A parasitological survey and the molecular detection of Entamoeba species in pigs of East Java, Indonesia.

Background and Aim: In several countries, two Entamoeba porcine species, Entamoeba suis and Entamoeba polecki (subtype 1 and 3), have been detected using molecular methods and identified pathogenicity associated with enteritis. However, globally, Entamoeba infection prevalence in pigs is extremely limited. This study aimed to coprologically and genetically examine pig parasites to estimate prevalence of Entamoeba in three pig farms in East Java, Indonesia. Materials and Methods: Hundred porcine fecal samples (Landrace) were collected from three East Javan farms in well-known swine industry regions. Fecal samples were examined under a microscope after sugar-flotation centrifugation, and molecular species and subtype identification were performed using polymerase chain reaction (PCR) and primer pairs targeting small-subunit ribosomal RNA. Results: Microscopy examinations identified parasites in 89/100 fecal samples; Entamoeba spp. cysts were the most frequent in these samples. Polymerase chain reaction showed that 58 samples were comprised of mixed Entamoeba suis and Entamoeba polecki, 22 E. suis alone, and nine E. polecki alone infections. Epolec F6-Epolec R6 primers successfully amplified E. polecki ST1-4 subtypes, while Epolecki 1-Epolecki 2 amplified only the E. polecki ST1 subtype. Entamoeba polecki ST1-specific primers successfully detected the ST1 subtype in 19/67 E. polecki positive samples. Conclusion: Entamoeba spp. prevalence in Indonesian pigs was previously shown to be high. On coprological examination of East Javan pigs, we detected high Entamoeba spp. levels, in which we genetically identified as E. suis (80.0%), E. polecki (67.0%), and E. polecki ST1 (19%).

Occurrence and biodiversity of Eimeria spp. (Apicomplexa: Eimeriidae) in Madura cattle reared on Kamal Subdistrict, Madura Island, Indonesia.

Background and Aim: In Indonesia, Madura cattle are native breeds that are expected to contribute to the improvement of regional meat self-sufficiency. Eimeria spp. are protozoans that are commonly found in ruminants. This study aimed to identify the occurrence and diversity of Eimeria spp. in Madura cattle. Materials and Methods: In this study, fresh fecal samples were collected from 100 cattle in Kamal Subdistrict, Bangkalan District, Madura Island, Indonesia. Morphological detection was performed using a light microscope, and molecular identification was performed using a polymerase chain reaction. DNA amplification was conducted using various species-specific primers for Eimeria bovis, Eimeria zuernii, Eimeria auburnensis, Eimeria alabamensis, Eimeria ellipsoidalis, and Eimeria cylindrica. Results: The results obtained 21% (21/100) of Eimeria spp. based on morphological detection. A total of 15 positive samples with 500-25,000/mL oocysts were selected for DNA extraction and amplification, resulting in 12 positive samples. Four Eimeria spp. were obtained based on molecular identification: E. bovis, E. zuernii, E. auburnensis, and E. cylindrica. Conclusion: Four species of Eimeria namely E. bovis, E. zuernii, E. auburnensis, and E. cylindrica were identified from fecal sample of Madura cattle using PCR method in this study. Further comprehensive studies are required to investigate the pathogenicity of Eimeria spp. in Madura cattle. Therefore, improved and integrated management practices should be strengthened by local governments to prevent pathogenic diseases and increase national livestock productivity in Indonesia.

Coproparasitological examinations and molecular determination of Eimeria species in Madura cattle reared on Madura Island, Indonesia.

Madura cattle, which are native to Indonesia and mainly kept on Madura Island, East Java, are expected to contribute to improving the regional meat self-sufficiency. Eimeria spp. are the most pathogenic protozoans among gastrointestinal parasites in livestock but no molecular surveys of Eimeria spp. in Madura cattle have been conducted to date. In this study, a total of 183 fecal samples were collected from Madura cattle and 60 (32.8%) were positive for parasites of protozoans and nematodes by the sugar floatation method. Among the samples with parasites, Eimeria spp. oocysts were detected in 50 samples (27.3%) with an average OPG value of 1686.1. Eimeria spp. were successfully identified to the species level in 26 samples with Eimeria bovis being the most prevalent, followed by E. zuernii and E. aubrunensis. A total of 21 samples showed mixed infection of more than two species of Eimeria. E. bovis and E. zuernii have been recognized as having high virulency and, thus, these parasites are potential sources of severe coccidiosis and the cause of infections in other cattle. Although additional studies are warranted, these results can be helpful for improving the management and productivity of Madura cattle.

