Isospora atrata n. sp.(Apicomplexa, Eimeriidae): a new coccidium isolated from Carduelis atrata (Passeriformes, Fringillidae)

Zsospora atrata N. Sp. (Apicomplexa, Eimeriidae): a New Coccidium Isolated from Carduelis atrata (Passeriformes, Fringillidae) GIACOMO ROSSI,' STEFANIA PERRUCCI and GUIDO MACCHIONI Dipurtimento Di Putologiu Animale Proflassi ed lgiene Degli Alimenti, Facolta Di Medicinu Veterinuriu, Universitu Di Pisa, Viu le delle Piufige, 2, 56/00 Pisu, Ita1.v ABSTRACT. Large numbers of coccidian oocysts belonging to the genus lsospora were obtained from the intestinal contents of 98 Carduelis utrafu imported into Italy from South America during the months of August through December 1994. The oocysts are subspherical and average 21 X 20.3 pm (19.4-23.5 pm X 18.5-22 &in), have a bilayered wall, and an oval polar granule (rarely two). The sporocysts are elliptical and measure 18.8 pm X 10.3 pm (17.5-18.94 pm X 9.5-11.0 pm). The Stieda body protrudes slightly from the end of the sporocyst. A large sporocyst residuum is present, consisting of many granules that may be in a compact mass or scattered. Since this Isospora sp.does not resemble any other species of Isospora previously described from birds of the genus Carduelis. it has been named Isosporu utratu n. sp. after the host. Disseminated asexual stages were found in mononuclear cells derived from formalinfixed post mortem material, suggesting this coccidian may represent an Atoxripplusma-like parasite. Four coccidia-free Serinus cunarius L. cohabitated for a long period (4 mo) with infected C. utrata but oocysts were never found in the stool of these birds. Supplementary key words. Curduelis atratu, black siskin, lsospora atruta n. sp., Aroxoplasma-like parasite.

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ARDUELIS atruta (La Fresnaye and D'Orbigny 1937) is a species belonging to the Family Fringillidae, subfamily Carduetinae. Native to South America, this species is found in a well-defined area of the Andean region that extends from southern Peru to northern Chile and to western Argentina in a range from 1,800-4,800 m above sea level. During the last 10 yr this species has been heavily imported into Europe for reproduction purposes, both pure breed and hybridization. During the months of August through December 1994, 98 Carduelis atrutu were imported into Italy from Bolivia and Peru by two dealers (from Pisa and Alessandria). On arrival, all birds had diarrhoea, swollen and congested abdomen, breast muscle atrophy, and an abnormally increased appetite. No bacterial a n d or viral organisms were isolated from culture prepared with material taken from recently deceased birds. Eighty birds died within 72 h of arrival, 15 died within 5 wk of arrival and only 3 survived. Great quantities of coccidial oocysts were found, both by fresh fecal and flotation tests, in the stool samples of recently imported birds. Coccidial oocysts were also present in the intestine of the deceased subjects immediately autopsied. Since there was no mention in the existing literature of Isospora sp. isolated from C. atrutu, we examined the various species of Isosporu isolated from other birds of the genus Carduelis in order to classify our findings. MATERIALS AND METHODS Stool samples were taken from newly arrived subjects and from the intestinal lumen of the deceased C. utratu. More than one single sample from each of the 98 birds was always observed. During the observation period (August-December 1994), four coccidia-free Serinus cunarius L. lived with the infected lots of C. utrutu. Our interest was to verify whether there would be any cross infection of Isospora between these two genera of passeriformes. Infected birds were identified by microscopically observing stool samples of fresh materials or material obtained after fecal flottation using a 33% zinc sulphate solution. Both the fecal material and scrapings from the intestinal mucosa of infected birds were dissolved in a 2% K,Cr,O, solution and maintained at room temperature (22" C -t 1" C) to allow sporulation of the oocysts. Oocysts have been checked every 12 h to evaluate the sporulation time. Ninety-five of the 98 subjects of C. utrata were autopsied upon death; samples were taken from various sections of the intestines, i.e. duodenal loop, jejunum, ileum and the rest of the gut, to determine which was most affected. In addition, the

