The epidemiology of canine leishmaniasis: transmission rates estimated from a cohort study in Amazonian Brazil

We estimate the incidence rate, serological conversion rate and basic case reproduction number (R0) of Leishmania infantum from a cohort study of 126 domestic dogs exposed to natural infection rates over 2 years on Marajó Island, Pará State, Brazil. The analysis includes new methods for (1) determining the number of seropositives in cross-sectional serological data, (2) identifying seroconversions in longitudinal studies, based on both the number of antibody units and their rate of change through time, (3) estimating incidence and serological pre-patent periods and (4) calculating R0 for a potentially fatal, vector-borne disease under seasonal transmission. Longitudinal and cross-sectional serological (ELISA) analyses gave similar estimates of the proportion of dogs positive. However, longitudinal analysis allowed the calculation of pre-patent periods, and hence the more accurate estimation of incidence: an infection–conversion model fitted by maximum likelihood to serological data yielded seasonally varying per capita incidence rates with a mean of 8·66×10[minus sign]3/day (mean time to infection 115 days, 95% C.L. 107–126 days), and a median pre-patent period of 94 (95% C.L. 82–111) days. These results were used in conjunction with theory and dog demographic data to estimate the basic reproduction number, R0, as 5·9 (95% C.L. 4·4–7·4). R0 is a determinant of the scale of the leishmaniasis control problem, and we comment on the options for control

The epidemiology and control of canine visceral leishmaniasis in Amazon Brazil.

This thesis describes a study of the dog and fox reservoir populations of Leishmamainfantum in an endemic focus in Amazon Brazil Following a brief review of the relevant literature on canine visceral leishmaniasis in Chapter 1, the aims of the study are described Broadly they were to (I) quantify the courses (and their inter-relationships) of infection, disease and infectiousness in dogs, (2) investigate the implications for culling strategies, (3) assess the relative role of dogs and foxes in transmission. Chapter 2 describes the overall study design. A cohort of 126 previously unexposed native dogs was established in 24 endemic study communities and monitored at bimonthly intervals over a period of 24 months. A total of 756 sera were tested by ELISA, and 514 bone marrow samples were examined either as smears, or following inoculation into hamsters or culture medium. Clinical examinations were performed on 116 dogs on 562 independent occasions, and 50 dogs were experimentally exposed to colony bred Lu. longipalpis (the vector) in 173 xenodiagnostic trials. Longitudinal demographic and serological data were also collected on the resident dogs of 15 communities and on a sympatric free-ranging population of crab-eating foxes, Cerdocyon thous. These data were added to those for the same populations collected during a previous study (by the author) giving 5 years of data, all of which is described in this thesis. Using comparable methods as for dogs, 37 foxes were clinically, serologically and parasitologically sampled on 74 occasions, 26 of which were also examined by xenodiagnosis in 44 trials. The principal results of this thesis are presented in Chapters 3- 6. Chapter 3 describes the demographic parameters relevant for transmission dynamics. These showed that the resident dog population had a high turnover rate (0 42 per year) characterised by a mortality rate of 0.40 per year. Dog abundance was sustained by immigration (owner-mediated) rather than by birth. The mortality rate was positively associated with seroprevalence, incidence, and sandfly abundance in houses and animal sheds. The seroconversion rate between villages was positively associated with sandfly abundance in houses and animal sheds, and negatively associated with the mean number of dogs per household The fox population replacement and mortality rates were similar to dogs, but with no evidence of mortality due to Leishmania. Chapter 4 describes the courses of infection and disease in the sentinel population. By the end of the study 80 dogs were identified as infected, representing 93% of the 86 dogs which remained in the study for > 3 sample rounds The seroconversion and serological recovery rates were 0 269 and 0 006 per month, respectively, the average time to infection was 115 days, and time from infection to seroconversion was 94 days All dogs developed one or more clinical signs of CVL by the end of 24 months, with an average time from infection to clinical onset of 66 days. Only 9% of dogs classified as symptomatic by their longitudinal clinical profile fully recovered from the disease. Clinical severity outcome was positively associated with antibody titre, parasite isolation success, and mortality. Forty-nine percent of dogs with confirmed Leishmania infections died by the end of the study. The risk of mortality was positively associated with the severity of infection and disease. In Chapter 5, the results from serial xenodiagnosis of the sentinel population revealed that the onset of infectiousness occurred a median 128 days after seroconversion with a latent period (time from infection to infectiousness) of 222 days. The heterogeneity of infectiousness was extensive both between and within dogs: 45% of seropositive dogs were observed to become infectious; 20% of the infectious dogs were responsible for 80% of all sandfly infections. Infectiousness was positively associated with high antibody titres, and severe clinical signs. However, neither antibody titre, clinical signs, biochemical parameters, nor any combinations of these, proved to be reliable surrogate markers of infectiousness. Chapter 6 provides comparable longitudinal serological and parasitological data for the fox population. Fox infection rates were similar to those for dogs, though there was an absence of clinical signs. No foxes were observed to be infectious to Lu. longipalpis by xenodiagnosis. Chapter 7 concludes that (1) the dynamics of canine L. infantum infection and disease in Marajo is similar to that in Europe, (2) the detection of potential clinically severe cases may be possible in early infection (e g for treatment by the veterinarians), however (3) selection of infectious dogs for targeted control (treatment, elimination or other) is not possible using the immunological and clinical parameters as surrogate markers, (4) foxes are not important for human transmission in the presence of infected (infectious) dogs

