vector - borne hemoparasites , including the apicomplexan protozoa babesia sp . and plasmodium sp . , trypanosomes and filarial nematodes , are important pathogens in humans and domestic animals , causing babesiosis , malaria , sleeping sickness , lymphatic filariasis and canine heartworm disease . endemic as well as introduced hemoparasites may also impact health and fitness of wildlife ( e.g. custer and pence , 1981 , atkinson et  al . , 2000 , garvin et  al . , 2003 , donahoe et  al . , furthermore , babesiosis is an emerging zoonosis worldwide , with wildlife reservoirs playing a particular role in its epidemiology ( gray et  al . , 2010 , yabsley and shock , 2013 ) . thus , characterizing hemoparasite infections in wildlife and understanding patterns of prevalence can potentially reveal emerging disease risks , especially in the light of ecological instabilities as a result of human encroachment into wildlife habitats ( daszak et  al . , 2001 , keesing et  al . , 2010 ) and climate change ( daszak et  al . , wild primate populations are of particular interest in this context , because nonhuman primates have played a major role in the emergence of human diseases , including malaria , in the past ( wolfe et  al . , 2007 , pedersen and davies , 2009 , pacheco et  al . , 2011 ) . age , sex or seasonal effects may have an influence on exposure and susceptibility to infection with many parasites . age effects have been demonstrated for plasmodium sp . infections in chimpanzees and humans ( doolan et  al . infections in dogs and cattle ( boozer and macintire , 2003 , bock et  al . , 2004 ) , with decreasing prevalence in older animals being most likely due to the development of an effective adaptive immune response ( frolich et  al . , 2012 ) . additionally , the innate immune system is an important controlling factor of apicomplexan parasite infectivity ( frolich et  al . , 2012 ) and sex differences in hemoparasite infections have been found in several vertebrates , including higher prevalences in male penguins ( merkel et  al . , 2007 ) , lizards ( schall et  al . , 2000 ) and these sex differences could either be due to physiological differences , e.g. immunosuppression caused by higher testosterone levels in males ( klein , 2004 , roberts et  al . , 2004 ) , or due to differences in parasite exposure . for example , a sex - bias in vector feeding rates towards males was recently demonstrated in birds ( burkett - cadena et  al . , 2014 ) . although hemoparasite infections have been investigated in several wild primate species ( de thoisy et  al . , 2001 , maamun et  al . , 2011 , de nys et  al . , 2013 , thurber et  al . , 2013 ) , no sex - differences in prevalence have been reported so far . furthermore , it has been proposed that group - living may decrease the risk of infection with vector - borne pathogens by means of an encounter - dilution effect , analogous to a decrease of predation risk ( freeland , 1976 , mooring and hart , 1992 , kappeler et  al . , 2015 ) . ( 2014 ) recently demonstrated that sentinel hosts caged inside large roosts of american robins seroconverted to west nile virus more slowly than those held outside of roosts , suggesting that exposure of individual hosts can indeed be reduced through group - formation . however , larger groups may also be more conspicuous , and thus attract more vectors . in a comparative study , colonially - breeding bird species were shown to experience both higher prevalences and higher species diversity of blood parasites than solitarily breeding species ( tella , 2002 ) . two studies on neotropical primates found that prevalence of plasmodium sp . increases with group size ( davies et  al . , 1991 , nunn and heymann , 2005 ) , but studies on the effect of within - species variability of group size on hemoparasite infections in primates are lacking . furthermore , infections with vector - borne parasites are likely to covary with environmental conditions ( altizer et  al . , rainfall and temperature affect mosquito abundance , biting rates and parasite development within mosquitoes ( altizer et  al . , 2006 , additionally , dry , cold conditions as well as frequent temperature fluctuations can reduce the abundance of host - seeking ticks ( sutherst and bourne , 2006 , swai et  al . , 2006 , herrmann and gern , 2013 ) . despite this seasonality in vector biology , empirical evidence regarding seasonal variation in host infection rates is contradictory and mainly limited to malaria in humans ( smith et  al . , 1993 , koram et  al . , 2003 , mabaso et  al . , 2007 ) . if transmission results in persistent infections , little seasonal variation in prevalence can be expected ( govender et  al . , finally , in natural systems co - infections with multiple parasites are common , potentially resulting in complex interspecific interactions . it has been shown that parasite community interactions may explain more variation in infection risk than the effects associated with host and