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Dientamoeba fragilis in swine population: A preliminary investigation
Veterinary Parasitology 145 (2007) 349–351 www.elsevier.com/locate/vetpar
Short communication
Dientamoeba fragilis in swine population: A preliminary investigation D. Crotti a,b, M. Sensi a, S. Crotti a, V. Grelloni a, E. Manuali a,* a
Istituto Zooprofilattico Sperimentale dell’Umbria e delle Marche, Via G. Salvemini 1, 06126 Perugia, Italy b Libero professionista in Parassitologia e Microbiologia Medica, Perugia, Italy Received 8 November 2006; received in revised form 20 December 2006; accepted 9 January 2007
Abstract Dientamoeba fragilis, a protozoan with worldwide distribution is considered to be responsible for enteric disease in humans. A wide spectrum of clinical symptoms including; diarrhoea (acute or prolonged), flatulence, abdominal pains and other unspecific bowel symptoms have been ascribed to this parasite. Asymptomatic infection has also been reported. Dientamoeba fragilis is as its name indicates an extremely delicate protozoon and only the trophozoite has ever been demonstrated in stool samples. The definitive diagnosis of this infection is based on demonstration in permanently stained stool samples. In Italy examination of ova and parasite (O&P) samples are not currently performed. This protozoan is extremely difficult to cultivate but molecular techniques such as the Polymerase Chain Reaction offer promise as a means of diagnosing infection. The epidemiology of Dientamoebiasis is not clear. This paper will present preliminary results from a study looking for the parasite’s presence in swine faeces. The possible role of pigs as a reservoir of infection was studied; 121 faecal samples from breeding and fattening pigs were examined using a Giemsa permanent stain. Dientamoeba fragilis was found in 53 (43.8%) of the stool samples examined. # 2007 Elsevier B.V. All rights reserved. Keywords: Dientamoeba fragilis; Swine; Epidemiology; Giemsa stain
1. Introduction Dientamoeba fragilis (D. fragilis) is recognised as a pathogenic human intestinal protozoan parasite throughout the world (Windsor et al., 1998; Johnson et al., 2004). In Italy has been isolated (Crotti et al., 2005) from patients with clinical disease. A wide spectrum of symptoms including diarrhoea (acute or prolonged), flatulence, abdominal pains and unspecific bowel syndrome have been described (Crotti and D’Annibale, 2001).
* Corresponding author at: Laboratory of Histopathology and Electron Microscopy, Via G. Salvemini 1, 06126 Perugia, Italy. Tel.: +39 075 343227; fax: +39 075 35047. E-mail address: [email protected] (E. Manuali). 0304-4017/$ – see front matter # 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.vetpar.2007.01.006
D. fragilis a flagellate protozoan that lacks flagella has no cystic form. It can be readily detected as a trophozoite in faecal specimens by direct macroscopic examination of fresh-Giemsa stained-samples (Windsor and Johnson, 1999) but is difficult to cultivate although a wide variety of culture systems have been used (Sawangjaroen et al., 1993; Windsor et al., 2003). These techniques are usually restricted to selected parasitology laboratories and are not used as routine diagnostic methods. Molecular techniques, such as conventional Polymerase Chain Reaction (PCR) and real-time PCR have promise as a means of diagnosing this protozoan infection (Stark et al., 2005, 2006). In the scientific literature there are many reports regarding D. fragilis detection in species other than humans, including macaques, baboons and sheep (Windsor and Johnson, 1999). Because of the fragile
350
D. Crotti et al. / Veterinary Parasitology 145 (2007) 349–351
nature of this organism and the fact that no cyst stages were observed, the mode of transmission of D. fragilis is unknown. It was believed to be through direct faecaloral route but the question arises as to how the trophozoites, the only stage found in stools, escape destruction by gastric acidity. For this reason, recent studies have postulated the possible role of Enterobius vermicularis as a vector (Menghi et al., 2005) and speculated that D. fragilis may transit the stomach inside the pinworm eggs. In the present paper, the preliminary results of an epidemiological survey performed on swine herds in Central Italy are briefly reported. 2. Materials and methods The study was carried out between February and October 2006. The coproparasitological investigation included 121 faecal samples from breeding (No. 94) and fattening (No. 27) pigs in 11 farrow-to-finish herds. All samples were collected directly from the rectum, cooled and sent to the laboratory where they were examined for D. fragilis by direct microscopic examination. Faeces (5 g) were diluted in 10 ml of saline solution, shaken vigorously and then 250 ml of supernatant was smeared onto a glass slide. Since the trophozoites degenerate rapidly, the samples were fixed with methanol for 1 min. The fresh faecal smears were stained with a 10% Giemsa solution in distilled water for 30 min. Statistical analysis has been performed using the Fisher’s exact test. 3. Results and discussion In this preliminary investigation, D. fragilis was detected in 53 (43.8%) stool specimens from 30 breeding (31.9%) and 23 fattening (85.2%) pigs. On the whole, 10/11 (90.9%) herds resulted positive. A statistically significant difference has been observed between the two groups ( p < 0.005), showing a possible effect of age on the infection rate of animals. However, considering the limited number of samples collected in this preliminary investigation, further studies have to be planned to better investigate this relationship. The pleomorphic trophozoites, most of which had two nuclei (Fig. 1), ranged in size from 4 to 20 mm in diameter (typically between 5 and 15 mm). Nuclear chromatin was usually fragmented and the pale greyblue cytoplasm appeared finely vacuolated. Furthermore, degenerated forms appearing full of small vacuoles which coalesced into a single large vacuole
Fig. 1. A typical binucleate form of Dientamoeba fragilis (Giemsa stain, bar 10 mm).
