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Morphology and molecular identification of Eurytrema spp. worm in Aceh cattle, Indonesia

Hanafiah M, Helmi TZ, Sutriana A, Bahi M. 2021. Morphology and molecular identification of Eurytrema spp. worm in Aceh cattle, Indonesia. Biodiversitas 22: 5654-5661. The objective of this research was to determine the species of Eurytrema spp. in Aceh cattle which were slaughtered in slaughterhouses located in Banda Aceh, Indonesia. Identification of Eurytrema spp worm species was performed by Semichon’s Carmine staining, SEM and molecular method. The result from Semichon’s Carmine staining and SEM method showed that worms coded E2 and E3 were Eurytrema cladorchis indicated by clearly visible protruding cirrus while worms coded E1, E4 and E5 were E. pancreaticum indicated by the absence of protruding cirrus. Molecular characterization of Eurytrema spp. produced fragments of approximately 700 bp in length. The phylogenic tree showed that Eurytrema spp. from Aceh cattle consisted of 2 clades: clade 1 was E. pancreaticum, while clade 2 was E. cladorchis. DNA sequencing result showed that the similarity of worm samples coded 1, 4, and 5 were 98.90% similar to E. pancreaticum (KJ767631.1 and KC535543.1), and 96. 33% similar to E. cladorchis (MN566134.5 and MN566140.1), whereas the worm samples coded 2 and 3 were 97.53% similar to E. pancreaticum (KY490000.1 until KY490004.1), and 97.55 % similar to E. cladorchis (MN566135.1 until MN566140.1). This research was able to identify E. pancreaticum and E. cladorchis in Aceh cattle slaughtered in Banda Aceh slaughter houses.


INTRODUCTION
The Aceh cattle breed came from a cross between local cattle (assumed to be a descendant of Bos sondaicus) and zebu cattle from India (Bos indicus) which occurred hundreds of year ago (Sutarno and Setyawan 2016). At present time, Aceh cattle population has reached an alarmingly low number, so Aceh cattle feedstock needs to be improved by increasing birth rate and lowering death rate percentage (Widyaningrum et al. 2021). The cause of this diminishing number is due to the low growth rate of Aceh cattle, which is much slower than the rate of crossbred cattle (Agus et al. 2018). Other than the slow growth, according to Schwertz et al. (2015) parasitic disease caused by digenetic trematode Eurytrema worm, which is a pancreas parasite with low pathogenicity, could be related to the decrease of livestock productive performance and may end in death.
One species of parasitic worm that infect the pancreas and biliary tract of cattle is Eurytrema spp. The genus Eurytrema appears to be confined to parts of South East Asia, East Asia and Latin America (Mohanta et al. 2015), where annual mortality caused by this pancreatic fluke ranges between 1-3% (Ilha et al. 2005;Okajima et al. 2016). Eurytrema pancreaticum infection can be debilitating towards animal health and can cause clinical and subclinical diseases, as well as causing great economic losses and medical costs and lowering overall productivity , it may be associated with mortality and loss of productive performance in animals due to chronic pancreatitis (Su et al. 2018). The parasites have also been known to impair calf growth, cause progressive weight loss, lower feed conversion, malnutrition, and impair milk production. Eurytrematosis case in cow has been reported in Brazil (Rachid et al. 2011; where clinical and pathological features observed shows chronic and progressive wasting related to interstitial pancreatitis. The prevalence of parasitism in Brazil varied between regions. Lucca et al. (2015) reported in the western region of Santa Catarina State, the prevalence of Eurytrema was as high as 69%. Xu et al. (2015) has researched the expression profile of global MicroRNAs (miRNAs) from adult E. pancreaticum utilizing next gen sequencing technology combined with real-time quantitative PCR. Mohanta et al. (2015) was to identify Eurytrema flukes both by morphology and molecular properties on the basis of 18-subunit ribosomal RNA (18S rRNA) gene as well as internal transcribed spacer 2 (ITS2) to clarify their phylogenetic status. Figueira et al. (2014) also identified unique morphological characteristics and molecular features in E. coelomaticum worms found in cattles in South Brazil. Mohanta et al. (2015) confirmed the presence of E. cladorchis in the biliary tract of cattle (Bos indicus) in Bandarban a mountainous district in Bangladesh. Research on the differences of trematode worm parasites Eurytrema spp. until now had not been reported for Aceh cattle. Considering the lack of information on species and the morphology of Eurytrema spp., this research may aid in better understanding eurytrematiasis in an effort to find efficient strategies to prevent and control infestations of the parasite in cattle from Aceh. In order to better manage cattle in Aceh, more accurate identification of species and morphology of the worms is required.
The purpose of this research is to determine the difference in species of Eurytrema spp. worms obtained from Aceh cattle slaughtered in Banda Aceh slaughterhouses by using Semichon's Carmine staining, scanning electron microscopy (SEM) and molecular method.

