Prevalence of Amphistomes and Fasciola in large ruminants reared by smallholders in Lampung and Banten Provinces, Indonesia.

Background and Aim: Parasitic diseases, including trematode invasions, result in losses to livestock in Indonesia, hindering the government's efforts to achieve meat self-sufficiency. This study aimed to estimate the prevalence of Amphistomes and Fasciola in large ruminants reared by smallholder farmers. Materials and Methods: Fecal samples from 199 buffalo and cattle were collected from the districts of East Lampung (Lampung Province) and Lebak (Banten Province). Fecal samples were examined for the presence of trematode eggs using a sedimentation technique. Results: Parasite invasion rate was 48.2% (95% confidence interval [CI]: 41.3%-55.2%). Rate of invasion was 63.3% (95% CI: 52.7%-73.9%) in Lampung and 38.3% (95% CI: 29.6%-47.0%) in Lebak-Banten. The prevalence of multiple invasions of both Amphistomes and Fasciola was 20% in buffalo and local cattle, whereas invasion rate was 12.8% in crossbred cattle. Invasion rate of Amphistomes alone was 27.1%, and that of Fasciola was 4.5%. A higher invasion rate of Amphistomes (29.8%) occurred in crossbred animals. There were no significant differences between age groups for trematode invasion. The Chi-square test showed that the prevalence of trematode invasion in females was significantly higher than in males (51.5% and 30.0%, respectively). Amphistomes more commonly infected females than males (29.0% and 16.7%, respectively). Conclusion: All breeds were vulnerable to invasion by both trematode species and single invasions with different invasion rates. These findings contribute to determining the magnitude of the disease and provide a basis for studies on prevention and treatment of trematode invasion.

Serological survey of immunoglobulin G from Toxoplasma gondii infection in dairy goats in East Java, Indonesia.

Background and Aim: Toxoplasma gondii infection is a significant issue of veterinary public health because it is potentially transmitted through goat milk. Therefore, the use of control measures and routine monitoring of toxoplasmosis in dairy goats is necessary. Serological analysis using antibodies can detect T. gondii infection. This study aimed to conduct an epidemiological study of T. gondii in dairy goats using antibody detection and risk factor identification. Materials and Methods: This was a cross-sectional study. We performed a serological analysis of T. gondii infection in dairy goats to evaluate the prevalence of toxoplasmosis. Random sampling was performed, including 132 lactating dairy goats. Toxoplasma-modified agglutination test was used as a serological test for immunoglobulin G with a sensitivity of 98.55%, specificity of 86.21%, and accuracy of 94.9%. A structured questionnaire was used to collect risk factor data, which were analyzed using the Chi-square and Fisher's exact tests. The statistical package for the social sciences v. 21 was used for statistical analyses. Results: The seroprevalence of T. gondii in Malang and Lumajang Regency was 100% and 90.7%, respectively. A significant difference in prevalence of T. gondii was observed between the two districts. Livestock management practices that significantly influenced T. gondii seroprevalence included water sources (p < 0.05; relative risk [RR] = 1.151; 95% confidence interval [CI]: 1.044-1.269). Farmers' characteristics that significantly influenced T. gondii seroprevalence included education (p < 0.05; RR = 1.125; 95% CI: 1.037-1.221), main occupation (p < 0.05; RR = 1.118; 95% CI: 1.035-1.207), and position in the organization of dairy goats farmers (p < 0.05; RR = 1.141; 95% CI: 1.022-1.274). Conclusion: In East Java, the prevalence of T. gondii in dairy goats is high. This study provides detailed information regarding risk factors associated with T. gondii seroprevalence in dairy goats in East Java, Indonesia.

DOES TRYPANOSOMA EVANSI HAVE THE MAXICIRCLE GENE, OR CAN TRYPANOSOMA EQUIPERDUM BE ISOLATED FROM BOVINES?

Identifying a trypanosome isolate is generally based on morphological observations and molecular identification of one of the genes, usually internal transcribed spacer 1 and 2 of ribosomal DNA (ITS1 rDNA, ITS2 rDNA), a variant surface glycoprotein of Rode Trypanozoon antigen type 1.2 (VSG RoTat 1.2), or expression site-associated genes (ESAG). However, this identification is insufficient because these genes cannot distinguish organisms in the subgenus Trypanozoon to the species level. A molecular approach using at least 5 sets of primers is needed, namely, ITS1, ESAG6/7, MINI, RoTat 1.2, and ND5, for stratified selection to obtain more targeted and conclusive results. Using this method to analyze isolates from Indonesia provided unexpected results: 9 isolates previously identified as Trypanozoon were found to have the kDNA maxicircle gene. Nine isolates of Trypanosoma equiperdum were identified for the first time in Indonesia, isolated from bovine (cattle and buffaloes). The identification of T. equiperdum in the 9 isolates was confirmed by analysis of the nucleotide sequence identity of the nad5-kDNA maxicircle gene.

