Description of the Life-Cycle Stages of Brachylaima Cribbi N. Sp. (Digenea: Brachylaimidae) Derived from Eggs Recovered from Human Faeces in Australia
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Systematic Parasitology 49: 211–221, 2001. 211 © 2001 Kluwer Academic Publishers. Printed in the Netherlands. Description of the life-cycle stages of Brachylaima cribbi n. sp. (Digenea: Brachylaimidae) derived from eggs recovered from human faeces in Australia A. R. Butcher & D. I. Grove Institute of Medical and Veterinary Science, The Queen Elizabeth Hospital, Department of Clinical Microbiology and Infectious Diseases, 28 Woodville Road, Woodville, South Australia 5011, Australia Accepted for publication 23rd October, 2000 Abstract The life-cycle of Brachylaima cribbi n. sp. was established in the laboratory. Asymmetrical brachylaimid eggs, measuring 26-32 µm (29.1µm) long and 16 -17.5 µm (16.6µm) wide, were recovered from human faeces and fed to the helicid land snail Theba pisana as the first intermediate host. Sporocysts and cercariae were recovered from the T. pisana eight weeks after infection. The cercariae were used to infect the helicid land snails Cernuella virgata and Helix aspersa as second intermediate hosts. Metacercariae were recovered from the kidneys of these snails and used to infect mice. Adults of Brachylaima cribbi n. sp. were recovered from the small intestine of the mice. The differential features of B. cribbi n. sp. are the specificity for helicid snails as first and second intermediate hosts; characteristic ventral sucker and body cercarial chaetotaxy; and a long slender adult worm with equal size suckers in the first quarter of the worm, the ventral sucker occupying 41 % of the body width, the uterus extending anterior to the ventral sucker and the vitelline follicles falling short of the posterior margin of the ventral sucker. No other known Brachylaima species exhibits all of these features. B. cribbi n. sp. is the first brachylaimid known to have infected humans and is probably of European origin, as the intermediate host snails were all introduced into Australia from Europe. Introduction In South Australia, other than the human infec- tions, a species of Brachylaima has been reported from Species of Brachylaima Dujardin, 1843 (Digenea: the house mouse Mus domesticus Linnaeus (Angel & Brachylaimidae) are trematodes of mammals and birds Mutze, 1987; Cribb, 1990), the native greater stick- and have land snails as the first and second intermedi- nest rat Leporillus conditor Sturt (Cribb, 1990) and ate hosts (Yamaguti, 1971; Yamaguti, 1975). There domestic free-range chickens (Cribb & O’ Callaghan, had been no reported human infections until those de- 1992). Cribb & O’ Callaghan (1992) could find no scribed in two children (Butcher et al., 1996) and an differences between the specimens from these reports adult (Butcher et al., 1998) from South Australia. The and considered them to be all the same species. Cribb description of these infections was based on the find- (1990) described the life-cycle of this Brachylaima ing of eggs which were characteristic of brachylaimids sp. in naturally infected Mus domesticus and Lep- in the patients’ faeces, the recovery after anthelmintic orillus conditor and in experimentally infected Rat- therapy of a partly decomposed worm that was typi- tus fuscipes Waterhouse (Australian bush rat). Intro- cal of a Brachylaima sp. and the finding of larvae of duced European helicid land snails Cernuella virgata Brachylaima sp. in local helicid land snails that have da Costa, Cochlicella barbara Ferussac and Theba been introduced from Europe. Nevertheless, insuffi- pisana Müller are both first and second intermediate cient material was available to definitively determine hosts (Cribb, 1990; Cribb & O’ Callaghan, 1992). the species of brachylaimid. Butcher et al. (1996, 1998) determined that these same 212 snails were involved in the human infections as one of cover-glass pressure with Bouin’s fluid and stained the children infected ate these snails. with borax-carmine or Mayer’s haematoxylin stains, The family Brachylaimidae is large with many as described by Pritchard & Kruse (1982). Argen- poorly described species dating back to the early 19th tophilic papillae of the cercaria were stained with 5% Century. Brachylaima is particularly confusing with silver nitrate for 5 minutes then washed in distilled considerable variation in the spelling of the taxon water and mounted in Puri’s chloral-gum medium in the literature, poorly or incompletely described (Upton, 1993). Cercarial chaetotaxy was determined life-cycles and a number of morphologically similar microscopically and figures prepared with the aid of species (Mas Coma & Montoliu, 1986; Cribb, 1992; a camera lucida. Argentophilic papillae nomenclature Ubelaker & Dailey, 1966). It is essential to study the followed that described by Bayssade-Dufour (1974) complete life-cycle, describing all stages and hosts and Mas-Coma & Montoliu (1986) for brachylaimid before