Infectiousness in a Cohort of Brazilian Dogs: Why Culling Fails to Control Visceral Leishmaniasis in Areas of High Transmission

The elimination of seropositive dogs in Brazil has been used to control zoonotic visceral leishmaniasis but with little success. To elucidate the reasons for this, the infectiousness of 50 sentinel dogs exposed to natural Leishmania chagasi infection was assessed through time by xenodiagnosis with the sandfly vector, Lutzomyia longipalpis. Eighteen (43%) of 42 infected dogs became infectious after a median of 333 days in the field (105 days after seroconversion). Seven highly infectious dogs (17%) accounted for >80% of sandfly infections. There were positive correlations between infectiousness and anti-Leishmania immunoglobulin G, parasite detection by polymerase chain reaction, and clinical disease (logistic regression, r2 = 0.080.18). The sensitivity of enzyme-linked immunosorbent assay to detect currently infectious dogs was high (96%) but lower in the latent period (<63%), and specificity was low (24%). Mathematical modeling suggests that culling programs fail because of high incidence of infection and infectiousness, the insensitivity of the diagnostic test to detect infectious dogs, and time delays between diagnosis and culling

Tissue Cytokine Responses in Canine Visceral Leishmaniasis

To elucidate the local tissue cytokine response of dogs infected with Leishmania chagasi, cytokine mRNA levels were measured in bone marrow aspirates from 27 naturally infected dogs from Brazil and were compared with those from 5 uninfected control animals. Interferon-γ mRNA accumulation was enhanced in infected dogs and was positively correlated with humoral (IgG1) but not with lymphoproliferative responses to Leishmania antigen in infected dogs. Increased accumulation of mRNA for interleukin (IL)4, IL-10, and IL-18 was not observed in infected dogs, and mRNA for these cytokines did not correlate with antibody or proliferative responses. However, infected dogs with detectable IL-4 mRNA had significantly more severe symptoms. IL-13 mRNA was not detectable in either control or infected dogs. These data suggest that clinical symptoms are not due to a deficiency in interferon-γ production. However, in contrast to its role in human visceral leishmaniasis, IL-10 may not play a key immunosuppressive role in dogs

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

Variations in visceral leishmaniasis burden, mortality and the pathway to care within Bihar, India

BACKGROUND: Visceral leishmaniasis (VL) has been targeted by the WHO for elimination as a public health problem (< 1 case/10,000 people/year) in the Indian sub-continent (ISC) by 2020. Bihar State in India, which accounts for the majority of cases in the ISC, remains a major target for this elimination effort. However, there is considerable spatial, temporal and sub-population variation in occurrence of the disease and the pathway to care, which is largely unexplored and a threat to achieving the target. METHODS: Data from 6081 suspected VL patients who reported being clinically diagnosed during 2012-2013 across eight districts in Bihar were analysed. Graphical comparisons and Chi-square tests were used to determine differences in the burden of identified cases by season, district, age and sex. Log-linear regression models were fitted to onset (of symptoms)-to-diagnosis and onset-to-treatment waiting times to estimate their associations with age, sex, district and various socio-economic factors (SEFs). Logistic regression models were used to identify factors associated with mortality. RESULTS: Comparisons of VL caseloads suggested an annual cycle peaking in January-March. A 17-fold variation in the burden of identified cases across districts and under-representation of young children (0-5 years) relative to age-specific populations in Bihar were observed. Women accounted for a significantly lower proportion of the reported cases than men (41 vs 59%, P < 0.0001). Age, district of residence, house wall materials, caste, treatment cost, travelling for diagnosis and the number of treatments for symptoms before diagnosis were identified as correlates of waiting times. Mortality was associated with age, district of residence, onset-to-treatment waiting time, treatment duration, cattle ownership and cost of diagnosis. CONCLUSIONS: The distribution of VL in Bihar is highly heterogeneous, and reported caseloads and associated mortality vary significantly across different districts, posing different challenges to the elimination campaign. Socio-economic factors are important correlates of these differences, suggesting that elimination will require tailoring to population and sub-population circumstances

Heterogeneities in leishmania infantum infection : using skin parasite burdens to identify highly infectious dogs

