MOLECULAR AND BIOCHEMICAL CHARACTERIZATION OF ECHINOCOCCUS SPP. IN HYDATID CYST FLUID COLLECTED FROM HUMAN AND LIVESTOCK IN NORTHERN KHYBER PAKHTUNKHWA AND GILGIT BALTISTAN

M. Abdullah¹, I. Ali², K. S. Haleem³, A. U. Rehman¹, S. Qayyum³, Z. Niaz³, S. Ahmed³, I. Khan⁴, M. N. K. Khattak⁵, N. Sultana⁶ and I. Tauseef^3*

¹Department of Zoology, Hazara University Mansehra, 21300, KPK, Pakistan; ²Institute of Biotechnology & Genetic Engineering, University of Agriculture, Peshawar, Pakistan; ³Department of Microbiology, Hazara University Mansehra, 21300, KPK, Pakistan; ⁴Department of Microbiology, AUST, Abbottabad; ⁵Department of Applied Biology, College of Sciences, University of Sharjah, United Arab Emirates; ; ⁶Department of Biochemistry, Hazara University Mansehra, 21300, KPK, Pakistan.

^*Corresponding Author’s E-mail: isfahan@hu.edu.pk

ABSTRACT

The Echinoccocus spp. infection results in cystic or alveolar echinococcosis causing billions of dollars loss to the international economy in respect of expensive medical treatments, permanent loss of entire or part of organ, and a substantial decrease in livestock productivity. Therefore, the study was designed with objectives to know existence of the species, their genotypes and biochemical profile of hydatid cyst fluids of Echinococci in the samples collected from northern Khyber Pakhtunkhwa and Gilgit Baltistan. Cyst samples (n = 46) were collected from two different sources i.e. livestock and human (n=34 and n=12, respectively). PCR-RFLP based analysis was used to investigate the mitochondrial gene (rrnL, large subunit of ribosomal RNA) based species detection and genotyping. Biochemical assay of all hydatid cyst fluid was performed to analyze the concentration of various parameters i.e. uric acid, glucose and triglyceride etc. One-way ANOVA followed by Tukey’s post-hoc test was also performed to determine the statistically significant differences among the biochemical parameters. Among all collected cysts, 56.5% (n=26) were positive for Echinococcus granulosus while 43.4% (n=20) were detected as Echinococcus multilocularis. Genotyping of the positive E. granulosus revealed the highest frequency (>90%) of G1–3 genotype while G6 genotype was found at low level (<10%). In case of human, all samples were positive for G1–3 genotype. Biochemical analysis of cyst fluids showed a significant (P ≤ 0.05) interspecies variation in all tested parameters. In conclusion, E. granulosus and E. multilocularis are found frequently in northern KP and Gilgit Baltistan. Further, E. granulosus sensu stricto (G1-3) and G6 genotypes are responsible for infections in both human and animals. A comprehensive surveillance program in whole northern Pakistan is needed for effective prevention/control of Echinococcus species.

Keywords: Cyst, Echinococcus, Human, Livestock, Genotypes of E. granulosus

https://doi.org/10.36899/JAPS.2021.5.0330
Published online January 21, 2021

INTRODUCTION

Echinococcus, an important cyclophyllid cestode tapeworm of canids and other animals, causes a zoonotic disease known as cystic echinococcosis (CE) (Rojas et al. 2014). Among all known species of Echinococci, E. granulosus and E. multilocularis are clinically important and cause cystic echinococcosis or alveolar echinococcosis, respec­tively (McManus et al. 2012; Shanshan et al. 2018). Globally, the annual economic losses due to this disease have been estimated as reaching up to 3 billion US dollars (WHO, 2013 and 2017). The worm resides in the digestive tract of the definitive host (domestic/wild canids) whereas the oncosphere produces hydatid cyst in the intermediate hosts (wild or livestock mammals). Human CE, reported worldwide, occurs by accidental ingestion of eggs leading to development of fluid-filled hydatid cysts which multiplicate there asexually (Rokni, 2009). Cyst fluid contains a number of organic and inorganic constituents i.e. protein, lipid, carbohydrates, electrolytes and trace elements which may have a key role in the immunology, metabolism and physiology of cystic echinococcosis (Shanshan et al. 2018). Demographic changes and a variation in genotypes of Echinococcus results in a change in the level/concentration of these biochemical parameters (Eslami et al. 2016).

