DOI: https://doi.org/10.21203/rs.3.rs-930890/v1
Fasciolosis is a neglected trematode infection of public health and veterinary importance caused by Fasciola gigantica and Fasciola hepatica. Molecular analysis using the internal transcribed spacers’ ITS-1 and ITS-2 of nuclear ribosomal DNA is useful in distinguishing Fasciola species. This study aimed to characterize liver flukes from sheep, goats and cattle using these genetic markers. Fifty nine adult Fasciola specimens were collected from livers of naturally infected sheep, goats and cattle at selected abattoirs in Kisumu, Baringo and Narok Counties. Sequence comparison of ITS-1 and ITS-2 sequences of Fasciola isolates from this study and sequences in Genbank was carried out. A maximum likelihood tree was constructed for phylogenetic analysis. Analysis of ITS-1 and ITS-2 rDNA sequences revealed that F. hepatica and F. gigantica caused infection in both cattle and sheep and in goats only F. gigantica caused infection. The sequenced PCR amplicons showed a close relationship between Fasciola species in this study with Fasciola isolates from other regions in the world. Phylogenetic analysis showed that sequences of F. hepatica are similar to the sequence from Spain, China and Tunisia obtained from GenBank. The sequences of F. gigantica in this study have similarity to the sequence from Iran and Burkina Faso. Data from this study provides information that serves as basis for further studies on the distribution of F. gigantica and F. hepatica in other localities in Kenya, and is also important in designing epidemiological and control programmes for zoonotic fascioliasis.
The most widespread liver flukes are of the genus Fasciola and include two species: Fasciola hepatica and Fasciola gigantica causing fasciolosis (Wongkham et al. 2005), but Fasciola hepatica is majorly prevalent in temperate regions while Fasciola gigantica is commonly distributed in tropical climates (Walker et al. 2008; Yakhchali et al. 2015). Fasciola infection is of veterinary importance, especially in regions majorly practicing sheep or cattle farming, resulting in massive economic burdens (Hussein and Khalifa 2010). The World Health Organization has recognized Fasciola infection as a Neglected Topical Disease due to its public health significance (Takeuchi-Storm et al. 2017; Sah et al. 2018) and is now considered as an emerging infection in different regions of the world, especially in South America, Africa and Asia (Hussein and Khalifa 2010, Salahi-Moghaddam and Arfaa 2013).
The two Fasciola species coexist in many African and Asian countries (Sumruayphol et al. 2020), and can also occur in the same region, although ecological factors influence the transmission of trematodes and the abundance of their freshwater snail intermediate host (Mas-Coma et al. 2005). Fasciola parasites can be distinguished using morphological features (Ashrafi et al. 2006), but this can lead to uncertainty in identification of species with intermediate morphological features (Itagaki et al. 2005). Molecular methods have been employed to distinguish between Fasciola hepatica and Fasciola gigantica (Marcilla et al. 2002), these are the most reliable and sensitive methods for the exploration of genetic variability among flukes (Mas-Coma et al. 2007). They are also very useful tools for epidemiological survey and diagnosis of Fasciola species (Mas-Coma et al. 2005).
Molecular analysis was carried out in this study to differentiate adult Fasciola species obtained from naturally infected liver of sheep, goats and cattle at selected slaughter houses within the study area. The nucleotide sequences of the ITS-1 and ITS-2 of the nuclear ribosomal DNA (rDNA) of 59 Fasciola worms from Kisumu, Baringo and Narok Counties was determined in order to establish the prevalence of Fasciola species and identify the domestic animal species most infected by Fasciola parasite in the study area.
This study was carried out in Mara river basin in Narok County, Perkera irrigation scheme in Baringo County and Ahero irrigation scheme in Kisumu County, Kenya (Fig. 1). The residents of the study area practice irrigation and livestock farming for both beef and milk production. Perkera irrigation scheme lies in the lowland area with an average altitude of 1100 m above sea level and covers a total area of 2,350 hectares. The rainfall varies from 1000 to 1500 mm in the highlands to 600 mm per annum in the lowlands. The temperatures vary between 25°C to 30°C, however in January the temperatures rises up to 35°C on average. The Ahero irrigation scheme has an area of 2085.9 Km2 and annual relief rainfall between 1200 mm and 1300 mm with a mean annual temperature of 230C with a range of between 200C and 350C and the altitude of 1168 m above sea level. The 13,750 km2 drainage area of the Mara River basin covers the agricultural and forested areas in the upper basin, the open pastureland in the middle portion of the basin and the Masai Mara Game Reserve in Kenya (1718 km2, all of which is within the Mara River Basin). It lies at an altitude range of 1480-2280 m above sea level. The rainfall is bimodal and the highest annual rainfall amount is received in the high altitude areas with 1100 mm on the average. Livestock accounts for 30% of the agriculture in this region.