Morphology, Biochemical, and Molecular Characterization of Pasteurella multocida Causing Hemorrhagic Septicemia in Indonesia.

Pasteurella multocida is a Gram-negative bacterium that causes hemorrhagic septicemia (HS) in buffaloes and cattle. The disease causes serious problems in Indonesian livestock and is classified as a serious transmissible animal disease. Previous research has determined the diversity of P. multocida using a serotyping method based on the antigenic properties of capsule polysaccharides. An alternative method for analysis utilizes sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and random amplified polymorphic DNA (RAPD). This study aimed to characterize and determine P. multocida diversity in several regions of Indonesia based on phenotypic character, protein profile, and the band pattern of RAPD results. Bacterial identification was performed using traditional biochemical techniques and API® 20NE systems and then confirmed molecularly using polymerase chain reaction (PCR). The freeze-thawing technique was performed to obtain the bacterial protein extract, and DNA extraction was executed using DNAzol. The extracted protein and RAPD product were then electrophoresed on 12% polyacrylamide gel and 1.5% agarose gel, respectively. The results indicate that the molecular weight range of the protein bands is 12-209 kDa, and the band pattern of the RAPD results ranged from 307-3,100 bp. Based on phenotypical analysis, P. multocida from South Sulawesi Province exhibited a variety of growth characteristics in MacConkey agar media. Using the hierarchical clustering analysis of the band patterns of RAPD and the whole-cell protein profiles, four and five clusters were formed, respectively. These results indicate molecular diversity among P. multocida from several regions of Indonesia.

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.

Toxoplasma gondii virulence prediction using hierarchical cluster analysis based on coding sequences (CDS) of sag1, gra7 and rop18.

Toxoplasma gondii consists of three genotypes, namely genotype I, II and III. Based on its virulence, T. gondii can be divided into virulent and avirulent strains. This study intends to evaluate an alternative method for predicting T. gondii virulence using hierarchical cluster analysis based on complete coding sequences (CDS) of sag1, gra7 and rop18 genes. Dendrogram was constructed using UPGMA with a Kimura 80 nucleotide distance measurement. The results showed that the prediction errors of T. gondii virulence using sag1, gra7 and rop18 were 7.41%, 6.89% and 9.1%, respectively. Analysis based on CDS of gra7 and rop18 was able to differentiate avirulent strains into genotypes II and III, whereas sag1 failed to differentiate.

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.

Phylogenetic characterization of Isospora jaracimrmani oocysts from a veiled chameleon (family Chamaeleonidae; Chamaeleo calyptratus) reared at a zoo in Ishikawa, Japan.

Oocysts of Isospora sp. were detected in the feces of a veiled chameleon (family Chamaeleonidae; Chamaeleo calyptratus) kept at a zoo in Ishikawa, Japan. Phylogenetic analysis placed the sequence in the cluster of Isospora spp. isolated from reptiles. Based on a comparison of morphological data of ten previously reported Isospora species from the Chamaeleonidae family, this isolate was morphologically similar to I. jaracimrmani, which has been considered to be a virulent species. This case study suggests the possibility that species of Isospora might not always cause disease because the animal that shed these oocysts showed no symptoms for more than two months.

Occurrence and genetic identifications of porcine Entamoeba, E. suis and E. polecki, at Tangerang in West Java, Indonesia.

Entamoeba suis and E. polecki subtype (ST) 1 and ST3 recently have been inferred to be virulent in pigs. However, because relevant molecular epidemiological surveys have been limited, the prevalences of these species remain unknown and their pathogenicities are still controversial. We surveyed 196 fecal samples of pigs (118 of adults, 78 of piglets) at Tangerang in West Java, Indonesia, in 2017, employing PCR using porcine Entamoeba-specific primers. E. suis was the more frequently detected species, observed in 81.1% of samples, while E. polecki ST1 and ST3 were detected in 18.4% and 17.3% of samples, respectively; mixed infections (harboring 2-3 species or subtypes of Entamoeba) were confirmed in 29.3% of positive samples. Statistically significant differences in the positive rates were not seen between adult pigs and piglets, except for those of E. polecki ST3. The prevalences of Eimeria spp. and/or Cystoisospora suis (79.1%), strongyles (55.6%), and Strongyloides spp. (6.1%) were also observed morphologically in the samples. Further chronological or seasonal investigations of pigs and humans in these high-prevalence areas are needed to assess the virulence of the Entamoeba parasites, including the effects on pig productivity, and to evaluate the zoonotic impacts of these organisms.