Genetic diversity in E. granulosus species has been reported since long and it is suspected that this interspecies variation may influence the infectivity and various other characteristics i.e. morphology, specificity of host and epidemiology (Carmena and Cardona, 2014). Mitochondrial DNA sequence analysis has identified ten heterogeneous groups (G1-G10) of variants in E. granulosus. Among them, three genotypes, the G1-G3 (G1-G2 found in sheep while G-3 in buffalo), are grouped in the species E. granulosus sensu stricto while other genotypes are E. equinus which is found in horses (G4), E. ortleppi in cattle (G5), E. Canadensis (G6–10 found in camels, pigs and cervids, respectively) and the lion strain (E. felidis) (Nakao et al. 2007; Rojas et al. 2014; Amer et al. 2015).

Frequent screening for detection of species and knowledge of circulating genetic variants of E. granulosus from a range of hosts in a selected area could be a useful strategy for not only in understanding the disease dynamics but also for designing effective diagnosis, control, prevention measures and treatment (Shariatzadeh et al. 2015; Carmena and Cardona, 2014; Wahlers et al. 2012). Molecular based approaches are considered as a reliable tool to identify and differentiate the circulating Echinococcus spp. Among these, Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP) analysis is of paramount importance for genotyping with higher accuracy to confirm the distinctiveness among Echinococcus spp. based on sequence-specific endonucleases (Hanifian et al. 2013; Ali et al. 2015). Nucleotide alterations in the genetic variants of E. granulosus populations can readily be detected by this highly verified molecular diagnostic procedure enabling the molecular discernment of genotypes (Dousti et al. 2013).

Lack of awareness and meager sterile circumstances regarding the parasite life cycle have made the conditions favorable for the persistence of the infection in many regions of Pakistan (Latif et al. 2010; Khan et al. 2018). It is believed that Echinococcus species may have been introduced into Pakistan from China and Iran (Harandi et al. 2002; Xiao et al. 2005; Xiao, et al. 2006; Parsa et al. 2012). Being the closest region to China, having suitable environmental conditions and pastures for traditional rearing of domestic animals, the emergence of echinococcosis is very likely in northern Khyber Pakhtunkhwa and Gilgit Baltistan regions. Further, as the echinococcosis prevalence is linked with the economic dependence on livestock, the people of northern area are continuously exposed to Echinococcus species because they mainly rely on livestock for daily food requirements (Latif et al. 2010). Hence, the current study was designed to straight the loop and was aimed to determine the frequency of Echinococcus spp. and distribution of sub types of E. granulosus in livestock and human residing in the areas under investigation.

MATERIALS AND METHODS

Experimental subjects and sample collection: A total of 46 hydatid cysts samples (Sheep (n=15); goats (n=12); buffalo (n=7) and humans (n=12) were collected from the northern areas of KP (Chitral, Swat, Mingora and Mansehra) and Gilgit Baltistan. The inclusion criterion was “all patients/animals with one or more than one hydatid cysts and belonging to the selected areas”. Exclusion criteria were the “human/animals found negative for the presence of cysts or not belonging to the study areas”.

In case of humans, the cysts were removed surgically from infected patients while the cysts from animals i.e. buffaloes, goat and sheep were taken from the slaughterhouses. Briefly, for the collection animal’s cyst, different abattoirs of northern Khyber-Pakhtunkhwa and Gilgit Baltistan were visited once a week and the samples from liver, lungs or other visceral regions were collected. The level of damage to viscera was also considered for each animal. Similarly, for human cysts collection, the samples were obtained from surgery units of different northern area hospitals and/or from those northern area’s patients who were brought to Lady Reading Hospital Peshawar (LRH) for admission/treatment (Ethical Approval No: F. No.73/HU/ORIC/IBC/2015/05). The collected cyst samples were transferred into sterile capped containers having approximately one volume (v/v) of 70% ethanol and stored at 4°C until further processing.

