundice. However, only 5% of the surveillance cases are positive for yellow fever and thus indicate the involvement of other pathogens. Leptospira spp. are the causative agents of leptospirosis, a widespread bacterial zoonosis, a known cause of fever with jaundice. This study aimed to determine the seropositivity of anti-Leptospira antibodies among suspected yellow fever cases and map the geographical distribution of possible leptospirosis in the DRC.MethodsWe conducted a retrospective study using 1,300 samples from yellow fever surveillance in the DRC from January 2017 to December 2018. Serum samples were screened for the presence of IgM against Leptospira spp. by a whole cell-based IgM ELISA (Patoc-IgM ELISA) at the Institut National de Recherche Biomedicale in Kinshasa (INRB) according to World Health Organization (WHO) guidance. Exploratory univariable and multivariable logistic regression analyses were undertaken to assess associations between socio-demographic factors and the presence of Leptospira IgM.ResultsOf the 1,300 serum samples screened, 88 (7%) showed evidence of IgM against Leptospira spp. Most positive cases (34%) were young adult males in the 20–29-year group. There were statistically significant associations between having Leptospira IgM antibodies, age, sex, and living area. Observed positive cases were mostly located in urban settings, and the majority lived in the province of Kinshasa. There was a statistically significant association between seasonality and IgM Leptospira spp. positivity amongst those living in Kinshasa, where most of the positive cases occurred during the rainy season.ConclusionsThis study showed that leptospirosis is likely an overlooked cause of unexplained cases of fever with jaundice in the DRC and highlights the need to consider leptospirosis in the differential diagnosis of fever with jaundice, particularly in young adult males. Further studies are needed to identify animal reservoirs, associated risk factors, and the burden of human leptospirosis in the DRC.     

Leptospira interrogans biofilm formation in Rattus norvegicus (Norway rats) natural reservoirs

Rattus norvegicus (Norway rat) is the main reservoir host of pathogenic Leptospira, the causative agent of leptospirosis, in urban environments. Pathogenic Leptospira forms biofilms in the environment, possibly contributing for bacterial survival and maintenance. Nonetheless, biofilms have not yet been studied in natural animal reservoirs presenting leptospiral renal carriage. Here, we described biofilm formation by pathogenic Leptospira inside the renal tubules of R. norvegicus naturally infected and captured in an urban slum endemic for leptospirosis. From the 65 rats carrying Leptospira in their kidneys, 24 (37%) presented biofilms inside the renal tubules. The intensity of leptospiral colonization in the renal tubules (OR: 1.00; 95% CI 1.05–1.1) and the type of occlusion pattern of the colonized renal tubules (OR: 3.46; 95% CI 1.20–9.98) were independently associated with the presence of Leptospira biofilm. Our data showed that Leptospira interrogans produce biofilms during renal chronic colonization in rat reservoirs, suggesting a possible role for leptospiral biofilms in the pathogenesis of leptospirosis and bacterial carriage in host reservoirs.     

Comparison of the Serion IgM ELISA and Microscopic Agglutination Test for diagnosis of Leptospira spp. infections in sera from different geographical origins and estimation of Leptospira seroprevalence in the Wiwa indigenous population from Colombia

Leptospirosis is among the most important zoonotic diseases in (sub-)tropical countries. The research objective was to evaluate the accuracy of the Serion IgM ELISA EST125M against the Microscopic Agglutination Test (MAT = imperfect reference test); to assess its ability to diagnose acute leptospirosis infections and to detect previous exposure to leptospires in an endemic setting. In addition, to estimate the overall Leptospira spp. seroprevalence in the Wiwa indigenous population in North-East Colombia. We analysed serum samples from confirmed leptospirosis patients from the Netherlands (N = 14), blood donor sera from Switzerland (N = 20), and sera from a cross-sectional study in Colombia (N = 321). All leptospirosis ELISA-positive, and a random of negative samples from Colombia were tested by the MAT for confirmation. The ELISA performed with a sensitivity of 100% (95% CI 77% - 100%) and a specificity of 100% (95% CI 83% - 100%) based on MAT confirmed Leptospira spp. positive and negative samples. In the cross-sectional study in Colombia, the ELISA performed with a sensitivity of 100% (95% CI 2–100%) and a specificity of 21% (95% CI 15–28%). Assuming a 5% Leptospira spp. seroprevalence in this population, the positive predictive value was 6% and the negative predictive value 100%. The Leptospira spp. seroprevalence in the Wiwas tested by the ELISA was 39%; however, by MAT only 0.3%. The ELISA is suitable to diagnose leptospirosis in acutely ill patients in Europe several days after onset of disease. For cross-sectional studies it is not recommended due to its low specificity. Despite the evidence of a high leptospirosis prevalence in other study areas and populations in Colombia, the Wiwa do not seem to be highly exposed to Leptospira spp.. Nevertheless, leptospirosis should be considered and tested in patients presenting with febrile illness.     

