Morphological and molecular characteristics of Plasmodium juxtanucleare in layer chicken from three districts of Yogyakarta, Indonesia.

Background and Aim: Blood parasite infections in poultry, such as Plasmodium, are a serious threat to the poultry industry due to their potential to cause economic losses. To date, there has been inadequate research on the morphological and molecular detection of the different Plasmodium species that infect poultry in Indonesia. Therefore, this study aimed to analyze the morphological and molecular characteristics of Plasmodium spp. and the several predisposing factors for Plasmodium infection in layer chickens from three districts of Yogyakarta, Indonesia. Materials and Methods: One hundred and five blood samples from layer chickens were collected from 13 farms located in three districts of Yogyakarta (Sleman, Bantul, and Kulon Progo) between September and November 2022. Blood samples were subjected to microscopic and polymerase chain reaction (PCR) analyses. Sequencing was performed using basic local alignment search tools to identify the nucleotide structure of cytochrome b. Phylogenetic analysis of Plasmodium was performed using the MEGA-X software. Results: Microscopic examination revealed that 17/105 positives (16.19%) were positive for blood parasite infection. Trophozoites, erythrocytic meronts, and microgametocytes of Plasmodium were found in blood samples. Based on the morphological examination, the species found in the samples was close to Plasmodium juxtanucleare. Polymerase chain reaction examination revealed that 21/60 samples were positive for Plasmodium (35%). The Plasmodium species identified from the sequenced samples were proven to be P. juxtanucleare. The P. juxtanucleare from Thailand was closely related to samples (99.64%-100%) with a genetic distance of 0%-1%. In addition, age, population, and cage type were not significantly associated with Plasmodium infection. Conclusion: Based on microscopic and PCR examinations, the Plasmodium species found in the three districts of Yogyakarta was P. juxtanucleare. The genetic distance between samples from the three districts of Yogyakarta was closely related (0%-1%) to P. juxtanucleare from Thailand and Japan. There was no correlation between Plasmodium infection and age, cage type, or population.

Detection of Trypanosoma evansi in a naturally infected cat in Indonesia using bioassay and molecular techniques.

Background and Aim: The prevalence of surra in domestic cat is seldom and it is caused by Trypanosoma brucei and Trypanosoma evansi. However, molecular diagnostic approaches are required owing to similarities in their morphology. In Yogyakarta, a domestic cat was diagnosed with trypanosomiasis; however, the causative species was undetermined. Therefore, we aimed to molecularly and biologically identify the isolate. Materials and Methods: Approximately 1 mL of blood from an infected cat was collected into EDTA tube and separated for inoculation into donor mice, blood smear, and DNA isolation. Two donor mice was then used for increasing the number of parasite in order to infect 10 experimental mice. Parasitemia was monitored daily in each experimental mouse by preparing a wet mount and Giemsa-stained thin blood smear. The blood of experimental mice that reached the peak of parasitemia was then collected and used for DNA isolation. Each blood sample, which collected from infected cat and experimental mice, was then isolated and amplified the DNA by polymerase chain reaction using ITS-1. The parasitemia pattern and viability of the animals were observed to determine the biological characteristics of trypanosomatid, while to assess the molecular characteristics, the internal transcribed spacer (ITS)-1 amplification was used. Results: The prepatent period of this trypanosomatid is between 2 and 4 dpi, whereas the life span of mice is approximately 4-10 dpi. Morphologically, the trypomastigote in the cat blood smear had long slender and intermediate shapes. However, only the long slender form was detected. Among the total of 410 nucleotides (NT) of ITS-1 sequences, 25 NT substitutions differed between the cat and mouse isolates. Phylogenetic analysis revealed that both samples had a close genetic relationship with T. evansi. Conclusion: Trypanosoma evansi, a highly virulent trypanosomatid, was isolated from a cat in Yogyakarta.

Molecular and morphological identification of Lernaea spp. in Cyprinid fishes from two districts in Yogyakarta, Indonesia.

