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Libyostrongylus douglassii (Strongylida: Trichostrongylidae) in ostrich (Struthio camelus) farms from Colombia
Accepted Manuscript Title: Libyostrongylus douglassii (Strongylida: Trichostrongylidae) in Ostrich (Struthio camelus) Farms from Colombia Author: Guillermo A. Mari˜no-Gonz´alez Alejandro Ram´ırez-Hern´andez Jes´us A. Cort´es-Vecino PII: DOI: Reference:
S0304-4017(17)30007-9 http://dx.doi.org/doi:10.1016/j.vetpar.2017.01.007 VETPAR 8228
To appear in:
Veterinary Parasitology
Received date: Revised date: Accepted date:
10-10-2016 8-12-2016 9-1-2017
Please cite this article as: Mari˜no-Gonz´alez, Guillermo A., Ram´ırez-Hern´andez, Alejandro, Cort´es-Vecino, Jes´us A., Libyostrongylus douglassii (Strongylida: Trichostrongylidae) in Ostrich (Struthio camelus) Farms from Colombia.Veterinary Parasitology http://dx.doi.org/10.1016/j.vetpar.2017.01.007 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Libyostrongylus douglassii (Strongylida: Trichostrongylidae) in Ostrich (Struthio camelus) Farms from Colombia.
Guillermo A. Mariño-Gonzáleza, Alejandro Ramírez-Hernándeza,b; Jesús A. CortésVecinoa,* a. Grupo Parasitología Veterinaria, Departamento Salud Animal, Facultad de Medicina Veterinaria y de Zootecnia, Universidad Nacional de Colombia, Cra. 30 No. 45-03, Ciudad Universitaria, Edificio 481, Bogotá D.C., Colombia. b. Departamento de Medicina Veterinária Preventiva e Saúde Animal, Faculdade de Medicina Veterinária e Zootecnia, Universidade de São Paulo, Av. Prof. Orlando Marques de Paiva, 87, CEP 05508-000, Cidade Universitária, São Paulo, SP, Brazil.
*Corresponding author: Laboratorio de Parasitología Veterinaria, Facultad de Medicina Veterinaria y de Zootecnia, Universidad Nacional de Colombia, Cra. 30 No. 45-03, Ciudad Universitaria, Edificio 481-Laboratorio 4, Bogotá D.C., Colombia. Tel.:+571 3165000 (Ext. 15333-15314) E-mail address: [email protected] (J.A. Cortés-Vecino)
Highlights
Libyostrongylus douglassii detected for the first time in ostriches from Colombia.
Previous Libyostrongylus spp. infections in Meta department are confirmed.
Possible mixed infections with L. dentatus could not be totally discarded.
Abstract Ostrich farming is an important livestock industry in different world regions with a diverse offer of products and services. In Colombia, as in other countries, this market led the importation of animals from countries like Canada, United States of America and South Africa for breeding objectives. With the animals, specific pathogens for these ratites could be introduced. Libyostrongylus spp. is a strongylid nematode with worldwide distribution, which can induce a severe disease and mortality in infected animals. Limited studies in Colombia have identified parasites in ostrich farming systems. The aim of this study was to identify parasites of the genus Libyostrongylus to a species level in faecal samples from ostrich farms in three departments of Colombia. Five ostrich farms from Boyacá, Meta and Tolima were sampled in 2011 and in 2013 to obtain fresh faecal samples which were further processed by flotation tests for egg visualization and faecal culture for infective larvae identification by morphological and morphometric parameters. One from the five farms, located in Meta department, was positive for strongylid eggs in both sampling periods. After faecal culture, infective larvae were identified as Libyostrongylus douglassii. These results corroborate previous records of Libyostrongylus in Ostrich farms from Meta and confirms, for the first time, infection by L. douglassii in ratites from this region. Further studies must
identify associated determinants for infection and its effects on the flock health and production. Keywords: Nematoda; Parasitic Diseases; Ratites; South America 1. Introduction The physical and physiological characteristics of the ostrich (Struthio camelus) have led to a renewed global industrial interest in this species, which has in turn led to a proliferation of farms and animals in different countries, including Colombia. Different ecto and endoparasites like ticks, protozoan, cestodes and nematodes could affect ostriches (Ponce-Gordo et al., 2002; Huchzermeyer, 2002). An example of the latter is Libyostrongylus Lane, 1923, a typical strongylid nematode of this bird (Deeming, 1999), which has been detected in ostrich farming worldwide (Barton and Seward, 1993; Hoberg et al., 1995; Landman and Bronneberg, 2001; Mackereth, 2004; Bonadiman et al., 2006; Tisljar et al., 2007). The related parasitism has been tied to high youth morbidity and mortality rates, having its greatest impact on animals of up to three months (50%), and even up to six months (20-30%), and a low mortality rate in adult ostriches (<10%) which can maintain variable parasitic loads without manifestation of clinical signs (Mackereth, 2004; Bastianello et al., 2005; Bonadiman et al., 2006; de Andrade et al., 2011). The disease is characterized by a local gastric inflammatory process involving the proventriculus (Ederli and Oliveira, 2009) which can lead to ulcerative lesions of varying degree, which can perforate and liberate content into the coelomic cavity in a disease process known as ‘rotten stomach’ or ‘vrottmaag’ (Huchzermeyer, 2002).