describing a new species in order to prevent cercariae. All measurements are in micrometres (µm) the proliferation of confusing taxonomic data. These using the system of measurement proposed for brachy- problems presented considerable difficulties in identi- laimids by Mas-Coma et al. (1984) and are given fication of the species implicated in human infections. as a range and mean in parentheses. Line drawings A life-cycle was therefore established in the laboratory of life-cycle stages were prepared with the aid of a using eggs recovered from the faeces of an infected camera lucida. Representative specimens of all life- human. Host selection was based on the life-cycle de- cycle stages have been deposited in the Australian tails described by Cribb (1990) for Brachylaima sp. Helminthological Collection (AHC), South Australian in South Australia. We now report the morphological Museum, Adelaide, South Australia. features of the key stages of the worm which warrants the description of a new species, Brachylaima cribbi n. sp. Brachylaima cribbi n. sp. Type-material: Holotype ex Mus musculus C57BL/6J Materials and methods mice 4 weeks post-infection (wpi), experimental life- cycle derived from eggs from human faeces, Adelaide, Life-cycle South Australia, March 2000, coll. A.R. Butcher, Eggs were recovered from the faeces of a 78 year AHC 28351. Paratypes: same host and location, 7 old woman and placed on filter paper in a large plas- specimens from 1-6 wpi, AHC 28352-8. Metacer- tic petri dish. Helicid land snails, Theba pisana,that cariae ex Helix aspersa (Helicidae) experimental life- were free of Brachylaima sporocyst infection were al- cycle, AHC 28359 & 28360. Cercariae and sporocyst lowed to feed on the human faeces. Cercariae began ex Theba pisana (Helicidae) experimental life-cycle to emerge 8 weeks after exposure. Laboratory-reared AHC 28361-3. helicid snails, Cernuella virgata and Helix aspersa Müller, were exposed to these cercariae suspended in Description a thin film of water in a petri dish for 4-5 hours. After 8 weeks metacercariae were removed from the kid- Adult worm (Figures 1-7) ney of the snails and given to C57BL/6J black Mus Gravid adults were recovered from the small intestine musculus by intra-oesophageal inoculation. Develop- of C57BL/6J Mus musculus (mice). Live specimens ing and adult worms were removed from the intestinal were able to extend to a length of 10-13 mm or con- tract of the mice at weekly intervals. Eggs were re- tract to as small as 2 mm. Worms of varying ages covered from mouse faeces for morphological study from 1-7 wpi were compared to evaluate allometry and continuation of the life-cycle. Further details of (Table 1). Juvenile adults 1-3 wpi were found to have the establishment of the life-cycle in the laboratory are a lower width to length ratio, the forebody occupied detailed elsewhere (in preparation). a greater proportion of the total body length and the mid-body (posterior margin of the ventral sucker to Light microscopy the margin of the anterior testis) was less than in Eggs, sporocysts, cercariae, metacercariae and adult older worms. Worms of greater than 3 wpi were con- worms were examined alive using the vital stains sidered to be mature adults and showed consistent neutral red or methylene blue, or were fixed under morphological measurements. 213 Table 1. Measurements (in micrometres) of Brachylaima cribbi n. sp. adult worms ex Mus musculus C57BL/6J mice. Age as weeks post infection 1 2 3 4-7 4-7 Number measured 10 10 10 30 Mean Mean Mean Mean Range Body Length 2,619 4,135 4,561 5,004 (3,800-6,010) Width 485 629 603 678 (520-790) Length/width 5.5 6.6 7.6 7.4 (5.4-9.8) Oral sucker (OS) Length 198 234 244 259 (230-290) Width 218 248 257 279 (250-380) Ventral sucker (VS) Length 200 241 255 277 (240-320) Width 187 230 240 267 (230-300) Body width at VS 466 611 589 673 (520-790) VS width parallel to lateral margins 119 238 248 271 (240-320) Width as % of body width at VS 42% 39 42% 41% (32-54) Ratio area VS to OS 0.87 0.96 0.98 1.03 (0.66-1.17) Pharynx Length 111 137 148 157 (140-180) Width 124 156 154 169 (150-220) Forebody 558 737 795 831 (620-1,150) Forebody as a % of body length 21% 18% 18% 17% (13-20) Inter-sucker distance 320 472 528 537 (300-810) Inter-sucker distance as % of body length 12.0 11.4 11.5 10.7 (6.1-14.0) Midbody Posterior margin of VS to anterior testis 999 1,921 2,129 2,559 (1,760-3,235) Above as % of body length 38% 46% 47% 51% (43-59) Inter-testis distance 157 221 197 245 (120-440) Above as a % of body length 5.9% 5.3% 4.4% 4.9% (2.6-7.3) Posterior testis to posterior end of body 186 235 325 323 (200-680) Above as a % of body length 7.1% 5.6% 7.1% 6.5% (3.8-14.7) Uterus +/− from anterior margin of VS 111 268 194 230 (10-420) Above as a % of body length 4.1% 6.5% 4.3% 4.6% (0.2-8.2) Ovary Length 162 207 195 217 (150-260) Width 191 272 232 261 (170-320) Vitelline follicles Left 1,114 1,833 1,835 2,438 (1,650-3,650) Right 1,024 1,887 2,054 2,466 (1,670-3,590) Most anterior extent +/− from post.