Adult Fasciola specimens were collected from livers of naturally infected sheep, goats and cattle at selected abattoirs in three regions, Perkera Irrigation Scheme, Ahero Irrigation Scheme and Mara River Basin. A total of 59 infected livers were collected from three abattoirs from 20 livers (8 sheep, 12 goats) from Perkera, 19 livers (7 sheep, 12cattle) fromAhero, and 20 livers (7 sheep, 4 goats, 9 cattle) from Narok (Table 1). The livers along with gall bladders including the bile duct of all cattle, sheep and goats slaughtered at the abattoir were thoroughly inspected for the presence of liver flukes and the infected ones were removed from the slaughtered animals. The bile ducts were incised longitudinally through the gall bladder and the parasites were removed with the help of fine forceps, taking all necessary precautions to avoid any damage to the parasite. The infected livers were squeezed manually to macerate the parenchyma and the flukes were carefully removed. A total of 354 of individual isolate were obtained (119 form Perkera, 133 form Ahero and 102 from Narok).All samples were thoroughly washed individually 2 to 3 times in physiological saline to remove debris and host cells, and subsequently fixed in 70% ethanol and were carried to the laboratory where they were identified as Fasciola species and stored at room temperature for DNA extraction.
| Location | Host | Species | Number of isolates |
|---|---|---|---|
| Perkera | Sheep | F. hepatica | 4 |
| Perkera | Sheep | F. gigantica | 4 |
| Perkera | Goat | F. gigantiga | 12 |
| Ahero | Sheep | F. gigantica | 7 |
| Ahero | Cattle | F. gigantica | 12 |
| Narok | Sheep | F. gigantica | 3 |
| Narok | Sheep | F. hepatica | 4 |
| Narok | Cattle | F. gigantica | 7 |
| Narok | Cattle | F. hepatica | 2 |
| Narok | Goat | F. gigantica | 4 |
One adult fluke from each liver was used for total Genomic DNA was extracted using QIAamp DNA Mini Kits (Qiagen, USA) following the manufacturer’s recommendations (Dar et al. 2012). The extracted DNA quality was assessed using 1% agarose gel examined in UV transilluminator and the bands were visualized and photographed.
PCR amplification was performed according to (Yuan et al. 2016). The internal transcribed spacer 1 (ITS1) and Internal transcribed spacer 2 (ITS2), regions were amplified by PCR using a set of BD1 5′-GTCGTAACAAGGTTTCCGTA-3′ and BD2 5′-TATGCTTAAATTCAGCGGGT-3′ as forward and reverse primers, respectively. PCR reaction was performed in a total volume of 50 µl containing 2 µl DNA template, 25 µl 2x master mix one taq Quick load, 21 µl of ddH2O and 1 µl of each primer in a thermocycler (BioRad®) under the following conditions: 94°C for 5 min as initial denaturation, followed by 30 cycles of 94°C for 20s (denaturation), 55°C for 30s (annealing), 72°C for 30s (extension) and final extension of 72°C for 10min. For detection of PCR results, 5µl of the PCR product was examined on 1% agarose gel in TAE buffer at 80V for 45min. The gels stained with ethidium bromide, visualized and photographed using a transilluminator (UVITEC). To estimate the size of the amplicons, a 100bp DNA ladder (Fermentas) was used in gels. The PCR products were sequenced (Sanger dideoxy sequencing, Ingaba biotec) from both directions, using the same primers used in the PCR amplication.
The sequences were aligned and compared with those of existing sequences related to Fasciola species ITS1 and ITS2, available in the GenBank, using the BLAST program (https://blast.ncbi.nlm.nih.gov/Blast.cgi). Multiple alignment was performed with data related to Fasciola species from other countries deposited in GenBank, using BioEdit Sequence Alignment Editor Version 7.1.3 software. A maximum likelihood tree was constructed using the MEGA X software and bootstrap analysis using 1,000 replicates (Shafiei et al. 2014; Kumar et al. 2018).
The PCR amplification of ITS-1 and ITS-2 rDNA yielded fragments of approximately 970 bp in length (Fig. 2). The 59 PCR amplicons of ITS-1 and ITS-2 were subjected to direct sequencing which yielded sequences of 946 bp in length. The sequence was composed of the complete ITS-1 sequence of 422 bp, complete 5.8S sequence of 158 bp, and complete ITS-2 sequence of 366 bp. Out of the 59 Fasciola samples sequenced, 17% were F. hepatica and 83% were F. gigantica, there were no intermediate forms of Fasciola species. Fasciola samples collected from cattle were F. hepatica 2 (9.5%), F. gigantica 19 (90.5%), sheep F. hepatica 8 (36.4%), F. gigantica 14 (63.6%) and all samples from goats were F. gigantica.