Three species of Libyostrongylus are known: L. douglassii (Cobbold, 1882) Lane, 1923, L. dentatus Hoberg, Lloyd and Omar, 1995 and L. magnus Gilbert, 1937 (Ederli and de Oliveira, 2015). L. douglassii, identified as the most pathogenic, has a worldwide distribution; contrarily, L. dentatus and L. magnus have been reported only in the American and African continent, respectively (Hoberg et al., 1995; Huchzermeyer, 1999; Ederli and de Oliveira, 2015). In Colombia, limited studies have identified its presence and there is a lack of knowledge about the species involved, its distribution and production impact. The aim of this study was to identify parasites of the genus Libyostrongylus to a species level in faecal samples from ostrich farms in three departments of Colombia. 2. Materials and methods Sampling was done on five ostrich farms in three different departments of Colombia: one from Villa de Leyva (Boyacá), one from Puerto López (Meta), one from Coello (Tolima) and two from Coyaima (Tolima) (see Supplement Material for additional information on localization of each farm). Samples were taken in two different periods as follows: between September and December 2011 in Coello, Coyaima, Puerto López and Villa de Leyva; and November 2013 in Puerto López. Fresh faecal samples were taken from the ground with new plastic bags within five minutes of defecation as previously recommended (Bonadiman et al., 2006; Lelis et al., 2014). They were transported in an insulated box, at a temperature of 4-7°C, to the Veterinary Parasitology Laboratory (Universidad Nacional de Colombia) and analysed within the following 24 hours. Approximately, 2 g of faecal matter were sieved to separate gross particles and centrifuged (1.500 r.p.m. for 5 minutes) to
perform a flotation test with Sheather’s sugar solution (specific gravity = 1.27). At the same time, a faecal culture was set up in which samples were homogenized in their respective transportation containers, and 6-8 grams (or 10 ml in liquid samples) were taken and placed at the bottom of a 300 ml disposable cup, creating a lightly compact form. The faecal sample was then moistened, but not diluted, using a fine atomized spray until a pool or layer of water of approximately 5 mm in height was formed. The samples were incubated at an average temperature of 27°C and relative humidity around 60%, as previously reported (Bonadiman et al., 2006). Incubation was controlled daily and involved rinsing the samples and observing the obtained matter under a light microscope in 100 and 400x magnification. In addition, serial flotation tests were performed to confirm changes in the eggs morphology throughout the study until the appearance of larvae. In order to limit their movement, they were refrigerated before being observed and measuring for genus and species classification. The following morphological variables, proposed by Ederli et al. (2008a) for species differentiation, were compared: total length of the larva (a), sheath tail length (b), and the ratio between both parameters (ratio = a/b) (c). Mean and standard deviations for morphometric data were calculated and further analysed using R software (version 3.0.1) (R-Core-Team, 2014). 3. Results
Analysis through flotation tests, confirmed the presence of strongyliform eggs in two different sampling periods (September 2011 and November 2013) from the ostrich farm located in Puerto López (Meta). Following the incubation period, larvae morphologically and morphometrically compatible with the genus Libyostrongylus were identified (Figure 1). The incubation period ranged from 6 to 22 days. A total of 102 larvae from both collections were identified as Libyostrongylus spp. mainly by the presence of the knob in the tail and were further measured, presenting an average total length of 888.02 μm (± SD: 25.53), an average sheath tail length of 26.88 μm (± 5.36) and an average ratio of 34.41 μm (± 7.10) (Table 1). Consequently, all larvae were morphologically identified as L. douglassii after comparing the latter measures with published data (Ederli et al., 2008c; Ederli et al., 2008d) and matched their morphologic characteristics (Table 1, Figure 1). In addition, different development steps of the parasite eggs, including some cell and larvae development phases, were recorded during the incubation phase (Figure 1). 4. Discussion Species level identification through routine flotation techniques is not possible due to undetectable differences between eggs of the Libyostrongylus genus and Codiostomum sp., a strongylid that parasitizes the distal third of the ostrich caecum and is apparently innocuous (Huchzermeyer, 2002; Ederli et al., 2008a; Ederli et al., 2008b; Ederli et al., 2008c; Ederli and Rodrigues de Oliveira, 2014). The use of these techniques, however, may be recommended as an approximation upon the
