Free Standard AU & NZ Shipping For All Book Orders Over $80!
Register      Login
Wildlife Research Wildlife Research Society
Ecology, management and conservation in natural and modified habitats
RESEARCH ARTICLE

Variation in Toxoplasma gondii seroprevalence: effects of site, sex, species and behaviour between insular and mainland macropods

Patrick L. Taggart https://orcid.org/0000-0001-9523-0463 A E , Bronwyn A. Fancourt https://orcid.org/0000-0003-2969-1530 B C , David Peacock https://orcid.org/0000-0003-2891-8238 A D , Charles G. B. Caraguel A * and Milton M. McAllister A *
+ Author Affiliations
- Author Affiliations

A School of Animal and Veterinary Sciences, The University of Adelaide, Mudla Wirra Road, Roseworthy, SA 5371, Australia.

B Pest Animal Research Centre, Biosecurity Queensland, Department of Agriculture and Fisheries, Toowoomba, Qld 4350, Australia.

C School of Environmental and Rural Science, University of New England, Armidale, NSW 2350, Australia.

D Biosecurity South Australia, GPO Box 1671, Adelaide, SA 5001, Australia.

E Corresponding author. Email: patrick.taggart@adelaide.edu.au

Wildlife Research 47(8) 540-546 https://doi.org/10.1071/WR19041
Submitted: 8 March 2019  Accepted: 10 June 2019   Published: 19 August 2019

Abstract

Context: Feral cats threaten wildlife conservation through a range of direct and indirect effects. However, most studies that have evaluated the impacts of feral cats on species of conservation significance have focussed on direct impacts such as predation; few studies have considered the indirect impacts of cat-borne disease. Toxoplasma gondii, a cat-borne parasite, causes both acute and latent disease in a range of wildlife species, and macropods are particularly susceptible. Kangaroo Island is Australia’s third largest island and supports a high density of feral cats and high seroprevalence of T. gondii in multiple species, relative to the mainland. This suggests that Kangaroo Island has a high environmental contamination with the parasite and a high risk of infection for other species.

Aims: We aimed to describe T. gondii seroprevalence in culled and road-killed macropods, so as to assess the effects of island versus mainland location, sex, species and behaviour.

Methods: Macropod sera were tested for T. gondii IgG antibodies using a commercially available modified agglutination test.

Key results: The seroprevalence of T. gondii in culled western grey kangaroos (Macropus fuliginosus) was significantly higher on the island (20%, 11/54 positive) than on the mainland (0%, 0/61 positive). There was no difference in T. gondii seroprevalence between culled and road-killed (21%, 21/102 positive) kangaroos from the island. The seroprevalence of T. gondii was significantly higher in female (32%, 12/38 positive) than in male (13%, 8/60 positive) kangaroos, but we observed no sex effect in tammar wallabies (Macropus eugenii), and no effect of species.

Conclusions: The higher T. gondii seroprevalence in insular macropods supports previous reports of higher T. gondii exposure in other Kangaroo Island fauna. The lack of difference in T. gondii seroprevalence between culled and road-killed kangaroos suggests that T. gondii-positive animals are not more vulnerable to road mortality, in contrast to that suggested previously.

Implications: Our findings suggest greater potential adverse conservation impacts owing to toxoplasmosis on the island than on the mainland. In light of a recent study demonstrating higher cat abundance on the island than on the mainland, the higher observed T. gondii seroprevalence in insular macropods is likely to be a consequence of higher cat density.

Additional keywords: carcass, feline, Felis catus, latent, marsupial, toxoplasmosis.


References

Ahlert, G. (2002). Longevity records: life spans of mammals, birds, amphibians, reptiles, and fish. Gerontology 48, 59.

Ahmad, N., Iqbal, Z., Mukhtar, M., Mushtaq, M., Khan, K. M., and Qayyum, M. (2015). Seroprevalence and associated risk factors of toxoplasmosis in sheep and goats in Pothwar region, northern Punjab, Pakistan. Pakistan Journal of Zoology 1, 161–167.