was observed. These resembled those of Blastocystis hominis. The relative frequency of D. fragilis found in faecal samples when compared to the frequency of other enteric-non-pathogens protozoa is shown in Table 1. Iodamoeba bu¨tschlii and Endolimax nana were prevalent in all the tested swine. Blastocystis hominis, Entamoeba suis, Entamoeba polecki, Chilomastix mesnili, Giardia spp. and Entamoeba dispar occurred only sporadically. D. fragilis and I. bu¨tschlii were associated in 41 (33.9%) samples, D. fragilis and E. nana in 29 (24%) samples and D. fragilis and B. hominis in 10 (8.3%) samples. To our knowledge, this is the first report of D. fragilis in swine faeces. The relationship if any between D. fragilis infection in pig and human dientamoebiasis remains to be clarified. It is possible that since there was such a high incidence of this protozoan in pigs, that pigs may well play a role as a reservoir of the parasite for humans, even if the mode of transmission remains unclear. Table 1 Intestinal protozoa isolated from swine faeces between February and October 2006 Protozoa
No. of samples
Positivity (%)
Iodamoeba bu¨tschlii Endolimax nana Dientamoeba fragilis Blastocystis hominis Entamoeba suis Entamoeba polecki Chilomastix mesnili Giardia spp. Entamoeba dispar
97 55 53 14 9 5 3 2 2
80.2 45.4 43.8 11.6 7.4 4.1 2.5 1.6 1.6
D. Crotti et al. / Veterinary Parasitology 145 (2007) 349–351
Acknowledgements The authors wish to thank Sonia Salamida and Angela Caporali for their contribution to sample collection and technical support. References Crotti, D., D’Annibale, M.L., 2001. Dientamoeba fragilis e dientamoebiasi: aspetti di parassitologia clinica e diagnostica di laboratorio. Parassitologia 43, 135–138. Crotti, D., D’Annibale, M.L., Fonzo, G., Lalle, M., Caccio`, S.M., Pozio, E., 2005. Dientamoeba fragilis is more prevalent than Giardia duodenalis in children and adults attending a day care centre in Central Italy. Parasite 12, 165–170. Johnson, E.H., Windsor, J.J., Graham Clark, C., 2004. Emerging from obscurity: biological, clinical, and diagnostic aspects of Dientamoeba fragilis. Clin. Microbiol. Rev. 17, 553–570. Menghi, C.I., Makiya, R., Gatta, C.L., Me´ndez, O.C., 2005. Dientamoeba fragilis: molecular biology techniques for the
351
Elucidation of hits mode of transmission. Parassitol. Latinoam. 60, 25–31. Sawangjaroen, N., Luke, R., Prociv, P., 1993. Diagnosis by faecal culture of Dientamoeba fragilis infections in Australian patients with diarrhoea. Trans. R. Soc. Trop. Med. Hyg. 87 (2), 163– 165. Stark, D., Beebe, N., Marriott, D., Ellis, J., Harkness, J., 2005. Detection of Dientamoeba fragilis in fresh stool specimens using PCR. Int. J. Parasitol. 35, 57–62. Stark, D., Beebe, N., Marriott, D., Ellis, J., Harkness, J., 2006. Evaluation of three diagnostic methods, including real-time PCR, for detection of Dientamoeba fragilis in stool specimens. J. Clin. Microbiol. 44 (1), 232–235. Windsor, J.J., Rafay, A.M., Shenoy, A.K., Johnson, E.H., 1998. Incidence of Dientamoeba fragilis in faecal samples submitted for routine microbiological analyses. Br. J. Biomed. Sci. 55, 172–175. Windsor, J.J., Johnson, E.H., 1999. Dientamoeba fragilis: the unflagellate human flagellate. Br. J. Biomed. Sci. 56, 293–306. Windsor, J.J., Macfarlane, L., Hughes-Thapa, G., Jones, S.K., Whiteside, T.M., 2003. Detection of Dientamoeba fragilis by culture. Br. J. Biomed. Sci. 60, 79–83.