Background: The relationships between heterogeneities in host infection and infectiousness (transmission to arthropod vectors) can provide important insights for disease management. Here, we quantify heterogeneities in Leishmania infantum parasite numbers in reservoir and non-reservoir host populations, and relate this to their infectiousness during natural infection. Tissue parasite number was evaluated as a potential surrogate marker of host transmission potential. Methods: Parasite numbers were measured by qPCR in bone marrow and ear skin biopsies of 82 dogs and 34 crab-eating foxes collected during a longitudinal study in Amazon Brazil, for which previous data was available on infectiousness (by xenodiagnosis) and severity of infection. Results: Parasite numbers were highly aggregated both between samples and between individuals. In dogs, total parasite abundance and relative numbers in ear skin compared to bone marrow increased with the duration and severity of infection. Infectiousness to the sandfly vector was associated with high parasite numbers; parasite number in skin was the best predictor of being infectious. Crab-eating foxes, which typically present asymptomatic infection and are non-infectious, had parasite numbers comparable to those of non-infectious dogs. Conclusions: Skin parasite number provides an indirect marker of infectiousness, and could allow targeted control particularly of highly infectious dogs

Pathogen quantitation in complex matrices: a multi-operator comparison of DNA extraction methods with a novel assessment of PCR inhibition

This is the final version of the article. Available from the publisher via the DOI in this record.BACKGROUND: Mycobacterium bovis is the aetiological agent of bovine tuberculosis (bTB), an important recrudescent zoonosis, significantly increasing in British herds in recent years. Wildlife reservoirs have been identified for this disease but the mode of transmission to cattle remains unclear. There is evidence that viable M. bovis cells can survive in soil and faeces for over a year. METHODOLOGY/PRINCIPAL FINDINGS: We report a multi-operator blinded trial for a rigorous comparison of five DNA extraction methods from a variety of soil and faecal samples to assess recovery of M. bovis via real-time PCR detection. The methods included four commercial kits: the QIAamp Stool Mini kit with a pre-treatment step, the FastDNA® Spin kit, the UltraClean™ and PowerSoil™ soil kits and a published manual method based on phenol:chloroform purification, termed Griffiths. M. bovis BCG Pasteur spiked samples were extracted by four operators and evaluated using a specific real-time PCR assay. A novel inhibition control assay was used alongside spectrophotometric ratios to monitor the level of inhibitory compounds affecting PCR, DNA yield, and purity. There were statistically significant differences in M. bovis detection between methods of extraction and types of environmental samples; no significant differences were observed between operators. Processing times and costs were also evaluated. To improve M. bovis detection further, the two best performing methods, FastDNA® Spin kit and Griffiths, were optimised and the ABI TaqMan environmental PCR Master mix was adopted, leading to improved sensitivities. CONCLUSIONS: M. bovis was successfully detected in all environmental samples; DNA extraction using FastDNA® Spin kit was the most sensitive method with highest recoveries from all soil types tested. For troublesome faecal samples, we have used and recommend an improved assay based on a reduced volume, resulting in detection limits of 4.25×10(5) cells g(-1) using Griffiths and 4.25×10(6) cells g(-1) using FastDNA® Spin kit.This work was supported by Biotechnology and Biological Sciences Research Council BBSRCBB/E020925/1 grant to OC and EMHW, and Department for Environment, Food and Rural Affairs, DEFRA SE3231 grant to EMHW and OC. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

The variability and seasonality of the environmental reservoir of Mycobacterium bovis shed by wild European badgers

This is the final version of the article. Available from the publisher via the DOI in this record.The incidence of Mycobacterium bovis, the causative agent of bovine tuberculosis, has been increasing in UK cattle herds resulting in substantial economic losses. The European badger (Meles meles) is implicated as a wildlife reservoir of infection. One likely route of transmission to cattle is through exposure to infected badger urine and faeces. The relative importance of the environment in transmission remains unknown, in part due to the lack of information on the distribution and magnitude of environmental reservoirs. Here we identify potential infection hotspots in the badger population and quantify the heterogeneity in bacterial load; with infected badgers shedding between 1 × 10(3)- 4 × 10(5) M. bovis cells g(-1) of faeces, creating a substantial and seasonally variable environmental reservoir. Our findings highlight the potential importance of monitoring environmental reservoirs of M. bovis which may constitute a component of disease spread that is currently overlooked and yet may be responsible for a proportion of transmission amongst badgers and onwards to cattle.We acknowledge funding from Defra, H.C.K. was in receipt of a BBSRC DTG studentship and E.M.W. and O.C. acknowledge support from BBSRC for collaboration with Eamonn Gormley, UCD. We are also grateful to the APHA field team at Woodchester Park for support during fieldwork, and to Defra who fund the long-term stud

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