GRA47 is important for the morphology and permeability of the parasitophorous vacuole in Toxoplasma gondii.

Establishing an intact intracellular parasitophorous vacuole (PV) that enables efficient nutrient uptake and protein trafficking is essential for the survival and proliferation of Toxoplasma gondii. Although the PV membrane (PVM)-localized dense granule protein 17 (GRA17) and GRA23 mediate the permeability of the PVM to small molecules, including nutrient uptake and excretion of metabolic by-products, the molecular mechanism by which T. gondii acquires nutrients remains unclear. In this study, we showed that the secreted protein GRA47 contributed to normal PV morphology, PVM permeability to small molecules, growth, and virulence in T. gondii. Co-immunoprecipitation analysis demonstrated potential interaction of GRA47 with GRA72, and the loss of GRA72 affected PV morphology, parasite growth and infectivity. To investigate the biological relationship among GRA47, GRA72, GRA17 and GRA23, attempts were made to construct strains with double gene deletion and overexpressing strains. Only Δgra23Δgra72 was successfully constructed. This strain exhibited a significant increase in the proportion of aberrant PVs compared with the Δgra23 strain. Overexpressing one of the three related GRAs partially rescued PVs with aberrant morphology in Δgra47, Δgra72 and Δgra17, while the expression of the Plasmodium falciparum PVM protein PfExp2, an ortholog of GRA17 and GRA23, fully rescued the PV morphological defect in all three Δgra strains. These results suggest that these GRA proteins may not be functionally redundant but rather work in different ways to regulate nutrient acquisition. These findings highlight the versatility of the nutrient uptake mechanisms in T. gondii, which may contribute to the parasite's remarkable ability to grow in different cellular niches in a very broad range of hosts.

Prevalence and Genetic Characterization of Giardia duodenalis and Blastocystis spp. in Black Goats in Shanxi Province, North China: From a Public Health Perspective.

Blastocystis spp. and Giardia duodenalis are two prevalent zoonotic intestinal parasites that can cause severe diarrhea and intestinal diseases in humans and many animals. Black goat (Capra hircus) farming is increasingly important in China due to the remarkable adaptability, high reproductive performance, rapid growth rate, and significant economic value of black goats. A number of studies have indicated that black goats are the potential reservoir of multiple zoonotic protozoans in China; however, the prevalence and zoonotic status of G. duodenalis and Blastocystis spp. in black goats in Shanxi Province is still unknown. Thus, a total of 1200 fecal samples of black goats were collected from several representative regions at different altitudes in Shanxi Province and were examined for the presence and genotypes of G. duodenallis and Blastocystis spp. by amplifying the beta-giardin (bg), glutamate dehydrogenase (gdh), and triosephosphate isomerase (tpi) loci of G. duodenalis and SSU rRNA of Blastocystis spp. using PCR and sequence analysis methods, respectively. The overall prevalence of G. duodenalis and Blastocystis spp. in black goats in Shanxi Province were 7.5% and 3.5%, respectively. Two assemblages (B and E) of G. duodenalis and four subtypes (ST5, ST10, ST14, and ST30) of Blastocystis spp. were identified, with assemblage E and ST10 as the prevalent genotype and subtype in black goats, respectively. One novel multilocus genotype (MLG) was identified in MLG-E and was designated as MLG-E12. For both G. duodenalis and Blastocystis spp., the prevalence was significantly related to the region and age groups (p < 0.05). This is the first report on the prevalence of G. duodenalis and Blastocystis spp. in black goats in Shanxi Province. These results not only provide baseline data for the prevention and control of both parasites in black goats in Shanxi Province, but also enhance our understanding of the genetic composition and zoonotic potential of these two parasites.

Proteomic change in the upper lobe of the left lung of Beagle dogs at the lung migration stage of Toxocara canis infection.

