to investigate the prevalence of different plasmodium spp . in wild great apes living in their natural habitat ( tropical rainforests ) , we analyzed tissue samples from 16 wild west african chimpanzees that died primarily of anthrax or respiratory disease in ta national park , cte divoire .
a generic real - time pcr that detects all known plasmodium spp . was used to test all samples for the parasite .
sequence analysis of the cytb gene and small subunit rrna genes was conducted for real - time pcr  positive samples to determine the strain present ; 1,140 bp of the cytb gene and 765 bp of the 18s gene of the plasmodium genome were amplified by classic pcr .
resulting products were sequenced either directly or after cloning for rrna gene and when initial sequence information showed the possible presence of 2 different species ( table ) . *
all chimpanzees were pan troglodytes verus from tai except p. t. schweinfuthii chimpanzees , which were from budongo forest .
parentheses indicate the number of samples for which sequences were obtained and used for phylogenetic tree analyses .
phylogenetic analyses of sequences obtained confirmed the presence of 5 species : p. reichenowi and p. gaboni , which had been found previously ( 2,3 ) ; but also p. vivax , p. ovale , and p. malariae  like strains ( figure 1 , figure 2 ) .
the most prevalent species was p. reichenowi ( 6/16 ) , which had representatives in subclusters p. gaboni and p. reichenowi .
the other species were rare , seen only 1 ( p. ovale and p. vivax ) or 2 ( p. malariae ) times .
( figure 1 , figure 2 ) , 1 infected with p. reichenowi and a p. malariae  like strain and the other with p. reichenowi and p. gaboni .
maximum - likelihood trees of plasmodium spp . obtained from the analysis of a 1,087-bp cytb alignment .
blue indicates sequences determined from chimpanzee hosts ; green , bonobos ; gray , gorillas ; and red , humans .
blue indicates sequences determined from chimpanzee hosts ; green , bonobos ; gray , gorillas ; and red , humans .
typical for wild chimpanzees or related to reduced immune function associated with the severe infection that was the primary cause of death in each case ? to investigate this question , we tested dna extracted from fecal samples of apparently healthy chimpanzees collected over the past 8 years ( n = 30 ) ( 11 ) of the same study population by using the generic real - time pcr followed by amplification of the cytb gene .
of these samples , 21 ( 70% ) were positive for plasmodium spp . by real - time pcr .
because of low copy numbers in feces , phylogentic analyses were limited to 2 samples in which p. reichenowi of the p. gaboni subcluster was confirmed . to determine if the observed high prevalence of plasmodia was a site- or chimpanzee subspecies  specific phenomenon , we tested 30 randomly selected fecal samples of individually known apparently healthy wild eastern chimpanzees from the budongo forest in uganda . overall prevalence of plasmodium spp .
was lower than in west african chimpanzees but still relatively high ( 40% ) ; p. reichenowi and p. gaboni were identified in 3 samples .
is similar to that of untreated human populations in sub - saharan africa ( www.who.int/malaria ) . throughout sub - saharan africa ,
p. falciparum is more predominant in humans than are other plasmodium spp . considering the lack of clinical signs of malaria in chimpanzees from which fecal samples were collected and those that had died of respiratory disease or anthrax , plasmodium spp .
however , signs of illness are rarely observed in wild primates because infected animals often mask weakness to maintain social position and avoid attack by predators ( 12 ) .
recently developed technologies for the noninvasive determination of temperature in wild chimpanzees may enable more effective examination of the relationship between the primary clinical feature of malaria ( i.e. , cyclical fevers ) and plasmodium spp .
p. ovale was previously described from captive chimpanzees and p. malariae from captive chimpanzees and captive bonobos have been described ( 58 ) .
our study results demonstrate that p. malariae and p. ovale occur in wild chimpanzees that inhabit pristine contiguous forest with extremely limited exposure to humans , suggesting the natural existence of these parasites in wild great apes . because of a duffy - negative condition in 95%99% of the human population in western and central continental africa , transmission of p. vivax does not seem to occur
. however , p. vivax infections are common in travelers returning from these areas ( 11 ) .
even though we can not totally exclude the possibility of introduction of p. vivax in the chimpanzee population through humans , our discovery of p. vivax in wild chimpanzees living exclusively within their natural habitat suggests that wild african apes may be a natural reservoir .
our study shows the existence of p. reichenowi and related strains in wild chimpanzees as described for chimpanzees and gorilla by others ( 24,6 ) .
infections with strains of the p. 
 reichenowi group ( sometimes referred to as the species p. gaboni , p. billbrayi , and p. billcollinsi ) appear to occur widely in wild and captive great apes in africa with some variation between chimpanzee subspecies from biogeographically distinct sites .
this lack of infection is likely caused by low human presence in their habitat and , consequently , few or no infected vectors , low sample size , or a missing receptor in chimpanzees ( 14 ) .
more investigations are needed because recently p. falciparum infections have been described for 2 captive chimpanzees ( 6 ) .
the situation is clearer for captive and wild lowland gorillas ( gorilla gorilla ) for which infections and receptors have recently been described ( 4 ) .
previous examination of the role of our closest phylogenetic relatives , the great apes , in the evolution and persistence of human plasmodia has been limited by a lack of data from wild ape populations where opportunities for human - mosquito - ape malaria exchange are minimal .
interpretation of patterns of malaria infection in captive ape populations , such as sanctuaries and zoos , must consider the ample opportunities for human - to - ape transmission of such parasites , negating the opportunity to investigate the evolutionary origins and public health  related risks of these parasites .
conversely , our examination of these parasites in wild chimpanzees with no contact to the periphery of the rainforest habitat ( technical appendix figure ) demonstrates that these apes are most likely naturally infected with p. ovale , p. vivax , and p. malariae , 3 types of plasmodia rarely observed in humans of the region . whether wild great apes are the origin or reservoirs of these plasmodium types requires further investigation .
these results may have implications for global efforts to eradicate malaria in humans , including vaccine development based on animal variants of human parasites .
 data are missing on the diversity of plasmodium spp . infecting apes that live in their natural habitat , with limited possibility of human - mosquito - ape exchange . 
 we surveyed plasmodium spp . 
 diversity in wild chimpanzees living in an undisturbed tropical rainforest habitat and found 5 species : p. malariae , p. vivax , 
 p. ovale , p. reichenowi , and p. gaboni . 
The Study
Conclusions
Supplementary Material