Ectoparasites Infestation to Small Ruminants and Practical Attitudes among Farmers toward Acaricides Treatment in Central Region of Java, Indonesia.

Ectoparasite infestations are one of the major problems affecting goat and sheep farming. Disease resulting from these infestations can cause changes in physical appearance, such as severe lesions on the skin, and economic consequences in the form of significantly reduced selling prices. This study aimed to determine the prevalence of ectoparasites in the Boyolali district, Central Java, Indonesia. A total of 651 sheep and goats were surveyed in this study. The parasites were collected via skin scraping, twister, or manually from clinically infected goats and sheep in traditional farms. All of the ectoparasites collected were successfully identified. The prevalence of ectoparasites in ruminants in Boyolali was 97.8% (637/651). The species make-up was as follows: Bovicola caprae 97.8% (637/651), Linognathus africanus 39% (254/651), Haemaphysalis bispinosa 3.5% (23/651), Ctenocephalides spp. 0.2% (1/651), and Sarcoptes scabiei 5.2% (34/651). The predilection sites were in the face, ear, and leg areas, and in the axillary, dorsal, abdomen, and scrotum regions of the surveyed animals. An evaluation of farmers' attitudes to ectoparasites was performed using a questionnaire. The findings of this study imply that animals in the investigated area are highly exposed to ectoparasite infestations. Given the importance of ectoparasites in both livestock and human communities, specifically in the health domain, more research into appropriate control strategies is necessary.

Perception of Ticks and Tick-Borne Diseases Worldwide.

In this comprehensive review study, we addressed the challenge posed by ticks and tick-borne diseases (TBDs) with growing incidence affecting human and animal health worldwide. Data and perspectives were collected from different countries and regions worldwide, including America, Europe, Africa, Asia, and Oceania. The results updated the current situation with ticks and TBD and how it is perceived by society with information bias and gaps. The study reinforces the importance of multidisciplinary and international collaborations to advance in the surveillance, communication and proposed future directions to address these challenges.

Artemisia vulgaris anthelmintic activities to ova and adult stages of Fasciola gigantica in vitro.

Background and Aim: Fasciolosis due to Fasciola gigantica is endemic to tropical countries and Fasciola hepatica in temperate climates, highly detrimental to livestock and known as foodborne zoonotic diseases. The strategic control of the disease is mainly the use of chemical anthelmintic. This study aimed to evaluate the anthelmintic properties of Artemisia vulgaris extract on the ova and adult stages of F. gigantica. Materials and Methods: Samples were collected from the Ampel Abbatoir, Boyolali District, Central Java, Indonesia. The ova from 20-gallbladders of cattle which were naturally infected with F. gigantica and 270 living F. gigantica worms were used in this study. The ovicidal assay was performed by incubating the ova with A. vulgaris in different concentrations, that is, 5%, 2.5%, and 1.25% for 5, 9, 11, 14, and 16 days. The efficacies were evaluated by quantification of ova degeneration during developmental stages in different time points and egg-hatch assay. The flukicidal effects were observed by mortality assay in 5, 10, 20, 40, 80, 160, 320, and 640 min incubations followed by scanning electron microscopy for surface morphology and histology of the fluke's transversal sections. Results: The concentration of 5% A. vulgaris showed the strongest ovicidal activities. The percentage of hatching ova on day 16 at concentrations of 5%, 2.5%, and 1.25% were 3.33%, 6.67%, and 16.67%. These ova hatch assay showed a significant reduction (p < 0.001) compared to untreated control. The flukicidal effect was significant (p < 0.001) at a concentration of 20%, with a mortality rate reaching 66.67% in the 40 min of incubation time. The surface properties of the adult worms, including the spine, tegument, acetabulum, intestine, and vitelline follicles, were disintegrated. Conclusion: The results showed that A. vulgaris has the potential ovicidal and flukicidal properties to F. gigantica. The active compounds remained necessary to be elucidated further and its modes of action would be interesting to be predicted by molecular docking modeling.

High prevalence of liver fluke infestation, Fasciola gigantica, among slaughtered cattle in Boyolali District, Central Java.