Study location and period
A total of 40 pancreas samples were collected from Aceh cattle from Banda Aceh, Indonesia slaughterhouses. The Aceh cattle that were slaughtered were mostly from Aceh Besar district, and partly from Pidie and Aceh Jaya districts. The samples were collected into plastics and labeled. The separation and identification of Eurytrema spp. worms were performed in the Laboratory of Parasitology, Faculty of Veterinary Medicine, Universitas Syiah Kuala, Banda Aceh. Eurtyrema spp. worms were put inside petri dishes, cleaned and later stained using Semichon's Carmine staining. SEM analysis were performed in the Center of Biology Research, LIPI Cibidong, Zoology Division, SEM Laboratory, Cibinong, Indonesia. For DNA extraction, 5 Eurytrema spp. were taken from the pancreas. The worms were rinsed in distilled water, fixed in absolute ethanol solution, and then stored at 4ºC. DNA extraction, electrophoresis and amplification of Eurytrema spp. worms was performed at Inter University Centre (PAU) Gadjah Mada University. This research was carried out between December 2020 and March 2021.
Semichon's Carmine Staining Method Eurytrema spp. was stained using Semichon's Carmine method which referred to Cable (1961). Eurytrema worm specimens were put into semichon's carmine staining solution barely covering the specimen thoroughly. The staining solution was previously diluted to an equal volume of 70% alcohol and stored for 1 to a few hours. The over stain color was removed by washing the specimen with 70% alcohol twice. Subsequently, the specimen was dipped in 0.5% to 1% HCL and 70% alcohol until the specimen is pink in color. Then, the specimen was washed twice or three times using 70% alcohol to remove acid and prevent further staining process. Subsequently, dehydration was carried out by dipping the specimens in 80% and 95% alcohol for 1 hour, each solution. Then, the specimen was dipped in another new 95% alcohol or in absolute alcohol if methyl salicylate was used to clean up the specimen. The next step was cleaning the specimen using neutral creosote, terpinol or methyl salicylate. The specimen was briefly washed in xylol then, attached to a glass object and added entellan, covered with a glass deck carefully to ensure the entellan is distributed thoroughly. The slide was stored for 24 hours to let the entellan dry before observing under a microscope using 40x magnification.

Scanning electron microscope (SEM) method to observe worm micro structure
Eurytrema spp. observations by SEM were performed to explore the small morphological features of Eurytrema spp. Specimen preparation was performed in the Center of Biology Research, LIPI Cibinong, Biology Division, SEM Laboratory, Cibinong, according to a particular protocol. Samples were processed at 4ºC in several stages: (i) Cleaning, the sample was soaked in cacodylate buffer (42.6 gr of 0.2 M sodium caccodylate + 1000 mL aquadest with a pH of 8.4) for approximately 2 hours, then agitated in an ultrasonic cleaner for 5 minute; (ii) Presification, the sample was put into a 2.5% glutaraldehyde solution (5 mL glutaraldehyde+caccodylate buffer up to 40 mL) for several hours to 2 days; (iii) Fixation, the sample was soaked in 2% tannic acid (2 g of tannic acid in 100 mL of caccodylate buffer) for 6 hours to several days, then washed with caccodylate buffer for 5 minutes for 4 times; (iv) Dehydration, the sample was soaked 5 minutes in 50% alcohol for 4 times, then immersed in 70% alcohol, 85% alcohol, and 95% alcohol for 20 minutes each (at room temperature), then with absolute alcohol for 10 minutes 2 times; (v) Drying, the sample was submerged for 10 minutes in tert butanol 2 times, then freezed until frozen and transferred to freeze drier/vacuum drier until dry; (vi) Installation, the specimen was fixed on the specimen stub according to the needs and stages; (vii) Coating, the specimen was coated with Au (Aurum) using the ION COATER tool. The microscope used was the JSM-IT200 InTouchScope™ Scanning Electron Microscope. Pictures were taken to observe morphological differences between the available worms using the appropriate magnification (Goldstein et al. 1992).

Sequencing
PCR product was sequenced by ABI PrismBigDye Terminator Cycle Sequencing Kit (Applied Biosystems, Foster City, CA, USA).