The sequences were deposited in the GenBank database (https://www.ncbi.nlm.nih.gov/genbank/) under the accession numbers MZ396875, MZ396876, MZ396877, MZ396878, MZ396885, MZ396925, MZ396926, MZ396928, MZ396929, MZ3969332 for Fasciola hepatica and MZ396874, MZ396879- MZ396884, MZ396886- MZ396924, MZ396927, MZ396930, MZ396931 Fasciola gigantica. The sequences were aligned with previously published Fasciola ITS-1, 5.8S and ITS-2 sequences retrieved from GenBank accession numbers JF432073, MK321604, JF496711, AM900371, MW046875, JF496709,KJ789338, HM746787, HQ197358, KF543340, AJ853848, JF432072, KJ789364, HM746785, MK321597, JF432075, GQ231547, AM709647, AM850107 and AM709621 (Alasaad et al. 2007; Ali et al. 2008; Farjallah et al. 2009; Rokni et al. 2010; Amor et al. 2011; Galavani et al. 2016; Evack et al. 2020; Omar et al. 2021).
The sequences of ITS-1 showed that F. hepatica differed form F. gigantica in five variable nucleotides, while the ITS-2 sequences of the examined F. hepatica was different from F. gigantica ITS-2 in seven nucleotides (Table 2). Among the 59 sequenced Fasciola isolates there was no nucleotide variation in the ITS-1 sequences. In ITS-2 sequences there was no nucleotide variation among F. hepatica but nucleotide variation was observed at two nucleotides of F. gigantica at sequence position 842 and 936, two and three Fasciola isolates respectively. This indicates the presence of two genotypes of F. gigantica isolates that were examines. Sequence variation in position 842 was examined only in Narok among sheep and cattle, while in position 936 it was examined in Fasciola samples from the three localities and it occurred in cattle and goat.
| Species | Variable sites in ITS-1 and ITS-2 sequences | Accession numbers | Location | |||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| ITS-1 ITS-2 | ||||||||||||||
| 18 | 108 | 202 | 280 | 300 | 815 | 842 | 854 | 860 | 911 | 918 | 936 | |||
| F. gigantica | T | T | T | A | T | C | G | T | T | - | A | G | JF432073 | Iran |
| T | T | T | A | T | C | G | T | T | - | A | G | MK321604 | Chad | |
| T | T | T | A | T | C | G | T | T | - | A | G | JF496711 | China | |
| T | T | T | A | T | C | G | T | T | - | A | G | AM900371 | Niger | |
| T | T | T | A | T | C | G | T | T | - | A | G | MW046875 | Zimbabwe | |
| T | T | T | A | T | C | G | T | T | - | A | G | MZ396874 | This study | |
| T | T | T | A | T | C | C | T | T | - | A | G | MZ396918 | This study | |
| T | T | T | A | T | C | C | T | T | - | A | G | MZ396931 | This study | |
| T | T | T | A | T | C | G | T | T | - | A | T | MZ396912 | This study | |
| T | T | T | A | T | C | G | T | T | - | A | T | MZ396916 | This study | |
| T | T | T | A | T | C | G | T | T | - | A | T | MZ396919 | This study | |
| F. hepatica | C | A | C | T | C | T | G | C | C | T | G | G | AM709647 | Spain |
| C | A | C | T | C | T | G | C | C | T | G | G | AM850107 | Niger | |
| C | A | C | T | C | T | G | C | C | T | G | G | JF432072 | Iran | |
| C | A | C | T | C | T | G | C | C | T | G | G | JF708027 | China | |
| C | A | C | T | C | T | G | C | C | T | G | G | MZ396875 | This study | |
Phylogenetic tree was constructed for the analysis of phylogenetic diversity of the liver flukes, using ITS1 and ITS2 sequences of F. gigantica and F. hepatica from this study along with available sequences in GenBank from other regions (Fig. 3). Fascioloides magna was used as an outgroup GenBank accession number EF534991 (Khalifa et al. 2016).
