Sequence and Morphology of Cysticercus pisiformis in Local Breed Rabbits

Since antiquity, tapeworm has caused considerable damage to wild and domestic animals, as well as human health, resulting in economic losses due to food safety, livestock output, and serious public health implications (Zhu et al., 2019). Rabbits are affected by many different species of parasites (El-Ashram et al., 2020). Taenia pisiformis which is one of these cestodes belonged to family Taeniidae and to the genus Taenia (Yang et al., 2013). The final hosts of the parasite are canines and rarely felines, while their intermediate hosts are some rodents (Karamon et al., 2019).This parasite distributed in different areas of the world, Lahmar et al. (2008) found the prevalence with T. pisiformis of stray dogs in Tunisia was 6.36%, 3.33% in East-Azerbaijan Province, north-west Iran (Yagoob and Hossein, 2011), 26% in New Zealand (Hajipour and Zavarshani, 2020) and 9.4% in Ukraine (Bogach et al., 2022) and in Iraq, 8. 6% in Mosul (Al-Moula, 2005), 38. 75% in Baghdad Province (Athraa, 2014) and 54.55% in Al-Diwaniyah Province (Marhoon et al., 2018).

The adult worm colonizes the small intestine of dogs (Hadi and Faraj, 2016), and the larval stage causes cysticercosis in rabbits (Overgaauw, 2020). It mostly produces blood poisoning, depression, lethargic, loss of appetite and emaciation, slate themselves from normal activities of the flock, a syndrome that has been defined as sickness behaviors, but it can also impair resistance to other illnesses and may even result in mortality (Hart, 1991; Kakai, 1994; Zhou et al., 2008).

Because there is no phylogenetic analysis of C. pisiformis in local bred rabbits in Iraq, this study was achieved for confirmation and identification of this parasite from rabbits.

Sixty local breed rabbits were purchased from Al-Ghazel market in the center of Baghdad city, visited weekly during a period 1/March/ until 31/May/2022, the animals were placed under the optimal conditions (room temperature regulated at 24˚C, dry food and clean tap water) of rearing the laboratory animals in the Animal House, College of Veterinary Medicine/ University of Baghdad.

Five rabbits humanely euthanized weekly. The skin and muscles of abdomen of necropsied rabbits were opened longitudinally and the looking for any cyst may be founded grossly on the abdominal cavity or on the visceral organs, recorded the location of cysts and numbers (Abdulkareem et al., 2022). The isolated cysts were stored in clean containers before being sent to the Parasitology Department, College of Veterinary Medicine, University of Baghdad Laboratory for further analysis.

All cysts were examined for their structures scolex, membrane and the size measurements. The fluid was collected from each cyst for measurement the volume (Abbas, 2017; Abbas and Abbas, 2017). The cysts were identified as C. pisiformis according to the Loos-Frank (2000) description, and some cysts were stained with a cetoacetic acid carmine stain to seen the structure of cysts (Abbas, 1998). To confirm the diagnosis of C. pisiformis (scolex and part of membrane) from these cysts were put in a 70% ethanol for further used in conventional molecular diagnosis.

Molecular confirmation was done on fourteen C. pisiformis isolated from naturally infected rabbits, they were achieved by DNA extraction from cyst tissue (scolex and part of membrane) by using commercially available kit (Add Prep Genomic DNA Extraction Kit/ Add Bio/ Korea) for tissue DNA extraction and according to instruction of manufacture’s (Al-Azawi and Al-Biatee, 2019; Mraidi and Lafta, 2021).

