Research Article | Open Access
Monika Dybicz, Piotr K. Borkowski, Maurycy Jonas, Dariusz Wasiak, Piotr Małkowski, "First Report of Echinococcus ortleppi in Human Cases of Cystic Echinococcosis in Poland", BioMed Research International, vol. 2019, Article ID 2474839, 5 pages, 2019. https://doi.org/10.1155/2019/2474839
First Report of Echinococcus ortleppi in Human Cases of Cystic Echinococcosis in Poland
Cystic echinococcosis is considered as an emerging zoonosis that can develop asymptomatically for years, clinically nonpathognomic. The disease is of public health importance due to often late, difficult diagnostics, uncertain results of treatment, the need to remove hydatid cysts surgically in advanced cases, and poor prognosis in untreated patients. Six Polish female patients with diagnosed cystic echinococcosis (CE) were examined. DNA extracted from the liver and lung samples served for amplification of mitochondrial nad1 gene fragment. Sequence alignments of 5 isolates showed identity with the pig strain, Echinococcus canadensis G7. One case was in 100% identical with Echinococcus ortleppi G5, the cattle strain. These data demonstrate first report of E. ortleppi, regarded as extinct species, causing human cystic echinococcosis in Poland, where the most frequent causative agent of human CE is E. canadensis.
Cystic echinococcosis (CE) is a globally distributed zoonotic disease caused by Echinococcus granulosus sensu lato which is a member of the Taeniidae family. In the life cycle of Echinococcus species, which is mostly domestic, dogs are the definitive hosts; however, wild canids (dingoes, wolves, jackals, coyotes, and red foxes), felids, and hyenids can also become definitive hosts in the transmission cycle. The adult worm lives in the small intestine and delivers eggs excreted with the stool. When intermediate hosts (sheep, goat, swine, cattle, or accidentally human) ingest eggs, they develop into hydatid cysts in the internal organs. The cysts in humans develop most commonly in the liver, and then in lungs, brain, heart, and bones. In humans, the disease usually develops asymptomatically for years until growing cysts start to press on the surrounding tissues and give nonspecific symptoms. Treatment of CE is considered difficult, because of the involvement of the vital organs and possibility of dissemination of fluid with protoscoleces during the surgery. Both deficient resection and bladder rupture may contribute to anaphylaxis, recurrence, and multiple secondary CE [1–3].
Echinococcosis is regarded as an emerging disease occurring worldwide with the highest prevalence in parts of Eurasia, North and East Africa, Australia, and South America [4–8]. Molecular analysis, based mostly on mitochondrial genes [9–13], have demonstrated that E. granulosus is a complex of many species differing in host specificity, pathology, development rate, and sensitivity to chemotherapeutics. The taxonomic revisions have led to revealing E. granulosus sensu stricto (G1-G3 genotypes), Echinococcus equinus (G4), Echinococcus ortleppi (G5), Echinococcus canadensis (G6-G10), and Echinococcus felidis (“lion strain”) [11, 14].
E. ortleppi was originally described as E. granulosus in dogs from South Africa . The essential differences of E. ortleppi in development and morphology, compared to other species, have resulted in the later description of the distinct genotype as a separate species, related most closely to E. canadensis. E. ortleppi is considered as the cattle strain (G5), because it predominantly develops in cattle as intermediate host ; however, it has also been recorded in other animals such as sheep, pigs, goats, camels, buffaloes, deer, monkeys, and porcupine [17–23]. The cases of E. ortleppi causing human CE have been reported in South America, South Africa, Asia, and Europe, where animal and human [24–29] E. ortleppi infections have been documented rarely.
It is the first report on E. ortleppi cases, causing CE in Polish patients in Poland, who underwent surgery to remove hydatid cysts.
