Cryptosporidium infection in ostriches ( Struthio camelus ) in Brazil : Clinical , Morphological and Molecular Studies

Avian cryptosporidiosis has been reported in more than 30 species of birds. To date, the species infecting birds are C. baileyi, C. galli and C. meleagridis. In this study, the morphological, clinical and molecular characteristics of a Brazilian ostrich isolate of Cryptosporidium are described. The oocysts of this Brazilian isolate are larger and more elongated than those of Cryptosporidium previously reported in ostriches, which were morphologically similar to C. meleagridis. Morphological, biological and molecular analyses demonstrated similarity of this ostrich isolate with C. baileyi, suggesting that there are at least two Cryptosporidium species infecting ostriches; one with molecular, biological and morphological characteristics related to C. baileyi, and another morphologically similar to C. meleagridis.

carried out cross transmission studies and morphological analysis of oocysts of a Cryptosporidium isolate recovered from ostriches, and there was no cross transmission to suckling mice, chicken, turkey and Japanese quail.Morphological analysis showed that oocysts diverged morphologically from that of C. baileyi and were similar to oocysts of C. meleagridis, and suggested that the ostrich isolate might represent another species of Cryptosporidium from birds.
In this study, the morphological, clinical and molecular characteristics of a Brazilian ostrich isolate of Cryptosporidium are described.

Birds
High mortality rates of unknown origin were recorded from seven to 30-days-old in ostriches raised at the facilities of the Veterinary Medicine Course of Universidade do Estado de São Paulo (UNESP), campus of Araçatuba, São Paulo, Brazil.Birds were thin and showed secondary bacterial infection sporadically, with yellow fatty liver and cloacal prolapse.From five birds which had cloacal prolapse, two were sent for necropsy and parasitological screening.

Parasites
Fresh harvested oocysts obtained from fecal samples of the examined birds were purified in discontinuous sucrose and cesium chloride gradients (Arrowood & Donaldson, 1996) and stored in 2.5% potassium dichromate at +4 C.

Morphological Analysis
The size and morphology of the oocysts were determined by evaluating 100 oocysts using malachite green (Elliot et al., 1999) with an Olympus BX-45 microscope equipped with a calibrated ocular micrometer.

Histopathology and Mucosal Smears
Fragments of proventriculum, duodenum, jejunum, ileum, ceca, proximal, medium and distal rectum, coprodeum, urodeum and bursa of Fabricius were collected, routinely processed for histopathology and stained with hematoxylin and eosin.Mucosal smears of the same organs were stained by modified Kinyoun acid-fast staining.

Genomic DNA extraction
DNA was extracted with phenol-chlorophorm as previously described (Sréter et al., 2000) and purified using the Prep-A Gene DNA purification system (Bio-Rad ® ).

Nested-PCR reaction
Cryptosporidium species were identified by a nested-PCR/RFLP (Xiao et al., 1999).Ultra-pure autoclaved water and DNA of a bovine C. parvum isolate which had been previously identified by PCR-RFLP were used as negative and positive controls, respectively.

Restriction
Fragment Length Polymorphism (RFLP) For RFLP analysis, 20 µl of secondary PCR product were digested with a mixture containing 20 U of restriction enzyme SspI or AseI (New England Biolabs ® ), and 5 µl of restriction buffer at 37 o C for 1 h, as recommended by the supplier.
The products of PCR/RFLP were analyzed by electrophoresis on a 1.5% agarose gel, stained with ethidium bromide and visualized under ultraviolet light.

RESULTS
Oocysts of the ostrich isolate measured 6.0 x 4.8 µm (5.0-6.5 X 4.2-5.3)with a shape index (length/width) of 1.31 (n=100) (Figure 1).The two birds with cloacal prolapse examined by histological sections had developmental stages of Cryptosporidium covering the border of cloacal epithelial cells.
Mucosal smears of naturally infected ostriches showed developmental Cryptosporidium stages mainly in the distal rectum and coprodeum.Slight infection was observed at the mucosa of proximal and medium rectum, urodeum, and rarely in the ceca and bursa of Fabricius.
Histological sections revealed the parasite only in distal rectum, coprodeum, urodeum and rarely in the bursa of Fabricius.Microscopic lesions were seen mainly in coprodeum and urodeum as an infiltrate of monoand polymorphonuclear cells, epithelial hyperplasia, mucosal swelling and many ovoid parasites in the border of epithelial cells (Figures 2 and 3).There was necrosis and atrophy of the bursa of Fabricius in the absence of developmental stages of Cryptosporidium in the bursal epithelium.Nested-PCR resulted in fragments of approximately 1,300 bp and 830 bp for primary and secondary reaction respectively.The digestion of the secondary product with enzymes SspI and AseI yielded bands of approximately 250 and 570 bp and 100 and 620 bp, respectively.

DISCUSSION
There were no evidences of relationships between ostrich mortality, bursal necrosis and Cryptosporidium infection.Stressing conditions leading to immunossupression or poor husbandry practices related to feed, water or hygiene apparently must have been present as predisposing factors to the onset of cloacal prolapse or other pathology related to the species of Cryptosporidium found in this experiment, since the improvement in husbandry practices has stopped the mortality and clinical signs, even in the presence of the parasite.
Oocysts of the Brazilian isolate are larger and more elongated than those of Cryptosporidium found in ostriches by Gajadhar (1993Gajadhar ( , 1994)).The author described the presence of oocysts related to the occurrence of subclinical cryptosporidiosis in adult ostriches, but no description of the development site of the parasite in the birds was available.Oocysts were spherical to subspherical and measured 4.6 x 4.0 mm; shape index (length/width) 1.15.
There is no information on which species of Cryptosporidium may infect ostriches.Probably the present ostrich isolate is the same species related to cloacal prolapse in other reports (Allwright & Wessels, 1993, Penrith et al., 994, Jardine & Verwoerd, 1997).Since the morphological data of the isolate described by Gajadhar (1994) are related to C. meleagridis, which are spherical and measure 5.2 x 4.6 µm; shape index 1.13 (Lindsay et al., 1989), and not to C. baileyi, probably there are 2 Cryptosporidium species infecting ostriches, one with molecular, biological and morphological characteristics related to C. baileyi and a second one morphologically related to C. meleagridis.
It is possible to differentiate C. baileyi from C. meleagridis by visualization of the electrophoresis profile of digested bands of the secondary reaction product.Nevertheless, it is not possible to differentiate between species/genotypes with similar digestion profiles without sequencing the amplified fragments (Xiao et al., 1999).Egyed et al., (2002) recommended that polyphasic typing must be accomplished for species not yet classified, with evaluation of biological characteristics, besides the molecular analysis of the parasite.
For a definitive classification of this isolate as C. baileyi or as a new species infecting ostriches, the comparison of the sequenced fragments would be necessary, followed by phylogenetic analysis.Besides, further studies involving its biological and morphological characteristics would also be necessary.

Figure 2 -
Figure 2 -Urodeum epithelium of an ostrich infected with Cryptosporidium sp.showing mononuclear cell infiltration in the submucosa and developmental stages of the parasite in the epithelial surface (arrowhead).

Figure 3 -
Figure 3 -Coprodeum epithelium of an ostrich infected with Cryptosporidium sp.showing severe infiltration of heterophils, macrophages, and mononuclear cells in the submucosa and developmental stages of the parasite in the epithelial surface (arrowhead).