Reproductive ecology of the black rat (Rattus rattus) in Madagascar: the influence of density-dependent and -independent effects.

The black rat (Rattus rattus) poses a severe threat to food security and public health in Madagascar, where it is a major cause of pre- and post-harvest crop losses and an important reservoir for many zoonotic diseases, including plague. Elsewhere, ecologically based rodent management (EBRM) strategies have been developed using ecological information to inform decisions on where and when to target control. EBRM could deliver improved health and well-being outcomes in Madagascar if adapted to the local ecological context. Using data collected from removal studies, we explored spatio-temporal patterns in the breeding activity of the black rat (R. rattus) in domestic and agricultural habitats across Madagascar and investigated to what extent these trends are influenced by rainfall and rat density. We identified clear spatio-temporal variation in the seasonality of R. rattus reproduction. Reproduction was highly seasonal both inside and outside of houses, but seasonal trends varied between these two habitats. Seasonal trends were explained, in part, by variation in rainfall; however, the effect of rainfall on reproductive rates did itself vary by season and habitat type. A decline in breeding intensity with increasing rat density was recorded outside of houses. This has important implications for control, as populations may compensate for removal through increased reproduction. We recommend that sustained control initiated before the main breeding season, combined with improved hygiene and adequate rodent-proofing in homes and grain stores, could curtail population growth and reduce pre- and post-harvest losses provided that these measures overcome the compensatory response of rodent populations.

Patterns and drivers of vector-borne microparasites in a classic metapopulation.

Many organisms live in fragmented populations, which has profound consequences on the dynamics of associated parasites. Metapopulation theory offers a canonical framework for predicting the effects of fragmentation on spatiotemporal host­parasite dynamics. However, empirical studies of parasites in classical metapopulations remain rare, particularly for vector-borne parasites. Here, we quantify spatiotemporal patterns and possible drivers of infection probability for several ectoparasites (fleas, Ixodes trianguliceps and Ixodes ricinus) and vector-borne microparasites (Babesia microti, Bartonella spp., Hepatozoon spp.) in a classically functioning metapopulation of water vole hosts. Results suggest that the relative importance of vector or host dynamics on microparasite infection probabilities is related to parasite life-histories. Bartonella, a microparasite with a fast life-history, was positively associated with both host and vector abundances at several spatial and temporal scales. In contrast, B. microti, a tick-borne parasite with a slow life-history, was only associated with vector dynamics. Further, we provide evidence that life-history shaped parasite dynamics, including occupancy and colonization rates, in the metapopulation. Lastly, our findings were consistent with the hypothesis that landscape connectivity was determined by distance-based dispersal of the focal hosts. We provide essential empirical evidence that contributes to the development of a comprehensive theory of metapopulation processes of vector-borne parasites.

Rodent control to fight plague: field assessment of methods based on rat density reduction.

Rodents represent a serious threat to food security and public health. The extent to which rodent control can mitigate the risk from rodent-borne disease depends on both the effectiveness of control in reducing rodent abundance and the impact on disease epidemiology. Focusing on a plague-endemic region of Madagascar, this study compared the effectiveness of 3 methods: live-traps, snap-traps, and rodenticides. Control interventions were implemented inside houses between May and October 2019. Tracking tiles monitored rodent abundance. Rodent fleas, the vector involved in plague transmission, were collected. Rodent populations consisted of Rattus rattus and Mus musculus. In terms of trap success, we found that our live-trap regime was more effective than snap-traps. While all 3 control strategies appeared to reduce in-house rodent activity in the short term, we found no evidence of a longer-term effect, with in-house rodent abundance in treated sites comparable to non-treatment sites by the following month. Endemic flea, Synopsyllus fonquerniei, is a key plague vector usually found on rats living outdoors. Although we found no evidence that its abundance inside houses increased following control, this may have been due to a lack of power caused by significant variation in S. fonquerniei abundance. The presence of S. fonquerniei in houses was more likely when S. fonquerniei abundance on outdoor rats was higher, which in turn correlated with high rat abundance. Our results emphasize that control strategies need to consider this connectivity between in-house rat-flea populations and the outdoor populations, and any potential consequences for plague transmission.

