First description of Echinococcus ortleppi infection in China

Background Echinococcosis has led to considerable social and economic losses in China, particularly in the endemic communities of the eastern Tibetan Plateau. In China, human cases of Echinococcus granulosus (sensu stricto), E. canadensis and E. multilocularis infections have been described, but no E. ortleppi (G5) infections in humans or animals have been reported. Results A case of E. ortleppi infection in a human from Guangxi, which is a non-endemic echinococcosis area in China, is described. A 17 × 12 × 20 cm (diameter) cyst was observed in the liver of the patient, and Echinococcus larvae were collected from the cyst. A morphological examination indicated that the larvae were E. ortleppi, and amplification and analysis of the nicotinamide adenine dinucleotide hydrogenase dehydrogenase subunit 1 (nad1) and cytochrome c oxidase subunit 1 (cox1) genes showed that the larvae had 99–100% homology with the corresponding E. ortleppi sequences on GenBank. Conclusions To our knowledge, this report describes the first identification of a human E. ortleppi infection in China. Our data broaden the geographical distribution of this rarely reported species of Echinococcus. Electronic supplementary material The online version of this article (10.1186/s13071-019-3653-y) contains supplementary material, which is available to authorized users.

Echinococcus ortleppi was formerly known as the cattle strain of E. granulosus (s.l.) or genotype G5 [6,7], and its morphology and developmental features show substantial differences compared with those of E. granulosus (s.s.) and other taxa. In addition to cattle, buffaloes, camels, sheep, pigs, goats, monkeys and deer can also be infected with E. ortleppi [8][9][10][11][12]. In recent years, several cases of E. ortleppi infection have been reported in Europe, South America, Africa and Asia [13]. Humans acquire the infection and become aberrant intermediate hosts through the accidental ingestion of parasite eggs and metacestodes develop in the liver or lungs.

Case presentation
The 65-year-old male described in this report was residing in Rongshui County, Liuzhou City, Guangxi Province, Southern China. At the time of study he was a teacher, was living in his place of birth, had never left the country prior to diagnosis and had not travelled to any echinococcosis-or schistosomiasis-endemic areas. In May 2017, he felt pain in the right upper abdomen; this pain was persistent and not accompanied by any paroxysmal aggravation, and all other clinical signs were not significant. He did not present with a fever, cough or other symptoms. He visited the People's Hospital of Miao Autonomous County in Rongshui County, and his clinical diagnosis and abdominal ultrasonography indicated a "hepatic cyst".

Morphological and molecular identification
The surgery was conducted laparoscopically, and Echinococcus was found in and collected from the fluid-filled cyst in the liver. The morphology of the collected Echinococcus was assessed. The total DNA of the Echinococcus parasite was extracted using a commercial DNeasy ® Blood & Tissue Kit (Qiagen, Hilden, Germany) in accordance with the manufacturer's instructions. Two mitochondrial genes, cytochrome c oxidase subunit 1 (cox1) and nicotinamide dehydrogenase subunit 1 (nad1), were amplified using the primers F/COI (5′-TTG AAT TTG CCA CGT TTG AA TGC-3′) and R/COI (5′-GAA CCT AAC GAC ATA ACA TAA TGA-3′) [20], and JB11 (5′-AGA TTC GTA AGG GGC CTA ATA-3′) and JB12 (5′-ACC ACT AAC TAA TTC ACT TTC-3′) [21], respectively. The PCRs were performed in a reaction volume of 25 μl containing 4 μl of DNA, 12.5 μl of PCR mix (Takara, Dalian, China), 1 μl of each primer (10 pmol/μl) and 6.5 μl of PCR-grade water. The amplification consisted of 94 °C for 3 min followed by 35 cycles of denaturation at 94 °C for 1 min, annealing at 50 °C (cox1)/54 °C (nad1) for 1 min and extension at 72 °C for 1 min, and a final extension step at 72 °C for 10 min. The PCR products were analysed by electrophoresis in 1.5% agarose gels. The amplified DNA was purified from the gel using an Agarose Gel DNA Extraction Kit (Takara), cloned into a PMD-19T vector (Takara) and transformed and propagated into DH5α-competent E. coli (Takara).
The T plasmid vector-conserved primer M13 was used to select the positive clones, and the plasmid DNA was isolated from single colonies using a MiniBEST Plasmid Purification Kit (Takara). The recombinant plasmids were sequenced at Sangon Biotech (Shanghai, China). The sequences were compared with those available in the GenBank database using the Basic Local Alignment Search Tool (BLASTn; http://blast .ncbi.nlm.nih.gov/ Blast .cgi). The phylogenetic trees of the nad1 and cox1 gene sequences of E. ortleppi China and other related strains were constructed using the maximum likelihood (ML) method with MEGA v.7.0 [22].