screening of new animals entering the production system (McKenna, 2005). The morphologic analysis of infective larvae, mainly with the evidence of the characteristic knob at the tail tip, allowed an initial differentiation between Libyostrongylus
and
Codiostomum,
and
subsequently,
with
morphometric
analyses, between L. douglassii and L. dentatus. During the sampling periods in the positive farm, one in 2011 and the second in 2013, an increase in the occurrence were observed with no evidence of illness even in younger animals, which probably suggests a parasite adaptation to the environment and a stable endemic condition, as already reported in other geographical areas (Barton and Seward, 1993; Bonadiman et al., 2006; de Andrade et al., 2011; Lelis et al., 2014). The duration of the pre-parasitic phase varies widely; under ideal oxygen, relative humidity, and temperature conditions, an egg can become an infective larva in a minimum of 60 hours and in an average of 6 days (Bonadiman et al., 2006; Ederli and Oliveira, 2009); which coincide with the values herein registered (day 6-22). Morphometric data of the observed infective larvae statistically differ from the published literature (Ederli et al., 2008b; Ederli et al., 2008c; de Andrade et al., 2011; Ederli and Rodrigues de Oliveira, 2014; Lelis et al., 2014). Although statistic differences were showed with L. douglassii in the three variables, data obtained (i.e. mean, minimum and maximum length and ratio) are close with published ranges. Besides, numerical differences with previous data for L. dentatus and Codiostomum spp. are notorious and the Student’s t-test showed higher statistical differences (i.e. through the p-value and 95% confidence intervals). These morphologic and morphometric observations, strongly suggest the identification of
L. douglassii in the positive samples in both sampling periods. This species has a worldwide distribution and has been previously reported in Australia (Barton and Seward, 1993), Belgium (Geurden et al., 2009), Brazil (Bonadiman et al., 2006), Croatia (Tisljar et al., 2007), Germany (Schulze et al., 2006), Iran (Eslami et al., 2007), Netherlands (Landman and Bronneberg, 2001), Portugal (Ponce-Gordo et al., 2002), Scotland (Pennycott and Patterson, 2001), South Africa (Cobbold, 1882), Spain (Ponce-Gordo et al., 2002), Sweden (Jansson and Christensson, 2000) and United States of America (Hoberg et al., 1995). Different studies (Hoberg et al., 1995; Ederli et al., 2008a; de Andrade et al., 2011; Lelis et al., 2014; Ederli and de Oliveira, 2015) have commonly identified mixed infections with L. douglassii and L. dentatus in ostrich farms. The latter species is apparently restricted to the American continent and has been only registered in USA (Hoberg et al., 1995) and Brazil (Bonadiman et al., 2006; Ederli et al., 2008a; de Andrade et al., 2011). Unless it was not identified in the samples, its presence could not be totally discarded due to the reported predominance of L. douglassii and the limited number of positive samples herein obtained. Libyostrongylus douglassii has been identified as the most pathogenic species within the genus, and infection in the host can cause clinical signs like anaemia, weight loss, anorexia, proventriculitis and mortality in younger animals (Reinecke, 1983). It is worth noting that the positive farm has no historical reports of clinical libyostrongylosis, which suggests a subclinical course of infection. Its effects on the production performance should be further analysed and monitoring of chronic infections implemented. Routine faecal parasitological screening and observation
of possible signs of disease (e.g. cachexia, generalized muscular atrophy, ascites, fat atrophy and fat deposition, generalized diffuse congestion, severe dilation of the proventriculus, gizzard impaction, proventricular necrosis and the presence of worms beneath the koilin lining of the gizzard and proventriculus) might aid in the surveillance process (Bastianello et al., 2005). Finally, this study confirms previous reports of Libyostrongylus spp. in ostrich farms from Meta department (Fernández-Manrique et al., 2011; Lozada et al., 2011) and identifies for the first time, through morphometric measurements, L. douglassii as the infective species. Acknowledgements The authors would like to thank Karol Bibiana Barragán Fonseca and Elkin Gustavo Forero Becerra for their help and guidance in the initial phases; the Ostrich
Farmers
Association
of
Colombia
(ASOAVESTRUCES)
for
their
cooperation and assistance; Alejandro Pacanchique for his willingness and help in sampling; and Carolina Alvarez Garzón for her assistance in the revision of the manuscript. References Barton, N.J., Seward, D.A., 1993. Detection of Libyostrongylus douglassi in ostriches in Australia. Aust. Vet. J. 70, 31-32. Bastianello, S., McKenna, P., Hunter, J., Julian, A., 2005. Clinical and pathological aspects of Libyostrongylus infection in ostriches. Surveillance 32, 3-6.