Background: Fasciolosis is a foodborne disease caused by Fasciola sp. infecting ruminants, especially cattle. Fasciolosis remains a significant concern for Veterinary Public Health because of its zoonosis risk and transmission mode. Aim: This study aimed to determine the prevalence and risk factors associated with Fasciola gigantica infestation in cattle at Ampel abbatoir, Central Java, Indonesia. Methods: A cross-sectional study was performed on 585 cattle from February to August 2022. Visual observation postmortem was used to assess Fasciola infection based on adult flukes in liver parenchyma and ductuli biliferi. Results: The overall prevalence of fasciolosis in Ampel abbatoir is high, reaching 25.12% (147/585). The highest prevalence was observed in the Ongole breed, 42.1% (24/57), female cattle, 38.72% (115/297), body condition score criteria of 2 50% (21/42), cattle aged >3.5 years 46.06% (82/178), and cattle originated from outside of Boyolali district 33.33% (71/213). Conclusion: This study showed a high prevalence of fasciolosis in Ampel abbatoir, as shown in the correlation between the risk factors of breed, sex, body condition score (BCS), origin, and age. Because of the high prevalence of fasciolosis in the abattoirs, it is essential to continue performing epidemiology studies in more expansive areas. The subsequent plans are important to reduce the risk of fasciolosis as a threat to productive cattle husbandry and warrant its transmission to humans as a foodborne-zoonotic disease.

Ethanolic extract of Etlingera elatior flower exhibits anthelmintic properties to Fasciola gigantica in vitro.

Background: Fasciolosis is a parasitic disease affecting the hepatobiliary system of livestock worldwide. The control of the fluke is important to be performed in endemic regions. Aim: This study aims to evaluate the effect of Etlingera elatior ethanolic extract on egg and adult stadia of Fasciola gigantica. Methods: Fasciola gigantica in different stages were incubated with E. elatior ethanolic extract in different concentrations and time points. Results: The number of developed eggs with different concentrations of 1.25%, 2.5%, and 5% was significantly decreased by 36.67%, 56.67%, and 56.67% on day 11 post-incubation, which showed an ovicidal effect of the herb. The developed eggs on day 14, which were represented by hatched larvae, were also decreased by 70%, 50%, and 13.33%, respectively. Significant flukicidal effects were observed in the incubation time of 80 minutes for the concentration of 20% (p = 0.007) and 640 minutes for 10% concentration (p = 0.003). Surface microscopy of adult F. gigantica showed damaged skin and spina with the erosion of the inner membrane and detached syncytium from the tegument. Conclusion: Overall, the results indicate that E. elatior has a promising anthelmintic property against F. gigantica in both ova and adult stages.

First Autochthonous Report on Cattle Babesia naoakii in Central Java, Indonesia, and Identification of Haemaphysalis bispinosa Ticks in the Investigated Area.

In tropical countries, clinical bovine babesiosis is a tick-borne disease primarily caused by Babesia bovis and Babesia bigemina. Here, we investigated 11 cattle with presumptive diagnosis of clinical babesiosis in Boyolali district, Central Java, Indonesia. The majority of the animals were anemic, as evidenced by lower hematocrit, hemoglobin concentration, and red blood cell counts than the normal ranges. Blood DNA was analyzed by a PCR assay targeting the 18S rRNA-ITS region of babesial origin, and the results confirmed that the cattle were infected with Babesia species. The sequencing and phylogenetic analyses demonstrated that the animals were infected with Babesia naoakii. This is the first report of B. naoakii in Indonesia and of B. naoakii-induced clinical bovine babesiosis outside of Sri Lanka. B. naoakii causes a persistent infection, as indicated by positive PCR results for serial blood samples of the circulatory system taken two weeks after treatment. Consequently, subclinical or newly recovered cattle may serve as potential intermediate hosts and infect ticks as definitive hosts to complete the life cycle. To identify potential tick vectors, we collected ticks from cattle, including 11 animals with clinical babesiosis. Based on the morphology and the mitochondrial cytochrome c oxidase subunit 1 (COX1) of collected ticks, we found that all of the collected ticks were Haemaphysalis bispinosa, identifying this tick species as a potential vector of B. naoakii in Indonesia. In this study, the evaluation of local farmers' awareness and practices regarding tick-borne diseases is presented, as disease prevention is also reliant on the implementation of strategies for vector control. Since livestock activities in Java represent the country's busiest animal trade, thereby the spread of disease to other regions is possible through anthropogenic factors. In conclusion, B. naoakii is a causative pathogen of clinical bovine babesiosis autochthonously occurred in this report and further research on B. naoakii-infection is required in other regions of the country. The prompt treatment of the disease seemed crucial for animal survival, which implies the necessity of early diagnosis and a sensitive detection method.