Sample Preparation: Cyst’s fluid was taken from all samples and the contents (fluid and germinal layer) were aspirated. Washing with phosphate buffer saline (PBS) was performed by spinning the mixture at 1200 g for 15 minutes. The pellets were then kept at -20°C for DNA extraction and the supernatant were used for biochemical analysis.

DNA Extraction:  All the procedures for extraction of DNA were carried out in the Institute of Biotechnology & Genetic Engineering, University of Agriculture, Peshawar, Pakistan. DNA from pellets was extracted using a Genomic DNA extraction kit (Biomatik; manufactured by Geneaid, USA Cat# GEB100) and the instruction manual provided by the manufacturer.

Mitochondrial gene (rrnL) amplification for genotyping of E. granulosus: A fragment of 570 bp of the mitochondrial gene was amplified using previously reported primers (Xiao et al. 2006), Ech-LSU/F (5'- GGTTTATTTGCCCTTTTGCATCATGC-3') and Ech-LSU/R (5'-ATCACGTCAAACCATTCAAACAAGC-3'), manufactured and supplied by Invitrogen (Carlsbad, California). Amplification of gene was performed using two μL template (DNA), one μL of each forward and reverse primer (10 μM), 0.5 μL Taq DNA polymerase (Fermentas USA; Cat# EP0401), one µL MgCl₂ (1.25 mM final concentration) and 0.5 µL dNTPs (2 mM). The PCR Grade water was used for the adjustment of the final volume up to 20 µL.

The entire amplification reactions were performed in a regular DNA thermal cycler (Bio Rad, Model#T100). The conditions for PCR were first optimized using a range of annealing and denaturation duration. Following optimized conditions were found suitable and selected for PCR of samples of this study; initial denaturation at 94°C for 5 min, denaturation at 94°C for 40 seconds, primer annealing at 66°C for 60 seconds and extension at 72°C for 60 seconds. The amplification reaction was repeated for 35 cycles followed by a final extension at 72°C for 7 minutes. Negative and positive controls were also set up in every reaction to check the reliability of the reaction. The amplified product (10 µl) was electrophoresed on agarose gel (2%) and visualized/photographed in UV trans-illuminator (UVitec, Cambridge; Model# Essential V6).

PCR-Restriction Fragment Length Polymorphism Analysis for identification of species: For removal of polymerase enzyme, nucleotides, primers and buffer components and avoid hinderance by these components during downstream processing, the cleansing and precipitation of PCR product were performed using ethanol and the resultant pellet was collected and re-suspended in 15 µ1 of PCR grade water. For identification, the purified PCR products of Echinococcus species were digested with the Ssp1 (New England Biolabs, UK; Cat# R0132S) restriction enzyme in a 50 µ1 mixture (Xiao et al. 2005; Xiao et al. 2006). The concentration of each ingredient was taken as per manufacturer’s protocol (10 µ1 PCR product; 1 µ1 restriction enzyme; 5 µ1 buffer and 34 µ1 nuclease free water) and the mixture was incubated for overnight (~ 16 hours) at 37 °C.

Genotyping of Echinococcus granulosus through PCR-RFLP: Genotyping was performed through digestion of PCR products of E. granulosus with BglII (New England Biolabs, UK; Cat# R0144S) restriction enzyme as described previously (Xiao et al. 2005; Xiao et al. 2006). In accordance with manufacturer’s instructions, a 50 µ1 mixture (10 µ1 PCR product; 1 µ1 BglII enzyme, 5 µ1 reaction buffer; 34 µ1 nuclease free water) was prepared in a PCR tube and incubated at 37°C for 16 hours. Finally, the digested products along with 50 bp ladders (Gene Ruler, Fermentas; Cat# SM0373), were visualized on 2% of pre-stained (ethidium bromide) agarose gel.