Bonadiman, S.F., Ederli, N.B., Soares, A.K.P., Neto, A.H.A.D.M., Santos, C.D.P., DaMatta, R.A., 2006. Occurrence of Libyostrongylus sp. (Nematoda) in ostriches (Struthio camelus Linnaeus, 1758) from the north region of the state of Rio de Janeiro, Brazil. Vet. Parasitol. 137, 175-179. Cobbold, T.S., 1882. New entozoon from the ostrich. J. Linnean Soc. 16, 18841888. de Andrade, J.G., Lelis, R.T., DaMatta, R.A., Santos, C.D., 2011. Occurrence of nematodes and anthelmintic management of ostrich farms from different Brazilian states: Libyostrongylus douglassii dominates mixed infections. Vet. Parasitol. 178, 129-133. Deeming, D.C., 1999. The ostrich: biology, production and health. CABI Publishing, 358 p. Ederli, N.B., Bonadiman, S.F., de Moraes, A.H.A., DaMatta, R.A., Santos, C.D., 2008a. Mixed infection by Libyostrongylus douglassii and L. dentatus (Nematoda
:
Trichostrongylidae)
in
Struthio
camelus
(Ratites
:
Struthioniformes) from Brazil with further morphological characterization of adults. Vet. Parasitol. 151, 227-232. Ederli, N.B., de Oliveira, F.C., Lopes, C.W., Damatta, R.A., Santos Cde, P., de Azevedo Rodrigues Mde, L., 2008b. Morphological diagnosis of infective larvae of Libyostrongylus douglassii (Cobbold, 1882) Lane, 1923 and L. dentatus Hoberg, Lloyd and Omar, 1995 (Nematoda: Trichostrongylidae) of ostriches. Vet. Parasitol. 155, 323-327. Ederli, N.B., de Oliveira, F.C., Rodrigues Mde, L., 2008c. Further study of Codiostomum struthionis (Horst, 1885) Railliet and Henry, 1911 (Nematoda,
Strongylidae) parasite of ostriches (Struthio camelus Linnaeus, 1758) (Aves, Struthioniformes). Vet. Parasitol. 157, 275-283. Ederli, N.B., de Oliveira, F.C.R., Lopes, C.W.G., DaMatta, R.A., Santos, C.D., Rodrigues, M.D., 2008d. Morphological diagnosis of infective larvae of Libyostrongylus douglassii (Cobbold, 1882) Lane, 1923 and L. dentatus Hoberg, Lloyd and Omar, 1995 (Nematoda : Trichostrongylidae) of ostriches. Vet. Parasitol. 155, 323-327. Ederli, N.B., Oliveira, F.C.R., 2009. Differential Localization of Libyostrongylus douglassii (Cobbold, 1882) Lane, 1923 and L. dentatus Hoberg, Lloyd, and Omar, 1995 (Nematoda: Trichostrongylidae) in Ostrich (Struthio camelus Linnaeus, 1758) Proventriculi. J. Parasitol. 95, 757-759. Ederli, N.B., Rodrigues de Oliveira, F.C., 2014. Comparative morphology of the species of Libyostrongylus and Codiostomum, parasites from ostriches, Struthio camelus, with a identification key to the species. Rev Bras Parasitol Vet 23, 291-300. Ederli, N.B., de Oliveira, F.C., 2015. Gastrointestinal nematodes in ostriches, Struthio camelus, in different regions of the state of Rio de Janeiro, Brazil. Rev Bras Parasitol Vet 24, 168-173. Eslami, A., Rahmat, H., Meshgi, B., Ranjbar-Bahadori, S., 2007. Gastrointestinal parasites of ostrich (Struthio camelus domesticus) raised in Iran. Iran J Vet Res 8, 80-82. Fernández-Manrique, J., Vargas, L., Melo-Guevara, J., Murillo-Pacheco, R., 2011. Reporte de nematodos gastrointestinales en una explotación de avestruces
(Struthio camelus) en el departamento del Meta, Colombia. Biomedica 31, 289-290. Geurden, T., van Leuven, M., Meeus, P., Vercruysse, J., Claerebout, E., 2009. Trichostrongylid nematode infections in ostriches (Struthio camelus) in Belgium. Vet. Rec. 164, 181-181. Ponce-Gordo, F., Herrera, S., Castro, A.T., García-Durán, B., Martínez-Díaz, R.A., 2002. Parasites from farmed ostriches (Struthio camelus) and rheas (Rhea americana) in Europe. Vet. Parasitol. 107, 137-160. Hoberg, E.P., Lloyd, S., Omar, H., 1995. Libyostrongylus dentatus n. sp. (Nematoda: Trichostrongylidae) from ostriches in North America, with comments on the genera Libyostrongylus and Paralibyostrongylus. J. Parasitol. 81, 85-93. Huchzermeyer, F.W., 1999. Patología de avestruces y otras ratites. S.A. MUNDIPRENSA LIBROS. Huchzermeyer, F.W., 2002. Diseases of farmed crocodiles and ostriches. Rev. Off. Int. Epizoot. 21, 265-276. Jansson, D., Christensson, D., 2000. Gastrointestinala parasiter hos strutsfaglar i Sverige I artikeln redovisas bland annat tva svenska fallrapporter och resultat fran en svensk studie av forekomsten av gastrointestinala parasiter hos strutsfaglar. SVENSK VETERINARTIDNING 52, 621-626. Landman, W.J.M., Bronneberg, R.G.G., 2001. Libyostrongylus douglassii in ostriches (Struthio camelus ssp.) in the Netherlands: case report and review. Tijdschr Diergeneeskd 126, 484-487.