Field assessment of dog as sentinel animal for plague in endemic foci of Madagascar.

The epidemiology of Yersinia pestis, the causative agent of plague, involves vectors and reservoirs in its transmission cycle. The passive plague surveillance in Madagascar targets mainly rodent and fleas. However, carnivores are routinely surveyed as sentinels of local plague activity in some countries. The aim of this study is to assess the use of domestic dog (Canis familiaris) as sentinel animal for field surveillance of plague in a highly endemic area in Madagascar. Cross-sectional surveys of plague antibody prevalence in C. familiaris were conducted in endemic areas with contrasting histories of plague cases in humans, as well as a plague free area. Rodent capture was done in parallel to evaluate evidence for Y. pestis circulation in the primary reservoirs. In 2 sites, dogs were later re-sampled to examine evidence of seroconversion and antibody persistence. Biological samplings were performed between March 2008 and February 2009. Plague antibody detection was assessed using anti-F1 ELISA. Our study showed a significant difference in dog prevalence rates between plague-endemic and plague-free areas, with no seropositive dogs detected in the plague free area. No correlation was found between rodents and dog prevalence rates, with an absence of seropositive rodents in some area where plague circulation was indicated by seropositive dogs. This is consistent with high mortality rates in rodents following infection. Re-sampling dogs identified individuals seropositive on both occasions, indicating high rates of re-exposure and/or persistence of plague antibodies for at least 9 months. Seroconversion or seropositive juvenile dogs indicated recent local plague circulation. In Madagascar, dog surveillance for plague antibody could be useful to identify plague circulation in new areas or quiescent areas within endemic zones. Within active endemic areas, monitoring of dog populations for seroconversion (negative to positive) or seropositive juvenile dogs could be useful for identifying areas at greatest risk of human outbreaks.

Multi-locus sequence analyses reveal a clonal L. borgpetersenii genotype in a heterogeneous invasive Rattus spp. community across the City of Johannesburg, South Africa.

BACKGROUND: Rattus spp. are frequently implicated as key reservoir hosts for leptospirosis, one of the most common, but neglected, bacterial zoonoses in the world. Although leptospirosis is predicted to be a significant public health threat in Africa, studies from the continent are limited. METHODS: Rattus spp. (n = 171) were sampled (January-May 2016) across the City of Johannesburg, South Africa's largest inland metropole. Rattus spp. genetic diversity was evaluated by full length (1140 bp) cyt b sequencing of 42 samples. For comparison, a further 12 Rattus norvegicus samples collected in Cape Town, South Africa's largest coastal metropole, were also genotyped. Leptospira infections were identified and genotyped using real-time PCR and multi-locus (lfb1, secY and lipL41) DNA sequencing. RESULTS: Five R. norvegicus haplotypes were identified across Johannesburg, four of which have not previously been detected in South Africa, and one in Cape Town. Across Johannesburg we identified a Leptospira spp. infection prevalence of 44% (75/171) and noted significant differences in the prevalence between administrative regions within the metropole. Multi-locus sequence analyses identified a clonal genotype consistent with L. borgpetersenii serogroup Javanica (serovar Ceylonica). DISCUSSION: The prevalence of infection identified in this study is amongst the highest detected in Rattus spp. in similar contexts across Africa. Despite the complex invasion history suggested by the heterogeneity in R. norvegicus haplotypes identified in Johannesburg, a single L. borgpetersenii genotype was identified in all infected rodents. The lack of L. interrogans in a rodent community dominated by R. norvegicus is notable, given the widely recognised host-pathogen association between these species and evidence for L. interrogans infection in R. norvegicus in Cape Town. It is likely that environmental conditions (cold, dry winters) in Johannesburg may limit the transmission of L. interrogans. Spatial heterogeneity in prevalence suggest that local factors, such as land use, influence disease risk in the metropole. CONCLUSIONS: In South Africa, as in other African countries, leptospirosis is likely underdiagnosed. The high prevalence of infection in urban rodents in Johannesburg suggest that further work is urgently needed to understand the potential public health risk posed by this neglected zoonotic pathogen.

