Veterinary Parasitology 92 (2000) 173179

Entamoeba sp. (Sarcomastigophora: Endamoebidae) from ostriches (Struthio camelus) (Aves: Struthionidae)
R.A. Martnez-Daz a, , S. Herrera a , A. Castro a , F. Ponce b
b a Departamento de Medicina Preventiva (Parasitologa), Facultad de Medicina, Universidad Autnoma de Madrid, C/Arzobispo Morcillo s/n, 28029 Madrid, Spain Departamento de Parasitologa, Facultad de Farmacia, Universidad Complutense de Madrid, Pza. Ramn y Cajal s/n (Ciudad Universitaria), 28040 Madrid, Spain

Received 23 February 2000; received in revised form 29 May 2000; accepted 6 June 2000

Abstract The rst case of Entamoeba of the 1-nucleate mature cyst group in birds is described. Trophozoites and cysts of Entamoeba have been found in ostriches (Struthio camelus) from farms located all over Spain. The cysts are large (13.47 m mean diameter); they possess one nucleus when mature, with a large endosome and peripheral chromatine arranged in small granules; chromatoid bodies, when present, are large and elongated. Trophozoites are large (19.88 m mean diameter), with a clear differentiation between ecto- and endoplasm, this containing numerous vacuoles; the nucleus is large and diffuse. The characteristics of this amoeba resembles but do not completely fulll those of E. suis and E. chattoni; also, these species are from mammals. 2000 Elsevier Science B.V. All rights reserved.
Keywords: Entamoeba sp.; 1-Nucleate mature cyst group; Morphology; Host range; Birds; Ostrich

1. Introduction The genus Entamoeba includes a number of commensal and parasitic species from shes, amphibians, reptiles, birds, mammals and some invertebrates (Wenyon, 1926; Neveaus-Lemaire, 1943; Neal, 1966; Kudo, 1972; Levine, 1985). These species can be divided into groups based on the number of nuclei in their mature cysts (Neal, 1966); the results from molecular biology analyses have conrmed the validity of this criterion
Corresponding author. Tel.: +34-1-397-53-11; fax: +34-1-397-53-53. E-mail address: rafael.martinez@uam.es (R.A. Martnez-Daz).

0304-4017/00/$ see front matter 2000 Elsevier Science B.V. All rights reserved. PII: S 0 3 0 4 - 4 0 1 7 ( 0 0 ) 0 0 3 1 4 - 9

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(Clarck and Diamond, 1997). According to Levine (1985), ve groups could be established: (a) the E. coli-like group, with 8-nucleate mature cysts; (b) the E. histolytica-like group, with 4-nucleate mature cysts; (c) the E. bovis-like group, with 1-nucleate mature cysts; (d) the E. gingivalis-like group, in which the cyst is unknown; (e) inadequately known species. There are only two species of Entamoeba described in birds: E. gallinarum, of the coli group, in chicken, turkey, guinea fowl, duck and goose; and E. anatis, of the histolytica group, in duck (Levine, 1985; Silvanose et al., 1998). No Entamoeba species have been cited from ostriches (Struthio camelus), although there are several observations on the presence of amoebae in this bird (Craig and Diamond, 1996; Carbajo et al., 1997). In samplings carried out during 19971999 in more than 80 ostrich farms from all over Spain, we have found over 80% of the samples positive to cysts (and occasionally, trophozoites) of amoebae belonging to the E. bovis-like group (Martnez Daz et al., 1999). In the present work, a study on the morphology and morphometry of the cysts and trophozoites of Entamoeba found in ostriches is performed, in order to compare this amoeba with the species cited in birds and with the species of the 1-nucleate mature cysts group (=bovis group).

2. Materials and methods The biological material analyzed has consisted of feces from farmed ostriches (255 samples, 86 farms from all over Spain) and feces and intestinal contents from slaughtered ostriches (52 samples, six farms from the central area of Spain), collected during the period 19971999. Most of the ostriches (about 95%) were born and raised in Spain, but some were imported directly from Africa, or from other European countries (Great Britain, Belgium, Holland and France). Special care was taken when sampling to avoid feces contamination with soil, or intestinal contents with other host uids. Samples from slaughtered animals were transported to the laboratory in less than 2 h after sacrice. Fecal material (both from farms and slaughterhouses) were processed using the formaline ethyl acetate stool concentration technique (Levine and Estevez, 1983). Cysts were detected under microscopy, at 200400, using wet mounts stained with Lugols iodine. From the samples found positive, smears of the intestinal contents and of the feces were made and stained with chlorazole black. Smears of intestinal contents and diarrheic feces were also stained with Giemsa. One-nucleate cysts were found in nearly 90% of the samples from farm and sacriced ostriches; a few positive and negative farm samples were diarrheic, but their etiology was unknown. Active trophozoites were observed only in two non-diarrheic, cyst-positive slaughtered animals. Both cysts and trophozoites from these two samples, and cysts from one diarrheic and two normal, randomly chosen, farm samples were examined in detail under optical microscopy, at 1000. Morphological and morphometric data on cysts and trophozoites were recorded from stained slides; also, data from Lugol-stained cysts were taken. Statistical analyses (ANOVA) were performed to detect differences between host samples and (with data from trophozoites) between staining methods.

