Infections by gastrointestinal parasites are found in a variety of animals worldwide. For the diagnosis of such infections, the flotation method is commonly used to detect parasitic microorganisms, such as oocysts or eggs, in feces. Instead of adding a flotation solution after the final centrifugation step and using a cover slip to collect the parasites, the method using a wire loop for the recovery of the organisms has been reported as one of alternative methods. However, the recovery rates of microorganisms from the flotation method have not been analysed. In the present study, the utility of a flotation method with the use of a wire loop of 8 mm in diameter (the loop method) was evaluated using different numbers of E. tenella oocysts and Heterakis gallinarum eggs, and chicken fecal samples collected at the farms. Consequently, we found that the oocysts and eggs in tubes could be collected at a ratio of 2.00 to 3.08. Thus, our results indicate that the loop method is a simple and time saving method, implicating the application for the estimated OPG/ EPG (Oocysts/Eggs per gram) of the samples.

Malaria drug research and development efforts have resurged in the last decade following the decelerating rate of mortality and malaria cases in endemic regions. The inefficiency of malaria interventions is largely driven by the spreading resistance of the Plasmodium falciparum parasite to current drug regimens and that of the malaria vector, the Anopheles mosquito, to insecticides. In response to the new eradication agenda, drugs that act by breaking the malaria transmission cycle (transmission-blocking drugs), which has been recognized as an important and additional target for intervention, are being developed. These drugs take advantage of the susceptibility of Plasmodium during population bottlenecks before transmission (gametocytes) and in the mosquito vector (gametes, zygotes, ookinetes, oocysts, sporozoites). To date, compounds targeting stage V gametocytes predominate in the chemical library of transmission-blocking drugs, and some of them have entered clinical trials. The targeting of Plasmodium mosquito stages has recently renewed interest in the development of innovative malaria control tools, which hold promise for the application of compounds effective at these stages. In this review, we highlight the major achievements and provide an update on the research of transmission-blocking drugs, with a particular focus on their chemical scaffolds, antiplasmodial activity, and transmission-blocking potential.

Coccidiosis is one of the most prevalent diseases found in local rabbits (Oryctolagus cuniculus), which is caused by the Eimeria. The study aimed to more reliably identify Eimeria species (Eimeria magna) infecting Local Rabbits in Alkarg City, Saudi Arabia, based the method on the molecular properties and morphological and molecular biological techniques. Sub-spheroidal oocysts measuring 21-27 × 12-16 (24 × 14.4) μm (20 n) and with a length/width (L/W) ratio of 0.9-1.1 (1.0) were identified by microscopic analysis of a fecal sample. Oocysts feature a bi-layered wall that is 1.0-1.2 (1.1) μm thick. About two-thirds of the wall\'s thickness is made up of a smooth outer layer. A polar granule is present, but neither a micropyle nor an oocyst residuum is present. The ovoidal sporozoites measure 15-18 × 8-11 (16.5 × 9.5) μm, have an L/W ratio of 1.6-1.8 (1.7), and take up around 21% of the oocyst\'s total surface. The mean size of the sub-Stieda body is 1.4 × 2.3 μm, while the average size of the Stieda body is 0.9 × 1.8 μm. The para-Stieda body is lacking. Sporocyst residuum appears membrane-bound and has an uneven form made up of several granules. With two refractile bodies below the striations and pronounced striations at the more pointed end, sporozoites are vermiform, measuring an average of 11.6 × 4.0 μm. The results of the sequencing for the 18S rDNA gene confirmed the species of Eimeria parasites found in the host (rabbits). The current parasite species is closely related to the previously described and deposited E. magna and deeply embedded in the genus Eimeria (family Eimeriidae). According to the findings, single oocyst molecular identification of Eimeria may be accomplished through consistent use of the morphological and molecular results. It is possible to draw the conclusion that the current research supplies relevant facts that help assess the potential infection and future control measures against rabbit coccidiosis to reduce the financial losses that can be incurred by the rabbit industry in Saudi Arabia.