Human plague: An old scourge that needs new answers.

Yersinia pestis, the bacterial causative agent of plague, remains an important threat to human health. Plague is a rodent-borne disease that has historically shown an outstanding ability to colonize and persist across different species, habitats, and environments while provoking sporadic cases, outbreaks, and deadly global epidemics among humans. Between September and November 2017, an outbreak of urban pneumonic plague was declared in Madagascar, which refocused the attention of the scientific community on this ancient human scourge. Given recent trends and plague's resilience to control in the wild, its high fatality rate in humans without early treatment, and its capacity to disrupt social and healthcare systems, human plague should be considered as a neglected threat. A workshop was held in Paris in July 2018 to review current knowledge about plague and to identify the scientific research priorities to eradicate plague as a human threat. It was concluded that an urgent commitment is needed to develop and fund a strong research agenda aiming to fill the current knowledge gaps structured around 4 main axes: (i) an improved understanding of the ecological interactions among the reservoir, vector, pathogen, and environment; (ii) human and societal responses; (iii) improved diagnostic tools and case management; and (iv) vaccine development. These axes should be cross-cutting, translational, and focused on delivering context-specific strategies. Results of this research should feed a global control and prevention strategy within a "One Health" approach.

Human Exposure to Hantaviruses Associated with Rodents of the Murinae Subfamily, Madagascar.

We conducted a national human serologic study of a hantavirus detected in Madagascar rodents using a commercial kit and a new ELISA targeting the virus. Our results suggest a conservative estimate of 2.7% (46/1,680) IgG seroprevalence. A second single-district study using the new ELISA revealed a higher prevalence (7.2%; 10/139).

Leptospira in livestock in Madagascar: uncultured strains, mixed infections and small mammal-livestock transmission highlight challenges in controlling and diagnosing leptospirosis in the developing world.

In developing countries, estimates of the prevalence and diversity of Leptospira infections in livestock, an important but neglected zoonotic pathogen and cause of livestock productivity loss, are lacking. In Madagascar, abattoir sampling of cattle and pigs demonstrated a prevalence of infection of 20% in cattle and 5% in pigs by real-time PCR. In cattle, amplification and sequencing of the Leptospira-specific lfb1 gene revealed novel genotypes, mixed infections of two or more Leptospira species and evidence for potential transmission between small mammals and cattle. Sequencing of the secY gene demonstrated genetic similarities between Leptospira detected in Madagascar and, as yet, uncultured Leptospira strains identified in Tanzania, Reunion and Brazil. Detection of Leptospira DNA in the same animal was more likely in urine samples or pooled samples from four kidney lobes relative to samples collected from a single kidney lobe, suggesting an effect of sampling method on detection. In pigs, no molecular typing of positive samples was possible. Further research into the epidemiology of livestock leptospirosis in developing countries is needed to inform efforts to reduce human infections and to improve livestock productivity.

Fast, Sensitive and Specific Detection of Thailand orthohantavirus and its Variants Using One-Step Real-Time Reverse-Transcription Polymerase Chain Reaction Assay.

Genetic variants of Thailand orthohantavirus (THAIV) have been recently reported from rodents in South-East Asia and in islands from the South-West part of the Indian Ocean. In order to detect THAIV and its variants, we developed a sensitive and specific real-time RT-PCR targeting the S segment. Our assay was developed in two different RT-PCR systems that gave similar results in terms of sensitivity. Moreover, our results demonstrated a specificity of 100%.

Geographical distribution and relative risk of Anjozorobe virus (Thailand orthohantavirus) infection in black rats (Rattus rattus) in Madagascar.