Alexander, J., and Stimson, W. (1988). Sex hormones and the course of parasitic infection. Parasitology Today (Personal Ed.) 4, 189–193.
Sex hormones and the course of parasitic infection.Crossref | GoogleScholarGoogle Scholar |

Aramini, J. J., Stephen, C., Dubey, J., Engelstoft, C., Schwantje, H., and Ribble, C. (1999). Potential contamination of drinking water with Toxoplasma gondii oocysts. Epidemiology and Infection 122, 305–315.
Potential contamination of drinking water with Toxoplasma gondii oocysts.Crossref | GoogleScholarGoogle Scholar | 10355797PubMed |

Campbell, I. (2007). Chi‐squared and Fisher–Irwin tests of two‐by‐two tables with small sample recommendations. Statistics in Medicine 26, 3661–3675.
Chi‐squared and Fisher–Irwin tests of two‐by‐two tables with small sample recommendations.Crossref | GoogleScholarGoogle Scholar | 17315184PubMed |

Canfield, P., Hartley, W., and Dubey, J. (1990). Lesions of toxoplasmosis in Australian marsupials. Journal of Comparative Pathology 103, 159–167.
Lesions of toxoplasmosis in Australian marsupials.Crossref | GoogleScholarGoogle Scholar | 2246391PubMed |

Courchamp, F., Chapuis, J.-L., and Pascal, M. (2003). Mammal invaders on islands: impact, control and control impact. Biological Reviews of the Cambridge Philosophical Society 78, 347–383.
Mammal invaders on islands: impact, control and control impact.Crossref | GoogleScholarGoogle Scholar | 14558589PubMed |

Dard, C., Bailly, S., Drouet, T., Fricker-Hidalgo, H., Brenier-Pinchart, M., and Pelloux, H. (2017). Long-term sera storage does not significantly modify the interpretation of toxoplasmosis serologies. Journal of Microbiological Methods 134, 38–45.
Long-term sera storage does not significantly modify the interpretation of toxoplasmosis serologies.Crossref | GoogleScholarGoogle Scholar | 28093212PubMed |

de Wit, L. A., Croll, D. A., Tershy, B., Correa, D., Luna-Pasten, H., Quadri, P., and Kilpatrick, A. M. (2019). Potential public health benefits from cat eradications on islands. PLoS Neglected Tropical Diseases 13, e0007040.
Potential public health benefits from cat eradications on islands.Crossref | GoogleScholarGoogle Scholar | 30763304PubMed |

Desmonts, G., and Remington, J. S. (1980). Direct agglutination test for diagnosis of Toxoplasma infection: method for increasing sensitivity and specificity. Journal of Clinical Microbiology 11, 562–568.
| 7000807PubMed |

Dietz, H. H., Henriksen, P., Bille-Hansen, V., and Henriksen, S. A. (1997). Toxoplasmosis in a colony of New World monkeys. Veterinary Parasitology 68, 299–304.
Toxoplasmosis in a colony of New World monkeys.Crossref | GoogleScholarGoogle Scholar | 9106950PubMed |

Dohoo, I. R., Martin, W., and Stryhn, H. (2009). Chapter 12: validity in observational studies. In ‘Veterinary Epidemiologic Research’. 2nd edn. (Ed. S. M. McPike) pp. 243–270. (AVC Incorporated: Charlottetown, Canada.)

Dubey, J. P. (2016a). ‘Toxoplasmosis of Animals and Humans.’ 2nd edn. (CRC Press: Boca Raton, FL.)

Dubey, J. P. (2016b). Chapter 1. General biology. In ‘Toxoplasmosis of Animals and Humans’. 2nd edn. pp. 1–72. (CRC Press: Boca Raton, FL.)

Dubey, J. P., and Crutchley, C. (2008). Toxoplasmosis in wallabies (Macropus rufogriseus and Macropus eugenii): blindness, treatment with atovaquone, and isolation of Toxoplasma gondii. The Journal of Parasitology 94, 929–933.
Toxoplasmosis in wallabies (Macropus rufogriseus and Macropus eugenii): blindness, treatment with atovaquone, and isolation of Toxoplasma gondii.Crossref | GoogleScholarGoogle Scholar | 18576797PubMed |

Dubey, J., and Desmonts, G. (1987). Serological responses of equids fed Toxoplasma gondii oocysts. Equine Veterinary Journal 19, 337–339.
Serological responses of equids fed Toxoplasma gondii oocysts.Crossref | GoogleScholarGoogle Scholar | 3622463PubMed |