Biochemical analysis of hydatid cyst fluids: The supernatants collected above were spun at 10000 g under low temperature conditions (4°C) for five minutes and analyzed for glucose, urea, uric acid, triglycerides and cholesterol through commercially available diagnostic Kits (ELITech clinical system; France) and the protocol contained within. Similarly, total protein, Creatinine, Calcium, Sodium and Potassium were estimated by Merck diagnostic kits (Merck, Germany).

Statistical Analysis: The data obtained for determination of biochemical parameters are presented as Mean±SD and were analyzed through one-way ANOVA followed by Tukey’s post-hoc test to see the statistical difference between the groups using webpage statpages.info free online calculator.

RESULTS

Observation of hydatid cysts from human and animals: In case of humans, the cysts were surgically removed and collected. Few representative images of hydatid cysts obtained from human liver and ovary are shown in Fig.1 (A-C). In case of animals, the images of infected viscera and hydatid cysts were also obtained. Representative images of both (viscera and cyst) obtained from a buffalo are shown in Fig. 1(D-E). The level and extent of damage caused by hydatid cysts was also observed and shown as Fig.1F.

Confirmation of Echinococcus in the samples through PCR: Molecular identification and confirmation of all hydatid cysts was performed using the conventional PCR. Samples from all sources (human and animals) were amplified and their products were run on 2% Agarose gel (Fig. 2). A distinctive fragment of ~ 565 base pair (bp) of the mitochondrial rrnL gene was identified for the conformation of presence of Echinococci. The confirmatory band was found in positive control and also in all tested samples of this study, hence all hydatid cysts collected in this study were found positive for Echinococci.

The figure shows only representative samples on gel after PCR amplification.The size of amplified product (~ 565 bp) was confirmed by comparing with marker DNA. Lane 1; amplified DNA from positive control; (Lane 2-5); Ladder DNA (Lane M); Negative control (Lane NC).

PCR-RFLPanalysis for differentiation between E. multilocularis and E. granulosusspecies of Echinococci: The differentiation between both species was performed using Ssp1 restriction analysis. Digestion of samples and presence of two fragments (295 and 270 bp) represented the E. multilocularis while presence of undigested (565 bp) fragment represented the E. granulosus (Fig. 3A). Out of 34 animal’s samples, 21 (61.7 %) and 13 (38.2 %) were confirmed E. granulosus and E. multilocularis, respectively (Figure 3b). There was no indication of Echinococcus oliogarthrus, Echinococcus shiquicus and Echinococcus vogeli, the remaining species of this genus. Likewise, in humans, out of 12 tested samples, five (41.6 %) were E. granulosus and seven (58.3 %) were E. multilocularis as shown in (Fig. 3B).

Genotyping of Echinococcus granulosus by PCR-RFLP: The PCR amplified products of confirmed positive samples of E. granulosus were subjected to another restriction digestion to determine their genotypes. The DNA samples were restricted by using enzyme BglII to determine the pattern of digestion. In case of successful digestion, two bands of sizes 158 bp and 407 bp were generated (Fig. 4). Digested samples represented the genotype-6 (lane 1-2) while undigested samples represented genotype 1-3 (G1-3). Out of 21 animal samples, 90.4% (n=19) were found to be E. granulosus G1-3 type while only 9.6% were identified as G6 strain. In case of human (n=5), 100% of E. granulosus were identified as G1 genotype.

Ten µL DNA amplified from Echinococcus granulosus digested with BglII was run on 2% agarose gel and the digestion pattern was observed. Lane 1-2 represent genotype G6 generating two bands (158 bp and 407 bp). Marker DNA (Lane M).

Biochemical profile of isolated cysts: The biochemical analysis among different animals (sheep, goat, buffalo) and human revealed that glucose concentration varies among cysts of different sources (Table 1).