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intestines and all other organs from infected birds were fixed in 10% buffered formalin and processed for routine histological tests. Voucher specimens are deposited at the Department of Animal Pathology, Profilaxis and Food Hygiene, Faculty of Veterinary Medicine, University of Pisa (Italy), no. 6243 and in the Museum of Veterinary School, University of Naples, Via E Delpino, 1/80137 Naples (Italy), no. 469/3A. RESULTS

Isosporu utruta n. sp. (Fig. 1-3) Description. Oocysts are spherical or subspherical and measure 21 X 20.3 pm (19.4-23.5 pm X 18.5-22 pm), with a IengtNwidth ratio of 1.03 (1-1.06 pm), and a bilayered wall 1.2 pm thick. Micropyle and oocyst residuum are absent while an oval polar granule (rarely two) is present. Sporocysts are elliptical and measure 18.8 X 10.3 pm (17.5-18.94 X 9.5-11.0 pm); with a IengtNwidth ratio of 1.82 (1.76-1.88 pm). They contain a sporocyst residuum, lacking a limiting membrane, consisting either of scattered granules or a compact mass. The Stieda complex, consisting of a slight thickening of the wall and of a substiedal body with a convex-linear base, is present. The sickle or spindle-shaped sporozoites have a smaller round anterior refractile body (about 1.8 X 1.8 pm) and a larger elliptical posterior refractile body (about 1.8 X 3 pm) and measure 7.64 X 2.2 pm after 48 h, and l l X 3 pm (10-12 pm X 2-4 pm) after 60 d. Sporulation time. Within 48 h at 22" C -C 1" C. Host. Curduelis atrutu (Passeriformes: Fringillidae), "black siskin." Geographical area. Imported into Italy from Peru, Bolivia, and Argentina. Prevalence. 98/98 (100%) of the examined subjects. Location in host. All of the intestinal tract, although the highest oocyst concentration and most significant lesions were found in the ileum. Extra-intestinal localization is presumable on the basis of schizont-like elements found in mononuclear phagocytes in blood and in various organs (i.e. liver, spleen, lungs, heart, and pancreas). DISCUSSION Several biomorphological differences emerged from a comparative study of I. utrutu and other species of Isosporu isolated from birds of the genus Curduelis (I. chloridis, I. carduelis, I. lacuzei, I. mcquistioni, I. bioccui, I. urrui, I. cunnubinu, I. cunnabinae). Isosporu utruta differs from I. chloridis [ l ] in that the latter presents larger ellipsoid oocysts with a single wall.

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Fig. 1, 2. Two photomicrographs of sporulated oocysts of Isospor-u ufr-u/un. sp.(after a week in potassium dichromate). Bars

Isospora chloridis sporocysts are piriform while those found in I. atrata are perfectly elliptical. More significantly, the Stieda complex is button-shaped in I. chloridis, while in I. atrata the Stieda complex consists only of a slight thickening of the sporocyst wall at its anterior pole. Lastly, the sporocyst residuum of I. chloridis is surrounded by a membrane which is always absent in I. atrata. There are several differences between I. atrata and I. carduelis [ll]; the latter is characterized by an oocyst wall made up of a single layer, is larger, and has morphologically different, tapering sporocysts. A comparison between I. atrata and I. lacazei [7] is more difficult because of the discrepancies in the biometric and morphological description given by various authors [6-91 I. Zacazei was first described by Labbk [7] from C. carduelis. Later, however, I. Zacazei was reported from in a large number of passeriformes and other birds, but Levine and Mohan [9] recognized that it might very well be a complex of several species. Later, Levine [8] considered the genuine I. lacazei to be that described by Hemandez-Rodriguez et al. [6] from C. carduelis in Spain,

Fig. 3. Line drawing of sporulated oocyst of Isosporu mrufu n. sp Bar = 10 pm.

