Paragonimus kerberti n.sp. (Digenea, Paragonimidae), parasite of carnivores in Cameroon

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1 ISSN (online) ISSN (print) The journal is available on line at Research Article Paragonimus kerberti n.sp. (Digenea, Paragonimidae), parasite of carnivores in Cameroon CHRISTIANE BAYSSADE-DUFOUR 1, RENÉ CHERMETTE 2, DANIJELA ŠUNDIĆ 3 & BRANKO M. RADUJKOVIĆ 4,* 1 Unité Molécules de Communication et Adaptation des Microorganismes (MCAM, UMR 7245), Sorbonne Univeristés, Muséum National d'histoire Naturelle, CNRS; CP52, 61, rue Buffon, Paris, France 2 Ecole Nationale Vétérinaire d'alfort, Unité de Parasitologie et Maladies Parasitaires, 7, av. du Général de Gaulle, Maisons-Alfort, France 3 Aerodromska 7, Podgorica, Montenegro. 4 University of Montenegro, Faculty of Sciences, Džordža Vašingtona bb, PoBox 5455, Podgorica, Montenegro. brradujkovic@yahoo.com. * Corresponding author: danijelasun@yahoo.com Received 21 March 2015 Accepted 25 May 2015 Published online 26 May Abstract Paragonimus kerberti n.sp. (hitherto undescribed) is reported from the lungs of artificially infected cat (Felis catus Linnaeus, 1758) from Cameroon. Its description is based on the morphology of adult worms but also, on the dimension of fresh, living eggs released from experimentally infected cats (in the metraterm of mature worms, or laid by living worm), length of prepatent period and duration of eggs incubation. Key words: Paragonimus kerberti n. sp., Paragonimidae, Digenea, Cameroon. Introduction Although the first report on paragonimiasis in Africa was published in the beginning of twentieth century (Onorato, 1920), the first description of African Paragonimidae species was done 45 years later. Voelker and Vogel (1965) described 2 species of Paragonimus, from different mammals, in Cameroon and Liberia (P. africanus and P. uterobilateralis). 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 (mentioned above) and two other, unknown and undescribed ones. Cabaret et al. (1999) conducted the study of 22 isolates of paragonimid eggs (13 newly collected) from Cameroon, Gabon, Liberia, Guinea and Congo. The authors confirmed the presence of 4 paragonimid species: P. africanus (in Cameroon), P. uterobilateralis (in Liberia, Gabon, Guinea and Congo), Euparagonimus sp. in Cameroon and Ivory Coast (provisionally named; later described as Paragonimus gondwanensis by Bayssade-Dufour, Chermette, Šundić & Radujković, 2014) and P. westermani-like in Cameroon and Gabon (provisionally named). We are going to describe the last one as a new species Paragonimus kerberti n. sp. Ecol. Mont., 2 (3), 2015,