Dubey, J., Bhaiyat, M., Macpherson, C. N. L., De Allie, C., Chikweto, A., Kwok, O. C. H., and Sharma, R. (2006). Prevalence of Toxoplasma gondii in rats (Rattus norvegicus) in Grenada, West Indies. The Journal of Parasitology 92, 1107–1108.
Prevalence of Toxoplasma gondii in rats (Rattus norvegicus) in Grenada, West Indies.Crossref | GoogleScholarGoogle Scholar | 17152961PubMed |

Dubey, J., Lago, E., Gennari, S., Su, C., and Jones, J. (2012). Toxoplasmosis in humans and animals in Brazil: high prevalence, high burden of disease, and epidemiology. Parasitology 139, 1375–1424.
Toxoplasmosis in humans and animals in Brazil: high prevalence, high burden of disease, and epidemiology.Crossref | GoogleScholarGoogle Scholar | 22776427PubMed |

Fancourt, B. A. (2015). Making a killing: photographic evidence of predation of a Tasmanian pademelon (Thylogale billardierii) by a feral cat (Felis catus). Australian Mammalogy 37, 120–124.
Making a killing: photographic evidence of predation of a Tasmanian pademelon (Thylogale billardierii) by a feral cat (Felis catus).Crossref | GoogleScholarGoogle Scholar |

Fancourt, B. A., and Jackson, R. B. (2014). Regional seroprevalence of Toxoplasma gondii antibodies in feral and stray cats (Felis catus) from Tasmania. Australian Journal of Zoology 62, 272–283.
Regional seroprevalence of Toxoplasma gondii antibodies in feral and stray cats (Felis catus) from Tasmania.Crossref | GoogleScholarGoogle Scholar |

Fancourt, B. A., Nicol, S. C., Hawkins, C. E., Jones, M. E., and Johnson, C. N. (2014). Beyond the disease: is Toxoplasma gondii infection causing population declines in the eastern quoll (Dasyurus viverrinus)? International Journal for Parasitology. Parasites and Wildlife 3, 102–112.
Beyond the disease: is Toxoplasma gondii infection causing population declines in the eastern quoll (Dasyurus viverrinus)?Crossref | GoogleScholarGoogle Scholar | 25161908PubMed |

Fancourt, B. A., Hawkins, C. E., Cameron, E. Z., Jones, M. E., and Nicol, S. C. (2015). Devil declines and catastrophic cascades: is mesopredator release of feral cats inhibiting recovery of the eastern quoll? PLoS One 10, e0119303.
Devil declines and catastrophic cascades: is mesopredator release of feral cats inhibiting recovery of the eastern quoll?Crossref | GoogleScholarGoogle Scholar | 26106887PubMed |

Frenkel, J., Ruiz, A., and Chinchilla, M. (1975). Soil survival of Toxoplasma oocysts in Kansas and Costa Rica. The American Journal of Tropical Medicine and Hygiene 24, 439–443.
Soil survival of Toxoplasma oocysts in Kansas and Costa Rica.Crossref | GoogleScholarGoogle Scholar | 1098494PubMed |

Glor, S. B., Edelhofer, R., Grimm, F., Deplazes, P., and Basso, W. (2013). Evaluation of a commercial ELISA kit for detection of antibodies against Toxoplasma gondii in serum, plasma and meat juice from experimentally and naturally infected sheep. Parasites & Vectors 6, 85.
Evaluation of a commercial ELISA kit for detection of antibodies against Toxoplasma gondii in serum, plasma and meat juice from experimentally and naturally infected sheep.Crossref | GoogleScholarGoogle Scholar |

Gondim, L. F., Mineo, J. R., and Schares, G. (2017). Importance of serological cross-reactivity among Toxoplasma gondii, Hammondia spp., Neospora spp., Sarcocystis spp. and Besnoitia besnoiti. Parasitology 144, 851–868.
Importance of serological cross-reactivity among Toxoplasma gondii, Hammondia spp., Neospora spp., Sarcocystis spp. and Besnoitia besnoiti.Crossref | GoogleScholarGoogle Scholar | 28241894PubMed |

Hardman, B., Moro, D., and Calver, M. (2016). Direct evidence implicates feral cat predation as the primary cause of failure of a mammal reintroduction programme. Ecological Management & Restoration 17, 152–158.
Direct evidence implicates feral cat predation as the primary cause of failure of a mammal reintroduction programme.Crossref | GoogleScholarGoogle Scholar |