Fascioliasis is a parasitic disease which is of concern to veterinary and public health sector worldwide (Sy et al. 2021). The genetic markers ITS1 and ITS2 genes of ribosomal DNA have been utilized to discriminate between Fasciola hepatica and Fasciola gigantica (Amor et al. 2011; Dar et al. 2019). There is limited data on the genetic characteristics of Fasciola species in Kenya. Out of the 59 Fasciola samples sequenced, 83% and 17% of the isolates were identified as F. gigantica and F. hepatica respectively. In the present study, F. gigantica caused majority of the infection in cattle compared to infection in sheep. Isolates for F. hepatica, 8 out of 10 were found in sheep. This was in agreement with studies from other regions of the world that shows F. hepatica are more prevalent in sheep while F. gigantica are more prevalent in cattle (Akhlaghi et al. 2017). A study in Tanzania showed that 41 Fasciola isolates were F. hepatica (Farjallah et al. 2009), in Iran showed that 96.8% of 31 sheep sampled were infected with F. hepatica (Rokni et al. 2010). A study in Niger also showed that 66.7% of 12 cattle isolates were F. gigantica (Ali et al. 2008). In the past, it was believed that F. hepatica was present primarily in the temperate regions, while F. gigantica is distributed in some countries in tropical region (Evack et al. 2020). This has been proved not to be the case because several studies have recently documented the presence of F. hepatica in Africa and Asia (Amer et al. 2011; Dar et al. 2012; Mucheka et al. 2015), including this present study that have shown the existence of both F. hepatica and F. gigantica in the three localities in Kenya. In addition, the present study is the first to demonstrate the presence of Fasciola hepatica in the study areas. This could be attributed to environmental and host-related factors that could affect the distribution of Fasciola flukes (Amer et al. 2016). Among the two Fasciola species, Fasciola hepatica has a selective advantage because it has been reported to adapt quickly to external selection pressures such as new hosts, new environments, and medications than Fasciola gigantica (Cwiklinski et al. 2015).
Alignment of the sequences of ITS-1 and ITS-2 rDNA with available sequences in GenBank showed ten DNA variable sites in which segregated the Fasciola isolates into two different genotypes, this is consistent with previous studies (Chougar et al. 2019). In this study, the sequences of ITS-1 showed five variable nucleotides that separated between F. hepatica from F. gigantica and there was no nucleotide variation in the ITS-1 sequences. ITS-2 sequences F. hepatica was different from F. gigantica in seven nucleotides. Also in ITS-2 sequences there was no nucleotide variation in F. hepatica but nucleotide variation was observed at two nucleotides of five F. gigantica at sequence position 842 and 936, two and three Fasciola isolates respectively. This indicates the presence of two genotypes of F. gigantica isolates in the three localities.
Phylogenetic tree constructed using ITS1 and ITS2 sequences of F. gigantica and F. hepatica from this study along with available sequences in GenBank from other regions showed a close relationship between Fasciola species in this study in comparison with Fasciola isolates from other regions in the world. All the sequences of F. hepatica are in the same group and are similar to the Fasciola species of Switzerland, Spain, China and Tunisia. The sequence of F. gigantica in this study, fall in the same group and are similar to the Fasciola species of Zimbabwe, Iran and Burkina Faso.
This present study has shown the existence of both F. hepatica and F. gigantica in Kenya. In addition, it is the first to demonstrate the existence of Fasciola hepatica in Narok, Kisumu and Baringo Counties in Kenya. Further studies involving more other mitochondrial polymorphic genes, like cytochrome c oxidase gene (CO1), can be carried out to assist understand more on genetic divergence of both Fasciola gigantica and Fasciola hepatica in the region and other parts of the world. This will also enable the assessment its zoonotic potential and epidemiological surveys especially in areas where there is limited resources making prevention and control strategies difficult to implement and sustain.
Funding information
This work was supported by the Kenya National Commission for Science, Technology and Innovation [NACOSTI] [grant number NACOSTI/RCD/ST&I/7TH CALL/PhD/168].
Conflicts of interests
The authors declare that there are no conflicts of interest.
Availability of data and material
The datasets generated during and/or analysed during the current study are available in the GenBank repository under the following accession numbers [MZ396875-78, MZ396885, MZ396925-26, MZ396928-29, MZ3969332, MZ396874, MZ396897-84, MZ396886-924, MZ396927, MZ396930-31], [https://www.ncbi.nlm.nih.gov/nuccore]
Code availability
Not applicable
Authors’ contributions
Cornelius Kibet Kipyegen designed the project, acquired and analysed data and wrote the first draft of the manuscript. Charle I. Muleke and Elick O. Otachi supervised the research, contributed to writing and reviewing of the manuscript. All authors read and approved the final manuscript.
Ethical approval
This study was approved by the Egerton University Research and Ethical Committee (EU/RE/DVC/009) and The Kenya National Commission for Science, Technology and Innovation (NACOSTI/P/15/8095/6943). “The authors assert that all procedures contributing to this work comply with the ethical standards of the relevant national and institutional committees on human experimentation and with the Helsinki Declaration 1975, as revised in 2008.”