Infection of sheep by Echinococcus multilocularis in Gansu, China: evidence from mitochondrial and nuclear DNA analysis

Background In the normal life cycle of the parasite (Echinococcus multilocularis) that causes alveolar echinococcosis, domestic and wild carnivores act as definitive hosts, and rodents act as intermediate hosts. The presented study contributes to the research on the distribution and transmission pattern of E. multilocularis in China having identified sheep as an unusual intermediate host taking part in the domestic transmission of alveolar echinococcosis in Gansu Province, China. Methods From 2020 to 2021, nine whitish different cyst-like were collected from the liver of sheep in Gansu Province for examination. A near complete mitochondrial (mt) genome and selected nuclear genes were amplified from the cyst-like lesion for identification. To confirm the status of the specimen, comparative analysis with reference sequences, phylogenetic analysis, and network analysis were performed. Results The isolates displayed ≥ 98.87% similarity to E. multilocularis NADH dehydrogenase sub-unit 1 (nad1) (894 bp) reference sequences deposited in GenBank. Furthermore, amplification of the nad4 and nad2 genes also confirmed all nine samples as E. multilocularis with > 99.30% similarity. Additionally, three nuclear genes, pepck (1545 bp), elp-exons VII and VIII (566 bp), and elp-exon IX (256 bp), were successfully amplified and sequenced for one of the isolates with 98.42% similarity, confirming the isolates were correctly identified as E. multilocularis. Network analysis also correctly placed the isolates with other E. multilocularis. Conclusions As a result of the discovery of E. multilocularis in an unusual intermediate host, which is considered to have the highest zoonotic potential, the result clearly demonstrated the necessity for expanded surveillance in the area. Graphical Abstract Supplementary Information The online version contains supplementary material available at 10.1186/s40249-023-01120-0.


Background
In the well-known wildlife transmission cycle of Echinococcus multilocularis, rodents and other small mammals (e.g., Microtus and Alticola voles; water voles, Arvicola spp.; shrews; muskrats, Ondatra zibethicus; nutria; Myocastor coypus) serve as intermediate hosts, while foxes such as red foxes (Vulpes vulpes), Arctic foxes (Alopex lagopus), Tibetan foxes (V.ferrilata) and sand foxes (V.corsac) serve as definitive hosts [1,2].In certain areas (such as sections of Alaska and China), a semidomestic or synanthropic cycle with domestic carnivores (dogs) as the definitive host has been demonstrated [3].In the intermediate host, E. multilocularis metacestodes produce a multilocular (multichambered) mass that contains hundreds to thousands of protoscolices in some and fewer to none in others [4].
In many countries, the distribution of E. multilocularis is not uniform, with areas with high and low prevalence zones.These disparities have been linked to the use and structure of the landscape (vegetation type, vegetation covering, grassland ratio, and elevation), as well as climatic elements (temperature and humidity), which determine the species range and abundance of rodent intermediate hosts [5].More so, there is mounting evidence that E. multilocularis is expanding both its geographic and host range as a result of anthropogenic influences and has been linked to changes in the distribution of definitive hosts [6][7][8].Depending on the location, E. multilocularis tapeworms can be found in 1-60% of wild canids (particularly foxes) and less than 1% of dogs and cats.In China, up to 12% of dogs can be infected with E. multilocularis, but the cysts are commonly found in less than 0.5% to 10% of livestock [9].
Alveolar echinococcosis (AE) is common in the northern hemisphere, including western, northern, and eastern Europe, Russia, western Alaska, the northwestern portion of Canada, central Asia, northern Japan, and western and northern China [17][18][19][20][21].In China, AE is endemic in 99 counties across six provincial-level administrative divisions, including Qinghai, Sichuan, Gansu, Tibet, Xinjiang, and Ningxia.Most of these counties are concentrated in the Qinghai-Tibet Plateau, especially in the border area between Qinghai and Sichuan provinces, as well as in the Tibet Autonomous Region, where 13 of the 15 counties with the highest prevalence of AE have been recorded [22].
Genetic characterization using mitochondrial deoxyribonucleic acid (mtDNA) markers, the microsatellite EmsB, and a multi-locus nuclear DNA markers were applied to E. multilocularis genotyping studies [23].Although E. multilocularis is less diverse than E. granulosus sensu lato, recent taxonomic studies have shown intra-specific variations within E. multilocularis at the global scale, allowing the identification of four genetic subgroups that are restricted to specific geographic regions: European, North American, Asian, and Mongolian [8,[24][25][26].
When a suspected or confirmed case of E. multilocularis infection is reported in any domestic animal, public health agencies must assess the risk of transmission to humans.Such proactive measures can help halt the spread of E. multilocularis [27].The aim of the current study was the molecular characterization, including phylogenetic analysis, genetic diversity, and network analysis, of E. multilocularis detected in sheep in Gansu, China.