Data analysis
The data was presented in picture and table format and then the accumulated data were analyzed descriptively. Phylogenetic tree and sequence alignments based on the 5.8S and ITS2 gene of the Eurytrema spp. were then created by using MEGA version 6 software. Constructed by the neighbor-joining method, based on 1.000 bootstrapped data sets; distance value was calculated by using the Kimura 2 parameter model. Fasciola hepatica was used as the out group to provide stability to the generated tree. The sequence identity was generated by using the software Bioedit (Tamura et al. 2013).

RESULTS AND DISCUSSION
Observation on 40 pancreases ( Figure 1) from Aceh cattle slaughtered in Banda Aceh slaughterhouses appeared to be infested by trematode Eurytrema spp. worms. Eurytrema are considered low pathogenic parasites (Ilha et al. 2005;Schwertz et al. 2016), yet are very frequently found at necropsy or at slaughterhouses in Aceh and other parts of West Indonesia.
The results of the examination on pancreas samples of Aceh cattle showed the presence of worms from the trematode class of the genus Eurytrema spp. (Figure 1). However, the species of these worms could not be determined due to the morphology of the worms can not be observed specifically. In general, the morphology of Eurytrema spp. was reddish in color and slightly oval in shape. This worm has a blunt and rounded anterior end, and has a short tongue-like shape at the posterior end. The body is thick and armed with spines, which often disappear as they mature. This worm has an oral sucker and a ventral sucker, pharynx, osophagus, testes and genital pore, and cirrus sac. The ovaries are near the midline, behind the testes, and the uterus is at the back of the body. The vitelline glands are follicular and located laterally. The species of Eurytrema worms found in the pancreas was still unknown and thus staining was required.

Morphology observation by worm staining
The results obtained in this research by Semichon's Carmine staining method showed that the worms found in the pancreas (Figure 1) of Aceh cattle were E. pancreaticum (Figure 2, E1, E4 and E5) and E. cladorchis (Figure 2, E2 and E3). E. pancreaticum worms found in this research morphologically were 8-16 mm in length and 5-8 mm in width. The body was flat and tapered. Oral sucker was located at the anterior region of the body while ventral sucker was located around the middle region of the body. Visible reproductive organs were a pair of testicles lobes located opposite of one another above ovarium lobulated with worm eggs situated inside the uterus. Ovarium with lobulated is an identifying characteristic of E. pancreaticum (Kumar et al. 2018), uterus, and glandula vitelline. Cirrus sac was located in the middle between oral and ventral sucker, cirrus was present but protruding ( Figure 2, E1, Inset). According to Mohanta et al. (2015), species identification of Eurytrema spp. worm has always been room for controversy especially related to the obtained morphologies, making interpretation differs between researchers. In the last several years, significant advancement in microscopic technology has led to new discoveries on the morphology and anatomy of parasitic worms (Faulwetter et al. 2013;Lopes-Torres et al. 2015;Gonçalves et al. 2016;Conn et al. 2018).
According Chai et al. (2013) morphologically E. pancreaticum worms have larger oral sucker compared to their ventral sucker, testes with lobes, ovarium with or without lobulated, and grouped vitellaria follicle with clear separation. Eurytrema cladorchis worms obtained in this research had morphological features such as protruding digitiform cirrus (Figure 2; E2 and E3. Inset), elongated cirrus sac, branching testicles which were located symmetrically at the posterolateral region towards the acetabulum. Ventral sucker (acetabulum) was larger (on average 322.67 µm) compared to the oval shaped oral sucker (on average 279.07 µm) similiar morphology was also observed previously in research (Figueira et al. 2014;Mohanta et al. 2015) which classifies the worms as the E. cladorchis species.
In China, there are at least seven species, including E. cladorchis, E. coelomaticum, E. fukienensis, E. hydropotes, E. minutum, E. pancreaticum and E. sphaeriorchis; however, E. pancreaticum is considered as predominant species in ruminants (Li et al. 2013). The study of Okajima et al. (2016) explained that eurytrematosis was endemic in Japan particularly in Kagoshima, Okinawa, and Shimane regions. In another study showed that E. pancreaticum eggs appeared in fecal samples of five camels in Karamoja sub region of North-eastern Uganda (Nakayima et al. 2017).
Eurytrema cladorchis was discovered living in the pancreatic tract of domestic ruminants. Afterward, the flukes were reported from both wild deer (Muntiacus muntjak, Hydropotes inermis) and domestic ruminants in the mountainous area of Fujian, Zhejiang, Jiangxi and Anhui Provinces, China (Cai et al. 2012). Morphological identification of E. cladorchis in Nepal has been performed by Mohanta et al. (2015). In this research we compared the morphometry features and morphology of Eurytrema worms from Bangladesh with obtained E. cladorchis. The result of this study which identifies Eurytrema cladorchis in cattle in Indonesia especially in Aceh cattle is the first to be conducted as far as our knowledge. It is the goal of the researchers that this research will add new information on the characteristic of Eurytrema worms especially E. cladorchis.
The prevalence of E. pancreaticum was detected in Aceh cattle with a prevalence rate of 0.4% (Hanafiah et al. 2019). Lucca et al. (2015) mentions that the prevalence of E. pancreaticum in cattle may occur, in extreme cases up to 100%, while (Okajima et al. 2016) reported that the prevalence of eurytrematiasis was less than 1% in animal slaughterhouses in Japan.