Background and Aim: Parasitic infection commonly affects freshwater ornamental fishes. Parasites in fish may impede their growth and even cause death, resulting in a decline in fecundity. The prevalence of lernaeosis in aquaculture ponds in Indonesia requires attention because of missing data, especially from Yogyakarta. Therefore, this study aimed to identify the Lernaea species found in fish in Indonesia, particularly in Yogyakarta, molecularly and morphologically, as well as an overview of their distribution and the water condition they inhabit. Materials and Methods: Lernaea species were collected from three different fish species in two districts of Yogyakarta, Indonesia, for precise identification. Lernaea specimens were characterized morphologically and subjected to molecular identification based on 18S rRNA and 28S rRNA genes. Results: Lernaea in this study was morphologically and genetically confirmed as Lernaea cyprinacea, and the infection rate in each fish species was different. Water conditions might have contributed to the differences in infection levels. Conclusion: This study characterized L. cyprinacea isolated from Yogyakarta. Future research should focus on sequencing as much molecular information as possible and carrying out more experimental infections.

Development of a multiplex polymerase chain reaction technique for detection and discrimination of Eimeria spp. in cattle in Indonesia.

Background and Aim: Bovine eimeriosis is a disease caused by apicomplexan parasites of the genus Eimeria. It is one of the most important and widespread bovine illnesses in the world. Some of the identified species of bovine eimeriosis have morphologically similar oocysts that are difficult to differentiate. For the identification of particular Eimeria spp., diagnostic laboratories are increasingly turning to DNA-based technology. This study aims to develop a multiplex polymerase chain reaction (mPCR) technique based on the internal transcribed spacer-1 (ITS-1) gene for the simultaneous identification of pathogenic Eimeria spp. in cattle from Sulawesi Island, Indonesia. Materials and Methods: Genomic DNA was extracted by the DNAzol reagent from the purified Eimeria oocysts. Species-specific primers targeting the ITS-1 region were used to amplify the distinct Eimeria spp. Results: Using PCR ITS-1, this study showed that 36 of 120 fecal samples (30%) were infected by Eimeria spp. The multiplex PCR assay allowed for the simultaneous identification of six major Eimeria spp. in a single-tube reaction. The proportion of mixed Eimeria spp. infections was 100% (36/36). The maximum number of Eimeria spp. was five, and the minimum number was two. Conclusion: Identification of six pathogenic Eimeria spp. in cattle was successfully carried out by nested multiplex PCR using ITS-1 gene. In the future, a procedure to detect pathogenic Eimeria spp. in one tube reaction will offer economical and save diagnostic time.

Molecular identification of cercaria Fasciola gigantica in lymnaeid snails in Kulon Progo, Yogyakarta.

Lymnaeid snails play an essential role in transmitting fasciolosis as intermediate hosts. Therefore, this study aims to use the molecular method to identify liver fluke in lymnaeid snails. A total of 320 lymnaeid snails were collected from a rice field. The samples were dissected to collect cercaria and identified using polymerase chain reaction. Furthermore, the internal transcribed spacer 2 (ITS2) was used as the target gene to identify the species of cercaria. The result showed that 3.75% (12/320) of the snails were infected by Fasciola gigantica, while the phylogenetic tree based on ITS2 showed that the cercaria in this study was monophyletic and similar to species from several countries in Southeast Asia, including China. Furthermore, the haplotype network showed that all four cercaria samples were similar with sequences from several countries. This study suggests that the F. gigantica cercaria isolated from lymnaeid snails in Kulon Progo, Yogyakarta, Indonesia, has a sequence similar to that of other species in Southeast Asian countries, although no hybrid type was detected in these sequences. This is the first report on the molecular identification of cercaria F. gigantica isolated from lymnaeid snails in Yogyakarta, Indonesia.

Prevalence and risk factors associated with Eimeria species infection in cattle of different geographical regions of Indonesia.