Lelis, R.T., de Andrade, J.G., Mendonca Vieira, R.A., DaMatta, R.A., Santos, C.d.P., 2014. Population dynamics of Libyostrongylus dentatus and L. douglassii of ostriches raised in the Northern Rio de Janeiro State, Brazil. Vet. Parasitol. 200, 147-152. Lozada, H., Rincón, J., López, V., 2011. Parásitos en avestruces en el Departamento
del
Meta.
Revista
de
Sistemas
de
Producción
Agroecológicos 2, 34-46. Mackereth, G., 2004. Libyostrongylus douglassii in New Zealand ostriches. Surveillance 31, 14-16. McKenna, P.B., 2005. Libyostrongylus infections in ostriches - a brief review with particular reference to their detection in New Zealand. N Z Vet J 53, 267270. Pennycott, T., Patterson, T., 2001. Gastrointestinal parasites in ostriches (Struthio camelus). Vet. Rec. 148, 155-156. R-Core-Team 2014. R: A language and environment for statistical computing. (Vienna, Austria, R Foundation for Statistical Computing). Reinecke, R., 1983. Veterinary helminthology. Butterworth & Co.(SA)(Pty.) Ltd., 392 p. Schulze, C., Grossmann, E., Krone, O., 2006. Case report: Libyostrongylus douglassii-associated proventriculitis in ostriches in Germany. Dtsch Tierarztl Wochenschr 113, 240-242. Tisljar, M., Beck, R., Cooper, R.G., Marinculic, A., Tudja, M., Lukac-Novak, I., Grabarevic, Z., Herak-Perkovic, V., Simpraga, B., 2007. First finding of libyostrongylosis in farm-reared ostriches (Struthio camelus) in Croatia:
Unusual histopathological finding in the brain of two ostriches, naturally infected with Libyostrongylus douglasi. Vet. Parasitol. 147, 118-124.
Figure 1. Some developmental phases of Libyostrongylus douglassii, from egg to infective larvae, under the light microscope. (A.) Undeveloped egg; (B.) Initial development with poles flattening; (C. and D.) Further egg development with polar organization of cells; (E.) Longitudinal development and coiled larva; (F. and G.) Larval organization inside the egg prior to hatching; (H.) Recently hatched larvae; (I.) Caudal end of hatched larva; (J.) Cranial end of hatched larva; (K.) Detail of larva tail knob; (L.) Bar indicating sheath length measurement (Bar = 10 µm.) Source: original
Table 1. Total and sheath length measurements (μm) and the ratio between them in infective larvae recovered from faecal cultures of ostriches from Puerto López (Meta, Colombia) and comparison with reference values reported for Libyostrongylus douglassii, L. dentatus and Codiostomum spp. Reference values for species Mean ± SD Parameters Libyostrongylus Libyostrongylus Codiostomum (Range) douglassii dentatus* spp.** Total length (a)
888.02 ± 25.53 (850 – 950)
784***± 43.63
598.25***± 25.15
(784.47 – 957.9)
(735.84 – 947.01)
(511.92 – 642.41)
874.33*** ± 33.8
Sheath tail length (b)
26.88 ± 5.36
29.52*** ± 4.11
61.2*** ± 2.17
110.74*** ± 13.46
(20 – 35)
(20.62 – 36.31)
(40.01 – 84.01)
(85.87 – 143.18)
Ratio (a)/(b)
34.41 ± 7.10
30.2*** ± 4.58
14.29*** ± 2.17
(23.25 – 42.38)
(10.81 – 20.32)
(24.57 – 47.50)
Values are reported as mean ± standard * (Ederli et al., 2008c; Ederli and Rodrigues de Oliveira, 2014) **(Ederli et al., 2008d) ***Mean comparison with reference values (p-value <0.05)
S0304-4017(17)30007-9 http://dx.doi.org/doi:10.1016/j.vetpar.2017.01.007 VETPAR 8228
To appear in:
Veterinary Parasitology
Received date: Revised date: Accepted date:
10-10-2016 8-12-2016 9-1-2017
Please cite this article as: Mari˜no-Gonz´alez, Guillermo A., Ram´ırez-Hern´andez, Alejandro, Cort´es-Vecino, Jes´us A., Libyostrongylus douglassii (Strongylida: Trichostrongylidae) in Ostrich (Struthio camelus) Farms from Colombia.Veterinary Parasitology http://dx.doi.org/10.1016/j.vetpar.2017.01.007 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Libyostrongylus douglassii (Strongylida: Trichostrongylidae) in Ostrich (Struthio camelus) Farms from Colombia.
Guillermo A. Mariño-Gonzáleza, Alejandro Ramírez-Hernándeza,b; Jesús A. CortésVecinoa,* a. Grupo Parasitología Veterinaria, Departamento Salud Animal, Facultad de Medicina Veterinaria y de Zootecnia, Universidad Nacional de Colombia, Cra. 30 No. 45-03, Ciudad Universitaria, Edificio 481, Bogotá D.C., Colombia. b. Departamento de Medicina Veterinária Preventiva e Saúde Animal, Faculdade de Medicina Veterinária e Zootecnia, Universidade de São Paulo, Av. Prof. Orlando Marques de Paiva, 87, CEP 05508-000, Cidade Universitária, São Paulo, SP, Brazil.