Identification of an HLA-A*0201-restricted CD8+ T-cell epitope encoded within Leptospiral immunoglobulin-like protein A

Leptospirosis is an important zoonosis in humans. Immunity against leptospiral infection was thought to be primarily humoral, and limited studies have addressed the role of CD8⁺ T cells. Leptospiral immunoglobulin-like protein A (LigA) is an important protective antigen of Leptospira and a potential target for Leptospira-specific cell-mediated immunity. In this study, twenty LigA-derived peptides were tested their binding affinity and stability for the HLA-A*0201 molecule. Peptides with high binding affinity and stability for HLA-A*0201 were then assessed their capacity to elicit specific cytotoxic T-lymphocyte (CTL) responses using cytotoxicity, ELISPOT assays for IFN-γ and HLA-A*0201-peptide tetramer assays. We identified a HLA-A*0201-restricted epitope, LigA_305–313 KLIVTPAAL in Leptospira LigA. CTLs specific for LigA_305–313 were elicited both in HLA-A2.1/K^b transgenic mice and in patients with a clinical and/or laboratory diagnosis of leptospirosis. Staining of the HLA-A*0201–LigA_305–313 tetramer revealed the presence of LigA_305–313-specific CTLs in peripheral blood mononuclear cells (PBMCs) sourced from five patients infected with three different serovars of Leptospira. In conclusion, we report the existence of specific cytotoxic CD8⁺ T cells in patients with leptospirosis and we suggest that the newly identified epitope, LigA_305–313, will be helpful in enhancing the understanding of the mechanism of immunity to leptospirosis.     

Development and Validation of a Real-Time PCR for Detection of Pathogenic Leptospira Species in Clinical Materials

Available serological diagnostics do not allow the confirmation of clinically suspected leptospirosis at the early acute phase of illness. Several conventional and real-time PCRs for the early diagnosis of leptospirosis have been described but these have been incompletely evaluated. We developed a SYBR Green-based real-time PCR targeting secY and validated it according to international guidelines. To determine the analytical specificity, DNA from 56 Leptospira strains belonging to pathogenic, non-pathogenic and intermediate Leptospira spp. as well as 46 other micro-organisms was included in this study. All the pathogenic Leptospira gave a positive reaction. We found no cross-reaction with saprophytic Leptospira and other micro-organisms, implying a high analytical specificity. The analytical sensitivity of the PCR was one copy per reaction from cultured homologous strain M 20 and 1.2 and 1.5 copy for heterologous strains 1342 K and Sarmin, respectively. In spiked serum & blood and kidney tissue the sensitivity was 10 and 20 copies for M 20, 15 and 30 copies for 1342 K and 30 and 50 copies for Sarmin. To determine the diagnostic sensitivity (DSe) and specificity (DSp), clinical blood samples from 26 laboratory-confirmed and 107 negative patients suspected of leptospirosis were enrolled as a prospective consecutive cohort. Based on culture as the gold standard, we found a DSe and DSp of 100% and 93%, respectively. All eight PCR positive samples that had a negative culture seroconverted later on, implying a higher actual DSp. When using culture and serology as the gold standard, the DSe was lower (89%) while the DSp was higher (100%). DSe was 100% in samples collected within the first – for treatment important - 4 days after onset of the illness. Reproducibility and repeatability of the assay, determined by blind testing kidney samples from 20 confirmed positive and 20 negative rodents both appeared 100%. In conclusion we have described for the first time the development of a robust SYBR Green real-time PCR for the detection of pathogenic Leptospira combined with a detailed assessment of its clinical accuracy, thus providing a method for the early diagnosis of leptospirosis with a well-defined satisfactory performance.     