2. Materials and Methods
Fragments of cysts were collected from 6 female patients operated on in years 2016-2017 in the Department of Infectious Diseases, Tropical, and Hepatology, Department of Surgical and Transplantation Nursing, and Department of General and Transplantation Surgery, Medical University of Warsaw, Poland. The study was approved by the Bioethical Committee of Medical University of Warsaw, Poland. Certain patients with cystic echinococcosis had been under medical supervision for many years and were treated with albendazole. The cysts were removed surgically because of emerging complications. The collected patient data included age, gender, and place of residence. All 6 patients were females aged from 30 to 56 years. The examined samples represented by a fragment of a cyst were stored frozen at −20°C or fixed in 70% ethanol prior to molecular analysis.
2.1. DNA Extraction and PCR
The samples of examined isolates and E. canadensis G7 positive control (isolates JX266793 and JX266824) were rinsed with phosphate-buffered saline (PBS) several times to remove any ethanol residues and centrifuged at 5000 ×g for 10min. Each pellet was dissolved in 100 μL PBS and genomic DNA was extracted using a NucleoSpin kit (Macherey-Nagel, Düren, Germany) following the manufacturer’s instructions. Mitochondrial region of NADH dehydrogenase 1 (nad1) gene was amplified by PCR using primers JB11 (5’-AGATTCGTAAGGGGCCTAATA-3’) and JB12 (5’-ACCACTAACTAATTCACTTTC-3’) . The 50 μL reactions comprised of 1μL of DNA template, 50pM of each primer, 0.2mM of each dNTP, 1x PCR buffer containing 2.5mM MgCl2, and 1U of Taq DNA polymerase (Qiagen, Hilden, Germany). The following PCR was performed in a PTC-200 thermal cycler (MJ Research, Waltham, USA) in the following conditions: 3 min at 95°C followed by 35 cycles of 1 min at 95°C, 1 min at 50°C, 1 min at 72°C, and final extension at 72°C for 5 min . The PCR products were separated by electrophoresis on a 2% agarose gel (MetaPhor, FMC BioProducts, Philadelphia, USA) and then stained with ethidium bromide and observed on a UV transilluminator. The nad1 gene fragments were purified and then directly sequenced in both directions using a BigDye Ready Reaction Cycle Sequencing kit and an ABI 3730 Genetic Analyzer (Applied Biosystems, Foster City, USA). Chromatograms were manually checked and edited using Chromas 2.0. The obtained sequences were aligned with others retrieved from NCBI GenBank using ClustalW2 (http://www.ebi.ac.uk/Tools/clustalw2).
A total of 6 samples were obtained from female patients (at the age of 30-56 years) who underwent surgery to remove cysts located mostly in the liver, and a single cyst was found in the lung. The patients came from central Poland (4 patients) and the other 2 from villages of southeast and east Poland. The cysts ranged in size from 3 to 12 cm in diameter and were mainly sterile (type CE3 and CE4) (Table 1).
DNA extracted from all 6 samples and positive controls were used as the template in separate PCRs to amplify region of the mitochondrial NADH dehydrogenase 1. Each PCR produced a single band upon agarose gel electrophoresis. All isolates and controls were diagnosed as positive by amplification of nad1 fragment (~500 bp). The nad1 fragments were sequenced and compared with sequences of Echinococcus genotypes determined by using BLAST software (http://www.ncbi.nlm.nih.gov/blast). The sequences of 5 isolates showed 100% identity to E. canadensis G7, the pig strain. The sequence of 1 isolate was identical to the cattle strain E. ortleppi G5. All nad1 sequences were deposited in GenBank with accession numbers MH492787–MH492792.
Human cystic echinococcosis is an emerging zoonotic disease occurring worldwide caused by the metacestode stage of Echinococcus species. E. granulosus sensu lato is a complex of many genotypes differing in pathology, development, sensitivity to anthelmintics, and intermediate host specificity, including sheep, goats, horses, cattle, pigs, camels, and members of the cervid family. Many distinct genotypes of E. granulosus s.l., marked as G1-G10, have been identified based on genetic diversity of the mitochondrial genes, mainly NADH dehydrogenase (nad) and cytochrome C oxidase (cox) genes [11, 14, 32]. The genotype description of E. granulosus G1 revealed that the sheep strain is the cosmopolitan genotype most commonly distributed in areas of extensive sheep farming and the predominant strain infecting humans; however, some other strains are also infective. In Poland, molecular studies proved that E. canadensis G7, the pig strain, is the main genotype infecting humans [31, 33, 34]. In many European countries, G7 genotype has often been identified due to the fact that pigs are raised on small farms in close contact with the breeders and definitive hosts. In this analysis, 6 samples of human liver cyst fragments were examined. All patients were of Polish nationality, living in central and east regions of the country. Genetic study of 5 isolates also confirmed G7 genotype, affirming that this strain has a significant role as the main aetiological agent of human cystic echinococcosis in Poland.