Artemisia vulgaris efficacies against various stages of Aedes aegypti.

BACKGROUND AND AIM: Aedes aegypti is the vector of dengue fever, dengue hemorrhagic fever, chikungunya, and, most recently, Zika. Dengue fever is one of Indonesia's endemic diseases. The principal tool for preventing dengue is controlling Ae. aegypti by chemical insecticides since vaccine against dengue is still under research. However, Ae. aegypti developed resistance to various chemical insecticides worldwide. Therefore, research on alternate compounds as mosquito insecticides is urgently needed. This study demonstrated the efficacy of Artemisia vulgaris extract as larvicidal, ovicidal, adulticidal, repellency, and oviposition deterrent activity against Ae. aegypti. MATERIALS AND METHODS: A. vulgaris was obtained from Temanggung, Indonesia, while the eggs of Ae. aegypti were collected from Yogyakarta, Indonesia, and were hatched in Laboratory of Parasitology, Faculty of Veterinary Medicine, Universitas Gadjah Mada. Larvicidal activity was evaluated according to the WHO protocol; adulticidal activity was performed using the Centers for Disease Control protocol. Oviposition activity was evaluated using ovitraps added with A. vulgaris extract, complete protection time in the repellent assay was defined as the number of minutes elapsed between compound application and the landing of the first mosquito. RESULTS: A test of the larvicidal activity of A. vulgaris extract returned an LC50 of 65.8 ppm (r2=0.9014) in 1 h and 18.6 ppm (r2=0.575) in 24 h. A. vulgaris was effective as an adulticidal, demonstrating LC50 values of 11.35 mg (r2=0.875) in 90 min, 9.63 mg (r2=0.924) in 105 min, and 6.46 mg (r2=0.925) in 120 min. A. vulgaris at a concentration of 1000 ppm was able to reach 96% of oviposition deterrent effect. The ovicidal assay, a concentration of 1000 ppm resulted in 82.67% of eggs remaining unhatched. An extract concentration of 80 mg/ml achieved 63.3±3.5% biting repellency in adults. CONCLUSION: This study gives a clear indication that A. vulgaris extract acts on Ae. aegypti at various developmental stages and is a potential alternative bioinsecticide for controlling this disease vector.

Intestinal and hepatic coccidiosis among rabbits in Yogyakarta, Indonesia.

BACKGROUND AND AIM: The attention to rabbit meat production in Indonesia is comparatively less to other farm animals such as cattle and poultry industries. However, future prospect of rabbit to be seriously industrialized seemed quite promising due to rabbit is highly productive and has short reproduction cycle as well as generation interval. One of the diseases infecting many rabbits is coccidiosis caused by protozoan parasite, Eimeria spp. The infectious stage of Eimeria spp. presents ubiquitously in the environment and increases the risk of parasite transmission. Preventive methods such as vaccination are not yet fully developed, while sporadic treatment is not efficiently reduce the cases. In this study, Eimeria spp. infecting rabbits in Yogyakarta Province, Indonesia, were investigated with the aim for precise diagnosis to determine targeted treatment and as a baseline epidemiological data from rabbit in Indonesia. MATERIALS AND METHODS: Sample collection was performed randomly for 3 months, from March 2017 to May 2017 and covered areas in Yogyakarta, Indonesia. A total of 750 samples were collected. Eimeria species identification was determined morphologically from the samples after sporulation in 2.5% potassium dichromate byCOCCIMORPH. RESULTS: Ten species of Eimeria spp. were identified in this study from the positive samples (527/750; 70.3%). Eimeria flavescens was present in 80% of the positive samples, Eimeria coeciola in 78%, Eimeria perforans in 61%, Eimeria exigua in 37%, Eimeria media in 33%, Eimeria stiedae in 31%, Eimeria irresidua in 12%, Eimeria magna in 11%, Eimeria intestinalis in 10%, and Eimeria piriformis in 10%. Coinfection as noted in 80% of the positive samples with 2-6 species in a specimen. E. flavescens and E. coeciola were the most prevalent among all Eimeria spp. (p≤0.0001). CONCLUSION: Eimeria spp. is detected in high prevalence among rabbit in Yogyakarta, Indonesia, with commonly occurs in mixed infections. In this paper, we describe Eimeria spp. that are circulating in Indonesia and present it as updated information to farmers and veterinarians. To the best of our knowledge, we provide the first information about rabbit coccidiosis in Indonesia.