BACKGROUND: Hantavirus infection is a zoonotic disease that is associated with hemorrhagic fever with renal syndrome and cardiopulmonary syndrome in human. Anjozorobe virus, a representative virus of Thailand orthohantavirus (THAIV), was recently discovered from rodents in Anjozorobe-Angavo forest in Madagascar. To assess the circulation of hantavirus at the national level, we carried out a survey of small terrestrial mammals from representative regions of the island and identified environmental factors associated with hantavirus infection. As we were ultimately interested in the potential for human exposure, we focused our research in the peridomestic area. METHODS: Sampling was achieved in twenty districts of Madagascar, with a rural and urban zone in each district. Animals were trapped from a range of habitats and examined for hantavirus RNA by nested RT-PCR. We also investigated the relationship between hantavirus infection probability in rats and possible risk factors by using Generalized Linear Mixed Models. RESULTS: Overall, 1242 specimens from seven species were collected (Rattus rattus, Rattus norvegicus, Mus musculus, Suncus murinus, Setifer setosus, Tenrec ecaudatus, Hemicentetes semispinosus). Overall, 12.4% (111/897) of Rattus rattus and 1.6% (2/125) of Mus musculus were tested positive for THAIV. Rats captured within houses were less likely to be infected than rats captured in other habitats, whilst rats from sites characterized by high precipitation and relatively low seasonality were more likely to be infected than those from other areas. Older animals were more likely to be infected, with infection probability showing a strong increase with weight. CONCLUSIONS: We report widespread distribution of THAIV in the peridomestic rats of Madagascar, with highest prevalence for those living in humid areas. Although the potential risk of infection to human may also be widespread, our results provide a first indication of specific zone with high transmission. Gathered data will be helpful to implement policies for control and prevention of human risk infection.

Mixed Leptospira Infections in a Diverse Reservoir Host Community, Madagascar, 2013-2015.

We identified mixed infections of pathogenic Leptospira in small mammals across a landscape-scale study area in Madagascar by using primers targeting different Leptospira spp. Using targeted primers increased prevalence estimates and evidence for transmission between endemic and invasive hosts. Future studies should assess rodentborne transmission of Leptospira to humans.

The Asian house shrew Suncus murinus as a reservoir and source of human outbreaks of plague in Madagascar.

Identifying key reservoirs for zoonoses is crucial for understanding variation in incidence. Plague re-emerged in Mahajanga, Madagascar in the 1990s but there has been no confirmed case since 1999. Here we combine ecological and genetic data, from during and after the epidemics, with experimental infections to examine the role of the shrew Suncus murinus in the plague epidemiological cycle. The predominance of S. murinus captures during the epidemics, their carriage of the flea vector and their infection with Yersinia pestis suggest they played an important role in the maintenance and transmission of plague. S. murinus exhibit a high but variable resistance to experimental Y. pestis infections, providing evidence of its ability to act as a maintenance host. Genetic analyses of the strains isolated from various hosts were consistent with two partially-linked transmission cycles, with plague persisting within the S. murinus population, occasionally spilling over into the rat and human populations. The recent isolation from a rat in Mahajanga of a Y. pestis strain genetically close to shrew strains obtained during the epidemics reinforces this hypothesis and suggests circulation of plague continues. The observed decline in S. murinus and Xenopsylla cheopis since the epidemics appears to have decreased the frequency of spillover events to the more susceptible rats, which act as a source of infection for humans. Although this may explain the lack of confirmed human cases in recent years, the current circulation of plague within the city highlights the continuing health threat.

Temporal phylogeography of Yersinia pestis in Madagascar: Insights into the long-term maintenance of plague.