2 PARAGONIMUS KERBERTI N.SP. FROM CAMEROON Material and Methods Research on the intermediate hosts Collection of crabs from the family Potamonautidae, Sudanonautes africanus (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 E; altitude 636 m.a.s.l. In South West Province, at Bulutu, near Kumba (Mejeck Creek) the search was realized at N and E; altitude 2457 m.a.s.l. (Fig. 1). In the Minsolo creek, South Province (S), 18% of crabs were infected by metacercariae, i.e. 73 crabs out of 400 dissected ones. Infection rate was seasonal: 37.1% (February-March), 18.9% (April-May) and 13% (June-July). No significant difference in number of metacercariae in male and female crabs was stated. The mean number of metacercariae by infected crabs was about 4. The total of 118 metacercariae were isolated from 27 crabs: 82 (69.5%) was found in thoracic muscles, 24 (20.3%) in pereiopods, 6 (5.1%) in chelipeds, 5 (4.3%) in hemolymph and 1 (0.8%) in gills. In Bulutu, South West Province (SW), 95.6% of crabs were infected by metacercariae, i.e. 89 crabs out of 93 dissected ones. Infection rate was equal all over the year. The mean number of metacercariae by infected crabs was about 21. The total of 1,909 metacercariae were isolated from 89 crabs: 1,187 (62.1%) in thoracic muscles, 546 (28.6%) in pereiopods and 176 (9.2%) in chelipeds. Registration numbers for final hosts Cats: TM (from South West Province) - experimentally infected TM (from South Province) - experimentally infected TM (from South Province) - experimentally infected Registration numbers for adult parasites TM 1 (from South West Province) TM 1 (from South Province) TM 1 (from South Province) TM 2 (from South Province) Studied eggs are marked as: 187 TM n (where n=batches of eggs from different hosts) Experimental infection of laboratory animals (cats) The metacercariae, extracted from naturally infected crab Sudanonautes africanus (Milne Edwards, 1869), were used for experimental infection of cat (Felis catus Linnaeus, 1758). South Province: the first cat, registered as 186 TM, was infected with 19 metacercariae (14 with 2 cyst walls, 2 with 1 wall and 3 with one soft wall). The second one, registered as 187 TM, was infected with 34 metacercariae (6 with the soft wall, the wall number of other 28 not stated!). Experiments were realized in Veterinary School of Maisons-Alfort in Paris (Service of Prof. R. Chermette). The cat 187 TM was sacrificed 85 days after metacercarial meal (2 adult worms of P. kerberti n.sp. were collected) and the cat 186 TM after 331 day (15 adult worms of P. kerberti found, four stained or used for different analyses). South West Province: the cat, 101 TM, was infected in Faculty of Medicine Paris V with Bulutu strain metacercariae. The cat was infected with 26 metacrecariae with 2 cyst walls. This cat has been sacrificed 362 days after metacercarial meal and 4 adult worms of P. kerberti were collected. The specimen 101 TM 1, was used for serial longitudinal histological section, in order to reconstruct the shape and size of small organs and confirm the presence of new species in SW Province. The cats were tranquillized and anesthetized with Calmivet R and Dolethal, and then dissected by routine procedure. The adult parasites and eggs were isolated from the lungs. Study methods for adult worms Three studied worms from South Province (186 TM 1, 187 TM 1, 187 TM 2 ) were flattened between slide and coverslip and fixed in formaldehyde. Then, they were stained in toto by Chlorhydric Carmin, mounted in 272

3 BAYSSADE-DUFOUR ET AL. Canada basalm and drawn with camera lucida. The worm 101 TM 1 from South-West Province, was prepared for longitudinal sections and histological examination. Measurements of the worms are given in micrometers (µm). Forebody refers to the body region anterior to the ventral sucker, and hindbody refers to the body region posterior to the ventral sucker. Figure 1. Studied area in Cameroon: sampling sites. Ecol. Mont., 2 (3), 2015,

