Paragonimus gondwanensis n . sp . ( Digenea , Paragonimidae ) , parasite of mammals ( humans and carnivores ) in Cameroon

Paragonimus gondwanensis n. sp. (hitherto undescribed) is reported from the lungs of naturally infected cat ( Felis catus Linnaeus, 1758) and civet [ Civettictis civetta (Schreber, 1776)] from Cameroon. Its description is based on the morphology of adult worms but also, on the dimensio of fresh, living eggs released from naturally and experimentally infected cats and hospitalized patients (in the met rat rm of worms, emitted in the beginning of prepat nt period and laid by living worm), length of prepatent period and dur ation of eggs incubation. The presence of this spec ies in human patients is confirmed .


Introduction
The paragonimid species are known as an agent of paragonimiasis, the zoonotic parasite disease of humans and some mammals, reported from many subtropical and, especially, tropical countries.This disease is stated in 12 countries of Africa.
Paragonimiasis is a chronic condition, persisting sometimes for many years.Pathological effects are due to the presence of adult worms and their eggs in the lungs and other tissues of hosts, to the movement of parasites through tissues, and to their toxic and allergenic metabolites.The symptoms of paragonimiasis, such as bronchial rales and haemoptysis associated with paroxysms of coughing, are sometimes similar to those of tuberculosis, leading to misdiagnosis.
All paragonimid species have the complex life cycle, including two intermediate hosts (first: snail, second: crustacean) and several definitive hosts (mammals: humans, carnivores, rodents etc.).Adult worms lay eggs which are released outside in sputum or stools.In water, eggs develop in swimming miracidia which can penetrate molluscs, multiply and evolve until cercarial stages, which can infect Crustaceans.Humans and other mammals become infected when they eat raw or undercooked crustaceans harbouring metacercariae, or ingest uncooked meat of paratenic hosts such as pigs (Blair et al. 1999a).
Some authors (Zahra 1952;Vogel & Crewe 1965) reported the findings of Paragonimus westermani Kerbert, 1878; these findings were based on the identification only by measuring formalin-fixed eggs and have never been confirmed later.The presence of P. uterobilateralis was only suspected in Cameroon (Sam-Abbenyi 1985).Bayssade-Dufour et al. (1994) suggested, after specific identification by multivariate analysis of paragonimid eggs, the presence of four species of Paragonimus in Cameroon: two already known (P.africanus, P. uterobilateralis) and two undescribed ones.The later study of paragonimid eggs from 22 West-African strains, conducted by Cabaret et al. (1999), confirmed the previous findings.
Our investigations aimed to describe these two new species of Paragonimidae from Cameroon and to study their complete life cycle.In this paper, we will describe new species of Paragonimus; in the next successive publications, another new species and the life cycle of both of them will be presented.

Material and Methods
In order to describe a new species we used our original material and unpublished data and the material deposited by Cabaret et al. (1999) and by Ripert et al. (1981) in the Collection of MNHN Paris.

Research on the intermediate hosts
Collection of snails (first intermediate host) and crabs from the family Potamonautidae, such as Sudanonautes africanus, S. granulates and S. pelli (second intermediate hosts) and their dissection, in order to find paragonimid metacercariae cysts, were realized in the creeks of South Province (S) and South West Province (SW) of Cameroon.In South Province, the collection of crabs took place at Bikpwae, near Ebolowa (Minsolo Creek) at 2° 59' N and 11° 17' E; altitude 636 m.a.s.l.In South West Province, at Bulutu, near Kumba (Mejeck Creek) the search is realized at 4° 38' 25'' N and 9° 26' 21'' E; altitude 2457 m.a.s.l.(Fig. 1)