DNA prime-protein boost based vaccination with a conserved region of leptospiral immunoglobulin-like A and B proteins enhances protection against leptospirosis

Leptospirosis is a zoonotic disease caused by pathogenic spirochetes of theLeptospira genus. Vaccination with bacterins has severe limitations. Here, we evaluated the N-terminal region of the leptospiral immunoglobulin-like B protein (LigBrep) as a vaccine candidate against leptospirosis using immunisation strategies based on DNA prime-protein boost, DNA vaccine, and subunit vaccine. Upon challenge with a virulent strain ofLeptospira interrogans, the prime-boost and DNA vaccine approaches induced significant protection in hamsters, as well as a specific IgG antibody response and sterilising immunity. Although vaccination with recombinant fragment of LigBrep also produced a strong antibody response, it was not immunoprotective. These results highlight the potential of LigBrep as a candidate antigen for an effective vaccine against leptospirosis and emphasise the use of the DNA prime-protein boost as an important strategy for vaccine development.     

Leptospira-rat-human relationship in Luzon,Philippines

Leptospirosis is a zoonotic infection that is caused by the pathogenic species of Leptospira. Rats are the most important reservoirs of these organisms. Our study aimed to characterize Leptospira isolates from humans and rats and elucidate the Leptospira-rat-human relationship in Luzon, Philippines. Forty strains were isolated from humans and rats. The isolates were confirmed to be Leptospira and pathogenic through rrl- and flaB-PCR, respectively. Around 73% of the isolates were found to be lethal to hamsters. Serotyping showed that there were mainly three predominant leptospiral serogroups in the study areas namely Pyrogenes, Bataviae, and Grippotyphosa. Gyrase B gene sequence analysis showed that all the isolates belonged to Leptospira interrogans. Most had 100% similarity with serovar Manilae (15/40), serovar Losbanos (8/40), and serogroup Grippotyphosa (8/40). Strains from each group had highly identical pulsed-field gel electrophoresis patterns and were further grouped as A (Pyrogenes, 14), B (Bataviae, 8), and C (Grippotyphosa, 10). Results further revealed that similar serotypes were isolated from both humans and rats in the same areas. It is suggested that these three predominant groups with highly similar intra-group PFGE patterns may have been primarily transmitted by rats and persistently caused leptospirosis in humans particularly in the Luzon islands.     

Parasite Burden in Hamsters Infected with Two Different Strains of Leishmania (Leishmania) infantum: “Leishman Donovan Units” versus Real-Time PCR

To develop and test new therapeutics and immune prophylaxis strategies for visceral leishmaniasis (VL), understanding tissue parasitism evolution after experimental infection with Leishmania infantum is important. Experimental infection in a hamster model (Mesocricetus auratus) reproduces several typical aspects of canine and human VL that are closely related to the inoculum’s route. We quantified the parasitism in the liver and spleen of hamsters experimentally infected by various routes (intradermal, intraperitoneal, and intracardiac [IC]) and different strains of L. infantum (MHOM/BR/74/PP75 and Wild) and compared two different methodologies to evaluate tissue parasitism (Leishman Donovan units [LDU] and real-time qPCR). In addition, the quantification of specific total-IgG in the serum of uninfected and infected hamsters was determined by ELISA. The animals were followed for 1, 3, 6 and 9 months post-infection for survival analysis. We found that infection with the Wild strain by the IC route resulted in higher mortality. Positive antibody (IgG) responses were detected with higher peaks at 6 and 9 months in the IC group inoculated with PP75 strain. However, in animals infected with the Wild strain the IgG levels were elevated in all infected groups during all the time evaluated. We also observed by LDU analysis that the IC route lead to higher parasitism in the liver and spleen with both strains. Furthermore, qPCR showed higher sensitivity for identifying animals with low parasitic burden. In conclusion, qPCR can be useful for assessing parasitism in the spleen and liver of a hamster model infected with L. infantum independent of the route of infection, and this technique may become an essential tool for assessing parasite density in the hamster model after experimental treatment or immunization with potential vaccine candidates.