Bovine coccidiosis cases of beef and dairy cattle in Indonesia.

The number of bovine coccidiosis cases is generally high and distributed worldwide. It can reach up to 100% in the early weeks of age in calves and has a corresponding impact on further cattle development and the economic outcome. For these reasons, this study was conducted to detect the presence of Eimeria spp. oocysts followed by morphologically species characterization in different breeds of beef and dairy cattle. These breeds were reared by rural farmers in 9 provinces in Indonesia from March to October 2017. A total of 2150 fecal samples were collected, along with farmer knowledge about coccidiosis through interviews. Results show that prevalence varied as indicated by the following regions: Riau (68.73%), South Sumatera (78.34%), Banten (63.73%), Special Region of Yogyakarta (54.69%), East Java (63.89%), Bali (83.34%), West Nusa Tenggara (78.43%), Central Kalimantan (72.46%), and South Sulawesi (85.07%). The overall prevalence of bovine coccidiosis in all provinces sampled was 72.07%. Eight species of Eimeria spp. were identified, namely, E. auburnensis (52.11%), E. bovis (81.89%), E. bukidnonensis (18.44%), E. canadensis (38.22%), E. cylindrica (31.67%), E. zuernii (60%), E. ellipsoidalis (10.11%), and E. alabamensis (11.67%). The highest prevalence was found in E. bovis compared with any other Eimerias (P ≤ .0001) followed by E. zuernii. Most of the interviewed farmers in this study did not know about coccidia, especially Eimeria spp. in cattle. They were more familiar with worm infections because of both their experience and knowledge. The distribution of cattle with ages <6 months, 6-18 months, and >18 months exhibited 97%, 81%, and 38% infection, respectively. Based on these results, the authors saw the necessity to inform farmers about bovine coccidiosis as well as the veterinarian regarding better preventive and curative treatments.

Besnoitia besnoiti infection alters both endogenous cholesterol de novo synthesis and exogenous LDL uptake in host endothelial cells.

Besnoitia besnoiti, an apicomplexan parasite of cattle being considered as emergent in Europe, replicates fast in host endothelial cells during acute infection and is in considerable need for energy, lipids and other building blocks for offspring formation. Apicomplexa are generally considered as defective in cholesterol synthesis and have to scavenge cholesterol from their host cells for successful replication. Therefore, we here analysed the influence of B. besnoiti on host cellular endogenous cholesterol synthesis and on sterol uptake from exogenous sources. GC-MS-based profiling of cholesterol-related sterols revealed enhanced cholesterol synthesis rates in B. besnoiti-infected cells. Accordingly, lovastatin and zaragozic acid treatments diminished tachyzoite production.  Moreover, increased lipid droplet contents and enhanced cholesterol esterification was detected and inhibition of the latter significantly blocked parasite proliferation. Furthermore, artificial increase of host cellular lipid droplet disposability boosted parasite proliferation. Interestingly, lectin-like oxidized low density lipoprotein receptor 1 expression was upregulated in infected endothelial hostcells, whilst low density lipoproteins (LDL) receptor was not affected by parasite infection. However, exogenous supplementations with non-modified and acetylated LDL both boosted B. besnoiti proliferation. Overall, current data show that B. besnoiti simultaneously exploits both, endogenous cholesterol biosynthesis and cholesterol uptake from exogenous sources, during asexual replication.

Larvicidal, adulticidal, and oviposition-deterrent activity of Piper betle L. essential oil to Aedes aegypti.

BACKGROUND AND AIM: Aedes aegypti is a primary vector of many arthropod-borne diseases. One of the diseases, dengue fever, is an endemic disease in Indonesia causing high mortalities for decades. There are no preventive and specific treatments for dengue so far. Therefore, prevention of this disease largely depends on the mosquito control. Since resistance to chemical insecticides occurred worldwide, the study on alternate and new mosquito insecticides are mandatory. This study aimed to demonstrate the effect of essential oil from P. betle L. in the larval and adult stages, as well as its influence on oviposition activity of A. aegypti mosquito. MATERIALS AND METHODS: P. betle efficacy was evaluated in various stages of A. aegypti development. For the larvicidal activity, larvae instar III stage was used. Adulticidal assay in this experiment was performed using newly emerged A. aegypti. For oviposition assay, mated A. aegypti was tested for their responses to P. betle-treated and non-treated ovitraps. RESULTS: P. betle L. - adulticide activity was effective with a concentration of 2.5 µl/ml, caused 100% mortality within 15-30 min. Larvicide activity was observed after 1 h, 24 h, and 48 h post-treatment with LC50183, 92.7, and 59.8 ppm and LC90 637, 525, and 434.7 ppm, respectively. Oviposition activity index was -0.917 in 1000 ppm. In addition, the eggs number of A. aegypti oviposition with 100 ppm of essential oil P. betle L. was 5 times lower than the control. CONCLUSION: This study demonstrated clearly that essential oil derived from P. betle L. potentially acts as alternate bioinsecticide to control A. aegypti population. The application can be varied or combined in different stages of mosquito development.