Study area
Gansu is a landlocked province in the northwest of China located at 32°31′-42°57′N and 92°13′-108°46′E with a semi-arid to arid continental climate.There are fourteen administrative divisions in Gansu: 12 prefecture-level cities and two autonomous prefectures which are subdivided into 86 county-level divisions [28].Samples were collected by the Gansu Provincial Center for Animal Disease Control and Prevention from different counties in different prefecture-level cities: Minqin and Tianzhu counties (Wuwei City), Jingyuan and Pingchuan counties (Baiyin City), Hezheng county (Linxia City), Gaotai county (Zhangye City), and Jiuquan City.The geographic coordinates of the survey sites are shown in Table 1, and the study area is presented in Additional file 1: Fig. S1.

Parasitic material, DNA extraction, PCR amplification and sequencing of mitochondrial and nuclear DNA
During a routine cystic echinococcosis (CE) investigation suspected (whitish lesion) samples of nine sheep from different slaughterhouses were collected.The Thereafter, a near-complete mt genome (Table 2) and nuclear genes (Table 3) were amplified for confirmation obtained from previous published papers and newly designed primer pairs using Premier 5 software [30].
A Touchdown PCR with the following programs: initial denaturation at 98 ℃ for 3 min, 10 cycles of 95 ℃ for 30 s, 60 °C for 30 s (sequential decrease of 0.5 ℃ in each cycle), and 72 ℃ for 2 min; then 25 cycles of 95 ℃ for 30 s, 55 ℃ for 30 s, and 72 ℃ for 2 min; and a final elongation step at 72 ℃ for 10 min modified after Laurimäe et al. [31] was used.

Molecular analysis
Nucleotides were inspected for errors in UGENE program [32].The species of each isolate was confirmed through NCBI BLAST search.Sequence comparison was performed separately for each mitochondrial and nuclear gene and for the near-complete mitochondrial genome.Phylogenetic and evolutionary analyses were carried out in MEGA v7 [33].Furthermore, a Bayesian phylogenetic tree was constructed using MrBayes v. 3.1.2[34] with a Markov Chain Monte Carlo sampling method to determine the posterior distributions of parameters using a chain length of 4,000,000.The parameters were logged every 1000 generations, with the first 25% discarded as 'burn-in' .TreeView v. 1.6.6 (University of Glasgow, Glasgow, UK) was used to visualize the resulting trees.Phylogenetic analysis was repeated for nad1, cob, nad2, nad5, and the near-complete mitochondrial genome datasets.Taenia solium (accession number: AB086256 and NC_004022) was used as the outgroup.Computation of the population diversity and neutrality indices, such as Tajima's D [35] and Fu's Fs [36], was achieved by generating haplotype files in DnaSP v6 [37] and exporting them to the population genetics program Arlequin v35.2.2 [38].Taenia solium (NC_004022) was used as an outgroup for the estimation of Fu's indices.For the sequence data of the cob and nad2 genes, each of which has more than five haplotypes, diversity and neutrality indices were calculated.Finally, Pop-ART was used to visualize the haplotype and networks [39].

Cyst characteristics, PCR amplification and sequencing
The suspected specimens were distributed as follows: 3 from Jingyuan and one each from Pingchuan, Minqin, Tianzhu, Gaotai, Hezheng, and Jiuquan.The cysts' morphological characteristics differed from those of classical Echinococcus cysts.They were all located in the liver, small in size, and without cystic fluid (Fig. 1).
As shown in Table 4, employing the near-complete mt genes specific to E. multilocularis, at least three primers and as many as nine primers were successfully amplified and sequenced for a single sample.With nad4 and nad2, all nine specimens were successfully amplified and sequenced, but not with ATP synthase F0 subunit 6 gene (atp6) and cytochrome c oxidase subunit 1 (cox1) genes.Only one specimen was amplified using cox2, nonetheless sequencing was unsuccessful due to poor DNA quality.