Short communication
Dientamoeba fragilis in swine population: A preliminary investigation D. Crotti a,b, M. Sensi a, S. Crotti a, V. Grelloni a, E. Manuali a,* a
Istituto Zooprofilattico Sperimentale dell’Umbria e delle Marche, Via G. Salvemini 1, 06126 Perugia, Italy b Libero professionista in Parassitologia e Microbiologia Medica, Perugia, Italy Received 8 November 2006; received in revised form 20 December 2006; accepted 9 January 2007
Abstract Dientamoeba fragilis, a protozoan with worldwide distribution is considered to be responsible for enteric disease in humans. A wide spectrum of clinical symptoms including; diarrhoea (acute or prolonged), flatulence, abdominal pains and other unspecific bowel symptoms have been ascribed to this parasite. Asymptomatic infection has also been reported. Dientamoeba fragilis is as its name indicates an extremely delicate protozoon and only the trophozoite has ever been demonstrated in stool samples. The definitive diagnosis of this infection is based on demonstration in permanently stained stool samples. In Italy examination of ova and parasite (O&P) samples are not currently performed. This protozoan is extremely difficult to cultivate but molecular techniques such as the Polymerase Chain Reaction offer promise as a means of diagnosing infection. The epidemiology of Dientamoebiasis is not clear. This paper will present preliminary results from a study looking for the parasite’s presence in swine faeces. The possible role of pigs as a reservoir of infection was studied; 121 faecal samples from breeding and fattening pigs were examined using a Giemsa permanent stain. Dientamoeba fragilis was found in 53 (43.8%) of the stool samples examined. # 2007 Elsevier B.V. All rights reserved. Keywords: Dientamoeba fragilis; Swine; Epidemiology; Giemsa stain
1. Introduction Dientamoeba fragilis (D. fragilis) is recognised as a pathogenic human intestinal protozoan parasite throughout the world (Windsor et al., 1998; Johnson et al., 2004). In Italy has been isolated (Crotti et al., 2005) from patients with clinical disease. A wide spectrum of symptoms including diarrhoea (acute or prolonged), flatulence, abdominal pains and unspecific bowel syndrome have been described (Crotti and D’Annibale, 2001).
* Corresponding author at: Laboratory of Histopathology and Electron Microscopy, Via G. Salvemini 1, 06126 Perugia, Italy. Tel.: +39 075 343227; fax: +39 075 35047. E-mail address: [email protected] (E. Manuali). 0304-4017/$ – see front matter # 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.vetpar.2007.01.006
D. fragilis a flagellate protozoan that lacks flagella has no cystic form. It can be readily detected as a trophozoite in faecal specimens by direct macroscopic examination of fresh-Giemsa stained-samples (Windsor and Johnson, 1999) but is difficult to cultivate although a wide variety of culture systems have been used (Sawangjaroen et al., 1993; Windsor et al., 2003). These techniques are usually restricted to selected parasitology laboratories and are not used as routine diagnostic methods. Molecular techniques, such as conventional Polymerase Chain Reaction (PCR) and real-time PCR have promise as a means of diagnosing this protozoan infection (Stark et al., 2005, 2006). In the scientific literature there are many reports regarding D. fragilis detection in species other than humans, including macaques, baboons and sheep (Windsor and Johnson, 1999). Because of the fragile
350
D. Crotti et al. / Veterinary Parasitology 145 (2007) 349–351
nature of this organism and the fact that no cyst stages were observed, the mode of transmission of D. fragilis is unknown. It was believed to be through direct faecaloral route but the question arises as to how the trophozoites, the only stage found in stools, escape destruction by gastric acidity. For this reason, recent studies have postulated the possible role of Enterobius vermicularis as a vector (Menghi et al., 2005) and speculated that D. fragilis may transit the stomach inside the pinworm eggs. In the present paper, the preliminary results of an epidemiological survey performed on swine herds in Central Italy are briefly reported. 2. Materials and methods The study was carried out between February and October 2006. The coproparasitological investigation included 121 faecal samples from breeding (No. 94) and fattening (No. 27) pigs in 11 farrow-to-finish herds. All samples were collected directly from the rectum, cooled and sent to the laboratory where they were examined for D. fragilis by direct microscopic examination. Faeces (5 g) were diluted in 10 ml of saline solution, shaken vigorously and then 250 ml of supernatant was smeared onto a glass slide. Since the trophozoites degenerate rapidly, the samples were fixed with methanol for 1 min. The fresh faecal smears were stained with a 10% Giemsa solution in distilled water for 30 min. Statistical analysis has been performed using the Fisher’s exact test. 3. Results and discussion In this preliminary investigation, D. fragilis was detected in 53 (43.8%) stool specimens from 30 breeding (31.9%) and 23 fattening (85.2%) pigs. On the whole, 10/11 (90.9%) herds resulted positive. A statistically significant difference has been observed between the two groups ( p < 0.005), showing a possible effect of age on the infection rate of animals. However, considering the limited number of samples collected in this preliminary investigation, further studies have to be planned to better investigate this relationship. The pleomorphic trophozoites, most of which had two nuclei (Fig. 1), ranged in size from 4 to 20 mm in diameter (typically between 5 and 15 mm). Nuclear chromatin was usually fragmented and the pale greyblue cytoplasm appeared finely vacuolated. Furthermore, degenerated forms appearing full of small vacuoles which coalesced into a single large vacuole
Fig. 1. A typical binucleate form of Dientamoeba fragilis (Giemsa stain, bar 10 mm).