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10 pm

so we used only the Labbt: [7] and Hernandez-Rodriguez et al. [6] descriptions of this species. I. lacazei presents a double wall with a marked salmon-orange color; according to Labbk, the Stieda complex, at the apex of its piriform sporocysts, is characterized by a button-shaped thickening of the wall. Furthermore, according to the biometric description of HemandezRodriguez et al. [6], the oocyst, the sporocysts, and the sporozoites of I. Iacazei are all larger than those of I. atrata. A comparison of the sporulation times recorded by Labbk (4-5 d) for I. Zacazei implies significant biological differences between the two species. There are also marked differences between I. atrata, I. rncquistioni, and I. arrui [4, 101. The oocysts of the last two species are larger and have an ovoid shape, a Stieda complex with a prominent button, and a compact spherical sporocyst residuum. Isospora bioccai and I. cannabina [4, 101 most closely resembles I. atrata, although there are differences in size and in the morphology of the polar granule, which is oval in I. atrata and spindle-shaped in I. bioccai and I. cannabina. However, we can observe a Stieda complex with a large buttonshaped thickening in I. bioccai and I. cannabina, which is not present in I. atrata. Furthermore, while the sporocyst residuum is consistently large and contains many granules in I. atrata, it is diffuse with scattered granules in I. bioccai. Lastly, the sporozoites in I. bioccai and I. cannabina present a single refractile body while there are always two refractile bodies in I. atrata. Finally, also I. cannabinae [12] shows marked differences if compared to I. atrata for having larger and ovoid oocysts, pointed sporocysts that, using Gottschalk descriptions and figures of these species, completely lack of a Stiedal complex. From a comparative study of I. atrata and numerous other Isospora spp. isolated from passerine birds [ 5 ] ,it emerges that Isospora serini is the only Isospora species which is morphologically similar to I. atrata. Isospora serini was isolated from S. canarius by Box in 1975 [2]. There are several morphological similarities between these two Isospora species (the appearance of their Stieda complex and the dimensions of the I. serini sporozoites and those of I. atrata 60 d after sporulation).

ROSS1 ET AL.-ISOSPORA

ATKATA, A NEW COCCIDIUM

There are also biological similarities, such as sporulation time and an extra-intestinal cycle characterized by an asexual phase in mononuclear phagocytes. We can, however, rule out the possibility that I. utrata and I. serini are members of a single species, because they were isolated from hosts that do not belong to the same genus. Furthermore, because none of the four S. cunurius that cohabitated with infected C. atruta produced oocysts or developed organic lesions, particulary not in the intestinal tract. The histopathological exam of the various sections of intestine taken from infected C. atruta revealed serious lesions, due to deep enteritis, with catarrhal-hemorragic exudate and areas of necrosis in the mucosa. A heavy leucocyte infiltration, consisting mainly of limphomonocytic cells, of the extremely thickened mucosa was also detected. There were also grave hepato-toxic lesions due to the massive migration of bacteria and toxins through the heavily damaged intestinal wall, as well as spleen damage and congestive-hemorragic pneumonia. Schizonts, similar to those observed by Box [2, 31 in I. serini infections of S. canarius, were present in the monocytes of the intestinal lamina propria blood vessels, as well as in hepatic, splenic and pulmonary macrophages of all the autopsied animals. In spite of a lack of more complete data that would allow for a more specific comparison, we believe that the oocysts isolated from C. atrata may be considered a new species of Isospora, the first to be isolated from this animal species. The particular area in which this species lives (plateaus in the Cordillera of the Andes ranging from 1,800-4,800 m above sea level), however, is very different from that of the Carduelis spp. studied up to now (the Euro-Asian continent) for coccidia. Further study is required to verify the possibility of an extra-intestinal cycle of the parasite. ACKNOWLEDGMENTS We thank Dr. Capecchi Alamanno for his exhaustive taxonomical research on Curduelis atruta and Dr. Candela Rosa E. for translating this manuscript.

LITERATURE CITED 1. Anwar, M. 1966. Isosporu lucuzei (Lahhe. 1893) and I . chloridis sp. n. (Protozoa: Eimeriidae) from the English sparrow ( P c m e r domest i c ~ . and ~ ) chaffing (Frinxillu coe1eb.s). J. P rotozool.. 13:84-90. 2. Box, E. D. 1975. Exogenous stages of lsosporu serini (Aragao) and Iso.cporu canaria sp. n. in the Canary (Serinus cunurius L.). J . Prf(>Z