Population and diversity indices
There were 26 distinct haplotypes of the cob gene (1068 bp).The haplotype diversity index for the population (n = 47) was 0.932, the nucleotide diversity index was 0.01267 while Tajima's D (− 1.89298) had a statistically significant P-value (P < 0.05), and Fu's Fs (− 0.98675) were not statistically significant (P > 0.10) when T. solium (NC_004022) was used as an outgroup.Similarly, there were also 18 distinct haplotypes of the nad2 gene (874 bp).The haplotype diversity index for the population (n = 52) was 0.896, the nucleotide diversity index was 0.00633, Tajima's D (− 1.76799) had a statistically significant P-value (P < 0.05), and Fu's Fs (− 2.78572) were not statistically significant (P > 0.10) when T. solium (NC_004022) was used as an outgroup.
Previous findings have indicated that E. multilocularis metacestodes have been found in natural settings in a range of mammalian hosts that are not involved in the transmission cycle, such as horses, domestic and wild pigs, nutria (Myocastor coypus), various species of captive monkeys, and others [44,45].A study conducted in Japan between 1993 and 1994 showed 0.14% of one million slaughtered pigs and 0.82% of 1100 horses both had E. multilocularis lesions [46].The necropsy of those animals may reveal asymptomatic liver abnormalities (either calcified or deteriorated cysts or fertile cysts).Because it's difficult to distinguish these lesions apart from other, more typical lesions like "white spots" or "dense white foci, " molecular testing might be utilized to track suspected lesions [46].Similarly, domestic ruminants have reported extensive liver lesions that were mistaken for AE, but PCR revealed that these were unusual growth forms of CE [47], as stated in the Panel on Animal Health and Welfare [48].Molecular testing is therefore required to confirm liver lesions in these animals, particularly in regions where echinococcosis is endemic [49].In the meantime, unrelated animal species from outside the parasite's geographic range (such as wallabies and hyraxes) have unintentionally contracted the disease [48].Additionally, certain nonhuman primates, including monkeys and apes, are extremely vulnerable to AE [50].In the lab, large herbivore infections invariably failed, resulting in small, calcified liver lesions [51].
In the current finding, based on the near-complete mitochondrial and nuclear gene analysis, sheep were found to be infected by E. multilocularis.Inline to this, Zhao et al. [52] have reported transmission of CE and AE in domestic animals in Gannan Tibetan Autonomous Prefecture, China as "113 out of 1021 (11.1%) sheep were found infected with CE, and 3 (0.3%) with AE; and 126 out of 634 (19.9%) yaks were infected with CE, and 2 yaks (0.3%) with AE".Additionally, previous reports showed that dogs, zokors, and pikas (5%, 1% and 2%, respectively) were all found to have E. multilocularis infection in South Gansu Province, China [52], suggesting that a life cycle might continue in the area without the presence of wild canids [14].These findings might give an insight into the possibilities of transmission of E. multilocularis in domestic animals such as sheep in endemic areas.
On the other hand, previous reports showed that experimentally introduced eggs in lambs' lungs had the ability to hatch and develop into Echinococcus cysts, suggesting that aerosol transmission, which causes lung cysts, is a potential possibility [53,54].The anomalously high numbers of pulmonary CE may also be explained by the cold,   continental weather patterns that persist for a significant portion of the year in a particular region.These patterns may allow eggs to survive in dust samples, which would then facilitate transmission to humans [54].Meanwhile, dogs that consume the excrement of wild canids or other dogs, "graze" on grass, or drink impure surface water in areas that may be polluted with the feces of wild canids are at risk of contracting AE in areas where E. multilocularis is endemic [55].This could explain the transmission pattern of E. multilocularis in the present study.
Inferred from molecular and phylogenetic data, E. multilocularis isolates from several intermediate and final Asian hosts form a well-defined, geographically characteristic clade that is distinctly different from isolates from Europe and North America [25].As was already mentioned, the three significant Echinococcus species are getting established in western China [40][41][42], so it is vital to consider the possibility of coinfection.This claim is confirmed by Santa et al. [56] from Canada, who discovered that while other potential hosts, including coyotes and foxes, have yet to be documented, coinfections of E. canadensis and E. multilocularis have only recently been detected in wolves.
The current study's unfortunate limitations are the lack of histopathology of the lesion structure and the maintenance of the samples for further processing.The study also failed to provide a clear explanation of how the transmission might have occurred.

Conclusions
The cysts did not have the same morphological characteristics as conventional Echinococcus cysts.The cysts, which were discovered in the livers of sheep, were small white patches devoid of cystic fluid.The present isolates revealed that the infection in sheep was caused by E. multilocularis based on near-complete mitochondrial and nuclear gene analysis, but we were unable to provide a clear explanation for how the transmission could have occurred.In endemic areas where CE and AE are prevalent, it is recommended to take into account that domestic animals, such as sheep, could be infected by E. multilocularis.As a result, given the importance of the parasite to public health, there is a need for increased surveillance in the region in particular and in the country in general following the detection of E. multilocularis in an uncommon intermediate host, which is known to have the highest zoonotic potential.

Fig. 3 3 Fig. 4
Fig. 3 Bayesian phylogeny of the current Echinococcus multilocularis isolates inferred from the nad2 (874 bp) gene.Posterior probability values are depicted at the nodes.Hap1 and Hap2 indicates haplotypes representing isolates from this study.Taenia solium (NC_004022) was used as the out-group sequence data.Eg G1, Eg G3: Echinococcus granulosus genotype 1, 3

Fig. 5
Fig. 5 Molecular Phylogenetic analysis by Maximum Likelihood method of the current Echinococcus multilocularis isolates inferred from the nad5 (1575 bp) gene.Support value greater than 0.39 are displayed to show posterior probability.Taenia solium (AB086256) was used as the out-group sequence data.Eg G1, Eg G3: Echinococcus granulosus genotype 1, 3; Eg s.l.: Echinococcus granulosus sensu lato