Interestingly, one isolate was identical to the cattle strain G5; this is the first report representing E. ortleppi case causing human CE of the liver carried out in Poland. This isolate involved a 38-year-old female who lives in a house with her own vegetable garden in close proximity to the forest allowing the forest animals to trespass. The patient is an office work with not a rich travel history; she visited Germany once. The female patient was diagnosed to have a cyst (12 cm in diameter) showing thick wall of about 3 mm in the right liver lobe. Using computed tomography scan, the cyst was described as probably decaying calcified and did not show any specific features being different from the cysts of E. canadensis, diagnosed most commonly in Poland. Infection with E. ortleppi causing human CE is rarely diagnosed, although G5 genotype has already been reported from humans in Netherlands, France, Argentina, Brazil, Mexico, India, Vietnam, and South Africa [24–29]. As it has been suggested by some authors, E. ortleppi may become extinct in Europe because of fewer opportunities for its transmission between cattle and dogs . However, it has also been recorded in farm animals, ruminants, camels, buffaloes, deer, monkeys, and porcupine [17–23]; therefore, this Polish human CE case of E. ortleppi might have pig origin.
Molecular analysis of CE is not conducted frequently in Poland; therefore, it is impossible to determine if E. ortleppi is widespread in Poland or to evaluate the potential for human infection with this parasite. No studies were performed and the situation of genotype G5 in livestock in Poland is not known. Our identification of E. ortleppi infection in human enhances the need of systematic genotyping of Echinococcus cysts found in humans to determine the patterns of infection and pathology and expand the study of the strains being capable to infect human in Poland. In many areas of the country, pigs and cattle are bred on farms in close proximity to carnivorous definitive hosts, especially dogs, exposing humans to the eggs of the parasite. Genotypes E. ortleppi (G5) and E. canadensis (G7) are different in biology compared with some other strains of Echinococcus, for example, they have a shorter maturation period in dogs, which should be considered when planning the anthelmintic treatment regime for dogs. However, both strains can develop in humans, generating threats to the public health and the animal breeding.
In Poland, human CE is not frequent and mainly a result of infection with the pig strain of E. canadensis (G7). Our research represents the first record of human E. ortleppi infection, rarely detected in Europe. Furthermore, the future molecular studies of E. granulosus s.l. complex, inhabiting humans but also domestic and wildlife hosts, are attentive to provide important information on the population of Echinococcus genotypes and to better understand the aetiology and epidemiology of echinococcosis in Poland.
(1) The sequences (accession numbers MH492787–MH492792) used to support the findings of this study have been deposited in the GenBank repository. (2) The methods data used to support the findings of this study are included within the article. (3) Previously reported primers data were used to support this study and are available at https://doi.org/10.1016/0020-7519(93)90065-7. These prior studies (and datasets) are cited at relevant places within the text as references no. .
Conflicts of Interest
The authors declare that there are no conflicts of interest regarding the publication of this paper.
- Z. S. Pawlowski, J. Eckert, D. A. Vuitton et al., “Echinococcosis in humans: clinical aspects, diagnosis and treatment,” in WHO/OIE Manual on Echinococcosis in Humans and Animals: A Public Health Problem of Global Concern, J. Eckert, M. A. Gemmell, F. X. Meslin, and Z. S. Pawlowski, Eds., pp. 20–71, World Organisation for Animal Health, Paris, France, 2001.
- D. P. McManus, W. Zhang, J. Li, and P. B. Bartley, “Echinococcosis,” The Lancet, vol. 362, no. 9392, pp. 1295–1304, 2003.