*Corresponding author: Laboratorio de Parasitología Veterinaria, Facultad de Medicina Veterinaria y de Zootecnia, Universidad Nacional de Colombia, Cra. 30 No. 45-03, Ciudad Universitaria, Edificio 481-Laboratorio 4, Bogotá D.C., Colombia. Tel.:+571 3165000 (Ext. 15333-15314) E-mail address: [email protected] (J.A. Cortés-Vecino)
Highlights
Libyostrongylus douglassii detected for the first time in ostriches from Colombia.
Previous Libyostrongylus spp. infections in Meta department are confirmed.
Possible mixed infections with L. dentatus could not be totally discarded.
Abstract Ostrich farming is an important livestock industry in different world regions with a diverse offer of products and services. In Colombia, as in other countries, this market led the importation of animals from countries like Canada, United States of America and South Africa for breeding objectives. With the animals, specific pathogens for these ratites could be introduced. Libyostrongylus spp. is a strongylid nematode with worldwide distribution, which can induce a severe disease and mortality in infected animals. Limited studies in Colombia have identified parasites in ostrich farming systems. The aim of this study was to identify parasites of the genus Libyostrongylus to a species level in faecal samples from ostrich farms in three departments of Colombia. Five ostrich farms from Boyacá, Meta and Tolima were sampled in 2011 and in 2013 to obtain fresh faecal samples which were further processed by flotation tests for egg visualization and faecal culture for infective larvae identification by morphological and morphometric parameters. One from the five farms, located in Meta department, was positive for strongylid eggs in both sampling periods. After faecal culture, infective larvae were identified as Libyostrongylus douglassii. These results corroborate previous records of Libyostrongylus in Ostrich farms from Meta and confirms, for the first time, infection by L. douglassii in ratites from this region. Further studies must
identify associated determinants for infection and its effects on the flock health and production. Keywords: Nematoda; Parasitic Diseases; Ratites; South America 1. Introduction The physical and physiological characteristics of the ostrich (Struthio camelus) have led to a renewed global industrial interest in this species, which has in turn led to a proliferation of farms and animals in different countries, including Colombia. Different ecto and endoparasites like ticks, protozoan, cestodes and nematodes could affect ostriches (Ponce-Gordo et al., 2002; Huchzermeyer, 2002). An example of the latter is Libyostrongylus Lane, 1923, a typical strongylid nematode of this bird (Deeming, 1999), which has been detected in ostrich farming worldwide (Barton and Seward, 1993; Hoberg et al., 1995; Landman and Bronneberg, 2001; Mackereth, 2004; Bonadiman et al., 2006; Tisljar et al., 2007). The related parasitism has been tied to high youth morbidity and mortality rates, having its greatest impact on animals of up to three months (50%), and even up to six months (20-30%), and a low mortality rate in adult ostriches (<10%) which can maintain variable parasitic loads without manifestation of clinical signs (Mackereth, 2004; Bastianello et al., 2005; Bonadiman et al., 2006; de Andrade et al., 2011). The disease is characterized by a local gastric inflammatory process involving the proventriculus (Ederli and Oliveira, 2009) which can lead to ulcerative lesions of varying degree, which can perforate and liberate content into the coelomic cavity in a disease process known as ‘rotten stomach’ or ‘vrottmaag’ (Huchzermeyer, 2002).