was observed. These resembled those of Blastocystis hominis. The relative frequency of D. fragilis found in faecal samples when compared to the frequency of other enteric-non-pathogens protozoa is shown in Table 1. Iodamoeba bu¨tschlii and Endolimax nana were prevalent in all the tested swine. Blastocystis hominis, Entamoeba suis, Entamoeba polecki, Chilomastix mesnili, Giardia spp. and Entamoeba dispar occurred only sporadically. D. fragilis and I. bu¨tschlii were associated in 41 (33.9%) samples, D. fragilis and E. nana in 29 (24%) samples and D. fragilis and B. hominis in 10 (8.3%) samples. To our knowledge, this is the first report of D. fragilis in swine faeces. The relationship if any between D. fragilis infection in pig and human dientamoebiasis remains to be clarified. It is possible that since there was such a high incidence of this protozoan in pigs, that pigs may well play a role as a reservoir of the parasite for humans, even if the mode of transmission remains unclear. Table 1 Intestinal protozoa isolated from swine faeces between February and October 2006 Protozoa
No. of samples
Positivity (%)
Iodamoeba bu¨tschlii Endolimax nana Dientamoeba fragilis Blastocystis hominis Entamoeba suis Entamoeba polecki Chilomastix mesnili Giardia spp. Entamoeba dispar
97 55 53 14 9 5 3 2 2
80.2 45.4 43.8 11.6 7.4 4.1 2.5 1.6 1.6
D. Crotti et al. / Veterinary Parasitology 145 (2007) 349–351
Acknowledgements The authors wish to thank Sonia Salamida and Angela Caporali for their contribution to sample collection and technical support. References Crotti, D., D’Annibale, M.L., 2001. Dientamoeba fragilis e dientamoebiasi: aspetti di parassitologia clinica e diagnostica di laboratorio. Parassitologia 43, 135–138. Crotti, D., D’Annibale, M.L., Fonzo, G., Lalle, M., Caccio`, S.M., Pozio, E., 2005. Dientamoeba fragilis is more prevalent than Giardia duodenalis in children and adults attending a day care centre in Central Italy. Parasite 12, 165–170. Johnson, E.H., Windsor, J.J., Graham Clark, C., 2004. Emerging from obscurity: biological, clinical, and diagnostic aspects of Dientamoeba fragilis. Clin. Microbiol. Rev. 17, 553–570. Menghi, C.I., Makiya, R., Gatta, C.L., Me´ndez, O.C., 2005. Dientamoeba fragilis: molecular biology techniques for the
351
Elucidation of hits mode of transmission. Parassitol. Latinoam. 60, 25–31. Sawangjaroen, N., Luke, R., Prociv, P., 1993. Diagnosis by faecal culture of Dientamoeba fragilis infections in Australian patients with diarrhoea. Trans. R. Soc. Trop. Med. Hyg. 87 (2), 163– 165. Stark, D., Beebe, N., Marriott, D., Ellis, J., Harkness, J., 2005. Detection of Dientamoeba fragilis in fresh stool specimens using PCR. Int. J. Parasitol. 35, 57–62. Stark, D., Beebe, N., Marriott, D., Ellis, J., Harkness, J., 2006. Evaluation of three diagnostic methods, including real-time PCR, for detection of Dientamoeba fragilis in stool specimens. J. Clin. Microbiol. 44 (1), 232–235. Windsor, J.J., Rafay, A.M., Shenoy, A.K., Johnson, E.H., 1998. Incidence of Dientamoeba fragilis in faecal samples submitted for routine microbiological analyses. Br. J. Biomed. Sci. 55, 172–175. Windsor, J.J., Johnson, E.H., 1999. Dientamoeba fragilis: the unflagellate human flagellate. Br. J. Biomed. Sci. 56, 293–306. Windsor, J.J., Macfarlane, L., Hughes-Thapa, G., Jones, S.K., Whiteside, T.M., 2003. Detection of Dientamoeba fragilis by culture. Br. J. Biomed. Sci. 60, 79–83.