- H. Wen, R. New, and P. Craig, “Diagnosis and treatment of human hydatidosis,” British Journal of Clinical Pharmacology, vol. 35, no. 6, pp. 565–574, 1993.
- T. Romig, A. Dinkel, and U. Mackenstedt, “The present situation of echinococcosis in Europe,” Parasitology International, vol. 55, supplement, pp. S187–S191, 2006.
- D. J. Jenkins, T. Romig, and R. C. A. Thompson, “Emergence/re-emergence of Echinococcus spp. - a global update,” International Journal for Parasitology, vol. 35, no. 11-12, pp. 1205–1219, 2005.
- A. Seimenis, “Overview of the epidemiological situation on echinococcosis in the Mediterranean region,” Acta Tropica, vol. 85, no. 2, pp. 191–195, 2003.
- A. Ito, C. Urbani, Q. Jiamin et al., “Control of echinococcosis and cysticercosis: a public health challenge to international cooperation in China,” Acta Tropica, vol. 86, no. 1, pp. 3–17, 2003.
- P. Moro and P. M. Schantz, “Cystic echinococcosis in the Americas,” Parasitology International, vol. 55, supplement, pp. S181–S186, 2006.
- J. Bowles, D. Blair, and D. P. McManus, “Genetic variants within the genus Echinococcus identified by mitochondrial DNA sequencing,” Molecular and Biochemical Parasitology, vol. 54, no. 2, pp. 165–173, 1992.
- A. Lavikainen, M. J. Lehtinen, S. Laaksonen, E. Agren, A. Oksanen, and S. Meri, “Molecular characterization of Echinococcus isolates of cervid origin from Finland and Sweden,” Parasitology, vol. 133, no. 5, pp. 565–570, 2006.
- M. Nakao, D. P. McManus, P. M. Schantz, P. S. Craig, and A. Ito, “A molecular phylogeny of the genus Echinococcus inferred from complete mitochondrial genomes,” Parasitology, vol. 134, no. 5, pp. 713–722, 2007.
- S. Maillard, M. C. Benchikh-Elfegoun, J. Knapp et al., “Taxonomic position and geographical distribution of the common sheep G1 and camel G6 strains of Echinococcus granulosus in three African countries,” Parasitology Research, vol. 100, no. 3, pp. 495–503, 2006.
- L. Kinkar, T. Laurimäe, M. Sharbatkhori et al., “New mitogenome and nuclear evidence on the phylogeny and taxonomy of the highly zoonotic tapeworm Echinococcus granulosus sensu stricto,” Infection, Genetics and Evolution, vol. 52, pp. 52–58, 2017.
- R. C. A. Thompson, “The taxonomy, phylogeny and transmission of Echinococcus,” Experimental Parasitology emphasizes, vol. 119, no. 4, pp. 439–446, 2008.
- J. R. Ortlepp, “Echinococcus in dogs from Pretoria and vicinity,” Onderstepoort Journal of Veterinary Science and Animal Industry, vol. 3, no. 1, pp. 97–108, 1934.
- T. Romig, D. Ebi, and M. Wassermann, “Taxonomy and molecular epidemiology of Echinococcus granulosus sensu lato,” Veterinary Parasitology, vol. 213, no. 3-4, pp. 76–84, 2015.
- S. Amer, I. B. Helal, E. Kamau, Y. Feng, and L. Xiao, “Molecular characterization of Echinococcus granulosus sensu lato from farm animals in Egypt,” PLoS ONE, vol. 10, no. 3, Article ID 0118509, 2015.
- R. P. Pednekar, M. L. Gatne, R. C. Thompson, and R. J. Traub, “Molecular and morphological characterisation of Echinococcus from food producing animals in India,” Veterinary Parasitology, vol. 165, no. 1-2, pp. 58–65, 2009.
- H. Mbaya, J. Magambo, S. Njenga et al., “Echinococcus spp. in central Kenya: a different story,” Parasitology Research, vol. 113, no. 10, pp. 3789–3794, 2014.