Three species of Libyostrongylus are known: L. douglassii (Cobbold, 1882) Lane, 1923, L. dentatus Hoberg, Lloyd and Omar, 1995 and L. magnus Gilbert, 1937 (Ederli and de Oliveira, 2015). L. douglassii, identified as the most pathogenic, has a worldwide distribution; contrarily, L. dentatus and L. magnus have been reported only in the American and African continent, respectively (Hoberg et al., 1995; Huchzermeyer, 1999; Ederli and de Oliveira, 2015). In Colombia, limited studies have identified its presence and there is a lack of knowledge about the species involved, its distribution and production impact. The aim of this study was to identify parasites of the genus Libyostrongylus to a species level in faecal samples from ostrich farms in three departments of Colombia. 2. Materials and methods Sampling was done on five ostrich farms in three different departments of Colombia: one from Villa de Leyva (Boyacá), one from Puerto López (Meta), one from Coello (Tolima) and two from Coyaima (Tolima) (see Supplement Material for additional information on localization of each farm). Samples were taken in two different periods as follows: between September and December 2011 in Coello, Coyaima, Puerto López and Villa de Leyva; and November 2013 in Puerto López. Fresh faecal samples were taken from the ground with new plastic bags within five minutes of defecation as previously recommended (Bonadiman et al., 2006; Lelis et al., 2014). They were transported in an insulated box, at a temperature of 4-7°C, to the Veterinary Parasitology Laboratory (Universidad Nacional de Colombia) and analysed within the following 24 hours. Approximately, 2 g of faecal matter were sieved to separate gross particles and centrifuged (1.500 r.p.m. for 5 minutes) to
perform a flotation test with Sheather’s sugar solution (specific gravity = 1.27). At the same time, a faecal culture was set up in which samples were homogenized in their respective transportation containers, and 6-8 grams (or 10 ml in liquid samples) were taken and placed at the bottom of a 300 ml disposable cup, creating a lightly compact form. The faecal sample was then moistened, but not diluted, using a fine atomized spray until a pool or layer of water of approximately 5 mm in height was formed. The samples were incubated at an average temperature of 27°C and relative humidity around 60%, as previously reported (Bonadiman et al., 2006). Incubation was controlled daily and involved rinsing the samples and observing the obtained matter under a light microscope in 100 and 400x magnification. In addition, serial flotation tests were performed to confirm changes in the eggs morphology throughout the study until the appearance of larvae. In order to limit their movement, they were refrigerated before being observed and measuring for genus and species classification. The following morphological variables, proposed by Ederli et al. (2008a) for species differentiation, were compared: total length of the larva (a), sheath tail length (b), and the ratio between both parameters (ratio = a/b) (c). Mean and standard deviations for morphometric data were calculated and further analysed using R software (version 3.0.1) (R-Core-Team, 2014). 3. Results
Analysis through flotation tests, confirmed the presence of strongyliform eggs in two different sampling periods (September 2011 and November 2013) from the ostrich farm located in Puerto López (Meta). Following the incubation period, larvae morphologically and morphometrically compatible with the genus Libyostrongylus were identified (Figure 1). The incubation period ranged from 6 to 22 days. A total of 102 larvae from both collections were identified as Libyostrongylus spp. mainly by the presence of the knob in the tail and were further measured, presenting an average total length of 888.02 μm (± SD: 25.53), an average sheath tail length of 26.88 μm (± 5.36) and an average ratio of 34.41 μm (± 7.10) (Table 1). Consequently, all larvae were morphologically identified as L. douglassii after comparing the latter measures with published data (Ederli et al., 2008c; Ederli et al., 2008d) and matched their morphologic characteristics (Table 1, Figure 1). In addition, different development steps of the parasite eggs, including some cell and larvae development phases, were recorded during the incubation phase (Figure 1). 4. Discussion Species level identification through routine flotation techniques is not possible due to undetectable differences between eggs of the Libyostrongylus genus and Codiostomum sp., a strongylid that parasitizes the distal third of the ostrich caecum and is apparently innocuous (Huchzermeyer, 2002; Ederli et al., 2008a; Ederli et al., 2008b; Ederli et al., 2008c; Ederli and Rodrigues de Oliveira, 2014). The use of these techniques, however, may be recommended as an approximation upon the
screening of new animals entering the production system (McKenna, 2005). The morphologic analysis of infective larvae, mainly with the evidence of the characteristic knob at the tail tip, allowed an initial differentiation between Libyostrongylus
and
Codiostomum,
and
subsequently,
with
morphometric
analyses, between L. douglassii and L. dentatus. During the sampling periods in the positive farm, one in 2011 and the second in 2013, an increase in the occurrence were observed with no evidence of illness even in younger animals, which probably suggests a parasite adaptation to the environment and a stable endemic condition, as already reported in other geographical areas (Barton and Seward, 1993; Bonadiman et al., 2006; de Andrade et al., 2011; Lelis et al., 2014). The duration of the pre-parasitic phase varies widely; under ideal oxygen, relative humidity, and temperature conditions, an egg can become an infective larva in a minimum of 60 hours and in an average of 6 days (Bonadiman et al., 2006; Ederli and Oliveira, 2009); which coincide with the values herein registered (day 6-22). Morphometric data of the observed infective larvae statistically differ from the published literature (Ederli et al., 2008b; Ederli et al., 2008c; de Andrade et al., 2011; Ederli and Rodrigues de Oliveira, 2014; Lelis et al., 2014). Although statistic differences were showed with L. douglassii in the three variables, data obtained (i.e. mean, minimum and maximum length and ratio) are close with published ranges. Besides, numerical differences with previous data for L. dentatus and Codiostomum spp. are notorious and the Student’s t-test showed higher statistical differences (i.e. through the p-value and 95% confidence intervals). These morphologic and morphometric observations, strongly suggest the identification of