Registration numbers for final hosts
Humans: 1. 158 Other infections, with Bulutu strain metacercariae, were realized in Center of Tropical Medicine of Kumba, Cameroon, on 3 cats: 155 TM, 157 TM and 160 TM.The cats received unprecised number of metacercariae, without data on number of walls.These cats were sacrificed after confirmed emissions of paragonimid eggs in stools.
The cats were anesthetized with Imalgen or Penthotal, and then dissected by routine procedure.The adult parasites and eggs were isolated from the lungs.
Three individuals of Mus musculus were infected with 8, 16 and 10 metacercariae, respectively.Seven individuals of Rattus rattus were infected with 4, 7, 2, 4, 7, 20 and 8 metacercariae and five individuals of Acomys sp. with 14 metacercariae (2 with a cyst and 12 excysted).The individuals of M. musculus were sacrificed between 112 and 136 days after the metacercarial meal.Three individuals of R. rattus were sacrificed after 30 days, two individuals after 156 days and remaining two after 83 or 115 days.The five individuals of Acomys were sacrificed after 172 days.
The rodents were anesthetized with CO 2 inhalation, and then dissected by routine procedure.South Province: the specimen of one adult paragonimid species from Civettictis civetta, deposited by Ripert et al. (1981) in the collection of MNHN Paris (registered as 894 TA 1 ), was also used in description of new species.

Study methods for eggs
Five parameters were selected: 1. length of prepatent period: the cats 186 TM and 187 TM were monitored, 2. size of living eggs emitted at the beginning of the prepatent period (from 187 TM 3, and 187 TM 5 ), 3. size of eggs laid by a living worms from cat (187 TM n ) and humans (143 TM n and 158 TM-1017 n ), 4. size of eggs in the metraterm (terminal portion of the uterus) of worms (187 TM 3 and 187 TM 5 ), 5. duration of incubation, until hatching of eggs (from feces: 101 TM and 102 TM; from tracheal mucus: 186 TM and 187 TM n ).South West Province.One parameter was considered: the size of eggs in the metraterm of worms (187 TM 3,5 and 143 TM 1 ).

Study methods for adult worms
Three studied worms from South Province (894 TA 1 , 187 TM 3 , 187 TM 5 ) and one from South West Province (157 TM 4 ), were flattened between slide and coverslip and fixed in formaldehyde.Then, they were stained in toto by Chlorhydric Carmin, mounted in Canada basalm and drawn with camera lucida.

Etymology:
The specific name P. gondwanensis come from Gondwana, the name of ancient continent uniting Africa and South America.

Description of the adult worms
They came from the lungs of final hosts, naturally or experimentally infected.New species is described after 4 adult ovigerous worms: 894 TA 1 , 187 TM 3 , 187 TM 5 and 157 TM 4 .
The bodies of these worms (from fixed, stained and mounted specimens, more or less flattened), measure 7 x 4,5 mm for 894 TA 1 , 7,5 x 3,7 mm for 187 TM 3,5 , 7 x 3 mm for 157 TM 4 .All present a plump elongate body, with spined tegument.Oral sucker ventro-terminal, ventral sucker in the 1/3 of the body.Oral sucker diameter/ventral sucker diameter ratio (OS/VS): OS/VS>1 in adults; OS/VS=1 in young specimen; OS/VS<1 in metacercaria.Digestive apparatus with a mouth in oral sucker, pharynx well developed, oesophagus short, ondulating caeca large, reaching the hindbody.The genital apparatus with a lobed ovary opposite to uterus; uterus convoluted, ovary and uterus pretesticular, seminal receptacle small, profuse dendritic vitellaria, extending dorso-laterally, from the forebody to the hindbody, two transverse vitelloducts ending in a median rounded bag, metraterm opening in hermaphrodite pore, immediately posterior to the acetabulum, lobed testes in hindbody, characteristics of the Paragonimidae.
All four worms had a short excretory bladder, reaching the lower part of acetabulum.Ovary with five deep lobes.Mehlis gland small.Seminal receptacle small.Two large testes star-shaped with 4-6 deep lobes with basis in form of "corolla".Thin spermiducts, reaching a crook curved seminal vesicle, devoid of cirrus sac, near a short pars prostatica, lying on the left side, between the uterus and the acetabulum.Uterus overlapping, on the left side, the anterior part of testis and transversal part of vitelloducts.The Laurer's canal was not conspicuous.
The duration of incubation (35 eggs monitored), at 22 °C, was 55-56 days, with a very long period of morula stage.
Prepatent period (period between infection with a parasite and appearance of eggs in feces) was 66 days.