Chicken Coccidiosis in Central Java, Indonesia: A Recent Update.

Avian coccidiosis is a huge problem worldwide. Heavily infected animals that show severe clinical signs and coccidiostat resistance are causing important economic losses. The present study aimed to update the recent cases of coccidiosis in Central Java, Indonesia, and to show the importance of the disease in the region. A total of 699 samples were obtained from different chicken breed. Different Eimeria species were detected in 175 individuals (25.04%). Three different groups of chicken breed were considered: local chicken (autochthonous chickens of Sentul and Jawa), commercial broiler, and layer. Broiler chickens showed the highest prevalence of infection (34%), followed by layer (26.26%) and local chickens (10.45%). Mild to severe clinical signs of avian coccidiosis were observed in 42% of the infected animals, while 58% of the infected animals showed no clinical signs other than low feed conversion rates. Seven different Eimeria species were identified: E. tenella was the most prevalent (43.3%), followed by E. maxima (26.3%), E. necatrix (15.7%), E. acervulina (8%), E. praecox (3.1%), E. mitis (2.2%), and E. brunetti (1.3%). Coinfections with several Eimeria species were diagnosed. With this study we found massive usage of coccidiostat in the region even though its usage cannot guarantee coccidiosis-free chicken production.

Aedes aegypti resistance development to commonly used insecticides in Jakarta, Indonesia.

Aedes aegypti is the primary vector of various relevant arthropod-borne viral infectious diseases worldwide. The mosquito control is still mainly performed by using insecticides but their effectiveness is increasingly questioned nowadays. We here conducted a study on Ae. aegypti resistance development towards several commonly used insecticides in the capital city of Jakarta, Indonesia. In order to achieve this goal, Ae. aegypti eggs from Jakarta were collected with ovitraps and hatched in the insectary of the Gadjah Mada University, Indonesia. The F0 generations were used for WHO resistance tests and knockdown resistance (kdr) assays. Presented results clearly showed that there was resistance development of Ae. aegypti populations to the here tested pyrethroid insecticides (i. e. permethrin). Observed mortalities were less than 90% with highest resistance against 0.75% permethrin concentrations. Furthermore, a significant association of V1016G gene mutations with resistance phenotypes to 0.75% permethrin was observed. Nevertheless, the F1534C mutation did not show a significant correlation to resistance development. In conclusion, our results show that populations of Ae. aegypti mosquitoes within the city of Jakarta have developed resistance against several routinely used pyrethroid insecticides in local performed control programs. Thus, the regular verification/assessment of resistance development status will hopefully help in the future to assist local public health authorities in their mosquito control programs by recommending and managing the rotation of different routinely used insecticides with diverse effector mechanisms in order to delay Ae. aegypti resistance development.

Knockdown resistance (kdr) of the voltage-gated sodium channel gene of Aedes aegypti population in Denpasar, Bali, Indonesia.