BACKGROUND AND AIM: Eimeria spp. are gastrointestinal protozoans that affect animal productivity, thereby causing symptoms that range from bloody diarrhea to death. These symptoms cause economic losses to farmers. The distribution of Eimeria spp. in cattle has, therefore, been reported to have spread widely, especially in the tropics and subtropics. Indonesia is a tropical country at high risk of Eimeria infections. This study aimed to identify the prevalence and risk factors related to the levels of eimeriosis in beef cattle originating from different geographic areas in Indonesia. MATERIALS AND METHODS: Here, 817 fecal samples were collected from beef cattle in Indonesia, including 282 calves, 535 adults, 530 males, and 287 females. In addition, 156 semi-intensively and 661 intensively managed cattle were randomly collected. Then, fecal samples were analyzed by parasitology examinations. RESULTS: Screening examination using the sugar flotation modification method showed that Eimeria spp. were prevalent in Indonesia, as 65.4% of the bacterial strain was detected. The prevalence of identified Eimeria spp. in Indonesia was highest in North Maluku (Maluku Island) (94.1%), whereas the lowest levels were observed in West Java (24.0%) (Java Island). The prevalence was also found to be higher in males (79.3%) than females (51.9%). Similarly, levels in semi-intensively managed cattle (66.7%) were higher than those subjected to intensive management (65.9%). However, its prevalence in calf and adult cattle was similar. CONCLUSION: Bovine eimeriosis spp. were detected at high prevalence in Indonesia, and high-level risks were observed in infected males, including those under the semi-intensive management. In addition, although the results from oocyst examinations were based on qualitative analysis, the endemicity levels of Eimeria spp. among farms in Indonesia should be considered because Eimeria spp. were distributed in most parts of Indonesia. Based on the results of this study, we provide the first information about the prevalence of bovine eimeriosis from different geographical locations in Indonesia, which have differing climates associated with the level of the existing risk factors. Hence, farmers are advised to pay more attention to strict biosecurity techniques on their farms, thereby favoring the early control of bovine eimeriosis.

Genetic and parasitological identification of Trypanosoma evansi infecting cattle in South Sulawesi, Indonesia.

BACKGROUND AND AIM: Sulawesi is an Indonesian island located within the Wallacea region that contains a distinctive mix of Asian and Australasian species. This distinctiveness extends to parasites, including Trypanosoma evansi, the cause of surra. Surra has non-specific clinical signs such as anemia, anorexia, weight loss, drop in milk production, and reproductive disorders which cause economic losses. Due to the trade of livestock, surra has spread in Indonesia from one island to another. The aim of this study was to investigate the trypanosomes infecting cattle in South Sulawesi, using internal transcribed spacer (ITS2) ribosomal DNA (rDNA) sequencing. MATERIALS AND METHODS: A total of 100 whole blood samples were collected from cattle in Makassar, South Sulawesi Province, Indonesia. All samples were tested using conventional parasitological methods (CPT), namely, thin blood smear, buffy coat smears, and polymerase chain reaction (PCR) testing. Positive PCR results were sequenced and phylogenetically analyzed. RESULTS: Only one of the 100 samples was found to be positive with microscopic observation; however, PCR analysis revealed that 3% (3/100) of samples were positive. Sequencing identified the positive samples as T. evansi, China isolate (KU552344), with a homology of 99%. Two out of three sequences showed variations in ITS2 region. CONCLUSION: Based on CPT and molecular analysis, T. evansi isolates from infected cattle in South Sulawesi demonstrate genetic diversity of ITS2 sequences.

Cloning and expression of Toxoplasma gondii GRA-4 recombinant protein as a toxoplasmosis diagnostic kit candidate.