4 PARAGONIMUS KERBERTI N.SP. FROM CAMEROON Study methods for metacercariae The metacercariae were excysted spontaneously in 0.9 % NaCl solution. A part of them were excysted with needles, or by shaking cysts in water. Study methods for eggs Parameters selected: 1. length of prepatent period: the cat 187 TM was monitored, 2. size of living eggs emitted at the beginning of the prepatent period (from 187 TM 1, 187 TM 2 ), 3. size of eggs laid by a living worms from cat (187 TM n ) 4. size of eggs in the metraterm (terminal portion of the uterus) of worms (187 TM 1, 187 TM 2 ), 5. duration of incubation, until hatching of eggs (from feces: 101 TM; from tracheal mucus: 187 TM). Results New paragonimid species, Paragonimus kerberti n. sp., was found in different pulmonary cysts of infected cats. Adult worms were present in pair, in the same cyst. Family Paragonimidae Dollfus, 1939 [Syn. Euparagoniminae Chen, 1963] Genus Paragonimus Braun, 1899 [Syn. Polysarcus Looss, 1899 Euparagonimus Chen, 1962 Pagumogonimus Chen, 1963 Rodentigonimus Chen, 1963 Megagonimus Chen, 1963] Paragonimus kerberti n. sp. (Figs. 2A-B, 3) Material studied: Adult worms from experimentally infected cats were studied. The type specimens were deposited in National History Museum of Paris, France. Registration numbers: holotype 187 TM 1 ; paratype 187 TM 2. Etymology: The specific name Paragonimus kerberti was given in honour to Coenraad Kerbert, who described P. westermani, type species of the genus (in 1878). First intermediate host (snail): unknown. Second intermediate host (crustacean): Sudanonautes africanus (A. Milne-Edwards, 1869). Definitive hosts: Felis catus (experimental). Site: lungs. Type locality: Bikpwae, in South Province of Cameroon: 2 59 N, E, altitude: 636 m.a.s.l. (Fig. 1) Other localities: Bulutu, Béchati and Mahollé, near Tombel in South-West province of Cameroon. Bulutu is settled at N, E; altitude 2457 m.a.s.l. Mahollé is settled at N, E; altitude 523 m.a.s.l. (Fig. 1). Description of the adult worms The adult worms originated from the lungs of final hosts, experimentally infected. New species is described after 3 adult ovigerous worms: 186 TM 1, 187 TM 1, 187 TM 2. Measurements were done upon one young (85 days old) and one older (331 days old) specimen. 274

5 BAYSSADE-DUFOUR ET AL. The bodies of these worms (from fixed, stained and mounted specimens, more or less flattened), measured 7,692 µm x 4,600 µm for young specimen, and 10,900 µm x 6,000 µm for the old one. Forebody 2,840 3,400 long; hindbody 4,840 7,400 long. Ratio forebody/hindbody All present an elongate body, with spinous tegument. Oral sucker rounded, ventro-terminal, ventral sucker in the 1/3 of the body. Oral sucker 770 1,200. Ventral sucker rounded, Ratio oral sucker/ventral sucker (OS/VS) in adults: 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, with 5-10 thick lobes, like a hand, opposite to uterus (1,307 x 846 1,500 x 1,000). Uterus curled, drawing an 8 (visible in young specimen), the first curl crossing the metraterm, near the hermaphrodite pore (4,460 5,400). Ovary and uterus pretesticular. Metraterm opening in hermaphrodite pore, immediately posterior to the acetabulum. Mehlis gland voluminous, median (540 1,050). Vitellin system abundant, dendritic, extended in latero-dorsal fields of the body. Two transverse vitelloducts ending in a median rounded bag. Two large, unequal, triangular testes, larger anteriorly than posteriorly, with 6-10 small rounded lobes, both in hindbody (left testes 2,150x1,000 3,500x2,000; right testes 1,770x1,307 3,400x2,500). Seminal vesicle falciform, 1,000 1,600. Two spermiducts reaching the seminal vesicle under the acetabulum. The excretory bladder long, reaching the caecal bifurcation (6,460 9,100). Figure 2. Paragonimus kerberti n. sp., scale bars 1,000 µm (vitellaria not presented). A - young specimen, 85 days old (note: uterus in the form of 8 ); B - mature specimen, 331 days old. Ecol. Mont., 2 (3), 2015,

