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Morphological diagnosis of infective larvae of Libyostrongylus douglassii (Cobbold, 1882) Lane, 1923 and L. dentatus Hoberg, Lloyd and Omar, 1995 (Nematoda: Trichostrongylidae) of ostriches

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Abstract

The differentiation of the species of the Libyostrongylus genus is only possible with the obtainment of the adult parasites in the ostriches proventriculus and gizzard. The present work confirms that it is possible to differentiate the infective larvae of L. douglassii and L. dentatus allowing the differential diagnosis of these species by fecal culture. To show this, adult females from both species were collected from ten proventriculus from adult ostriches and separated by species. Both groups were macerated individually added to sterilized feces for standard fecal cultures. The infective larvae were recovered, identified, quantified and measured. All proventriculus analyzed were parasitized by Libyostrongylus spp. and a clear heterogeneous location for each species was observed. The infective larvae from the fecal cultures of macerated L. douglassii presented a mean total length of 874.3 ± 33.80 μm, and a short sheath tail (29.5 ± 4.11 μm) with acute termination. The infective larvae from the macerated L. dentatus presented mean total length of 856.0 ± 43.63 μm, long sheath tail (61.2 ± 9.52 μm) with filamentous termination. The mean measures of the tails of both species had a significant difference. The differentiation of the infective larvae of L. douglassii and L. dentatus by fecal cultures will facilitate the diagnosis of both species for further understanding the Libyostrongylus biology.

Introduction

Nematodes of the Libyostrongylus genus are small hematophagous worms found below the proventriculus membrane of ostriches (McKeena, 2005). These parasites are responsible for 50% mortality of juvenile ostriches, occasionally killing adults (Reinecke, 1983, Sotiraki et al., 2001). This genus is from the Trichostrongylidae family and is composed by the species L. douglassii (Cobbold, 1882) Lane, 1923, L. magnus Gilbert, 1937 and L. dentatus Hoberg, Lloyd and Omar, 1995. L. douglassii has been noticed in ostriches in Africa (Cobbold, 1882), Australia (Barton and Seward, 1993), Europe (Ponce Gordo et al., 2002), North (Hoberg et al., 1995) and South America (Bonadiman et al., 2006, Ederli et al., 2008), while L. dentatus only in the American continent (Hoberg et al., 1995, Bonadiman et al., 2006, Ederli et al., 2008) and L. magnus just in Africa (Hoberg et al., 1995).

Libyostrongylus spp. diagnosis includes clinical manifestations such as: anorexia, weight loss, anemia and proventricular impactation (Huchzermeyer, 1998). Confirmation is achieved by the finding of typical Trichostrongylidae eggs in feces, with subsequent fecal culture to differentiate infective larvae of species such as Codiostomum struthionis, a Strongylidae parasite of the cecum not considered pathogenic. The differentiation of the Libyostrongylus species is possible only by the characterization of adults that need to be collected during necropsy of the proventriculus or gizzard. However, our group has recently reported significant size differences of the sheath tail of two types of infective larvae obtained by fecal cultures (Bonadiman et al., 2006). It was suggested that the morphology of the infective larvae could be used for diagnosis and, thus, a mixed infection of L. douglassii and L. dentatus was probably present. The mixed infection was confirmed by necropsies of proventriculus and adult worm analyses (Ederli et al., 2008). Here, we confirmed the diagnosis hypothesis proposed before (Bonadiman et al., 2006). Female adults of L. douglassii and L. dentatus were speciated, macerated, eggs cultured, and infective larvae were analyzed morphologically. These results show that it is possible to differentiate both species without necropsy, thus, allowing the species diagnosis by fecal cultures, which will provide a better understanding of the biology of both species and its prevalence all over the world.

Section snippets

Adult nematode collection

Ten adult ostriches from Rio de Janeiro state, Brazil were sacrificed for the collection of adult worms. The proventriculus were separated from the digestive system, emptied, conditioned in plastic bags and transported to the laboratory in isothermal boxes. Species of the genus Libyostrongylus were collected with the aid of a brush under a stereo microscope and transferred to petry dishes with a 0.9% saline solution at room temperature until the separation by sex and species.

Obtainment of infective larvae and microscopic characterization

Females of L.

Results

All analyzed proventriculus had both species of Libyostrongylus. When the koilin layer was removed it was easy to observe L. douglassii at the inner surface of this membrane and at the tissue of the proventriculus. On the other hand, L. dentatus was inserted in the koilin layer with a reeled formation. The absolute number of both adults were not quantified, however, because adult females were separated in equal groups we can surely affirm that L. douglassii dominated the examined proventriculus.

Discussion

Infection by Libyostrongylus is a major parasitism of ostriches (Reinecke, 1983) spread all over the world (Cobbold, 1882, Barton and Seward, 1993, Ponce Gordo et al., 2002, Hoberg et al., 1995, Bonadiman et al., 2006, Ederli et al., 2008). Although there are three species of Libyostrongylus, only L. douglassii and L. dentatus have been reported outside Africa. In addition, L. dentatus has been detected only by two research groups (Hoberg et al., 1995, Ederli et al., 2008), showing that this

Acknowledgments

The authors would like to thank Andrèa Carvalho César for proof reading the manuscript and Fabiana Valadão Massad for technical assistance in the collection of the parasites. This study was supported by Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), and Fundação Carlos Chagas Filho do Rio de Janeiro (FAPERJ).

References (14)

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