in may 2009 , a 7-year - old female dog was admitted to a veterinary clinic in bijilo , the gambia , for elective ovariohysterectomy . the owner of the dog , a snake farm operator , reported late abortions during several pregnancies of the animal .
the dog had been kept on the farm premises , where adult snakes ( african rock pythons , python sebae ) had died several months before of infection with adult a. armillatus pentastomes ( figure 1 , panel a ) . during the dog
s surgery , hundreds of pentastomid larvae were seen on the enteral serosa , bladder , uterus , and in the omentum ( figure 1 , panels b , c ) . in april 2010
, a male stray dog , 6 months of age , was admitted to the veterinary clinic for elective neutering .
adult and larval parasite specimens were preserved in 100% ethanol for further parasitologic , histologic , and molecular examinations .
a ) ventral view of 2 adult a. armillatus parasites recovered from the lungs and trachea of a deceased rock python ; a gravid female ( bottom ) and a pre - adult female ( top ) are shown .
the parasites showed 20 and 18 marked body rings , and had a length of 10 cm and 9 cm and a body width of 58 mm and 35 mm , respectively .
b ) heavily parasitized omentum of a female stray dog , showing typical encapsulated c - shaped larval stages of a. armillatus parasites .
the larvae had a length of 1819 mm and a body width of 2 mm and showed 2022 rings .
scale bars = 1 cm . to investigate the extent of infection and determine whether transmission to humans on the snake farm grounds had occurred , we collected serum specimens from the 46-year - old male caucasian snake farm owner , his 28-year - old wife , his 3 children , and the infected female dog .
serum samples were transferred to the institute of hygiene and microbiology ( university of wrzburg , wrzburg , germany ) for analysis by elisa and western blot based on larval parasite antigens .
dna of the pentastome specimens ( 2 adults from the snakes , 1 larva from each dog ) was extracted by using the qiagen tissue kit ( qiagen , hilden , germany ) and subjected to 18s rrna and cytochrome c oxidase ( cox ) gene pcr with primers pent629f ( 5-cggttaaaaagctcgtagttgg-3 ) and pent629r ( 5-ggcatcgtttatggttagaactaggg-3 ) and primers cox1-f ( 5-ctgcgacaatgactattttcaac-3 ) and cox1-r ( 5-atatgggaagttctgagtagg-3 ) . after sequencing and blast ( www.ncbi.nlm.nih.gov/blast )
analysis of the partial 18s rrna gene amplicons , the sequences showed high homology with a. agkistrodontis and porocephalus crotali parasites .
however , no a. armillatus 18s rrna gene entry existed in genbank ; cox gene sequences showed high homology with a. armillatus , followed by a. agkistrodontis .
the 4 amplified partial 18s rrna gene sequences were 100% identical , as expected for the same species , but heterogenicity was seen in the cox sequences ( 99.7% identity ) .
when phylogenetic trees were constructed , the nearest neighbor of a. armillatus was a. agkistrodontis in both models for both genes , followed by p. crotali and linguatula serrata ( 18s rrna only as no cox entry existed ) .
all pentastomes analyzed clustered together and formed their own branch , with argulus sp . being the nearest maxillopodan / branchiuran neighbor in both models by using 18s rrna sequence data , and speleonectes tulumensis ( remipedia ) crustaceans when cox sequences were used ( figure 2 ) . the 18s sequence of a. armillatus was submitted to genbank ( accession no .
molecular phylogeny of 4 armillifer amillatus specimens based on partial 18s rrna and partial cytochrome c oxidase ( cox ) gene sequences .
panels a and c show cladograms based on maximum - likelihood ( ml ) ; panels b and d show minimum - evolution ( me ) . in the cladograms ,
the approximate likelihood ratios are given next to the branches to indicate the statistical support for the respective branches . in panels
b and d , the percentage of replicate trees in which the associated taxa clustered together in the bootstrap test ( 1,000 replicates ) is shown next to the branches , and the evolutionary distances are given as scale bars as the number of base substitutions per site .
the phylogram of the partial 18s rrna sequences ( b ) depicts respective host species for the parasitic pentastomida .