Conventional polymerase chain reaction (PCR) technique were performed for (14 DNA samples) by using specific primers for T. pisiformis, first amplification of gene NADH dehydrogenase subunit1 (NAD1) using the primers JB11F (5’-AGATTCGTAAGGGGCCTAATA-3’) forward and JB12R (5’-ACCACTAACTAATTCACTTTC-3’) reveres, that amplify a fragment of about 500bp., the second conventional PCR was carried out for amplification of cytochrome oxidase subunit1 (COX1) using the primers CO1F (5’-TTTTTTGGCCATCCTGAGGTTTAT-3’) forward and CO1R (5’-TAACGACATAACATAATGAAAATG-3’) reveres, which amplifies a fragment of 446bp. according to (Samorek-Pier et al., 2021). Reaction of PCR technique Master Mix were performed by added 10μl of Master Mix to 3μl DNA template, 1.5μl primer forward, 1.5μl reverse, and completed by 4μl distilled water to make total volume of 20μl (Table 1).

Table 1: Contents for reaction of PCR technique.

Thermocycler conditions were carried out as the following steps for both genes and for each sample (Table 2).

DNA sequencing was used to validate the presence of local C. pisiformis and to create a phylogenetic connection tree between local isolates and NCBI-Blast submissions, as well as to submit local isolates to NCBI-Gen Bank. Three PCR positive isolate results for each gene were delivered in an ice bag to Bioneer Company in Korea for DNA sequencing using Applied Biosystem (AB) of DNA sequencing system. The NCBI-Gen Bank submission was completed using the Ban kit submission tool. Six PCR products’ sequences were submitted to the National Center for Biotechnology Information (NCBI) under specific accession numbers for each strain. The sequences data were arranged into a FASTA format by saving the consensus as a single file (Chilton et al., 1997). The DNA sequencing analysis was conducted by using molecular evolutionary genetics analysis version 6.0. (Mega 6.0) and multiple sequence alignment analysis of the partial nicotinamide adenine dinucleotide dehydrogenase subunit1 (NAD1) and cytochrome oxidase subunit1 (COX1) genes, internal transcribed spacer each gene based Clustal W alignment analysis, the evolutionary distances were computed using the maximum composite likelihood method by phylogenetic tree UPGMA (unweighted pair group method with arithmetic mean) method (Tamura et al., 2011). Using molecular evolutionally genetics analysis version 6.0 (Mega 6.0) multiple sequencing alignment analysis of the partial NAD1 gene and COX1 gene. Sequence analysis of the NAD1 and COX1 genes has been used to show the similarity and differences between local and international genes sequence.

Table 2: Thermocycler conditions of PCR.

To determine the significance of different components in research parameters, the Statistical Analysis System- SAS 9.3 (2012) application was utilized. In this study, the Chi-square test was utilized to compare percentages (0.05 and 0.01 likelihood).

Fourteen of 60 (23.33%) necropsied local bred rabbits was found infected naturally with C. pisiformis (Table 3). The result of current study was less than result of Athraa (2014) who recorded 38.75% (31/80) infection rate of in local rabbits in Baghdad city-Iraq, also Jori (2016) who founded 40% (4/10) infection rate from domestic rabbits in Basrah city-Iraq, and less than Marhoon et al. (2018) recorded 54.55% (30/55) of C. pisiformis in a wild rabbits collected from the local animals market in Al-Diwaniyah Province-Iraq and less than Roldan (2019) in Spain 67.7% (21/31) infection rate with metacestodes C. pisiformis from the capturing wild rabbits, while the results enclose with study of Hajipour and Zavarshani (2020) in Iran which found the prevalence rate 26% (13/50) in new Zealand white rabbits. The results are higher than Al-Moula (2005) which recorded 8.6% (2/30) infection rate when examined domestic rabbits in Mosul city-Iraq. Also, more than Foronda et al. (2005) in Canary Islands which found the prevalence rate 16% in total 244 wild rabbits. Also higher than Yagoob and Hossein (2011) which isolated 3.33% (2/60) from abdominal cavity of wild rabbits in East Azerbaijan province of Iran.