BACKGROUND: Toxocara canis is considered one of the most neglected parasitic zoonoses and threatens the health of millions of people worldwide with a predilection for pediatric and adolescent populations in impoverished communities. Exploring the invasion and developmental mechanisms associated with T. canis infection in its definitive canine hosts will help to better control zoonotic toxocariasis. METHODS: Proteomic changes in samples from the upper lobe of the left lung of Beagle puppies were systematically analyzed by quantitative proteomic technology of data-independent acquisition (DIA) at 96 h post-infection (hpi) with T. canis. Proteins with P-values < 0.05 and fold change > 1.5 or < 0.67 were considered proteins with differential abundance (PDAs). RESULTS: A total of 28 downregulated PDAs and 407 upregulated PDAs were identified at 96 hpi, including RhoC, TM4SFs and LPCAT1, which could be associated with the maintenance and repair of lung homeostasis. GO annotation and KEGG pathway enrichment analyses of all identified proteins and PDAs revealed that many lung proteins have correlation to signal transduction, lipid metabolism and immune system. CONCLUSIONS: The present study revealed lung proteomic alterations in Beagle dogs at the lung migration stage of T. canis infection and identified many PDAs of Beagle dog lung, which may play important roles in the pathogenesis of toxocariasis, warranting further experimental validation.

Molecular Identification and Survey of Trichomonad Species in Pigs in Shanxi Province, North China.

Several trichomonad species have already been identified in pigs, and their pathogenic potential may not be ruled out. To date, however, no information is available regarding the prevalence of trichomonads in pigs in Shanxi Province, North China. In the present study, a total of 362 fecal samples collected from pigs in three representative counties (Qi, Jishan, and Shanyin) in this province were examined for Tetratrichomonas buttreyi, Tritrichomonas foetus, and Pentatrichomonas hominis using a nested polymerase chain reaction (PCR) with primers targeting the small subunit ribosomal RNA (SSU rRNA) gene. The overall prevalence of T. buttreyi was 49.72%, and region and age were found to be significantly associated with T. buttreyi infection, respectively. Only one pig fecal sample from Qi County was found to be positive for T. foetus, and all samples were negative for P. hominis. Molecular evolutionary analysis revealed that some T. buttreyi isolates showed complete genetic identity with those reported previously, and some T. buttreyi isolates and one T. foetus isolate showed minor allelic variations compared with those reported previously. This is the report of the molecular epidemiology of T. foetus and T. buttreyi in pigs in Shanxi Province, North China. These findings not only enrich the knowledge on the distribution of these trichomonad species in pigs in China but also provide baseline information for planning future research and control strategies.

Trx4, a novel thioredoxin protein, is important for Toxoplasma gondii fitness.

BACKGROUND: To successfully replicate within the host cell, Toxoplasma gondii employs several mechanisms to overcome the host cell defenses and mitigate the harmful effects of the free radicals resulting from its own metabolic processes using effectors such as thioredoxin proteins. In this study, we characterize the location and functions of a newly identified thioredoxin in T. gondii, which was named Trx4. METHODS: We characterized the functional role of Trx4 in T. gondii Type I RH and Type II Pru strains by gene knockout and studied its subcellular localization by endogenous protein HA tagging using CRISPR-Cas9 gene editing. The enzyme-catalyzed proximity labeling technique, the TurboID system, was employed to identify the proteins in proximity to Trx4. RESULTS: Trx4 was identified as a dense granule protein of T. gondii predominantly expressed in the parasitophorous vacuole (PV) and was partially co-localized with GRA1 and GRA5. Functional analysis showed that deletion of trx4 markedly influenced the parasite lytic cycle, resulting in impaired host cell invasion capacity in both RH and Pru strains. Mutation of Trx domains in Trx4 in RH strain revealed that two Trx domains were important for the parasite invasion. By utilizing the TurboID system to biotinylate proteins in proximity to Trx4, we identified a substantial number of proteins, some of which are novel, and others are previously characterized, predominantly distributed in the dense granules. In addition, we uncovered three novel proteins co-localized with Trx4. Intriguingly, deletion of trx4 did not affect the localization of these three proteins. Finally, a virulence assay demonstrated that knockout of trx4 resulted in a significant attenuation of virulence and a significant reduction in brain cyst loads in mice. CONCLUSIONS: Trx4 plays an important role in T. gondii invasion and virulence in Type I RH strain and Type II Pru strain. Combining the TurboID system with CRISPR-Cas9 technique revealed many PV-localized proximity proteins associated with Trx4. These findings suggest a versatile role of Trx4 in mediating the processes that occur in this distinctive intracellular membrane-bound vacuolar compartment.

Seroprevalence and molecular detection of Toxoplasma gondii and Neospora caninum in beef cattle and goats in Hunan province, China.