, reighardia sternae , and hispania vulturis belong to the order cephalobaenida , whereas snake parasites armillifer spp . and porocephalus spp . and dog parasites linguatula spp .
note that not all species have entries for 18s rrna and cox genes in genbank .
genbank accession numbers of the species depicted can be found in the table ( table ) .
muscle ( www.ebi.ac.uk/tools/muscle/index.html ) and revtrans 1.4 ( www.cbs.dtu.dk/services/revtrans/ ) were used for the alignment of partial 18s rdna and cox sequences , respectively .
ml analyses of the 18s rdna sequences were performed with phyml 3.0 ( http://atgc.lirmm.fr/phyml/ ) under the tn93 + i +  substitution model ( 6 rate classes and nni algorithm for tree searching ) .
ml analyses of the cox sequences were performed under the generalized time reversible + i +  substitution model .
approximate likelihood ratios were used to estimate the branch supports of the inferred ml phylogeny , which was visualized with treegraph2 .
mega4 ( www.megasoftware.net ) was used for me analysis of the 18s rdna sequences under the tn93 + i +  substitution model , and with maximum composite likelihood for cox sequences .
the close neighbor interchange algorithm was used for tree searching , with pair - wise deletion of sequence gaps and considering differences in the substitution pattern among lineages .
the crude parasite antigen elisa was set up in a similar manner to an in - house echinococcus multilocularis elisa ( 11 ) by using larvae from the canine omentum . because no serum specimens from persons with proven armillifer spp .
infections were available as positive controls , a stored serum sample was used from a patient with a histologically confirmed l. serrata infection ( 12 ) .
ten serum samples from healthy german blood donors served as negative controls , and a standardized threshold index of 1.0 was calculated ( 11 ) .
in addition , the serum of the infected female dog was tested , as well as 10 serum samples from uninfected dogs from germany .
the crude larval antigen was also used in a western blot ( 2 g / slot ) , and all serum samples from the gambia and the serum sample from the linguatula spp.infected patient were analyzed . of the serum samples tested , only the serum sample from the snake farm owner was positive for pentastomiasis in the elisa , index 1.2 .
the control serum sample from the patient with linguatuliasis exhibited an index of 1.3 . when tested by western blot , the serum of the farm owner demonstrated a banding pattern similar to the l. serrata  positive control serum . both serum samples
exhibited 97-kda and 37-kda bands , and the serum from the patient with linguatuliasis had an additional 50-kda band ( not shown ) .
when dog serum samples were tested , only the sample from the gambia showed a positive reaction in the elisa , with an index of 1.0 .
pentastomiasis is a parasitic zoonosis with an increasing number of recognized human infections in west africa .
our investigation highlights the local transmission of infective a. armillatus ova to dogs and , by serologic evidence , also to 1 human , and thus demonstrates a public health concern .
possibly because of their eating habits ( e.g. , consumption of dead snakes ) , dogs seem to be at high risk and could function as sentinel animals . in this study
, we set up serologic assays for pentastomiasis based on raw larval a. armillatus antigens and screened the farm workers for past infection .
the infection of 1 person , the snake farm owner , could be demonstrated by elisa and western blot for human serum samples . in 1982 ,
an indirect immunofluorescence assay based on a. armillatus larvae was used for a survey in the ivory coast ; results indicated a low seroprevalence ( 13 ) . in most human cases ,
pentastomiasis is asymptomatic and is an incidental finding during surgery or autopsy , and diagnosis largely relies on parasitologic and histopathologic examination ( 2,14,15 ) .
recently , pcrs have been developed for canine pentastomiasis ( 9 ) , but dna sequences in the databases are limited to a few species of pentastomes only .
we have provided partial 18s rrna gene sequences of a. armillatus pentastomes and used pcr for the diagnosis of pentastomiasis from a clinical sample ( 9 ) .
we also constructed phylogenetic trees for all pentastome species infecting humans and animals from which sequence data were available .
phylogenetic analysis showed that pentastomes formed their own branch in proximity to the branchiura and remipedia , a finding which is consistent with results of a previous study by others who investigated a. armillatus as a sole member of the pentastomes in comparison with pancrustaceans ( 1 ) .
the nearest phylogenetic relatives of a. armillatus are a. agkistrodontis and p. crotali , 2 species of the porocephalida , followed by l. serrata and by the members of the cephalobaenida ( pentastomes that infect birds ) .
the phylogenetic trees constructed here indicate a coevolution of the pentastomes and other maxillopodan / branchiuran parasites with their vertebrate hosts ( birds , snakes , mammals , and fish ) .
we demonstrated that the serum of a patient with linguatuliasis markedly cross - reacted on the elisa and western blot based on armillifer spp .
personal hygiene measures were implemented , such as thorough hand cleansing after handling snakes and avoidance of contact with snake excretions .
public health institutions have been informed , and future studies will address the extent of seroprevalence in the local population .
 visceral pentastomiasis caused by armillifer armillatus larvae was diagnosed in 2 dogs in the gambia . 
 parasites were subjected to pcr ; phylogenetic analysis confirmed relatedness with branchiurans / crustaceans . 
 our investigation highlights transmission of infective a. armillatus ova to dogs and , by serologic evidence , also to 1 human , demonstrating a public health concern . 
The Study
Conclusions