Repetitive R529 sequence amplification
Amplification results of DNA from blood, pancreas, and Eurytrema spp. samples were obtained by using forward primer 3SC and reverse primer JB9 neu with different master mix, in which mix PCR Go Tag specifically attached to sample DNA copy of D1, E1, E2 as well as J1 dan J2 producing 700 bp in length, while D2 did not show any band. Using master mix PCR Bio Line, E1, E2 and J1 samples produced 700 bp in length, while D1, D2 the band is amplified in an unspecified region with 5.8 S and IT2 primers (Figure 4). Meanwhile, worm samples E1, E2, E3, E4 and E5 produced 700 bp in length ( Figure  5).
The result of amplification of Polymerase chain reaction (PCR) on 5.8S and ITS2 site in this research showed that the DNA length of the Eurytrema spp. found was the same with research conducted by Mirza and Kurniasih (2002) which was 700 bp in samples from Makassar, Yogyakarta, and Aceh, while using ITS2, 15 samples of Eurytrema spp. worms on agarose gel 1% which produced 1500 bp. Different results were also obtained in research by Mohanta et al. (2015) who used 8S rRNA on Eurytrema spp. worms which showed 1784 bp DNA, while the ITS2 was 229 bp in length.
The results of nucleotide BLAST (Basic Alignment Search Tool) which are shown in NCBI showed the homology between Eurytrema spp. worms in this research (sequenced product) with the sequence of several worms in GenBank. For worm samples E1, E4, and E5 they were 98.90% homologous with E. pancreaticum, and 96. 33% homologous with E. cladorchis. Worm samples E2 and E3 were 97.53% homologous with E. pancreaticum, and 97. 55 % homologous with E. cladorchis. Estimated evolution differences with the E. cladorchis genotype from Bangladesh according to research by Cai et al. (2012) stated that the difference is low in values between 0,000 (identical) up to 0,002 (similarities 99.8%) for 18S rRNA and for ITS2 was 0,000. In 18S region rRNA, the estimated divergent evolution between E. cladorchis from Bangladesh and E. pancreaticum

Phylogenetic analysis result
Sequence results obtained were later used to construct a phylogenetic tree which was then compared to determine the difference level between isolates. Eurytrema spp. sequences from Aceh cattle slaughtered in Banda Aceh slaughterhouses were then compared to sequence data from 10 different strain/isolates from the GenBank. The sequence organizing and phylogenetic tree were performed by MEGA X.0 software ( Figure 6).    (700 bp Cai et al. (2012) in China. The estimated evolutionary divergent value showed that the genetic distance between E. cladorchis from Bangladesh and E. cladorchis reported from China was 1.7-1.9%. This showed that E. cladorchis in Bangladesh and in China are genetically quite different. E. pancreaticum Aceh isolates from this research when compared to E. pancreaticum from Bangladesh, India, has an evolutionary divergence value of 1.8%, while the E. cladorchis Aceh isolate and E. cladorchis from Liangshan, China, showed the genetic distance of 3.2%. This result showed that E. pancreatium from Aceh cattle when compared with those from Bangladesh are genetically quite different, while the E. cladorchis from Aceh cattle when compared with China and Bangladesh are genetically vastly different.
In conclusion, this research was able to identify E. pancreaticum and E. cladorchis in Aceh cattle obtained from Banda Aceh slaughterhouses. E. cladorchis indicated by clearly visible protruding cirrus while worms E. pancreaticum indicated by the absence of protruding cirrus. The DNA amplification of Eurytrema spp. worm was 700 bp in length. The phylogenetic tree showed that Eurytrema spp. from Aceh cattle formed 2 clades, clade 1 consisted of Eurytrema pancreaticum, while clade 2 consisted of Eurytrema cladorchis.