Table 1: Biochemical parameters of Hydatid cysts fluid from human and animal sources

The glucose level of hydatid cyst fluid of sheep and goats was significantly higher (P ≤0.05) compared to buffalo and humans while it was higher in sheep compared to goats. The total protein contents were significantly higher (P ≤0.05) in the cyst fluid of buffalo compared to other sources. Similarly, the estimated level of Urea concentration was significantly higher (P ≤0.05) in the cyst fluid of human when compared to others (Table 1). For uric acid, the concentration was significantly higher (P ≤0.05) in the Echinococcus cyst fluid of human compared to the other hosts. Further, triglycerides in the cyst fluid of buffalo and sheep were found significantly higher (P ≤0.05) when compared with the other hosts.

In contrast, the concentration of Cholesterol in human cyst was higher (P ≤0.05) than all other sources. For Creatinine, a significantly higher concentration (P ≤0.05) was found in the cyst obtained from sheep compared to others. Creatinine was also statistically higher (P ≤0.05) in goats samples compared to humans. Calcium level in hydatid cyst fluid collected from buffalo was significantly high (P ≤0.05) compared to other hosts. The sodium and potassium concentrations of Echinococcus cyst fluid were significantly higher (P ≤0.05) in sheep compared to rest of the hosts (For detailed comparison, please refer to Table 1).

DISCUSSION

Pakistan is one of the neglected endemic regions for Echinococci where majority of population (~96%) have not even heard about the worm (Khan et al. 2018). Although few studies in Punjab and Sindh provinces have produced some valuable data, frequency of Echinococcus species and the distribution of genotypes in northern Khyber Pakhtunkhwa and Gilgit Baltistan is not well documented. Therefore, an investigation concerning local Echinococcus species and their genotypes was required. Hence, the results of this study document the first-hand information on these aspects.

The PCR-RFLP data revealed that the species circulating in human and animals of study areas are either E. granulosus or E. multilocularis, though E. granulosus was predominant. These findings are in accordance with previous studies conducted locally and worldwide (Muqaddas et al. 2019; Guo et al. 2019). Another local study which used same primers and enzyme for RFLP based analysis has also reported the abundance of E. granulosus in the collected samples (Ali et al. 2015). High frequency of this strain suggests its high transfer potential between animals and other hosts (Latif et al. 2010).

Analysis of genetic diversity in E. granulosus showed that genotype G1-3 is the main causative agent for Echinococcus related infections in human and animals of the selected areas. These results are in agreement with various studies conducted in different countries for genotypic analysis of E. granulosus and reported G1-3 as the most prevalent source of infection both in animals and human (Euzeby, 1991; Breyer et al. 2004; M’rad et al. 2005; Romig et al. 2006; Varcasia et al. 2007; Busi et al. 2007; Schneider et al. 2008; Pezeshki et al. 2013). A score of other genotyping studies based on PCR-RFLP method have also reported the predominance of G1-3 strain in tested population (Tigre et al. 2016; Fallahizadeh et al. 2019). This validates that in addition to cost-effectiveness, this method is authentic as well. Results of this study are also in good agreement with few studies conducted at local level. Studies conducted in Punjab and Sindh Provinces have reported either exclusive or predominant presence of G1-3 genotype (Ehsan et al. 2017; Latif et al. 2010; Mehmood et al. 2020). Similarly, a local RFLP based study with same primers and restriction enzymes, reported the over-representation of G1-3 in the population (Ali et al. 2015). This necessitates the launching of extensive awareness, prevention and control programs.

The exact reason for the predominance of G1-3 genotype in the study area is unclear. One reasonable explanation for higher frequency of G1-3 genotypes in northern areas of Pakistan is that “as the G1-3 genotypes are prevalent in Chinese region sharing boundary with northern Pakistan” (Xiao et al. 2006; Guo et al. 2019), demographic expansions of the parasite might have resulted due to the movements of carrier hosts. Further, as Iran is a hyperendemic area with G1-3 predominant genotypes and has shared borders, it is quite likely that the spread of G1-3 genotypes may have occurred from this region (Xiao et al. 2006; Parsa et al. 2012; Khademvatan et al. 2013). In addition, the presence of Echinococcus infected stray dogs with local migratory tribes, might have further added to the potential of parasitic migration and transmission, as reported elsewhere (Daryani et al. 2007).