environmental factors . for example , in field voles ( microtus agrestis ) chronic infection with babesia microti reduces susceptibility to bartonella spp . parasites may directly affect the fitness of co - infecting species through interference competition , they may compete for the same resources within a host , or affect each other 's abundance via interaction with the host 's immune system ( pedersen and fenton , 2006 ) . cross - immunity between co - infecting parasite species might limit prevalence at the population level , whereas parasite - induced immunosuppression may lead to synergistic effects ( cox , 2001 , telfer et  al . , 2008 ) . we present the first study which systematically characterizes infections with several co - occurring hemoparasites in a population of malagasy primates , verreaux 's sifakas ( propithecus verreauxi ) . madagascar faces acute risks of species extinctions as well as disease emergence in humans and wildlife due to intense human disruption of natural ecosystems ( harper et  al . , 2007 , junge , 2007 , barrett et  al . , 2013 , ratsimbazafy et  al . , 2013 , verreaux 's sifakas are diurnal , sexually monomorphic lemurs which live in multi - male multi - female groups of varying size in seasonal habitats , in which mosquitoes are virtually absent for several months of the year , in western and southern madagascar ( kappeler and fichtel , 2012 ) . the life expectancy of sifakas in the wild can exceed 20 years ( richard et  al . , 2002 , kappeler and fichtel , 2012 ) . we used samples collected from members of all age classes from 10 adjacent groups ranging in size from 2 to 7 individuals during annual captures to detect infections with plasmodium sp . , babesia sp . and filarial nematodes using a combination of a pcr - based approach and microscopical examination of blood smears . these parasites have previously been reported to occur in blood samples of sifakas ( uilenberg et  al . , 1972 , junge and louis , 2005 , duval et  al . , 2010 , pacheco et  al . , 2011 , rasambainarivo et  al . , 2014 ) , but systematic investigations of infection patterns are lacking . anopheline mosquitos and haemaphysaline ticks , are present in the study region ( davidson , 1966 , rodriguez et  al . , 2012 ) , whereas the vectors for filarial parasites of lemurs are unknown ( irwin and raharison , 2009 ) . to illuminate natural drivers of infection patterns , we tested the influence of host age and sex , host group size and seasonality on individual infection status and hemoparasite species richness in our study population . to assess possible health consequences , we also tested the influence of these infections on packed cell volume , to assess possible anemia , and total plasma protein as well as the neutrophil - lymphocyte ratio , which are usually increased during inflammatory processes ( thrall et  al . based on results of previous studies with other vertebrates , we predicted that infection probabilities would decrease with age and that males would harbor more hemoparasites than females . furthermore , we predicted that group size would negatively affect the probability of testing positive for each hemoparasite , as well as individual hemoparasite species richness , in case of an encounter - dilution effect . alternatively , we predicted that group size would have a positive effect on these measures if larger groups attract more vectors . we also expected to find lower prevalences of hemoparasite infections during the dry season than during the hot , wet season . the study was carried out in kirindy forest , western madagascar , located at approximately 4439e , 2003s . the study area is part of a field site operated by the german primate center ( dpz ) since 1993 and is situated within a forestry concession managed by the centre national de formation , detudes et de recherche en environnement et foresterie ( cnferef ) . kirindy forest is a dry deciduous forest and subject to pronounced seasonality , with a dry season from april to october and a hot , wet season from november to march ( kappeler and fichtel , 2012 ) . as part of an ongoing long - term study ( kappeler and fichtel , 2012 ) , several social groups of verreaux 's sifakas have been habituated to human observers and individually marked with microchips and unique collars . a total of 45 blood samples were taken from 36 individual verreaux 's sifakas belonging to 10 social groups during routine immobilization procedures in april 2013 , august 2013 , march 2014 and april 2014 . group sizes ranged from 2 to 7 individuals , and a minimum of 43% of individuals were sampled from each group . seven animals were repeatedly sampled at intervals of 45 months , resulting in a maximum of 3 samples per individual ( table  1 ) . packed cell volume was determined by centrifuging edta blood in a microhematocrit capillary using a sigma 114 centrifuge ( sigma laborzentrifugen gmbh , osterode am harz , germany ) and total plasma protein was estimated using a hand - held refractometer . two to 3 blood smears were prepared per individual , air dried , stained ( diff quick stain , eberhard lehmann gmbh , berlin , germany ) and preserved with mounting medium ( eukitt , fluka analytics , sigma  aldrich chemie gmbh , munich , germany ) and a cover slip . blood smears were scanned for the presence of hemoparasites and used for a differential white blood cell count to assess the percentages of the following leukocyte categories : neutrophils , banded neutrophils , lymphocytes , monocytes , eosinophils and basophils . photographs were taken with a zeiss axiocam erc 5s fitted to a zeiss primo star microscope ( carl zeiss ag , oberkochen , germany ) . measurements were made using the zeiss zen lite 2012 software ( carl zeiss ag ) after calibration with a stage micrometer . an aliquot of blood was mixed with the same amount of rnalater and frozen at  20  c until shipment to germany and further analysis . all necessary research permits were obtained from the malagasy and german authorities and the study was approved by the ethics committee of the german primate center . dna was extracted from rnalater - preserved blood samples using the qiamp blood mini kit ( qiagen , hilden , germany ) according to the manufacturer 's instructions . to test for the presence of plasmodium spp . in blood samples , a semi - nested pcr was carried out targeting an approximately 1000  bp long fragment of the parasite 's cytochrome b gene . in the first amplification round , primers p.sp.cytb f1 ( 5-tgc cta gac gta ttc ctg att atc cag ; kaiser et  al . ( 2010 ) ) and p.sp.cytb r1 ( 5- ctt gtg gta att gac atc cwa tcc ; kaiser et  al . ( 2010 ) ) were used , followed by p.sp.cytb f2 ( 5-att ggd tca acw atg act tta ttt gg ) and p.sp.cytb r1 in the second round . the 25  l reaction mixture contained 1  l of dna - extract or pcr - product ( diluted 1:40 ) from the first round , respectively , 2.5  l 10x pcr buffer ( invitrogen , karlsruhe , germany ) , 2  l of 50  mm mgcl2 ( invitrogen , karlsruhe , germany ) , 2  l of 2.5  mm deoxynucleotide triphosphates ( thermo scientific fermentas , st . leon - rot , germany ) , 0.5  l of each primer ( 10  m ) and 0.2  l of platinum taq polymerase ( invitrogen , karlsruhe , germany ) . the thermal profile was the same for both rounds , with an initial denaturation step at 95  c for 5  min followed by 40 cycles of 95  c for 30  s , 58  c for 45  s and 72  c for 60  s , and a final elongation step at 72  c for 10  min . we tested for babesia spp . using primers bj1 ( 5-gtc ttg taa ttg gaa tga tgg-3 ) and bn2 ( 5-tag ttt atg gtt agg act acg-3 ) , targeting a 500  bp long fragment of the 18s rrna gene ( casati et  al . we used 5  l of dna - extract in a 25  l reaction mixture containing the same quantities of reagents as stated above . the thermal profile consisted of an initial denaturation step at 94  c for 10  min followed by 40 cycles of 94  c for 60  s , 55  c for 60  s and 72  c for 2  min , and a final elongation step at 72  c for 5  min . to generate sequences for microfilaria observed in blood smears , we employed a nested pcr targeting an approximately 900  bp long fragment spanning part of the 18s rrna gene , the internal transcribed spacer 1 ( its1 ) and part of the 5.8s rrna gene . primers used were nf1 ( 5-ggt ggt gca tgg ccg ttc tta gtt-3 ) ( porazinska et  al . , 2009 ) and nc2 ( 5-tta gtt tct ttt cct ccg ct-3 ) ( gasser et  al . , 1993 , chilton et  al . , 2003 ) in the first round and a modification of its1-f ( 5- ttg att acg tcc ctg ccc-3 ) ( vrain et  al . , 1992 , 2014 ) and the filaria - specific di5.8s - r ( 5-acc ctc aac cag acg tac-3 ) ( nuchprayoon et  al . , 2003 , nuchprayoon et  al . , 2005 ) in the second round . the 25  l reaction mixture contained 5  l of dna - extract in the first round and 1  l pcr - product ( diluted 1:40 ) in the second round , and the same quantities of reagents as in the other pcrs . the thermal profile of the first round consisted of an initial denaturation step at 95  c for 5  min followed by 35 cycles of 94  c for 60  s , 58  c for 30  s and 72  c for 60  s , and a final elongation step at 72  c for 10  min . the thermal profile of the second round consisted of an initial denaturation step at 94  c for 10  min followed by 35 cycles of 94  c for 30  s , 55  c for 30  s and 72  c for 60  s , and a final elongation step at 72  c for 10  min . pcrs were run in a flexcycler thermal cycler ( analytic jena , jena , germany ) and amplification products were detected by electrophoresis on 1.5% agarose gels . pcr products of the corresponding size were purified from 2% agarose gels using the jetquick gel extraction spin kit ( genomed , lhne , germany ) and sanger sequencing of both strands was performed by seqlab sequence laboratories gttingen gmbh ( gttingen , germany ) . auckland , new zealand ) and compared to publicly available sequences using blast ( altschul et  al . , new sequences were deposited in the embl nucleotide sequence database ( kulikova et  al . , 2004 ) , under accession numbers ln869519  ln869522 . for plasmodium sequences the two unique sequences identified in this study were put together with the representative sequences selected by pacheco et  al . ( 2011 ) , which includes all sequences derived from lemur plasmodium available to this date . we used jmodeltest v2.1.4 to identify the model of nucleotide substitution with the best fit to the data ( gtr  +  i  +  g4 ; ( darriba et  al . , 2012 ) ) . we then reconstructed a maximum likelihood tree under this model using phyml v3 ( guindon et  al . , 2010 ) , as implemented on the phyml webserver ( guindon et  al . , 2005 ) . branch robustness was assessed through non - parametric bootstrapping ( 500 bootstrapped pseudo - replicates ; bp ) . we used generalized linear - mixed models ( glmms ) with binomial error structure and logit link function to analyse which factors influenced the probability of testing positive for each parasite species . we included sex , age , season ( wet or dry ) and size of the animal 's social group as fixed effects and individual identity nested in group as a random effect . furthermore , we ran a glmm with poisson error structure and log link function to test influences on hemoparasite species richness , including the same fixed and random effects . based on results of the glmms for plasmodium and babesia infections , we used another glmm with binomial error structure to test whether babesia infection status had an effect on plasmodium infection , again controlling for individual identity nested in group . to assess potential health impacts , we first tested whether age affected hematology values , using spearman rank correlation , and then used linear mixed models ( lmms ) to test influences of infection with each hemoparasite and hemoparasite species richness on log - transformed packed cell volume , total plasma protein and the log - transformed neutrophil : lymphocyte ratio , controlling for animal age and individual identity nested in group as a random effect . we compared each full model to a null model comprising only the random effect in a likelihood ratio test using the r - function anova with the argument statistical significance was inferred if the specific p - value of the factor as well as the p - value of the likelihood ratio test were < 0.05 . seven individuals were tested more than once , and repeated sampling at different times of the year almost always gave identical results for these animals . only results for one animal ( age 9 ) switched from negative to positive during the study period . we did not observe any parasite stages in diff - quick - stained blood smears . only two unique sequences were amplified in this study , which showed 95100% sequence identity to publicly available plasmodium sequences . lld-2010 which has previously been found in verreaux 's sifakas ( duval et  al . , 2010 ) . ln869522 ) , which unambiguously nested within the clade of malaria parasites infecting lemurs ( bp 100 ; fig .  1 ) . c - map-2012 ( which has been found in hapalemur griseus ( pacheco et  al . , 2011 ) ) , although the statistical support for this association was moderate ( bp 59 ; fig .  1 ) . the patristic distance between these two sequences was 0.21 substitution per site ( s.s ) , which is both higher than the 0.17  s  s observed between the two most closely related lemur parasite sequences known to date ( plasmodium sp . b - map.2012 and e - map-2011 ) and higher than the maximum 0.14  s  s observed between p.  reichenowii ( clade c1 , infecting chimpanzees ) and p.  praefalciparum sequences ( clade g1 , infecting gorillas ; rayner et  al . , 2011 ) . test outcome , with older animals being more likely to test positive ( fig .  2 ) . in fact , none of the animals up to the age of 5 years were infected ( n  =  18 ) , 50% of animals between age 6 and 10 were infected ( n  =  10 ) and all animals over the age of 10 tested positive ( n  =  8) . however , in the glmm including age as well as sex , season and group size as fixed factors , age only tended to have a statistically significant effect ( table  2 ) . again , test results for only 1 individual ( age 7 ) changed from negative to positive during the study period . the babesia sequence amplified ( embl accession no . ln869519 ) was the same in all samples and showed 94% identity to different babesia canis isolates . we did not microscopically observe any babesia parasite stages in diff - quick - stained blood smears . infections , young animals were more likely to test positive ( table  3 , fig .  