Hill, D., and Dubey, J. (2002). Toxoplasma gondii: transmission, diagnosis and prevention. Clinical Microbiology and Infection 8, 634–640.
Toxoplasma gondii: transmission, diagnosis and prevention.Crossref | GoogleScholarGoogle Scholar | 12390281PubMed |

Hillman, A. E., Lymbery, A. J., and Thompson, R. A. (2016). Is Toxoplasma gondii a threat to the conservation of free-ranging Australian marsupial populations? International Journal for Parasitology. Parasites and Wildlife 5, 17–27.
Is Toxoplasma gondii a threat to the conservation of free-ranging Australian marsupial populations?Crossref | GoogleScholarGoogle Scholar | 27141439PubMed |

Hollings, T., Jones, M., Mooney, N., and McCallum, H. (2013). Wildlife disease ecology in changing landscapes: mesopredator release and toxoplasmosis. International Journal for Parasitology. Parasites and Wildlife 2, 110–118.
Wildlife disease ecology in changing landscapes: mesopredator release and toxoplasmosis.Crossref | GoogleScholarGoogle Scholar | 24533323PubMed |

Horby, P. W., Laurie, K. L., Cowling, B. J., Engelhardt, O. G., Sturm‐Ramirez, K., Sanchez, J. L., Katz, J. M., Uyeki, T. M., Wood, J., and Van Kerkhove, M. D. (2017). CONSISE statement on the reporting of Seroepidemiologic Studies for influenza (ROSES‐I statement): an extension of the STROBE statement. Influenza and Other Respiratory Viruses 11, 2–14.
CONSISE statement on the reporting of Seroepidemiologic Studies for influenza (ROSES‐I statement): an extension of the STROBE statement.Crossref | GoogleScholarGoogle Scholar | 27417916PubMed |

Hutchison, W. M., Dunachie, J. F., Work, K., and Siim, J. C. (1971). The life cycle of the coccidian parasite, Toxoplasma gondii, in the domestic cat. Transactions of the Royal Society of Tropical Medicine and Hygiene 65, 380–398.
The life cycle of the coccidian parasite, Toxoplasma gondii, in the domestic cat.Crossref | GoogleScholarGoogle Scholar | 4104434PubMed |

Inns, R. (1982). Age determination in the Kangaroo Island wallaby, Macropus eugenii (Desmarest). Wildlife Research 9, 213–220.
Age determination in the Kangaroo Island wallaby, Macropus eugenii (Desmarest).Crossref | GoogleScholarGoogle Scholar |

Jenkins, R. B. (1985). ‘Parks of the Fleurieu Peninsula: Draft Management Plan. Part 2: Fleurieu Peninsula – The Region.’ (National Parks and Wildlife Service, Department of Environment and Planning SA: Adelaide, SA.)

Johnson, A., Roberts, H., Statham, P., and Munday, B. (1989). Serodiagnosis of acute toxoplasmosis in macropods. Veterinary Parasitology 34, 25–33.
Serodiagnosis of acute toxoplasmosis in macropods.Crossref | GoogleScholarGoogle Scholar | 2588468PubMed |

Jones, J. L., Kruszon-Moran, D., Wilson, M., McQuillan, G., Navin, T., and McAuley, J. B. (2001). Toxoplasma gondii infection in the United States: seroprevalence and risk factors. American Journal of Epidemiology 154, 357–365.
Toxoplasma gondii infection in the United States: seroprevalence and risk factors.Crossref | GoogleScholarGoogle Scholar | 11495859PubMed |

Kittas, S., Kittas, C., Paizi-Biza, P., and Henry, L. (1984). A histological and immunohistochemical study of the changes induced in the brains of white mice by infection with Toxoplasma gondii. British Journal of Experimental Pathology 65, 67–74.
| 6365146PubMed |

Lynch, M., Obendorf, D., Statham, P., and Reddacliff, G. (1993). An evaluation of a live Toxoplasma gondii vaccine in Tammar wallabies (Macropus eugenii). Australian Veterinary Journal 70, 352–353.
An evaluation of a live Toxoplasma gondii vaccine in Tammar wallabies (Macropus eugenii).Crossref | GoogleScholarGoogle Scholar | 8240176PubMed |