L. douglassii in the positive samples in both sampling periods. This species has a worldwide distribution and has been previously reported in Australia (Barton and Seward, 1993), Belgium (Geurden et al., 2009), Brazil (Bonadiman et al., 2006), Croatia (Tisljar et al., 2007), Germany (Schulze et al., 2006), Iran (Eslami et al., 2007), Netherlands (Landman and Bronneberg, 2001), Portugal (Ponce-Gordo et al., 2002), Scotland (Pennycott and Patterson, 2001), South Africa (Cobbold, 1882), Spain (Ponce-Gordo et al., 2002), Sweden (Jansson and Christensson, 2000) and United States of America (Hoberg et al., 1995). Different studies (Hoberg et al., 1995; Ederli et al., 2008a; de Andrade et al., 2011; Lelis et al., 2014; Ederli and de Oliveira, 2015) have commonly identified mixed infections with L. douglassii and L. dentatus in ostrich farms. The latter species is apparently restricted to the American continent and has been only registered in USA (Hoberg et al., 1995) and Brazil (Bonadiman et al., 2006; Ederli et al., 2008a; de Andrade et al., 2011). Unless it was not identified in the samples, its presence could not be totally discarded due to the reported predominance of L. douglassii and the limited number of positive samples herein obtained. Libyostrongylus douglassii has been identified as the most pathogenic species within the genus, and infection in the host can cause clinical signs like anaemia, weight loss, anorexia, proventriculitis and mortality in younger animals (Reinecke, 1983). It is worth noting that the positive farm has no historical reports of clinical libyostrongylosis, which suggests a subclinical course of infection. Its effects on the production performance should be further analysed and monitoring of chronic infections implemented. Routine faecal parasitological screening and observation
of possible signs of disease (e.g. cachexia, generalized muscular atrophy, ascites, fat atrophy and fat deposition, generalized diffuse congestion, severe dilation of the proventriculus, gizzard impaction, proventricular necrosis and the presence of worms beneath the koilin lining of the gizzard and proventriculus) might aid in the surveillance process (Bastianello et al., 2005). Finally, this study confirms previous reports of Libyostrongylus spp. in ostrich farms from Meta department (Fernández-Manrique et al., 2011; Lozada et al., 2011) and identifies for the first time, through morphometric measurements, L. douglassii as the infective species. Acknowledgements The authors would like to thank Karol Bibiana Barragán Fonseca and Elkin Gustavo Forero Becerra for their help and guidance in the initial phases; the Ostrich
Farmers
Association
of
Colombia
(ASOAVESTRUCES)
for
their
cooperation and assistance; Alejandro Pacanchique for his willingness and help in sampling; and Carolina Alvarez Garzón for her assistance in the revision of the manuscript. References Barton, N.J., Seward, D.A., 1993. Detection of Libyostrongylus douglassi in ostriches in Australia. Aust. Vet. J. 70, 31-32. Bastianello, S., McKenna, P., Hunter, J., Julian, A., 2005. Clinical and pathological aspects of Libyostrongylus infection in ostriches. Surveillance 32, 3-6.
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Lelis, R.T., de Andrade, J.G., Mendonca Vieira, R.A., DaMatta, R.A., Santos, C.d.P., 2014. Population dynamics of Libyostrongylus dentatus and L. douglassii of ostriches raised in the Northern Rio de Janeiro State, Brazil. Vet. Parasitol. 200, 147-152. Lozada, H., Rincón, J., López, V., 2011. Parásitos en avestruces en el Departamento
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Figure 1. Some developmental phases of Libyostrongylus douglassii, from egg to infective larvae, under the light microscope. (A.) Undeveloped egg; (B.) Initial development with poles flattening; (C. and D.) Further egg development with polar organization of cells; (E.) Longitudinal development and coiled larva; (F. and G.) Larval organization inside the egg prior to hatching; (H.) Recently hatched larvae; (I.) Caudal end of hatched larva; (J.) Cranial end of hatched larva; (K.) Detail of larva tail knob; (L.) Bar indicating sheath length measurement (Bar = 10 µm.) Source: original
Table 1. Total and sheath length measurements (μm) and the ratio between them in infective larvae recovered from faecal cultures of ostriches from Puerto López (Meta, Colombia) and comparison with reference values reported for Libyostrongylus douglassii, L. dentatus and Codiostomum spp. Reference values for species Mean ± SD Parameters Libyostrongylus Libyostrongylus Codiostomum (Range) douglassii dentatus* spp.** Total length (a)
888.02 ± 25.53 (850 – 950)
784***± 43.63
598.25***± 25.15
(784.47 – 957.9)
(735.84 – 947.01)
(511.92 – 642.41)
874.33*** ± 33.8
Sheath tail length (b)
26.88 ± 5.36
29.52*** ± 4.11
61.2*** ± 2.17
110.74*** ± 13.46
(20 – 35)
(20.62 – 36.31)
(40.01 – 84.01)
(85.87 – 143.18)
Ratio (a)/(b)
34.41 ± 7.10
30.2*** ± 4.58
14.29*** ± 2.17
(23.25 – 42.38)
(10.81 – 20.32)
(24.57 – 47.50)
Values are reported as mean ± standard * (Ederli et al., 2008c; Ederli and Rodrigues de Oliveira, 2014) **(Ederli et al., 2008d) ***Mean comparison with reference values (p-value <0.05)