BACKGROUND: Toxoplasma gondii and Neospora caninum are closely related protozoan parasites that are considered important causes of abortion in livestock, causing huge economic losses. Hunan Province ranks 12th in the production of beef and mutton in China. However, limited data are available on the seroprevalence, risk factors and molecular characterization of T. gondii and N. caninum in beef cattle and goats in Hunan province, China. METHODS: Sera of 985 beef cattle and 1147 goats were examined for the presence of specific antibodies against T. gondii using indirect hemagglutination test (IHAT) and anti-N. caninum IgG using competitive-inhibition enzyme-linked immunoassay assay (cELISA). Statistical analysis of possible risk factors was performed using PASW Statistics. Muscle samples of 160 beef cattle and 160 goats were examined for the presence of T. gondii DNA (B1 gene) and N. caninum DNA (Nc-5 gene) by nested PCR. The B1 gene-positive samples were genotyped at 10 genetic markers using the multilocus nested PCR-RFLP (Mn-PCR-RFLP). RESULTS: Specific IgG against T. gondii were detected in 8.3% (82/985) and 13.3% (153/1147) and against N. caninum in 2.1% (21/985) and 2.0% (23/1147) of the beef cattle and goats, respectively. Based on statistical analysis, the presence of cats, semi-intensive management mode and gender were identified as significant risk factors for T. gondii infection in beef cattle. Age was a significant risk factor for T. gondii infection in goats (P < 0.05), and age > 3 years was a significant risk factor for N. caninum infection in beef cattle (P < 0.05). PCR positivity for T. gondii was observed in three beef samples (1.9%; 3/160) and seven chevon samples (4.4%; 7/160). Genotyping of PCR positive samples identified one to be ToxoDB#10. The N. caninum DNA was observed in one beef sample (0.6%; 1/160) but was negative in all chevon samples. CONCLUSIONS: To our knowledge, this is the first large-scale serological and molecular investigation of T. gondii and N. caninum and assessment of related risk factors in beef cattle and goats in Hunan Province, China. The findings provide baseline data for executing prevention and control of these two important parasites in beef cattle and goats in China.

The Toxoplasma monocarboxylate transporters are involved in the metabolism within the apicoplast and are linked to parasite survival.

The apicoplast is a four-membrane plastid found in the apicomplexans, which harbors biosynthesis and organelle housekeeping activities in the matrix. However, the mechanism driving the flux of metabolites, in and out, remains unknown. Here, we used TurboID and genome engineering to identify apicoplast transporters in Toxoplasma gondii. Among the many novel transporters, we show that one pair of apicomplexan monocarboxylate transporters (AMTs) appears to have evolved from a putative host cell that engulfed a red alga. Protein depletion showed that AMT1 and AMT2 are critical for parasite growth. Metabolite analyses supported the notion that AMT1 and AMT2 are associated with biosynthesis of isoprenoids and fatty acids. However, stronger phenotypic defects were observed for AMT2, including in the inability to establish T. gondii parasite virulence in mice. This study clarifies, significantly, the mystery of apicoplast transporter composition and reveals the importance of the pair of AMTs in maintaining the apicoplast activity in apicomplexans.

Immunization with Live-Attenuated RHΔhad2a Strain Confers Partial Protective Immunity against Acute and Chronic Infection of Toxoplasma gondii in Mice.

Toxoplasmosis caused by Toxoplasma gondii is an important zoonosis of human and animal health significance. Current chemical therapeutics have side effects, and no commercially available vaccine is licensed for the prevention of toxoplasmosis in humans and most animals. Developing a safe and effective vaccine with long-term protection against T. gondii infection is necessary to control toxoplasmosis. HAD2a is a key member of the haloacid dehalogenase (HAD) phosphatase family, which is essential for T. gondii daughter budding. However, the role of HAD2a in T. gondii virulence remains unknown. In this study, we successfully constructed the had2a gene knockout strain in the T. gondii-type I RH strain (RHΔhad2a) and determined its role in virulence and vaccination. These results demonstrate that HAD2a played an important role in parasite daughter budding and in vitro replication. Knockout of the had2a gene attenuated the virulence of the T. gondii-type I RH strain. Vaccination with RHΔhad2a tachyzoites induced a Th1-biased immune response, provided partial protection against acute T. gondii infection in mice by highly virulent tachyzoites of RH and PYS (ToxoDB#9, Chinese I) strains, and conferred strong protection against challenge infection by cysts and oocysts of the less virulent type II Pru strain. These results demonstrate that T. gondii had2a is important for its in vitro proliferation and virulence in mice and that RHΔhad2a may be used as a candidate strain to generate a multiple gene knockout live-attenuated strain or be collaboratively applied with other live-attenuated strains to confer more effective protection against T. gondii infection.