Mainar-Jaime, R., and Barberan, M. (2007). Evaluation of the diagnostic accuracy of the modified agglutination test (MAT) and an indirect ELISA for the detection of serum antibodies against Toxoplasma gondii in sheep through Bayesian approaches. Veterinary Parasitology 148, 122–129.
Evaluation of the diagnostic accuracy of the modified agglutination test (MAT) and an indirect ELISA for the detection of serum antibodies against Toxoplasma gondii in sheep through Bayesian approaches.Crossref | GoogleScholarGoogle Scholar | 17624672PubMed |

Medina, F. M., Bonnaud, E., Vidal, E., and Nogales, M. (2014). Underlying impacts of invasive cats on islands: not only a question of predation. Biodiversity and Conservation 23, 327–342.
Underlying impacts of invasive cats on islands: not only a question of predation.Crossref | GoogleScholarGoogle Scholar |

Murata, F. H., Cerqueira-Cézar, C. K., Kwok, O. C., Tiwari, K., Sharma, R. N., Su, C., and Dubey, J. (2018). Role of rats (Rattus norvegicus) in the epidemiology of Toxoplasma gondii infection in Grenada, West Indies. The Journal of Parasitology 104, 571–573.
Role of rats (Rattus norvegicus) in the epidemiology of Toxoplasma gondii infection in Grenada, West Indies.Crossref | GoogleScholarGoogle Scholar | 29986158PubMed |

Newsome, A. (1980). Differences in the diets of male and female red kangaroos in central Australia. African Journal of Ecology 18, 27–31.
Differences in the diets of male and female red kangaroos in central Australia.Crossref | GoogleScholarGoogle Scholar |

Norbury, G., Coulson, G., and Walters, B. (1988). Aspects of the Demography of the Western grey-kangaroo, Macropus–Fuliginosus–Melanops, in semiarid northwest Victoria. Wildlife Research 15, 257–266.
Aspects of the Demography of the Western grey-kangaroo, Macropus–Fuliginosus–Melanops, in semiarid northwest Victoria.Crossref | GoogleScholarGoogle Scholar |

Ntafis, V., Xylouri, E., Diakou, A., Sotirakoglou, K., Kritikos, I., Georgakilas, E., and Menegatos, I. (2007). Serological survey of antibodies against Toxoplasma gondii in organic sheep and goat farms in Greece. Journal of the Hellenic Veterinary Medical Society 58, 22–33.
Serological survey of antibodies against Toxoplasma gondii in organic sheep and goat farms in Greece.Crossref | GoogleScholarGoogle Scholar |

O’Donoghue, P. J., Riley, M. J., and Clarke, J. F. (1987). Serological survey for Toxoplasma infections in sheep. Australian Veterinary Journal 64, 40–45.
Serological survey for Toxoplasma infections in sheep.Crossref | GoogleScholarGoogle Scholar | 3606503PubMed |

Obendorf, D. L., and Munday, B. L. (1983). Toxoplasmosis in wild Tasmanian wallabies. Australian Veterinary Journal 60, 62.
| 6838441PubMed |

Pan, S., Thompson, R. A., Grigg, M. E., Sundar, N., Smith, A., and Lymbery, A. J. (2012). Western Australian marsupials are multiply infected with genetically diverse strains of Toxoplasma gondii. PLoS One 7, e45147.
Western Australian marsupials are multiply infected with genetically diverse strains of Toxoplasma gondii.Crossref | GoogleScholarGoogle Scholar | 23185317PubMed |

Parameswaran, N., O’Handley, R. M., Grigg, M. E., Fenwick, S. G., and Thompson, R. C. A. (2009b). Seroprevalence of Toxoplasma gondii in wild kangaroos using an ELISA. Parasitology International 58, 161–165.
Seroprevalence of Toxoplasma gondii in wild kangaroos using an ELISA.Crossref | GoogleScholarGoogle Scholar | 19567231PubMed |

Poirotte, C., Kappeler, P. M., Ngoubangoye, B., Bourgeois, S., Moussodji, M., and Charpentier, M. J. (2016). Morbid attraction to leopard urine in Toxoplasma-infected chimpanzees. Current Biology 26, R98–R99.
Morbid attraction to leopard urine in Toxoplasma-infected chimpanzees.Crossref | GoogleScholarGoogle Scholar | 26859275PubMed |

R Core Team (2018). ‘R: a Language and Environment for Statistical Computing.’ (R Foundation for Statistical Computing: Vienna, Austria.) Available at https://www.R-project.org/ [verified 12 January 2019].