Detections of gastrointestinal parasites, including Giardia intestinalis and Cryptosporidium spp., in cattle of Banten province, Indonesia.

Gastrointestinal parasites can induce low productivity in livestock by causing acute or chronic enteritis. Veterinarians make great efforts to design rational and effective hygienic protocols for both the prevention and treatment of diarrhea. Although prevalences can vary depending on the examined areas or the ages of the hosts, and the methods used for detections, it is helpful to accumulate data across many areas to evaluate parasitic distribution. A coprological survey in cattle was conducted in Tangerang, Banten Province of Indonesia, in order to determine the prevalence of the parasites, including those of diarrhea-associated diseases. Furthermore, the risk of transmission of Giardia intestinalis and Cryptosporidium spp. to human was genetically analyzed. Gastrointestinal parasites were detected in 87 of 109 cattle samples, including 85 carrying Eimeria spp., 36 carrying Fasciola gigantica, 35 carrying Strongyloides spp., 33 carrying Paramphistomum spp., and 15 carrying Capillaria spp. Giardia intestinalis and Cryptosporidium spp., parasites with zoonotic potential, were detected in 9 and 1 cattle samples, respectively. Molecular analyses identified the G. intestinalis isolate as a member of Assemblage E, which has been recently detected in humans in another country. These results may be helpful in understanding the hygienic risk affecting the livestock productivity and zoonotic potential of cattle in Indonesia.

Molecular identification of Eimeria hestermani and Eimeria prionotemni from a red-necked wallaby (Macropodidae; Macropus rufogriseus) in Japan.

To date, more than 50 Eimeria spp. have been isolated from marsupials of the family Macropodidae. Although 18 species of Eimeria have been previously detected from multiple animal species belonging to the genus Macropus of the family, limited genetic analyses of the parasites are available, and their pathogenicity remains unclear. Here, we report the isolation of Eimeria spp. from a zoo specimen of red-necked wallaby (Macropodidae; Macropus rufogriseus). Specifically, two distinct types of Eimeria oocysts were recovered, one from the feces before treatment with an anthelmintic and the second from the intestinal contents after death of the animal. The oocysts obtained from the two sources were morphologically identified as E. hestermani and E. prionotemni, respectively. We successfully determined partial gene sequences from the two isolates, including segments of the 18S rRNA genes, and for the first time have used phylogenetic analyses of these sequences to assign the species to distinct clades. In combination with further genetic data, these results are expected to help elucidate the pathogenicity and host ranges of Eimeria spp. within the respective family and genus.

Molecular characterization of highly pathogenic Eimeria species among beef cattle on Java Island, Indonesia.

Gastrointestinal parasites including Eimeria spp. are known to affect domestic animal productivity causing watery or lethal bloody diarrhea. However, there are few reports on the detailed distribution of bovine Eimeria spp. in cattle, particularly in developing tropical and sub-tropical areas. Using a total of 289 fecal samples collected from beef cattle on Java Island, one of the five main islands of Indonesia, fecal examinations by the Whitlock and sugar flotation methods and molecular surveys were conducted to reveal the prevalence of 6 Eimeria spp. As a result of morphological screening using Whitlock methods and sugar flotation, Eimeria spp. prevalences of 9.4% and 52.3% were confirmed, respectively. The prevalence was higher in younger cattle [under 1 year (63.9%), 1-2 years (75.0%) and more than in 2 year old cattle (42.3%)]. The prevalences of identified species were as follows: 10.4% for E. bovis, 2.8% for E. ellipsoidalis, 2.1% for E. alabamensis, 1.4% for E. zuernii, 1.1% for E. auburnensis, and 0.4% for E. cylindrica. Moreover, prevalences of 12.8% for Strongyloides papillosus, 7.3% for Trichuris globulosa, and 0.3% for Capillaria bovis were detected. Although the average number of oocysts per gram of feces was <100 among the positive samples, and cases of heavy infection were limited, the endemicity of these pathogenic Eimeria species among farms in Indonesia should be noted.