BACKGROUND: Yersinia pestis appears to be maintained in multiple, geographically separate, and phylogenetically distinct subpopulations within the highlands of Madagascar. However, the dynamics of these locally differentiated subpopulations through time are mostly unknown. To address that gap and further inform our understanding of plague epidemiology, we investigated the phylogeography of Y. pestis in Madagascar over an 18 year period. METHODOLOGY/PRINCIPAL FINDINGS: We generated whole genome sequences for 31 strains and discovered new SNPs that we used in conjunction with previously identified SNPs and variable-number tandem repeats (VNTRs) to genotype 773 Malagasy Y. pestis samples from 1995 to 2012. We mapped the locations where samples were obtained on a fine geographic scale to examine phylogeographic patterns through time. We identified 18 geographically separate and phylogenetically distinct subpopulations that display spatial and temporal stability, persisting in the same locations over a period of almost two decades. We found that geographic areas with higher levels of topographical relief are associated with greater levels of phylogenetic diversity and that sampling frequency can vary considerably among subpopulations and from year to year. We also found evidence of various Y. pestis dispersal events, including over long distances, but no evidence that any dispersal events resulted in successful establishment of a transferred genotype in a new location during the examined time period. CONCLUSIONS/SIGNIFICANCE: Our analysis suggests that persistent endemic cycles of Y. pestis transmission within local areas are responsible for the long term maintenance of plague in Madagascar, rather than repeated episodes of wide scale epidemic spread. Landscape likely plays a role in maintaining Y. pestis subpopulations in Madagascar, with increased topographical relief associated with increased levels of localized differentiation. Local ecological factors likely affect the dynamics of individual subpopulations and the associated likelihood of observing human plague cases in a given year in a particular location.

Genetic structure and gene flow of the flea Xenopsylla cheopis in Madagascar and Mayotte.

BACKGROUND: The flea Xenopsylla cheopis (Siphonaptera: Pulicidae) is a vector of plague. Despite this insect's medical importance, especially in Madagascar where plague is endemic, little is known about the organization of its natural populations. We undertook population genetic analyses (i) to determine the spatial genetic structure of X. cheopis in Madagascar and (ii) to determine the potential risk of plague introduction in the neighboring island of Mayotte. RESULTS: We genotyped 205 fleas from 12 sites using nine microsatellite markers. Madagascan populations of X. cheopis differed, with the mean number of alleles per locus per population ranging from 1.78 to 4.44 and with moderate to high levels of genetic differentiation between populations. Three distinct genetic clusters were identified, with different geographical distributions but with some apparent gene flow between both islands and within Malagasy regions. The approximate Bayesian computation (ABC) used to test the predominant direction of flea dispersal implied a recent population introduction from Mayotte to Madagascar, which was estimated to have occurred between 1993 and 2012. The impact of this flea introduction in terms of plague transmission in Madagascar is unclear, but the low level of flea exchange between the two islands seems to keep Mayotte free of plague for now. CONCLUSION: This study highlights the occurrence of genetic structure among populations of the flea vector of plague, X. cheopis, in Madagascar and suggests that a flea population from Mayotte has been introduced to Madagascar recently. As plague has not been reported in Mayotte, this introduction is unlikely to present a major concern for plague transmission. Nonetheless, evidence of connectivity among flea populations in the two islands indicates a possibility for dispersal by fleas in the opposite direction and thus a risk of plague introduction to Mayotte.

Molecular and serological evidence of flea-associated typhus group and spotted fever group rickettsial infections in Madagascar.

BACKGROUND: Rickettsiae are obligate intracellular bacteria responsible for many febrile syndromes around the world, including in sub-Saharan Africa. Vectors of these pathogens include ticks, lice, mites and fleas. In order to assess exposure to flea-associated Rickettsia species in Madagascar, human and small mammal samples from an urban and a rural area, and their associated fleas were tested. RESULTS: Anti-typhus group (TGR)- and anti-spotted fever group rickettsiae (SFGR)-specific IgG were detected in 24 (39%) and 21 (34%) of 62 human serum samples, respectively, using indirect ELISAs, with six individuals seropositive for both. Only two (2%) Rattus rattus out of 86 small mammals presented antibodies against TGR. Out of 117 fleas collected from small mammals, Rickettsia typhi, a TGR, was detected in 26 Xenopsylla cheopis (24%) collected from rodents of an urban area (n = 107), while two of these urban X. cheopis (2%) were positive for Rickettsia felis, a SFGR. R. felis DNA was also detected in eight (31%) out of 26 Pulex irritans fleas. CONCLUSIONS: The general population in Madagascar are exposed to rickettsiae, and two flea-associated Rickettsia pathogens, R. typhi and R. felis, are present near or in homes. Although our results are from a single district, they demonstrate that rickettsiae should be considered as potential agents of undifferentiated fever in Madagascar.