Reddacliff, G., Hartley, W., Dubey, J., and Cooper, D. (1993). Pathology of experimentally‐induced, acute toxoplasmosis in macropods. Australian Veterinary Journal 70, 4–6.
Pathology of experimentally‐induced, acute toxoplasmosis in macropods.Crossref | GoogleScholarGoogle Scholar | 8460990PubMed |

Schwerdtfeger, P. (2002). Chapter 5: Climate. In ‘Natural History of Kangaroo Island’. (Eds M. Davies, C. R. Twidale and M. J. Tyler) (Royal Society of South Australia: Adelaide, SA.)

Sedlák, K., Literák, I., Faldyna, M., Toman, M., and Benák, J. (2000). Fatal toxoplasmosis in brown hares (Lepus europaeus): possible reasons of their high susceptibility to the infection. Veterinary Parasitology 93, 13–28.
Fatal toxoplasmosis in brown hares (Lepus europaeus): possible reasons of their high susceptibility to the infection.Crossref | GoogleScholarGoogle Scholar | 11027857PubMed |

Taggart, P. L., Fancourt, B. A., Bengsen, A. J., Peacock, D. E., Hodgens, P., Read, J. L., McAllister, M. M., and Caraguel, C. G. B. (2019). Evidence of significantly higher island feral cat abundance compared with the adjacent mainland Wildlife Research 46, 378–385.

Telfer, W. R., and Bowman, D. M. (2006). Diet of four rock‐dwelling macropods in the Australian monsoon tropics. Austral Ecology 31, 817–827.
Diet of four rock‐dwelling macropods in the Australian monsoon tropics.Crossref | GoogleScholarGoogle Scholar |

Teshale, S., Dumetre, A., Dardé, M.-L., Merga, B., and Dorchies, P. (2007). Serological survey of caprine toxoplasmosis in Ethiopia: prevalence and risk factors. Parasite (Paris, France) 14, 155–159.
Serological survey of caprine toxoplasmosis in Ethiopia: prevalence and risk factors.Crossref | GoogleScholarGoogle Scholar |

Tryland, M., Handeland, K., Bratberg, A.-M., Solbakk, I.-T., and Oksanen, A. (2006). Persistence of antibodies in blood and body fluids in decaying fox carcasses, as exemplified by antibodies against Microsporum canis. Acta Veterinaria Scandinavica 48, 10.
Persistence of antibodies in blood and body fluids in decaying fox carcasses, as exemplified by antibodies against Microsporum canis.Crossref | GoogleScholarGoogle Scholar | 16987389PubMed |

van der Puije, W., Bosompem, K., Canacoo, E., Wastling, J., and Akanmori, B. (2000). The prevalence of anti-Toxoplasma gondii antibodies in Ghanaian sheep and goats. Acta Tropica 76, 21–26.
The prevalence of anti-Toxoplasma gondii antibodies in Ghanaian sheep and goats.Crossref | GoogleScholarGoogle Scholar | 10913761PubMed |

Vyas, A., Kim, S.-K., Giacomini, N., Boothroyd, J. C., and Sapolsky, R. M. (2007). Behavioral changes induced by Toxoplasma infection of rodents are highly specific to aversion of cat odors. Proceedings of the National Academy of Sciences, USA 104, 6442–6447.
Behavioral changes induced by Toxoplasma infection of rodents are highly specific to aversion of cat odors.Crossref | GoogleScholarGoogle Scholar |

Wann, J., and Bell, D. (1997). Dietary preferences of the black-gloved wallaby (Macropus irma) and the western grey kangaroo (M. fuliginosus) in Whiteman Park, Perth, Western Australia. Journal of the Royal Society of Western Australia 80, 55–62.

Yilmaz, S. M., and Hopkins, S. H. (1972). Effects of different conditions on duration of infectivity of Toxoplasma gondii oocysts. The Journal of Parasitology 58, 938–939.
Effects of different conditions on duration of infectivity of Toxoplasma gondii oocysts.Crossref | GoogleScholarGoogle Scholar | 5078600PubMed |