Second internal transcribed spacer (ITS-2) as genetic marker for molecular characterization of Sarcoptes scabiei in rabbits from several areas of East Java, Indonesia.

OBJECTIVES: The purpose of this study is to use the second internal transcribed spacer (ITS-2) to determine the molecular characteristics of Sarcoptes scabiei in rabbits from several areas of East Java. METHODS: Collecting S. scabiei mites from rabbits with clinical signs of scabies; DNA extraction with minikit QIAamp DNA; polymerase chain reaction amplification; nucleotide sequence analysis; homology and phylogenetic tree using the Neighbor-Joining method in the program molecular evolutionary genetics analysis-7 (MEGA-7). RESULTS: Sequence analysis of ITS-2 S. scabiei from five regions in East Java showed an identity >91.23% with isolates from China (KX695125.1). The phylogenetic analysis of ITS-2 S. scabiei from Mojokerto rabbits has a close relationship with AB82977.1; Surabaya and Nganjuk rabbits are closely related to KX695125.1; while Sidoarjo and Pasuruan rabbits are closely related to EF514469.2. and AB369384.1. CONCLUSIONS: The homology analysis of all samples showed identity of more than 91.23% with isolate China (KX695125.1). The sequences of ITS-2 gen of S. scabiei from rabbits in several areas were relatively close to S. scabiei obtain various hosts from National Centre for Biotechnology Information (NCBI) data.

Zoonotic and other gastrointestinal parasites in cats in Lumajang, East Java, Indonesia.

Relationship between humans and cats has negative impact associates with zoonotic diseases. It is the reason why studies on the prevalence of gastrointestinal (GI) parasites in cats are important. Some of zoonotic GI parasites in cats are Toxocara spp, Ancylostoma sp, and Toxoplasma gondii. The current study was conducted to investigate the prevalence of GI parasites in owned and stray cats in Lumajang East Java Indonesia. One hundred and twenty fecal samples were collected from owned and stray cats on November 2018 to January 2019. The samples were examined by direct smears, sedimentation and flotation techniques. Identification of parasites was determined based on the morphology of worm eggs and protozoan cysts. The results showed that gastrointestinal parasites were found in 68.33% (82/120) examined samples, respectively, 48.33% (29/60) and 88.33% (53/60) from owned cats and stray cats. We found 7 genera of parasites, 4 genera of worm eggs and 2 genera protozoan oocyst. The egg worm were Toxocara cati (40 %), Toxocara leonina. (10.33%), Ancylostoma sp. (18.33%), Diphylobothrium sp. (3.33%) and Dipylidium caninum (1.67%). The protozoan oocyst were Isospora felis (27.5%), Isospora rivolta (13.33%) and Eimeria spp. (8.33%). Toxocara cati, Ancylostoma sp. (hookworm), Diphylobothrium sp. and Dipylidium caninum were zoonotic parasites. Rate infection in younger and older cat were no significant difference. One cat can be infected one or more parasite. To conclude, the prevalence of zoonotic GI parasites both in owned and stray cats were high. It is necessary to plan a program to control this zoonotic parasites.

Blastocystis spp. subtype 10 infected beef cattle in Kamal and Socah, Bangkalan, Madura, Indonesia.

BACKGROUND AND AIM: Blastocystis spp. is a gastrointestinal parasite that can infect both humans and animals and has the potential to become a zoonotic parasite. This study analyzed a subtype (ST) of Blastocystis spp. that had infected beef cattle in Kamal and Socah, Bangkalan, Madura, Indonesia. MATERIALS AND METHODS: Fresh stool samples were collected from 108 beef cattle at Kamal and Socah, Bangkalan, Madura, Indonesia. Blastocystis spp. were detected both morphologically and genetically based on the 18S rRNA gene. The morphology of Blastocystis spp. from the stool samples and cultured samples were observed under a light microscope. Blastocystis spp. from 20 positive cultures were amplified through polymerase chain reaction, and the resultant sequences were identified by ST. RESULTS: One hundred and eight (100%) fecal samples from the fresh or cultured stools were positive morphologically for Blastocystis spp. Molecularly, all 20 of the samples selected for DNA analysis were found to be Blastocystis spp. ST 10. CONCLUSION: Based on morphological and molecular detection, the prevalence of Blastocystis spp. infection in beef cattle within Kamal and Socah, Bangkalan, Madura, Indonesia, was high. About 100% were non-zoonotic parasites. This was the first report of Blastocystis spp. ST 10 found in infected beef cattle in Kamal and Socah, Bangkalan, Madura, Indonesia.