BACKGROUND: Aedes aegypti is the main vector of several arthropod-borne viral infections in the tropics profoundly affecting humans, such as dengue fever (DF), West Nile (WN), chikungunya and more recently Zika. Eradication of Aedes still largely depends on insecticides, which is the most cost-effective strategy, and often inefficient due to resistance development in exposed Aedes populations. We here conducted a study of Ae. aegypti resistance towards several insecticides regularly used in the city of Denpasar, Bali, Indonesia. METHODS: Aedes aegypti egg samples were collected with ovitraps and thereafter hatched in the insectary of the Gadjah Mada University. The F0 generation was used for all bioassay-related experiments and knockdown resistance (kdr) assays. RESULTS: Results clearly showed resistance development of Ae. aegypti against tested insecticides. Mortalities of Ae. aegypti were less than 90% with highest resistance observed against 0.75% permethrin. Mosquitoes from the southern parts of Denpasar presented high level of resistance pattern in comparison to those from the western and northern parts of Denpasar. Kdr analysis of voltage-gated sodium channel (Vgsc) gene showed significant association to S989P and V1016G mutations linked to resistance phenotypes against 0.75% permethrin. Conversely, Ae. aegypti F1534C gene mutation did not result in any significant correlation to resistance development. CONCLUSIONS: Periodically surveillance of insecticide resistances in Ae. aegypti mosquitoes will help local public health authorities to set better goals and allow proper evaluation of on-going mosquito control strategies. Initial detection of insecticide resistance will contribute to conduct proper actions in delaying mosquito resistance development such as insecticide rotation or combination of compounds in order to prolong chemical efficacy in combating Ae. aegypti vectors in Indonesia.

Eimeria bovis infection modulates endothelial host cell cholesterol metabolism for successful replication.

During first merogony Eimeria bovis forms large macromeronts in endothelial host cells containing >120 000 merozoites I. During multiplication, large amounts of cholesterol are indispensable for the enormous offspring membrane production. Cholesterol auxotrophy was proven for other apicomplexan parasites. Consequently they scavenge cholesterol from their host cell apparently in a parasite-specific manner. We here analyzed the influence of E. bovis infection on endothelial host cell cholesterol metabolism and found considerable differences to other coccidian parasites. Overall, free cholesterol significantly accumulated in E. bovis infected host cells. Furthermore, a striking increase of lipid droplet formation was observed within immature macromeronts. Artificial host cell lipid droplet enrichment significantly improved E. bovis merozoite I production confirming the key role of lipid droplet contents for optimal parasite proliferation. The transcription of several genes being involved in both, cholesterol de novo biosynthesis and low density lipoprotein-(LDL) mediated uptake, was significantly up-regulated at a time in infected cells suggesting a simultaneous exploitation of these two cholesterol acquisition pathways. E. bovis scavenges LDL-derived cholesterol apparently through significantly increased levels of surface LDL receptor abundance and LDL binding to infected cells. Consequently, LDL supplementation significantly improved parasite replication. The up-regulation of the oxidized LDL receptor 1 furthermore identified this scavenger receptor as a key molecule in parasite-triggered LDL uptake. Moreover, cellular cholesterol processing was altered in infected cells as indicated by up-regulation of cholesterol-25-hydroxylase and sterol O-acyltransferase. Overall, these results show that E. bovis considerably exploits the host cell cholesterol metabolism to guarantee its massive intracellular growth and replication.

Differential inhibition of host cell cholesterol de novo biosynthesis and processing abrogates Eimeria bovis intracellular development.

Eimeria bovis macromeront formation in bovine endothelial host cells is an energy- and nutrient-demanding process. Obligate intracellular replicating coccidians are generally considered as auxotrophic for cholesterol synthesis and scavenge cholesterol from the host cell by either enhancing the uptake of extracellular cholesterol sources or by upregulating the host cellular de novo biosynthesis. We here focused on the latter mechanism and analyzed the effects of several inhibitors targeting the host cellular mevalonate biosynthesis pathway and cholesterol processing. The following inhibitors were used: lovastatin, squalestatin, CI976 and C75 targeting HMG-CoA reductase, squalene synthase, acyl-CoA:cholesterol acyltransferase, and fatty acid synthase, respectively. In summary, all inhibitors significantly interfered with E. bovis meront formation and merozoite production in a dose-dependent manner. Dose effect responses identified lovastatin as the most effective compound, followed by CI976, C75, and squalestatin, respectively. Overall, merozoite production was inhibited by 99.6, 99.7, 84.6, and 70.2% via lovastatin (1 µM), CI976, C75, and squalestatin (all 5 µM) treatments, respectively. Concerning macromeront formation, both the rate and size of developing meronts were affected by inhibitor treatments. The effects were characterized by developmental arrest and meront degradation. In the case of CI976 treatment, we additionally observed detrimental effects on host cellular lipid droplet formation leading to meront developmental arrest irrespective of the time point of treatment onset. These analyses clearly indicate that successful E. bovis intracellular development strictly depends on the host cellular de novo biosynthesis of cholesterol and on the adequate subsequent processing thereof.