2 ) . only 3 animals ( 6 , 7 and 11 years old ) were co - infected with plasmodium sp . and babesia sp . based on this complementary age pattern , the low number of co - infections , and because neither age , sex , season nor group size influenced plasmodium spp . infections , we tested babesia infection status as a predictor of plasmodium infection , and found a highly significant negative effect ( table  4 ) . two to 3 blood smears per sample were scanned for microfilaria and on average 4.2 microfilaria were measured per sample . microfilaria of species 1 were 268    10.1  m long with the nerve ring located at 53    4.5  m from the anterior end . microfilaria of species 2 were considerably shorter , 222    18.6  m , with the nerve ring located at 43    5.4  m . species 1 produced a shorter band of about 850  bp and species 2 produced a longer band of about 900  bp . ln869520 and ln869521 ) showed 99100% sequence identity to different filarial nematodes such as mansonella sp . , dirofilaria sp . and onchocerca sp . in the conserved 18s and 5.8s regions , but only 8490% similarity to other filarial nematodes in the variable its1 region . in 33 of 45 samples , the pcr results exactly confirmed the morphological diagnosis ( negative sample , presence of one or both species ) . in 7 cases , a species was morphologically identified but not detected by pcr , while in 5 cases an infection was revealed by pcr only . thus , neither method seems to be 100% sensitive . for statistical analyses , we considered an infection present if it was detected by one of the methods . prevalence of species 1 was 50% , while species 2 was found in 33.3% of animals . probability of infection with species 1 significantly increased with age ( table  5 ) . we did not find any significant influences on infection with the second species ( table  5 ) . we tested whether host age , sex , season or group size affected the number of hemoparasite species found in each individual , but we did not find any statistically significant influences ( table  6 ) . hematology results are summarized in table  7 packed cell volume and total plasma protein were significantly positively correlated with the animals age ( spearman rank correlation , n  =  44 ,   =  0.44 , p  =  0.003 , and n  =  43 ,   =  0.4 , p  =  0.008 , respectively ) and the ratio of neutrophils to lymphocytes tended to be positively correlated with age ( spearman rank correlation , n  =  45 ,   =  0.26 , p  =  0.08 ) . controlling for age , we did not find any significant influences of infection with any single parasite species on packed cell volume ( pcv ; table  8) , and hemoparasite richness also did not influence pcv ( table  9 ) . likewise , total plasma protein ( tp ) was neither significantly influenced by infection with any single parasite species ( table  8) , nor by hemoparasite species richness ( table  9 ) . the ratio of neutrophils to lymphocytes ( nlr ) was 0.47    0.22 ( range : 0.151.25 ) . no single infection influenced log - transformed nlr values , ( table  8) , and hemoparasite richness was also not a significant predictor ( table  9 ) . the percentage of eosinophils was 0.96    0.91 ( range : 04.5 ) , which is in the range of published values for propithecus sp . ( bergeron and buettner - janusch , 1970 , junge and louis , 2005 , irwin et  al . , 2010 , we identified 5 putative species of hemoparasites in verreaux 's sifakas in kirindy forest : 2 species of malaria parasites , only one of which has been previously identified in verreaux 's sifakas by means of pcr ( duval et  al . , 2010 ) , the other one likely standing for a yet undetected and undescribed species ; one species of babesia ; and 2 species of filarial nematodes . as sequences of babesia parasites and filarial nematodes from madagascar are not publicly available to date , we could not assign species names to our sequences . morphological descriptions of microfilaria found in lemurs were ambiguous ( irwin and raharison , 2009 ) . of the 4 species described in lemurs , which all belong to the family onchocercidae , species 1 could either be dipetalonema petteri or protofilaria furcata , while measurements for species 2 fall into the range described for paulianfilaria pauliani . we did not recover any adult worms , as dissections of animals were not possible in this study , and the insufficient quality of blood smears did not allow us to use further microfilaria characteristics for identification . however , genetic analyses confirmed the presence of two different filarial nematode species in the population . we found high prevalence of infection with babesia sp . in wild verreaux 's sifakas under the age of 5 , while infections with plasmodium sp . were absent from this age class , although most , but not all individuals of this age class were sampled during the wet season , when mosquitoes are abundant . this is unusual , as it has been found in humans and chimpanzees that younger animals usually have higher plasmodium sp . prevalences and that prevalence decreases with age , possibly due to naturally acquired immunity ( doolan et  al . , 2009 , de nys et  al . , 2013 ) . the age - infection patterns we observed give no indication of age - related immunity to plasmodium sp . in verreaux 's sifakas . in our study , animals older than approximately 10 years showed 100% plasmodium sp . juveniles under the age of one year were never pcr - positive for filariasis , nor were microfilaria observed in their blood - smears . however , we can not exclude the possibility that these animals were already infected , as the prepatent period of filarial nematodes , i.e. the period from infection to appearance of reproductive parasite stages in the blood , may span several months ( bowman and georgi , 2009 ) . therefore , the result that age significantly affects filarial nematode infections should be treated with caution . neither pcr nor microscopic examinations of blood smears seem to be sensitive enough to reliably detect filarial nematodes infections in all cases , so we combined the results of both methods . in cases of low parasitemia , pcrs produced some false - negatives although single microfilaria were identified in the corresponding blood smears , and species differentiation based on microfilaria morphology only is difficult , because the length ranges of both species may potentially overlap . this could have led us to misdiagnose infections in some cases and we might have missed statistically significant effects on onchocercidae infections or hemoparasite species richness , as well as the effects of those on hematology values . sex did not influence infection with any single species , nor species richness , indicating that males are neither more susceptible to infections nor more exposed . although sex - biased patterns of parasitism , including the parasite taxa examined in this study , seem to exist in many mammals ( klein , 2004 ) , this effect does not seem to be universal ( kiffner et  al . , 2013 ) . specifically , sifakas do not display sexual size dimorphism ( kappeler , 1990 ) , which has been proposed to relate to sex differences in parasite susceptibility in general ( moore and wilson , 2002 ) , while larger bodied hosts may also attract more vectors ( davies et  al . , 1991 ) . furthermore , male and female sifakas , like other lemurs with female dominance , exhibitit smaller differences in androgen levels than other mammals ( von engelhard et  al . , 2000 , drea , 2007 ) , we tested whether host social group size influenced infection with each single hemoparasite species as well as hemoparasite species richness , but we did not find any significant effect . group sizes in this study ranged from 2 to 7 individuals , and this range may have been too small to detect any differences , although small group - size differences should have a more pronounced effect on individual infection risk in small groups . thus , our data do not support the encounter - dilution hypothesis , nor the opposing prediction that larger groups attract more vectors , which was supported by two studies of plasmodium sp . prevalences across neotropical primates with group sizes ranging from 3 to 29 individuals ( davies et  al . infections nor on infections with any of the other parasites , although fewer mosquitoes are present during the dry season . persistent infections as well as long developmental times of the parasite within the host , as might be the case for filarial nematodes , may mask seasonal effects . because of the opposite patterns of age - dependency of plasmodium and babesia infections , and because babesia sp . infections were a significant negative predictor for plasmodium sp . infections , we suggest that infection with babesia sp . may be a natural protectant against plasmodium sp . in this lemur species . as sifakas in kirindy give birth during the dry season , when almost no mosquitoes are present , juveniles likely acquire babesia sp . infections via tick - bites , probably of haemaphysaline ticks , which occur in western madagascar ( rodriguez et  al . , 2012 ) and have been detected on sifakas during animal captures , before they become exposed to plasmodium sp . until babesiosis resolves , e.g. due to antibody - mediated immunity ( frolich et  al . , 2012 ) the suppression of malaria infections in the course of ongoing babesiosis has been investigated in rhesus macaques ( macaca mulatta ) in laboratory settings ( van duivenvoorde et  al . , 2010 ) , after it was found that a macaque infected with a b.  microti - like parasite showed a suppressed plasmodium cynomolgi infection ( wel et  al . , heterologous immunity between the two parasites has also been observed in mice ( cox , 1978 , zivkovic et  al . , 1984 ) . it has been proposed that the cross - protection is most likely due to immune responses such as the activation of a distinct class of pro - inflammatory monocytes , release of pro - inflammatory cytokines , and increased c - reactive protein levels , which potentially play an important protective role in malaria infections ( clark , 2001 , ansar et  al . , 2006 , it has also been proposed that this heterologous immunity might be exploited for the development of malaria vaccines . however , an interaction between plasmodium and babesia could also arise due to resource competition within the host . in fact , in humans it has been shown that co - infecting parasites mostly interact in this way ( griffiths et  al . , 2014 ) . both parasites invade and replicate in erythrocytes and both of them modify the erythrocytic membrane to achieve higher permeability , but do so using different mechanisms and at different time points after invasion ( alkhalil et  al . , 2007 ) . changes in membrane permeability induced by babesia infection may thus make subsequent infection with plasmodium sp . impossible and lead to a competitive advantage for babesia sp . however , b.  microti parasites suppressed p.  cynomolgi parasitemia without altering the induction of initial anemia in rhesus macaques , suggesting the malaria parasites could enter erythrocytes during an infection with babesia ( van duivenvoorde et  al . , 2010 ) . the lack of positive microscopic observation of both babesia and plasmodium stages in the blood smears of infected individuals may partially result from technical limitations , but probably also indicates that parasitemia was generally low . this may suggest that if competition occurs , the parasites only compete for a narrow erythrocyte subset . while the precise mechanism of interaction remains unclear , the pattern found in our study provides the first indication of a possible protective effect of babesiosis on malaria infections in a wild population of primates naturally infected with both parasites , thus adding external validity to the above observation . finally , we did not find any signs of morbidity caused by the parasites tested in this study . only animal age affected hematology values , a common pattern also in primates ( mcpherson , 2013 ) . packed cell volume was not significantly affected by infection with any single hemoparasite species nor by hemoparasite species richness , indicating that these parasites do not cause clinically relevant anemia . the absence of anemia may be explained by the long coevolutionary history between hemoparasites and their hosts , leading to a complete dependence of the parasite on the host for survival , which favours long - lasting asymptomatic infections with low parasitemia ( chauvin et  al . , 2009 , furthermore , evidence has been found that co - infection with filarial nematodes may attenuate anemia associated with clinical malaria in humans ( dolo et  al . , 2012 ) . total plasma protein and the neutrophil - lymphocyte ratio were not affected in a statistically significant way . acute inflammation usually causes an increase in plasma globulin concentrations , which may reflect in increased total plasma protein levels , as well as an increase in neutrophilic granulocytes ( thrall et  al . , 2006 ) . however , the neutrophil - lymphocyte ratio may be affected in both directions by an excitement response with epinephrine release due to animal capture , as leucocytes are released from the marginal into the circulating pool ( thrall et  al . , 2006 ) , which may have masked effects due to parasitism . eosinophils typically show an increase as a response to helminth infection ( nutman , 2007 ) , and high values have been measured in the course of filarial infections in dogs and humans ( mackenzie , 1980 , niwetpathomwat et  al . , 2007 ) . elevations may also occur in the course of babesiosis ( vercammen et al . , 1997 ) . the relative counts of eosinophils were not markedly elevated in any of the animals in this study , judging by comparison with previously reported values from both captive and wild propithecus sp . populations ( bergeron and buettner - janusch , 1970 , junge and louis , 2005 , irwin et  al . , 2010 , rasambainarivo et  al . , 2014 ) . however , this conclusion remains preliminary as we were not able to obtain absolute leucocyte counts due to restrictions of fieldwork . in conclusion , prevalences of hemoparasite infections in verreaux 's sifakas in kirindy forest , madagascar , are significantly related to host age , probably shaped by interaction between babesia sp . and plasmodium sp . , but independent of host sex , host group size and season . the possible cross - protection between babesia sp . and plasmodium sp . is a prime example of how biodiversity may affect the ecology of infectious diseases . increasing evidence has accumulated that biodiversity may have a buffering role on disease transmission , while biodiversity loss is thought to increase transmission , disease incidence and disease emergence ( daszak et  al . , 2001 , ostfeld , 2009 , keesing et  al . , 2010 , vourch et  al . , 2012 ) . in this case , the presence of tick - borne babesiosis seems to reduce plasmodium sp . prevalence in a natural system , illustrating how within - host pathogen diversity may regulate the abundance of parasites . infection status did not influence hematology values , suggesting that the parasites considered in this study do not cause clinically relevant anemia or inflammation , but that hosts and parasites are well - adapted to each other , resulting in persistent infections with low pathogenic potential .