Effect of temperature and relative humidity on the development times and survival of Synopsyllus fonquerniei and Xenopsylla cheopis, the flea vectors of plague in Madagascar.

BACKGROUND: Plague, a zoonosis caused by Yersinia pestis, is found in Asia, the Americas but mainly in Africa, with the island of Madagascar reporting almost one third of human cases worldwide. In the highlands of Madagascar, plague is transmitted predominantly by two flea species which coexist on the island, but differ in their distribution. The endemic flea, Synopsyllus fonquerniei, dominates flea communities on rats caught outdoors, while the cosmopolitan flea, Xenopsylla cheopis, is found mostly on rats caught in houses. Additionally S. fonquerniei seems restricted to areas above 800 m. Climatic constraints on the development of the two main vectors of plague could explain the differences in their distribution and the seasonal changes in their abundance. Here we present the first study on effects of temperature and relative humidity on the immature stages of both vector species. METHODS: We examined the two species' temperature and humidity requirements under experimental conditions at five different temperatures and two relative humidities. By employing multivariate and survival analysis we established the impact of temperature and relative humidity on development times and survival for both species. Using degree-day analysis we then predicted the average developmental threshold for larvae to reach pupation and for pupae to complete development under each treatment. This analysis was undertaken separately for the two relative humidities and for the two species. RESULTS: Development times and time to death differed significantly, with the endemic S. fonquerniei taking on average 1.79 times longer to complete development and having a shorter time to death than X. cheopis under adverse conditions with high temperature and low humidity. Temperature had a significant effect on the development times of flea larvae and pupae. While humidity did not affect the development times of either species, it did influence the time of death of S. fonquerniei. Using degree-day analysis we estimated an average developmental threshold of 9 °C for S. fonquerniei, and 12.5 °C for X. cheopis. CONCLUSIONS: While many vector-borne diseases are limited to warm, low-lying regions, plague in Madagascar is unusual in being most prevalent in the cool, highland regions of the country. Our results point towards the possibility that this is because the endemic flea vector, S. fonquerniei, is better adapted to cool temperatures than the exotic flea vector, X. cheopis. Future warming caused by climate change might reduce the area suitable for S. fonquerniei and may thus reduce the incidence of plague in Madagascar.

Spatial analyses of wildlife contact networks.

Datasets from which wildlife contact networks of epidemiological importance can be inferred are becoming increasingly common. A largely unexplored facet of these data is finding evidence of spatial constraints on who has contact with whom, despite theoretical epidemiologists having long realized spatial constraints can play a critical role in infectious disease dynamics. A graph dissimilarity measure is proposed to quantify how close an observed contact network is to being purely spatial whereby its edges are completely determined by the spatial arrangement of its nodes. Statistical techniques are also used to fit a series of mechanistic models for contact rates between individuals to the binary edge data representing presence or absence of observed contact. These are the basis for a second measure that quantifies the extent to which contacts are being mediated by distance. We apply these methods to a set of 128 contact networks of field voles (Microtus agrestis) inferred from mark-recapture data collected over 7 years and from four sites. Large fluctuations in vole abundance allow us to demonstrate that the networks become increasingly similar to spatial proximity graphs as vole density increases. The average number of contacts, 〈k〉, was (i) positively correlated with vole density across the range of observed densities and (ii) for two of the four sites a saturating function of density. The implications for pathogen persistence in wildlife may be that persistence is relatively unaffected by fluctuations in host density because at low density 〈k〉 is low but hosts move more freely, and at high density 〈k〉 is high but transmission is hampered by local build-up of infected or recovered animals.

A non-stationary relationship between global climate phenomena and human plague incidence in Madagascar.