OBJECTIVE: To investigate the development and dynamic changes of cysts in the brain of mice following infection with different forms of Toxoplasma gondii, so as to provide insights into for toxoplasmosis prevention and control.
METHODS: ICR mice at ages of 6 to 8 weeks, each weighing 20 to 25 g, were intraperitoneally injected with tachyzoites of the T. gondii PRU strain at a dose of 1 × 105 tachyzoites per mouse, orally administered with cysts at a dose of 20 oocysts per mouse or oocysts at a dose of 200 oocysts per mouse for modeling chronic T. gondii infection in mice, and the clinical symptoms and survival of mice were observed post-infection. Mice were orally infected with T. gondii cysts at doses of 10 (low-dose group), 20 (medium-dose group), 40 cysts per mouse (high-dose group), and the effect of different doses of T. gondii infections on the number of cysts was examined in the mouse brain. Mice were orally administered with T. gondii cysts at a dose of 20 cysts per mouse, and grouped according to gender (female and male) and time points of infections (20, 30, 60, 90, 120, 150, 180 days post-infection), and the effects of gender and time points of infections on the number of cysts was examined in the mouse brain. In addition, mice were divided into the tachyzoite group (Group T), the first-generation cyst group (Group C1), the second-generation cyst group (Group C2), the third-generation cyst (Group C3) and the fourth-generation cyst group (Group C4). Mice in the Group T were intraperitoneally injected with T. gondii tachyzoites at a dose of 1 × 105 tachyzoites per mouse, and the cysts were collected from the mouse brain tissues 30 days post-infection, while mice in the Group C1 were orally infected with the collected cysts at a dose of 30 cysts per mouse. Continuous passage was performed by oral administration with cysts produced by the previous generation in mice, and the effect of continuous passage on the number of cysts was examined in the mouse brain.
RESULTS: Following infection with T. gondii tachyzoites, cysts and oocysts in mice, obvious clinical symptoms were observed on days 6 to 13 and mice frequently died on days 7 to 12. The survival rates of mice were 67.0%, 87.0% and 53.0%, and the mean numbers of cysts were (516.0 ± 257.2), (1 203.0 ± 502.0) and (581.0 ± 183.1) in the mouse brain (F = 11.94, P < 0.01) on day 30 post-infection with T. gondii tachyzoites, cysts and oocysts, respectively, and the numbers of cysts in the brain tissues were significantly lower in mice infected with T. gondii tachyzoites and oocysts than in those infected with cysts (all P values < 0.01). The survival rates of mice were 87.0%, 87.0% and 60.0%, and the mean numbers of cysts were (953.0 ± 355.5), (1 084.0 ± 474.3) and (1 113.0 ± 546.0) in the mouse brain in the low-, medium- and high-dose groups on day 30 post-infection, respectively (F = 0.42, P > 0.05). The survival rates of male and female mice were 73.0% and 80.0%, and the mean numbers of cysts were (946.4 ± 411.4) and (932.1 ± 322.4) in the brain tissues of male and female mice, respectively (F = 1.63, P > 0.05). Following continuous passage, the mean numbers of cysts were (516.0 ± 257.2), (1 203.0 ± 502.0), (896.8 ± 332.3), (782.5 ± 423.9) and (829.2 ± 306.0) in the brain tissues of mice in the T, C1, C2, C3 and C4 groups, respectively (F = 4.82, P < 0.01), and the number of cysts was higher in the mouse brain in Group 1 than in Group T (P < 0.01). Following oral administration of 20 T. gondii cysts in mice, cysts were found in the moues brain for the first time on day 20 post-infection, and the number of cysts gradually increased over time, peaked on days 30 and 90 post-infection and then gradually decreased; however, the cysts were still found in the mouse brain on day 180 post-infection.
CONCLUSIONS: There is a higher possibility of developing chronic T. gondii infection in mice following infection with cysts than with oocysts or tachyzoites and the most severe chronic infection is seen following infection with cysts. The number of cysts does not correlate with the severity of chronic T. gondii infection, and the number of cysts peaks in the mouse brain on days 30 and 90 post-infection.
［摘要］ 目的 观察不同形态刚地弓形虫感染后小鼠脑内包囊形成及其动态变化, 为弓形虫病防控提供依据。方法 取 6~8周龄ICR小鼠 (20~25 g) 建立慢性弓形虫感染模型, 其中弓形虫PRU株速殖子按1 × 105个/只剂量腹腔注射感染小鼠, 包囊和卵囊分别按20、200个/只剂量通过灌胃针口服感染小鼠, 观察感染后小鼠临床症状和存活情况。分别以10 (低剂 量组) 、20 (中剂量组) 、40个包囊/只 (高剂量组) 剂量感染小鼠, 观察弓形虫不同感染剂量对小鼠脑内包囊数量的影响。 将小鼠按性别 (雌、雄性) 、感染时间 (感染后20、30、60、90、120、150、180 d) 分组, 按20个/只剂量口服弓形虫包囊后, 分别 观察性别和感染时间对小鼠脑内包囊数量的影响。将小鼠分成速殖子组 (T组) 、包囊1代组 (C1组) 、包囊2代组 (C2 组) 、包囊3代组 (C3组) 、包囊4代组 (C4组); T组小鼠按1 × 105个/只剂量腹腔注射弓形虫速殖子, 感染后第30天处死小 鼠并收集其脑组织内包囊, 再按20个/只感染C1组小鼠。此后每一代小鼠均采用上一代所产生包囊进行口服连续传代, 观察连续传代对小鼠脑内弓形虫包囊数量的影响。结果 以弓形虫速殖子、包囊、卵囊分别感染小鼠, 感染第6~13天 小鼠出现明显临床症状、感染第 7~12 天小鼠出现集中死亡。感染第 30 天时, 感染速殖子、包囊、卵囊的小鼠存活率分 别为67.0%、87.0%、53.0%, 平均脑内包囊数量分别为 (516.0 ± 257.2) 、 (1 203.0 ± 502.0) 、 (581.0 ± 183.1) 个, 差异有统计 学意义 (F = 11.94, P < 0.01), 感染速殖子、卵囊的小鼠脑内包囊数低于感染包囊的小鼠 (P 均< 0.01) 。感染后第30天, 低、中、高剂量组小鼠存活率分别为87.0%、87.0%、60.0%, 平均脑内包囊数量分别为 (953.0 ± 355.5) 、 (1 084.0 ± 474.3) 、 (1 113.0 ± 546.0) 个, 差异无统计学意义 (F = 0.42, P > 0.05); 雄、雌性组小鼠存活率分别为73.0%和80.0%, 平均脑内包 囊数量分别为 (946.4 ± 411.4) 、 (932.1 ± 322.4) 个, 差异无统计学意义 (F = 1.63, P > 0.05) 。通过连续传代感染后, T、C1、 C2、C3、C4组小鼠平均脑内包囊数量分别为 (516.0 ± 257.2) 、 (1 203.0 ± 502.0) 、 (896.8 ± 332.3) 、 (782.5 ± 423.9) 、 (829.2 ± 306.0) 个, 差异有统计学意义 (F = 4.82, P < 0.01); C1组小鼠脑内包囊数高于速殖子组, 差异有统计学意义 (P < 0.01) 。 小鼠口服20个包囊后, 感染第20天首次查见脑内弓形虫包囊, 随感染时间延长脑内包囊数量逐渐增加; 至感染第30、90 天时, 脑内包囊数量分别达峰值, 此后逐步下降, 至感染第180天时仍能查见脑内包囊。结论 刚地弓形虫包囊较速殖 子、卵囊感染后形成慢性感染的可能性更高, 且慢性感染程度亦最严重; 感染弓形虫包囊数量与慢性感染严重程度无关; 脑内包囊形成数量于感染第30天和90天时达高峰。.