Characterization of mitochondrial COX-1 gene of Sarcoptes scabiei from rabbits in East Java, Indonesia.

OBJECTIVE: The purpose of this study was to characterize the mitochondrial COX-1 gene of Sarcoptes scabiei in rabbits from three districts of Malang, Nganjuk, and Kediri, East Java, Indonesia. The gene was aligned with a DNA isolated from S. scabiei of Chong'qing rabbit (accession number: EU256388.1) to construct a molecular analysis of phylogenetic in S. scabiei COX-1 gene. MATERIALS AND METHODS: This study has been verified by the Committee Ethics (Faculty of Veterinary Medicine, Universitas Airlangga). The mites were collected and identified from rabbits that have an indication of scabies infection. DNA was extracted with QIAamp DNA mini kit and polymerase chain reaction (PCR) analysis was done. The PCR products were purified with the protocol of the BigDye XTerminator™ Purification Kit (Thermo Scientific) and were double-sequenced with the forward and reverse PCR primers of ABI PRISM 310 Genetic Analyzer. The sequence product was confirmed with Clone Manager Professional 9 (Sci-Ed Software) and the Neighbor-Joining method was done with MEGA6 to build a phylogenetic tree. RESULTS: The target product of DNA amplification in this PCR was around 290-bp. The amplicon was visualized in 2% of agarose gel electrophoresis. The homology analysis of these sequences showed that it had more than 99% similarity. CONCLUSION: COX-1 gene sequences of S. scabiei from rabbits in Malang, Nganjuk, and Kediri were very similar to COX-1 gene sequences in S. scabiei acquired from several hosts according to NCBI data.

Sequence analysis of the cytochrome c oxidase subunit 1 gene of Sarcoptes scabiei isolated from goats and rabbits in East Java, Indonesia.

AIM: This study aimed to sequence the Cytochrome c oxidase (COX-1) gene sequence from mitochondrial DNA of Sarcoptes scabiei isolated from Lamongan goats and Mojokerto rabbits, align it with DNA isolated from Zi'gong rabbit (GenBank accession No. EU256389.1), and produce a phylogenetic analysis of S. scabiei COX-1 gene. MATERIALS AND METHODS: S. scabiei mites were obtained from goats and rabbits, and DNA was extracted using QIAamp DNA Mini Kit. The forward and reverse primer sequences were designed based on the DNA sequence of an S. scabiei COX-1 gene isolated from the Zi'gong rabbit (5'-TCTTAGGGGCTGGATTTAGTATG-3' and 5'-AGTTCCTCTACCAGTTCCAC-3', respectively). To confirm sequencing output, the sequence resulting from the reverse primer was inverted and aligned to the sequence from the forward primer using Clone Manager Professional Version 9 for Windows (Scientific & Educational Software; http://www.scied.com). This alignment was subsequently used to build a phylogenetic tree, using the Neighbor-Joining method, in the MEGA6 program (https://www.megasoftware.net/). RESULTS: Polymerase chain reaction (PCR) products from S. scabiei isolates from Lamongan goats and Mojokerto rabbits produced bands of around 290 bp with 2% agarose gel electrophoresis. Comparing the DNA sequences of the S. scabiei COX-1 gene with those isolated from Lamongan goats and Mojokerto rabbits showed 99% homology. CONCLUSION: PCR products of the S. scabiei COX-1 gene isolated from Lamongan goats and Mojokerto rabbits were around 290 bp long. The sequences had more than 99% homology. The sequences of the COX-1 gene of S. scabiei from Lamongan goats and Mojokerto rabbits were relatively close to the sequence of the gene in S. scabiei obtained from various hosts according to National Center for Biotechnology Information data.

Prevalence and diversity of gastrointestinal protozoa in Madura cattle at Bangkalan Regency, East Java, Indonesia.