According to the sex a total rate of infection in 27.90% (12/43) females was which was more than males’ infection 11.76% (2/17) without significant differences (P≥0.05) (Table 3). This result agree with Roldan (2019) in Spain which recorded high infection rate in females 80% (17/21) than males 40% (4/10) with significant difference (P≤0.05).In contrast to Hajipour and Zavarshani (2020) in Iran who found males affected more than females and recorded infection rate 23.80% (5/21) in females and 27.58% (8/29) males without significant difference (P≥0.05). The disparities between the current study and prior studies might be attributed to variances in the quantity of samples obtained between investigations, difference in type of animals (domestic or wild), as well as differences in the distribution and proliferation of stray dogs.

Table 3: Total Infection rate with C. pisiformis in naturally infected rabbits according to the sex.

Fifty-five cysts were isolated from peritoneal cavity of 14 positive samples (single or group) as balloon filled with clear fluid with white floating scolex (evagenated or invagenated) or pus-filled in others. The range of diameters of cyst between 7 to 21 in length and 4 to 10 mm in width. (Table 4) and (Figure 1), this cysts were confirmed to the Loos-Frank (2000) description as C. pisiformis.

According to the location of cyst into internal organs of 55 cysts, large number of larval cysts attached to the mesentery 47.27% (26/55), liver 21.82% (12/55), other cysts found free within the abdominal cavity 21.82% (12/55) and on omentum only 9.09% (5/55) with Significant deffrences (P<0.05) (Table 5).

Table 4: Measurements of cysts size and volume.

Table 5: The site and number of cysts in fourteen naturally infected rabbits.

Cysts staining with acetoacetic carmine stain appeared scolex with armed rostellum with tow raw of hooks and four suckers (Figure 2) like the method of (Abbas, 1998).

The result of convential PCR technique to which used for confirm C. pisiformis based on using (NAD1) genes and primer 500bp the result was 100(14/14) (Figure 3) , also 14 DNA sample were subgected to PCR to amplification (COX1) genes with primer 446 bp the result was was 100(14/14) (Figure 4).

The nucleotide sequences of 3 local C. pisiformis isolates for each genes were submitted to GenBank under the accession numbers (LC731847.1, LC731848.1 and LC731849.1) for NAD1 gene (Table 6) and (OP274120.1, OP277616.1 and OP277618.1) for COX1 gene (Table 7).

Three sequences of NAD1 gene (LC731847.1, LC731848.1 and LC731849.1) was 100% identical to the China sequence JN870149.1 while less identically (97.15-99.47%) in Australia and Poland (Table 6 and Figure 5), and sequences of COX1 gene (OP274120.1, OP277616.1 and OP277618.1) were matching variance between 100% to 99.56% in the China, Portugal and Poland (Table 7 and Figure 6).

Table 6: Isolates of T. pisiformis from natural infected by NAD1 gene sequencing ID in gene bank and nucleotide sequence identity from NCBI.

Table 7: Isolates of T. pisiformis from natural infected by COX1 gene sequencing ID in gene bank and nucleotide sequence identity from NCBI.

This study is the first one by using conventional PCR and sequence identification of Cysticercus. pisiformis in Baghdad, Iraq.

Authors would like to express their special thanks and gratitude to the Department of Parasitology/College of Veterinary Medicine/University of Baghdad for all their kind and helpful support during the study period.

This study is the first one by using conventional PCR and sequence identification of Cysticercus pisiformis in Baghdad, Iraq and study the highly distribution of the infection of local bred rabbits in Souq al-Ghazal in the Baghdad city.

Both authors conceived and designed the study, reviewed and approved the final manuscript. Kadhim Kh. K. Al-Khayat performed the experiments. Athmar K. A. Al-Azawi analyzed the data. Kadhim Kh. K. Al-Khayat contributed materials and wrote the manuscript.

All animals in this study were handled and cared for according to the appropriate biosecurity procedures. The study was performed in accordance with the rules of the ‘Guide for the Care and Use of Laboratory Animals that were approved by the Ethics Committee of the College of Veterinary Medicine, University of Baghdad, Iraq (Number 472 P.G at 28/2/2023) before starting this study.

The authors have declared no conflict of interest.