BACKGROUND: Plague, a zoonosis caused by Yersinia pestis, is found in Asia and the Americas, but predominantly in Africa, with the island of Madagascar reporting almost one third of human cases worldwide. Plague's occurrence is affected by local climate factors which in turn are influenced by large-scale climate phenomena such as the El Niño Southern Oscillation (ENSO). The effects of ENSO on regional climate are often enhanced or reduced by a second large-scale climate phenomenon, the Indian Ocean Dipole (IOD). It is known that ENSO and the IOD interact as drivers of disease. Yet the impacts of these phenomena in driving plague dynamics via their effect on regional climate, and specifically contributing to the foci of transmission on Madagascar, are unknown. Here we present the first analysis of the effects of ENSO and IOD on plague in Madagascar. METHODOLOGY/PRINCIPAL FINDINGS: We use a forty-eight year monthly time-series of reported human plague cases from 1960 to 2008. Using wavelet analysis, we show that over the last fifty years there have been complex non-stationary associations between ENSO/IOD and the dynamics of plague in Madagascar. We demonstrate that ENSO and IOD influence temperature in Madagascar and that temperature and plague cycles are associated. The effects on plague appear to be mediated more by temperature, but precipitation also undoubtedly influences plague in Madagascar. Our results confirm a relationship between plague anomalies and an increase in the intensity of ENSO events and precipitation. CONCLUSIONS/SIGNIFICANCE: This work widens the understanding of how climate factors acting over different temporal scales can combine to drive local disease dynamics. Given the association of increasing ENSO strength and plague anomalies in Madagascar it may in future be possible to forecast plague outbreaks in Madagascar. The study gives insight into the complex and changing relationship between climate factors and plague in Madagascar.

To be or not to be associated: power study of four statistical modeling approaches to identify parasite associations in cross-sectional studies.

A growing number of studies are reporting simultaneous infections by parasites in many different hosts. The detection of whether these parasites are significantly associated is important in medicine and epidemiology. Numerous approaches to detect associations are available, but only a few provide statistical tests. Furthermore, they generally test for an overall detection of association and do not identify which parasite is associated with which other one. Here, we developed a new approach, the association screening approach, to detect the overall and the detail of multi-parasite associations. We studied the power of this new approach and of three other known ones (i.e., the generalized chi-square, the network and the multinomial GLM approaches) to identify parasite associations either due to parasite interactions or to confounding factors. We applied these four approaches to detect associations within two populations of multi-infected hosts: (1) rodents infected with Bartonella sp., Babesia microti and Anaplasma phagocytophilum and (2) bovine population infected with Theileria sp. and Babesia sp. We found that the best power is obtained with the screening model and the generalized chi-square test. The differentiation between associations, which are due to confounding factors and parasite interactions was not possible. The screening approach significantly identified associations between Bartonella doshiae and B. microti, and between T. parva, T. mutans, and T. velifera. Thus, the screening approach was relevant to test the overall presence of parasite associations and identify the parasite combinations that are significantly over- or under-represented. Unraveling whether the associations are due to real biological interactions or confounding factors should be further investigated. Nevertheless, in the age of genomics and the advent of new technologies, it is a considerable asset to speed up researches focusing on the mechanisms driving interactions between parasites.

A coupled hidden Markov model for disease interactions.

To investigate interactions between parasite species in a host, a population of field voles was studied longitudinally, with presence or absence of six different parasites measured repeatedly. Although trapping sessions were regular, a different set of voles was caught at each session, leading to incomplete profiles for all subjects. We use a discrete time hidden Markov model for each disease with transition probabilities dependent on covariates via a set of logistic regressions. For each disease the hidden states for each of the other diseases at a given time point form part of the covariate set for the Markov transition probabilities from that time point. This allows us to gauge the influence of each parasite species on the transition probabilities for each of the other parasite species. Inference is performed via a Gibbs sampler, which cycles through each of the diseases, first using an adaptive Metropolis-Hastings step to sample from the conditional posterior of the covariate parameters for that particular disease given the hidden states for all other diseases and then sampling from the hidden states for that disease given the parameters. We find evidence for interactions between several pairs of parasites and of an acquired immune response for two of the parasites.