6 PARAGONIMUS KERBERTI N.SP. FROM CAMEROON Description of the metacercariae Excysted living metacercariae measured x µm. They had a long excretory bladder, filled with dark granules, extending either between the two suckers, or close to the pharynx. The excretory pore was terminal. Suckers subequal; OS/VS ratio range from 0.75 to Figure 3. Metacercariae of P. kerberti n. sp. (scale bar 100 µm). Measurement of eggs The metraterm eggs measure in length 98 µm, based on 20 specimens. First living laid eggs measure 102 (85-115) x 60 (48-75) µm (20 specimens). The duration of incubation (35 eggs monitored), at 22 C, was 37 days, with a very long period of morula stage. Prepatent period (period between infection with a parasite and appearance of eggs in feces) unknown, probably about days. In cat 187 TM all the mated worms after 85 days were ovigerous. Remarks Paragonimus kerberti n.sp. is the fourth paragonimid species described from Africa. This species can be distinguished from other African paragonimids by four main characteristics: triangular testes, larger anteriorly than posteriorly, with small rounded lobes on the inner part, hand shape ovaries with 5-10 lobes, falciform seminal vesicle, posterior to the ventral sucker, utrerus in the form of 8. Acknowledgements The authors give their thanks to Dr. J.P. Chippaux, Dr. J. Prod hon and Dr. M. Boussinesq (Institute of Research for Development, Yaoundé, Cameroon), who sent the paragonimid metacercariae, collected from crabs of South Province to the Museum of Paris and National Veterinary School of Maisons-Alfort, in order to infest the cats and determine the adult Paragonimidae; to Dr. C. Ripert who sent us two adult paragonimids from naturally infested civet from South Province; to Dr. R. Moyou-Somo (Institute of Medical Research and the Study of Medicinal Plant, Cameroon) who kindly provided us with several adult specimen of Paragonimidae from South-West Province in Cameroon; to Miss G. Guemgne Simo (now G. Tami) who identified three types of paragonimid metacercariae and two adults. We give our thanks to Dr. N. Cumberlidge (Northern Michigan University) for identification of crabs from South Province; to Dr. N. Guinot (MNHN Paris) for identification of crabs from South-West Province; to Professor J. Diebold (Faculty of Medicine Cordeliers, Paris) and Dr N.P. Vuong (Saint-Michel Hospital, Paris) for their histological slides and Mrs. D. Guillaumin (Faculty of Sciences, Paris-Raspail) who performed S.E.M. and photographs of metacercariae and adult paragonimids. We are also grateful to our colleagues Dr. J. Albaret (MNHN, Paris), Dr. H. Picot (Faculty of Medicine Cordeliers, Paris) and Dr. J. Cabaret (National Institute of Agronomic Research, Nouzilly), for their valuable scientific 276

7 BAYSSADE-DUFOUR ET AL. support during our investigation; also to Dr. M. Kombila and her team (B. Mabika and S. Ogoula-Gerbeix) for providing us with parasite eggs from Gabonese patient and his clinical data. We express the special thanks to ing. Soraya Chaouch for her appreciable assistance and Jelena Tomić for the English text redaction of this paper. References Bayssade-Dufour, C., Cabaret, J., Tami, G. & Chippaux, J.P. (1994) Classification problems in Paragonimus spp., Trematoda. Specific identification by multivariate analysis of the eggs. International Congress of Parasitology, Izmir, Turkey. Abstracts 1, p Bayssade-Dufour, C., Chermette, R., Šundić, D. & Radujković, B.M. (2014) Paragonimus gondwanensis n. sp. (Digenea, Paragonimidae), parasite of mammals (humans and carnivores) in Cameroon. Ecologica Montenegrina, 1 (4), Cabaret, J., Bayssade-Dufour, Ch., Tami, G. & Albaret, J.L. (1999) Identification of African Paragonimidae by multivariate analysis of the eggs. Acta Tropica, 72, Onorato, R. (1920) La paragonimiasi in Tripolitania. Archivio Italiano di Scienze Mediche Coloniali, Tripoli, 1 (1), Voelker, J. & Vogel, H. (1965) Zwei neue Paragonimus-Arten aus West-Africa: Paragonimus africanus und Paragonimus uterobilateralis (Troglotrematidae, Trematoda). Zeitschrift für Tropenmedizin und Parasitologie, 16, Ecol. Mont., 2 (3), 2015,