- M. E. Ahmed, K. H. Eltom, N. O. Musa et al., “First Report on Circulation of Echinococcus ortleppi in the one Humped Camel (Camelus dromedaries), Sudan,” BMC Veterinary Research, vol. 9, no. 127, 2013.
- B. Boufana, M. Stidworthy, S. Bell et al., “Echinococcus and Taenia spp. from captive mammals in the United Kingdom,” Veterinary Parasitology, vol. 190, no. 1-2, pp. 95–103, 2012.
- R. Plesker, T. Nadler, A. Dinkel, and T. Romig, “A case of an Echinococcus ortleppi infestation in a red-shanked douc langur (Pygathrix nemaeus) in northern Vietnam,” Vietnamese Journal of Primatology, vol. 3, pp. 75–81, 2009.
- A. Hodžić, A. Alić, J. Šupić, V. Škapur, and G. G. Duscher, “Echinococcus ortleppi, the cattle strain in a crested porcupine (Hystrix cristata): A new host record,” Veterinary Parasitology, vol. 256, pp. 32–34, 2018.
- F. Grenouillet, G. Umhang, F. Arbez-Gindre et al., “Echinococcus ortleppi Infections in Humans and Cattle, France,” Emerging Infectious Diseases, vol. 20, no. 12, pp. 2100–2102, 2014.
- M. Sharma, R. Sehgal, B. A. Fomda, A. Malhotra, and N. Malla, “Molecular Characterization of Echinococcus granulosus Cysts in North Indian Patients: Identification of G1, G3, G5 and G6 Genotypes,” PLOS Neglected Tropical Diseases, vol. 7, no. 6, 2013.
- V. D. Nguyen and D. Le Van, “The first report of two cases of cystic echinococcosis in the lung by Echinococcus ortleppi infection, in Vietnam,” Research and Reports in Tropical Medicine, vol. 8, pp. 45–51, 2017.
- M. L. de la Rue, K. Takano, J. F. Brochado et al., “Infection of humans and animals with Echinococcus granulosus (G1 and G3 strains) and E. ortleppi in Southern Brazil,” Veterinary Parasitology, vol. 177, no. 1-2, pp. 97–103, 2011.
- H. G. Avila, G. B. Santos, M. A. Cucher et al., “Implementation of new tools in molecular epidemiology studies of Echinococcus granulosus sensu lato in South America,” Parasitology International, vol. 66, no. 3, pp. 250–257, 2017.
- B. K. Mogoye, C. N. Menezes, M. L. Wong et al., “First insights into species and genotypes of Echinococcus in South Africa,” Veterinary Parasitology, vol. 196, no. 3-4, pp. 427–432, 2013.
- J. Bowles and D. P. McManus, “NADH dehydrogenase 1 gene sequences compared for species and strains of the genus Echinococcus,” International Journal for Parasitology, vol. 23, no. 7, pp. 969–972, 1993.
- M. Dybicz, A. Gierczak, J. Dabrowska, Ł. Rdzanek, and B. Michałowicz, “Molecular diagnosis of cystic echinococcosis in humans from central Poland,” Parasitology International, vol. 62, no. 4, pp. 364–367, 2013.
- T. Laurimäe, L. Kinkar, T. Romig et al., “The benefits of analysing complete mitochondrial genomes: Deep insights into the phylogeny and population structure of Echinococcus granulosus sensu lato genotypes G6 and G7,” Infection, Genetics and Evolution, vol. 64, pp. 85–94, 2018.
- A. H. Kȩdra, Z. Swiderski, V. V. Tkach et al., “Genetic analysis of Echinococcus granulosus from humans and pigs in Poland, Slovakia and Ukraine,” Acta Parasitologica, vol. 44, no. 4, pp. 248–254, 1999.
- M. Dybicz, P. K. Borkowski, J. Dąbrowska, and L. Chomicz, “Cases of Echinococcus granulosus sensu stricto isolated from polish patients: imported or indigenous?” BioMed Research International, vol. 2015, Article ID 728321, 5 pages, 2015.
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