Discussion
The paragonimid species with short excretory bladder seem to be very rare.Chen (1962) described, in China, the new species with that type of bladder (Euparagonimus cenocopiosus) and created new genus Euparagonimus; one year later (1963), Chen proposed new subfamily Euparagoniminae.Chen & Ho (1965) gave more details on adults, metacercaria and egg size of above mentioned species.The second species of the genus, E. hongzesienzis, was described by Hu et al. (1990) in China.Unfortunately, the authors did not give enough details or drawings.Miyazaki & Hendricks (1975) reported the finding, in Panama, of a very rare metacercaria with short bladder belonging, probably, to the genus Euparagonimus (the species was never described).(1999), modified; and C -P. uterobilateralis (scale bar 300 µm), after Voelker & Sachs (1985), modified.Kurochkin (1987) recognized E. cenocopiosus as a valid species, and Blair et al. (1999a) recognized both of described species (E.cenocopiosus and E. hongzesienzis).Later the same year, on the basis of DNA sequence data obtained by his group of authors and morphometric analysis of 16 paragonimid species (Zhan et al. 1997), Blair et al. (1999b) placed E. cenocopiosus within the genus Paragonimus and concluded "it seems that this species should not be placed in a distinct genus".Their conclusion was confirmed by Blair (2008): "the former genus (Euparagonimus) must fall as a synonym and the subfamily Euparagoniminae Chen, 1963 is not recognized".
So, the present new species, Paragonimus gondwanensis n. sp., differs from all other Paragonimus species (with exception of P. cenocopiosus) in the length of excretory bladder (Fig. 3).This species is the third paragonimid species described from Africa.
New species differs from P. uterobilateralis by: -the body size (Table 1) -the size of oral sucker and OS/VS ratio (Table 1) -the shape and size of excretory bladder (Figs.3A, 3C) -the shape and size of testes and ovaries (Figs.4A, 4C, 5A, 5C) -the uterus overlapping the left testis (Fig. 2) -the size of eggs (Table 1).
P. uterobilateralis differs from all African paragonimds by an uterus extending into both halves of the body.
South West Province: one cat, registered as 156 TM, native of Kumba, naturally infected, releasing Paragonimidae eggs in stools, was examined, too.Only one adult worm remained for identification at the Faculty of Medicine Paris V.The sputum of several hospitalized children, suffering from paragonimiasis, was studied and the paragonimid eggs were stated.The 35 eggs from the sputum of two boys from Béchati (registered as 143 TM and 158 TM-1017), where measured (143 TM n and 158 TM-1017 n ).
The metacercariae, extracted from naturally infected crab Sudanonautes africanus(Milne Edwards, 1869), were used for experimental infection of cat (Felis catus Linnaeus, 1758), Rattus rattus (Linnaeus, 1758), Mus musculus Linnaeus, 1758 and Acomys sp.South Province: the first cat, registered as 186 TM, was infected with 19 metacercariae from Bikpwae strain (14 with 2 cyst walls, 2 with 1 wall and 3 without wall); the second cat, 187 TM, with 34 metacercariae, from the same strain (28 with 2 cyst walls, 6 with 1 wall).The cats 186 TM and 187 TM were sacrificed 334 and 85 days after metacercarial meal, respectively.Both experiments were realized in Veterinary School of Maisons-Alfort in Paris.South West Province: Two other cats, 101 TM and 102 TM, were infected in Faculty of Medicine Paris V with Bulutu strain metacercariae.The cat, registered as 101 TM, was infected with 15 metacercariae with 1 cyst wall; the cat 102 TM, with 26 metacrecariae with 2 cyst walls.They have been sacrificed 136 and 362 days after metacercarial meal, respectively.
TM-1017 (patient from South West Province) 2. 143 TM (patient from South West Province) Cats: 1. 186 TM (from South Province) 2. 187 TM (from South Province) 3. TM (from South West Province) 4. TM (from South West Province) 5. TM (from South West Province) 6. TM (from South West Province) 7. TM (from South West Province) Civet: 1. TA (from South Province) Figure 1.Studied area in Cameroon: -sampling sites.n (where n=batches of eggs from different hosts) Experimental infection of laboratory animals (cats and rodents)

Table 1 .
Comparative measurements of adult African paragonimids and their eggs (OS-oral sucker; VS-ventral sucker; mean values in brackets).