Another likely explanation for spread is the higher susceptibility of human to G1-3 genotypes than G6 genotype, as proposed by a previous study (Piccoli et al. 2013). The third possibility for the low presence of G-6 genotype (and higher frequency of G1-3) may be due to the fact that its transmission is low due to some unidentified factors responsible for the reduction of contamination by this specific genotype. Though the effect of low contamination on prevalence of G-6 has been hypothesized in a number of previous investigations (Ahmadi and Dalimi 2006; Utuk et al. 2008; Vural et al. 2008; Šnábel et al. 2009), a concrete evidence of its low infectivity is still missing.

Echinococcus cyst fluid contains a score of organic and inorganic constituents (Juyi et al. 2013). The ingredients in the cyst play a key role in the immunology, metabolism and physiology of cystic echinococcosis (Siracusano et al. 2012). This implies that the function and biochemical nature of the specific organ involved might have an effect on the nature of cyst fluid. Some electrolytes of different organs actively diffuse through cyst layers which easily change the biochemical parameters and alter the cyst fertility and expansion (Conchedda et al. 2016). Therefore, chemical estimation can provide useful information about E. granulosus sub types and identification of host (Sharma et al. 2013). The data of biochemical profiles presented significant (P ≤0.05) differences among livestock and human isolates. It was found that Glucose, Creatine, Sodium and Potassium are lower in the cyst fluid of buffalo, goat and human when compared with sheep. This demonstrates that the range of these biochemical profiles is not influenced by the previous hosts. Contrastingly, Triglyceride, Calcium and total protein were found lower in hydatid cyst fluid of sheep, goat and human as compared to buffalo. Though a previous study didn’t notice significant variations in the profiles of protein, lipid, cholesterol and glycerides in cysts isolated from liver and lungs of humans and sheep (Sharif et al. 2005), a recent study reported that hydatid cyst fluids contained significantly more glucose and Ca²⁺ (P ≤.05) as compared to liver simple cysts (Shanshan et al. 2018).

The level of Urea, Uric acid and Cholesterol in the Echinococcus cyst were lower in sheep, goat and buffalo as compared to human isolates. These results are consistent with a previous investigation which showed higher level of uric acid in human Echinococcus cyst when compared with livestock cyst fluid (Juyi et al. 2013). A high concentration of uric acid may be a sign of degenerative changes in hydatid cysts of humans. Surprisingly, in the isolates from goat, all biochemical parameters were lower than other animals. Such lower levels of biochemical constituents in goats have also been reported in a study conducted in Iran (Radfar and Iranyar, 2004). In our study, the level of potassium was also comparable to a couple of previous investigations (Izadi and Ajami, 2006). In summary, a significant difference of biochemical profile in Echinococcus cyst content of different secondary hosts (human and livestock) was observed. This difference in concentration of Echinococcus cyst contents in livestock and humans may be due to the fact that several genotypes of E. granulosus are circulating in Northern, Pakistan.

Conclusion The current results revealed that E. granulosus and E. multilocularis are the frequently found species of Echinococci in northern Khyber Pakhtunkhwa and Gilgit Baltistan regions. At genotypic level, E. granulosus sensu stricto (G1) and G6 genotypes are responsible for infection in both human and animal hosts. Further, the identification of species and genotypes of Echinococcus on the basis of PCR-RFLP is a functional and viable method. Moreover, biochemical investigations will be valuable for immunological studies, diagnostic tests and may help to confirm different genotypes of E. granulosus.

Author contributions MA and IA performed the experiments. SQ, ZN, IK, SA and MNK provided technical expertise in the experimentation and data analysis. NS, AR and KSH participated in biochemical analysis, and preparation/editing of the manuscript. IA and IT designed and supervised the experimental study and prepared the final manuscript.

Ethical Approval Statement The study was approved (F. No.73/HU/ORIC/IBC/2015/05) by the Ethical Committee of Hazara University, Mansehra, Pakistan.

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