AIM: This study aimed to describe the gastrointestinal protozoa in Madura cattle at Bangkalan Regency, East Java, Indonesia. MATERIALS AND METHODS: A total of 500 samples of Madura cattle feces were collected from 10 districts at Bangkalan Regency. Those ten districts represent the lowland and upland areas, and each district was represented by one village. The collected feces were examined using native, sedimentation, and floating methods. The species identification was determined by their morphology. RESULTS: There were 357 (71.4%) samples positively infected with protozoan. The highest rate of sample with protozoan infection was at Kamal District (88.23%), and Bangkalan District (52.83%) was the lowest one. There were six species of protozoa that infected gastrointestinal tract; those are Eimeria spp., Balantidium spp., Isospora spp., Blastocystis spp., Entamoeba spp., and Cryptosporidium spp. The highest number of protozoa found in this research was Eimeria (53.42%) followed by Blastocystis (14.43%). In this study, we found that 295 samples (58.76%) infected by one kind of protozoa, 53 samples (10.56%) infected by two kinds of protozoa, and 11 samples (2.19%) infected by three kinds of protozoa. In addition, there were 65.54% of bulls infected with protozoa, considerably lower than cows (72.97%). Cattle aged 6 months-2 years old (73.39%) and >2 years old (71.25%) are known more prone to protozoan infections than cattle aged <6 months (66.15%). CONCLUSION: The present study revealed that protozoan infection of cattle is common in Bangkalan Regency. Studies focused on determining that the prevalence of protozoan, risk factors for the parasitism, and the geographic distribution are needed and will be effective guide for prevention and control measures.

Exploration of Sarcoptes scabiei Antigenic Protein Which Play Roles in Scabies Pathogenesis in Goats and Rabbits.

BACKGROUND: Scabies or mange is an infectious skin disease caused by the mite Sarcoptes scabiei. This skin disease affects various livestock such as goats, sheep, swine, cattle, other animals like dogs, cats, wild animals and also affect human. This research aimed to explore the protein in mites S. scabiei which has antigenic character and play roles in scabies pathogenesis in goats and rabbits. METHODS: S.scabiei mites were isolated from goats and rabbits, and characterized using SDS-PAGE. In addition the protein was also analysed using Western Blot assay. The isolation and identification were carried out in 2015 at the Parasitology Laboratory of Veterinary Medicine Faculty, Universitas Airlangga, Surabaya, Indonesia. RESULTS: The identification results using SDS-PAGE of mites S. scabiei var. caprae expressed 12 protein bands between 26,7 kDa and 205,8 kDa, continued by Western Blot showed 3 protein bands, after being reacted with blood serum from scabies infected goat, it could be identified antigenic protein with molecule weight 205.8 kDa, 57.3 kDa, and 43 kDa. While protein in mites S. scabiei var. cuniculi identified 9 protein bands between 24 kDa and 75 kDa by SDS-PAGE, and the Western Blot assay identified antigenic protein with molecule weight 62 kDa and 51 kDa. CONCLUSION: The antigenic protein of S. scabiei var. caprae and S. scabiei var. cuniculi showed that they are probably involved in the scabies pathogenesis in goats and rabbits.

Humoral and cellular immune response induced by antigenic protein of Sarcoptes scabiei var. caprae.

AIM: Scabies is one of the most important diseases in goats and caused by a complex hypersensitivity process that involves both humoral and cell-mediated immune responses. This phenomenon shows that the variety of Sarcoptes scabiei has different characteristics of specific antigenic protein or different immune-dominant. This research aims to detect the humoral and cellular immune response of rabbits which were immunized with the protein of S. scabiei var. caprae. MATERIALS AND METHODS: This research was done as follows, identification and collection of Sarcoptes scabiei var caprae from goat infected with scabies, separation of protein antigen from S. scabiei mites with ultrasonic sonicator, measurement of protein content with spectrophotometry, rabbit injection with 500 µg dose of antigen protein which was repeated 5 times (5x booster) every 2 weeks. Measurement of IgG titer using indirect ELISA, whereas to detect the expression of cellular immune response (TLR-9, CD4, and CD8) using Direct Immunofluorescence assay. RESULTS: Based on the statistical analysis, it showed that there was a significant enhancement (p<0.05) for optical density value or antibody titer and cellular immune response was shown by TLR-9, CD4, and CD8 expression in rabbit T lymphocytes which appear yellow to green fluorescent color using fluorescence microscope. The amount of fluorescence T lymphocytes showed a significant difference (p<0.05) between the control and various boosters. CONCLUSION: Antigenic protein of S. scabiei var. caprae contains ligands, which are involved in the pathogen-associated molecular pattern that has an ability to induce humoral and cellular immune response in rabbit. Specifically, that TLR-9 is not only involved in innate immunity but also in adaptive immunity and can be used as alternative adjuvant development research.