Toxoplasma gondii is a food-borne zoonotic parasite widespread in a variety of hosts, including humans. With a majority of infections in Europe estimated to be meat-borne, pork, as one of the most consumed meats worldwide, represents a potential risk for consumers. Therefore, we aimed to investigate the progress of T. gondii infection and tissue tropism in experimentally infected pigs, using different T. gondii isolates and infectious stages, i.e. tissue cysts or oocysts. Twenty-four pigs were allocated to treatment in four groups of six, with each group inoculated orally with an estimated low dose of either 400 oocysts or 10 tissue cysts of two European T. gondii isolates, a type II and a type III isolate. The majority of pigs seroconverted two weeks post-inoculation. Pigs infected with the type III isolate had significantly higher levels of anti-T. gondii antibodies compared to those infected with the type II isolate. Histopathological exams revealed reactive hyperplasia of the lymphatic tissue of all pigs. Additionally, a selected set of nine tissues was collected during necropsy at 50 dpi from each of the remaining 22 pigs for T. gondii DNA detection by quantitative real-time PCR. A positive result was obtained in 29.8 % (59/139) of tested tissues. The brain was identified as the most frequently positive tissue in 63.6 % (14/22) of the animals. In contrast, liver samples tested negative in all animals. The highest mean parasite load, calculated by interpolating the average Cq values on the standard curve made of ten-fold serial dilutions of the genomic DNA, corresponding to 100 to 104 tachyzoites/µL, was observed in shoulder musculature with an estimated concentration of 84.4 [0.0-442.5] parasites per gram of tissue. The study highlights the variability in clinical signs and tissue distribution of T. gondii in pigs based on the combination of parasite stages and strains, with type III isolates, particularly oocysts, causing a stronger antibody response and higher tissue parasite burden. These findings suggest the need for further investigation of type III isolates to better understand their potential risks to humans.

Background: The aim of the present study was to describe the presence of co-infection by Toxoplasma gondii and Neospora caninum in goats reared in extensive systems from Mexico. Materials and Methods: A cross-sectional study was conducted to determine the frequency of T. gondii and N. caninum, by detecting antibodies to each parasite by mean commercial ELISA kits. A total of 176 blood samples were randomly collected from mature females reared in extensive system herds from 20 municipalities of state of Guanajuato, Mexico. Results: The general seroprevalence was 23.9 and 21.0% for T. gondii and N. caninum, respectively, while co-infection rate was 3.6%. For geographic and environmental variables, no differences were observed among T. gondii and coinfection; however, it was observed that altitude, annual precipitation, annual average temperature, and rainy period showed significant differences with N. caninum seropositive goats. Conclusion: The seroprevalence of both parasites was appreciated in most of the studied herds. The present study is the first report of T. gondii and N. caninum co-infection in goats from extensive herds in Mexico.