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3. Results The morphometric data of trophozoites and cysts are presented in Table 1. Trophozoites stained with chlorazole black (Fig. 1) showed a low-dened nucleus that led to important observer bias when measuring its diameter. For this reason, the only character recorded from chlorazole black stained trophozoites is the diameter of the cell. No statistical differences (p < 0.05) between host samples nor between trophozoite staining methods were found. Trophozoites (Fig. 1) were usually large with a clear differentiation between endoplasm (very granular with vacuoles containing bacteria and yeast) and ectoplasm. The nucleus was large and diffuse; in vivo, it appeared as a hardly recognizable ring-like body. The movement varied depending on consistency and temperature of the sample: when most active, the trophozoites showed an almost unidirectional projection of pseudopodes, and a rapid locomotion; at low temperatures or with low water content in the medium, amoebae were very sluggish, with multidirectional emission of pseudopodes and tend to round up. Cysts were large and almost always spherical (Fig. 2), rarely ovoid or elongated. Binucleated cysts have been observed with a very low frequency (<1%). The endosome was very large, generally irregular (occasionally spherical) and usually eccentric. Peripheral chromatin was sometimes a rather homogeneous ring within the nuclear membrane, but in most of the cases this chromatin was arranged in granules of different size, giving the picture of an irregular ring (Fig. 2). Small chromatin granules were sometimes present between

Table 1 Morphometrical characteristics of trophozoites and cysts of Entamoeba from ostriches (measurements in m) Chlorazole black staining Trophozoites Cell Maximum diameter (mean S.D.) Size range Nucleus Maximum diameter (mean S.D.) Size range (n = 100) 19.72 5.38 835
a a

Giemsa staining (n = 100) 20.03 4.53 832 (n = 51) 5.17 0.65 47 Lugol staining (n = 185) 12.73 2.07 819 (n = 185) 3.95 0.83 26

Total (n = 200) 19.88 4.97 835 (n = 51) 5.17 0.65 47

Chlorazole black staining Cysts Cyst Maximum diameter (mean S.D.) Size range Nucleus Maximum diameter (mean S.D.) Size range Endosome Maximum diameter (mean S.D.) Size range
a

(n = 176) 13.47 1.98 820 (n = 171) 4.34 0.83 27 (n = 167) 1.73 0.43 13

Not determined owing to observer bias. See text for details.

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Fig. 1. Active trophozoites of Entamoeba from ostrich: (a) Giemsa staining; (b) chlorazole black staining. E ectoplasm; V vacuoles; N nucleus. Scale bar: 10 m.

the nuclear membrane and the endosome. With a low frequency, vacuoles and chromatoid bodies (very large, elongated and with rounded ends) were found in the cytoplasm (Fig. 2).

4. Discussion The only Entamoeba species cited in birds are E. gallinarum (with 8-nucleate mature cysts) and E. anatis (with 4-nucleate mature cysts) (Levine, 1985; Silvanose et al., 1998), neither of them in ratites (ostrich, rhea, emu, cassowary, etc.). These two species can be discarded as responsible for the ostrich infection, as it has never been observed cysts with more than two nuclei (and these, in less than 1% of the cysts). All Entamoeba species with 1-nucleate mature cysts have been described exclusively from mammals. These species are E. bovis, E. ovis, E. dilimani, E. polecki, E. suis, E. debliecki, E. bubalis and E. chattoni. Their differentiation was made on the basis

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Fig. 2. Cysts of Entamoeba from ostrich: (a) temporal Lugol staining; (b, c) chlorazole black permanent staining. Scale bar: 10 m.

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of their morphology and/or host species. Some problems have been pointed out regarding their use to validate specic status (Wenrich, 1941; Neal, 1966), and some synonyms may exist. For the comparison of the Entamoeba from ostrich with those of the bovis group, the species we consider are E. bovis, E. ovis, E. dilimani, E. suis (=E. polecki, =E. debliecki), E. chattoni (in part, possible synonym of E. suis) and E. bubalis (following Wenyon, 1926; Kudo, 1972; Levine, 1985). The morphology of both the trophozoites and cysts of Entamoeba from ostriches is compatible with those of the other species of the bovis group, except E. dilimani (with a smaller endosome) and E. bubalis (with the endosome giving the picture of a cluster of four granules; Levine, 1985). In relation to size, both trophozoites and cysts of Entamoeba from ostriches are larger than those of other species of the bovis group. It is well known that some environmental conditions (related to the nutritional richness of the media) may affect the size of the trophozoite, while the cyst size is relatively stable (Neal, 1966). In vivo, the nutritional characteristics of the intestinal content could be related not only to host lesions, but also to their different chemical compositions in different host species. In consequence, the in vivo greater size of the trophozoites from ostriches may not be a valid differential characteristic, and only the cyst size should be considered. Using this criterion, only E. suis and E. chattoni have approximate cyst sizes to that of Entamoeba from ostriches. The specic status of the Entamoeba from ostrich cannot be clearly established from the data available. Its morphology and size resemble to those of some of the described species but they do not completely fulll them, and this is the rst case of a bird as host of Entamoeba of the 1-nucleate mature cyst group. Further work on cross-transmission infections and on biochemical and genetic comparative analysis are needed to clarify their taxonomic status.

5. Conclusions This is the rst report of an Entamoeba of the 1-nucleate mature cyst group in birds. This amoeba has been observed infecting farmed ostriches in Spain. It is widely distributed and seems to be non-pathogenic. Both the trophozoites and cysts of this amoeba present some differences in morphology and size with the described Entamoeba species of the 1-nucleate mature cyst group. Further work on cross-transmission and on biochemical and genetic comparative analysis are needed in order to establish if it is a mammal Entamoeba affecting a bird, or if it is a new species.

Acknowledgements The authors wish to thank Carlos Prez Ruiz for his contribution to the collection of ostrich fecal samples from farms, and to Dr. Luis Zapatero Ramos for his critical comments. References
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