Results
Abdominal ultrasonography showed cystic-like low-density shadows in the right lobe of the liver; the boundary was clear, and the size of the shadow was approximately 17 × 12 × 20 cm (Fig. 1). No dilatation of the intrahepatic and extrahepatic bile ducts was observed, and no abnormal tissue was detected in the hilum of the liver. The size and shape of the gall-bladder and spleen were normal. According to the WHO ultrasound image classification guidelines [23], the CE type belongs to CE1-large (CE1l). A laparoscopic examination revealed compensatory enlargement of the left lobe of the liver, enlargement of the right lobe of the liver and morphological abnormalities. The cystic mass was approximately 17 × 12 × 20 cm in size and exhibited a clear cystic fluid, calcification of the cyst wall, and a layer of translucent white powdery material with a thickness of approximately 0.2-0.3 cm in the cyst. The cyst (cystic echinococcosis) contained many protoscoleces, each of which had many hooks (Fig. 2a-c). Each protoscolex appeared as a spherical body with a diameter of approximately 95 × 80 µm and an invaginated scolex with 30 hooks (Fig. 2b), and each hook was 20 μm (Fig. 2d).
A 935-bp nucleotide sequence for cox1 loci and a 530-bp sequence for nad1 loci were obtained through sequencing. The cox1 and nad1 sequences were submitted to the GenBank database under the accession numbers MN058591 and MN058592, respectively. The BLAST analysis showed that the cox1 sequence exhibited 99-100% homology with that of E. ortleppi in Gen-Bank isolates previously collected in France (GenBank: KU743919, KU743922 and KU743922), Estonia (Gen-Bank: KY766908, KY766906 and KY766907), Japan (Gen-Bank: AB235846), Africa (e.g. GenBank: KU842045) and the UK (GenBank: KU378107). The nad1 sequences showed 99-100% homology to the E. ortleppi sequences in GenBank obtained from Estonia (GenBank: KY766908 and KY766907) and Sudan (GenBank: KU842045). Both cox1 and nad1 sequences showed only 93-94% homology to those of E. granulosus. The phylogenetic tree of E. ortleppi and other related strains derived from part of the cox1 and nad1 genes showed that the E. ortleppi strain collected in China belongs to the same group as the strains of E. ortleppi in GenBank (Additional file 1: Figure  S1 and Additional file 2: Figure S2, respectively).
Echinococcus ortleppi-infected serum was detected using an ELISA kit (Combined Biotech Co. Ltd., Shenzhen, China) that could detect a wide range of Echinococcus [including E. granulosus (s.s.) and E. multilocularis] IgG antibodies and showed positive reactivity with E. ortleppi. To determine whether the animals and humans in the patient's residential area were infected with E. ortleppi, 10 dog faeces samples were collected and examined using the Kato-Katz technique. Forty human serum samples from families, neighbours and colleagues of patients were collected and analysed using an ELISA kit (Combined Biotech Co. Ltd., Shenzhen, China) to detect Echinococcus IgG antibodies. No positive samples were detected from dogs or humans.
China is one of the most important endemic regions of echinococcosis [43], and echinococcosis is a major parasitic problem in humans and livestock. It has been estimated that China has 0.6-1.3 million cases of human echinococcosis by 2000 [44]. Most of the provinces have reported human infections, and western and northwest China are the main endemic areas.
The eastern Tibetan Plateau, China, has the highest reported prevalence of echinococcosis in the world [45]. Echinococcus granulosus and E. multilocularis are common species in China, and five genotypes of E. granulosus (s.l.), namely G1, G3, G6, G7 and G10, have been reported [14][15][16][17][18]. Here, we report a case of E. ortleppi (G5) infection in a human residing in a nonendemic echinococcosis area in China. It is generally believed that humans who are infected with echinococcosis in non-endemic areas obtained the infection mainly from endemic areas when they visited these areas or ingested the eggs on contaminated fruits from endemic areas. In this study, we did not detect hydatid infection in dog or human samples collected near the patient's home. Because the incubation period of echinococcosis can be as long as 30 years, it is impossible to determine whether the reported case should be considered an imported or local case. Three confirmed human cases of hydatid infections in Guangxi have been reported, and the patients did not reside in echinococcosis-endemic areas. Because the parasites were not subjected to molecular analysis, their species and genotype were not identified. Some echinococcosis cases in non-endemic areas of China have been reported, and the infection source in some of these cases has not been identified. We hypothesize that these patients might also have been considered cases of local E. ortleppi infection. Therefore, we strongly suggest that the parasites from patients with echinococcosis residing in non-endemic areas should be subjected to genotyping and molecular analysis to identify the Echinococcus species.

Conclusions
This study reports a case of E. ortleppi infection that caused cystic echinococcosis in the liver of a Chinese patient. To our knowledge, this case constitutes the first detection of this species in China, and the identification of this first case highlights the need to enhance national surveillance efforts, particularly human, livestock and dog surveillance in non-endemic areas. Moreover, the Echinococcus species and genotype of infections should be detected.

Additional files
Additional file 1: Figure S1. Phylogenetic tree for Echinococcus spp. based on the cox1 gene including the sequence of E. ortleppi from China. Additional file 2: Figure S2. Phylogenetic tree for Echinococcus spp. based on the nad1 gene including the sequence of E. ortleppi from China.