Multi-protein complexes are crucial for various essential biological processes of the malaria parasite Plasmodium, such as protein synthesis, host cell invasion and adhesion. Especially during the sexual phase of the parasite, which takes place in the midgut of the mosquito vector, protein complexes are required for fertilization, sporulation and ultimately for the successful transmission of the parasite. Among the most noticeable protein complexes of the transmission stages are the ones formed by the LCCL domain-containing protein family that play critical roles in the generation of infective sporozoites. The six members of this protein family are characterized by numerous adhesive modules and domains typically found in secreted proteins. This review summarizes the findings of expression and functional studies on the LCCL domain-containing proteins of the human pathogenic P. falciparum and the rodent-infecting P. berghei and discusses the common features and differences of the homologous proteins.

Plasmodium sporozoites are the highly motile and invasive forms of the malaria parasite transmitted by mosquitoes. Sporozoites form within oocysts at the midgut wall of the mosquito, egress from oocysts and enter salivary glands prior to transmission. The GPI-anchored major surface protein, the circumsporozoite protein (CSP) is important for Plasmodium sporozoite formation, egress, migration and invasion. To visualize CSP, we previously generated full-length versions of CSP internally tagged with the green fluorescent protein, GFP. However, while these allowed for imaging of sporogony in oocysts, sporozoites failed to egress. Here, we explore different strategies to overcome this block in egress and obtain salivary gland resident sporozoites that express CSP-GFP. Replacing the N-terminal and repeat region with GFP did not allow sporozoite formation. Lowering expression of CSP-GFP at the endogenous locus allowed sporozoite formation but did not overcome egress block. Crossing of CSP-GFP expressing parasites that are blocked in egress with wild-type parasites yielded a small fraction of parasites that entered salivary glands and expressed various levels of CSP-GFP. Expressing CSP-GFP constructs from a silent chromosome region from promoters that are active only post salivary gland invasion yielded normal numbers of fluorescent salivary gland sporozoites, albeit with low levels of fluorescence. We also show that lowering CSP expression by 50% allowed egress from oocysts but not salivary gland entry. In conclusion, Plasmodium berghei parasites with normal CSP expression tolerate a certain level of CSP-GFP without disruption of oocyst egress and salivary gland invasion.

Cryptosporidium is a primary cause of waterborne epidemics, despite being previously considered only an opportunistic pathogen. The disease is associated with significant economic losses in humans and animals that are brought on by diarrhea, which frequently causes dehydration. Contact with diseased people or animals, as well as polluted water, is the major cause of infection. Different drugs are used to control the parasites. Nitazoxanide (NTZ), which is an anti-protozoan and anti-viral drug, can be used to control helminths, viruses, and protozoan parasites as a broad-spectrum antibiotic and has been approved by the food and drug authority (FDA). However, the problem is the development of resistance over a period of time in these parasites. Nanoparticles have received significant attention as possible anti-parasitic agents in recent years. By directing medications to specific cellular locations, targeted drug delivery minimizes the side effects of medications. Nanoparticles have demonstrated effectiveness against different Cryptosporidium species. Nanoparticles loaded with NTZ are found to be an effective remedy for C. parvum in young ones and decrease the oocyst count shed in the stools. Additionally, silver nanoparticles have proven to be effective against C. parvum by releasing silver ions that breach the cell wall of the oocyst, causing the escape of intracellular contents and the destruction of sporozoites within the oocyst. Implementing tiny particles for the purification of consuming water from Cryptosporidium is an economical and environmentally sustainable process. However, the use of nanoparticles in medicine requires more research.

The oocyst is a sporogonic stage of Plasmodium development that takes place in the mosquito midgut in about 2 weeks. The cyst is protected by a capsule of unknown composition, and little is known about oocyst biology. We carried out a proteomic analysis of oocyst samples isolated at early, mid, and late time points of development. Four biological replicates for each time point were analyzed, and almost 600 oocyst-specific candidates were identified. The analysis revealed that, in young oocysts, there is a strong activity of protein and DNA synthesis, whereas in mature oocysts, proteins involved in oocyst and sporozoite development, gliding motility, and invasion are mostly abundant. Among the proteins identified at early stages, 17 candidates are specific to young oocysts. Thirty-four candidates are common to oocyst and the merosome stages (sporozoite proteins excluded), sharing common features as replication and egress. Western blot and immunofluorescence analyses of selected candidates confirm the expression profile obtained by proteomic analysis.