AIM: The objective of this study was to produce recombinant protein GRA-4 (rGRA-4) of a local Toxoplasma gondii isolate as a candidate for a toxoplasmosis diagnosis kit in Escherichia coli BL21 (DE3) competent cells using pET SUMO plasmid. MATERIALS AND METHODS: Samples used were stock T. gondii tachyzoites DNA from the Parasitology Laboratory, Faculty of Veterinary Medicine, Gadjah Mada University, Yogyakarta. Amplified GRA-4 polymerase chain reaction product of T. gondii tachyzoite DNA was cloned in the pET-SUMO TAR cloning vector. The GRA-4 gene from T. gondii local isolate was sequenced, followed by plasmid transformation, recombinant plasmid DNA isolation, and recombinant protein expression in DE3 competent cells. RESULTS: The amplification product of GRA-4 T. gondii gene was 1036 bp, with 48 kDa molecular weight after expression in DE3 competent cells. An alignment of the amino acid sequence of GRA-4 from the local isolate which was cloned with GRA-4 was obtained from NCBI database and showed 99.61% homology to the predicted GRA-4 from the T. gondii Izatnagar isolate. Amino acid sequence of the predicted GRA-4 protein from local isolate was different at positions 19 and 304. CONCLUSION: This research cloned rGRA-4 in pET SUMO plasmid.

Ticks (Acari: Ixodidae) infestation on cattle in various regions in Indonesia.

BACKGROUND AND AIM: Ticks (Ixodidae) not only cause blood loss in cattle but also serve as vectors for various diseases, thus causing direct and indirect losses. Moreover, tick infestation can cause significant economic losses. This study aimed to identify the diverse species of ticks infesting cattle in five different regions in Indonesia. MATERIALS AND METHODS: Tick specimens were obtained from local cattle in five different areas in Indonesia. The morphology of the specimens was macroscopically and microscopically evaluated, and the resulting data were descriptively and qualitatively analyzed. RESULTS: In total, 1575 ticks were successfully collected from 26 animals. In total, two genera and three species, namely, Rhipicephalus microplus, Haemaphysalis bispinosa, and Rhipicephalus pilans, were identified. The cattle in Yogyakarta and Riau were infested by H. bispinosa, while the cattle in Sukabumi, Bali, and Lombok were infested by R. microplus and R. pilans. The level of infestation varied among regions, with R. microplus being the most commonly found species. CONCLUSION: The results of this study revealed that cattle in different regions of Indonesia were infested by variable numbers of tick species. In particular, the cattle in Yogyakarta and Riau were solely infested by H. bispinosa; this is a new finding in terms of the distribution of tick species in the country. Increased tick infestation in cattle decreases productivity and causes health problems; therefore, it deserves serious attention. Our findings can help in the formulation of an effective strategy for controlling and preventing cattle tick infestation in the country.

Lice infestation and diversity in turkeys (Meleagris gallopavo) in the Special Region of Yogyakarta and Central Java, Indonesia.

BACKGROUND AND AIM: Biting lice (Phthiraptera: Amblycera and Ischnocera) are ectoparasites that play important roles in the transmission of disease agents that infect turkeys and impact turkey productivity. This study aimed to determine the diversity of lice that infest turkeys in the Central Java Province and the Special Region of Yogyakarta, Indonesia. MATERIALS AND METHODS: Lice sampling was conducted at 16 different locations from April 2019 to June 2019 in turkeys aged 4 months to 2 years. The samples were stored in 70% alcohol and were identified using avian louse keys. The morphology of the specimens was macroscopically and microscopically evaluated, and the resulting data were descriptively and qualitatively analyzed. RESULTS: A total of 2505 lice were collected, and two families and five genera of lice were identified. Three lice genus members of the Philopteridae family (Lipeurus, Oxylipeurus, and Chelopistes) and two genera of the Menoponidae family (Colpocephalum and Menacanthus) were identified. Lipeurus was the most frequently identified genera in turkeys, whereas Menacanthus was the most rarely identified one. The White Holland breed had the highest number of lice infestations, whereas the Jersey Buff breed exhibited the highest diversity of lice genera. The average number of lice infestations was higher in male turkeys than in female turkeys. CONCLUSION: The occurrence of ectoparasites in domestic turkeys indicates that the existence and diversity of lice genera in the study location can be influenced by turkey type, turkey maintenance system, enclosure sanitation measures, lack of strategic ectoparasite control, and environmental factors.