Expression profiles of host miRNAs and circRNAs and ceRNA network during Toxoplasma gondii lytic cycle.

Toxoplasma gondii is an opportunistic protozoan parasite that is highly prevalent in the human population and can lead to adverse health consequences in immunocompromised patients and pregnant women. Noncoding RNAs, such as microRNAs (miRNAs) and circular RNAs (circRNAs), play important regulatory roles in the pathogenesis of many infections. However, the differentially expressed (DE) miRNAs and circRNAs implicated in the host cell response during the lytic cycle of T. gondii are unknown. In this study, we profiled the expression of miRNAs and circRNAs in human foreskin fibroblasts (HFFs) at different time points after T. gondii infection using RNA sequencing (RNA-seq). We identified a total of 7, 7, 27, 45, 70, 148, 203, and 217 DEmiRNAs and 276, 355, 782, 1863, 1738, 6336, 1229, and 1680 DEcircRNAs at 1.5, 3, 6, 9, 12, 24, 36, and 48 h post infection (hpi), respectively. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyses revealed that the DE transcripts were enriched in immune response, apoptosis, signal transduction, and metabolism-related pathways. These findings provide new insight into the involvement of miRNAs and circRNAs in the host response to T. gondii infection.

Prevalence and genotype/subtype distribution of Enterocytozoon bieneusi and Blastocystis in donkeys in Shanxi Province, north China.

Enterocytozoon bieneusi and Blastocystis may cause diarrhea in humans and various animals. However, little information is available regarding the prevalence and genetic diversity of E. bieneusi and Blastocystis in donkeys. To fill this gap, we molecularly assessed E. bieneusi and Blastocystis in fecal samples from donkeys (n = 815) in Shanxi Province, north China. The overall prevalence of E. bieneusi and Blastocystis in donkeys was 8.1% and 0.2%, respectively. Region and age were risk factors associated with E. bieneusi infection in donkeys. Three internal transcribed spacer (ITS) genotypes of E. bieneusi were identified in the current study, including two previously described genotypes (D and Henan-IV) and one novel genotype (named SXD1). Of which, genotype D was found to be the most prevalent. Phylogenetic analysis demonstrated that the three genotypes belonged to group 1, implying a potential of zoonotic transmission. Multilocus sequence typing showed that 19, 15, 13, and 22 types were identified at the loci MS1, MS3, MS4, and MS7, respectively, forming six multilocus genotypes (MLGs) distributed in the genotype D. One Blastocystis subtype (ST33) was identified, which has previously been reported only in horses. This is the first molecular-based description of E. bieneusi and Blastocystis infections in donkeys in Shanxi Province, north China, contributing to a better understanding of transmission dynamics and molecular epidemiological characteristics of the two intestinal protozoa.

Research Note: Preliminary functional analysis of EGF-like domains of Eimeria tenella microneme protein 7 identified in sporozoites and merozoites.

Eimeria tenella, an obligate intracellular apicomplexan parasite, is the major causative agent of chicken coccidiosis. Some epidermal growth factor (EGF)-like domain-containing proteins of other members of apicomplexan parasites have been reported to contribute to parasite survival. To date, however, EGF-like domain-containing proteins of E. tenella are not well studied. In this study, a gene fragment that encodes 4 EGF-like domains of E. tenella microneme protein 7 (EGF-EtMIC7) was amplified and expressed using an Escherichia coli expression system. Following generation of polyclonal antibodies that recognize recombinant EGF-EtMIC7 (rEGF-EtMIC7), the expression of EtMIC7 in sporozoites and merozoites was examined. Moreover, its roles in cellular regulation were investigated. The native EtMIC7 in E. tenella sporozoites and merozoites was detected by using Western blot and indirect immunofluorescence assays. rEGF-EtMIC7 could activate Akt, whereas blockade of EGF receptor (EGFR) failed to induce Akt phosphorylation. Compared with the control group, LMH cells treated with rEGF-EtMIC7 showed increased cell proliferation and expressed higher levels of B cell leukemia/lymphoma 2 (BCL-2). These findings contribute to the better understanding of parasite-host interactions at the molecular level during E. tenella infection.

The transcription factor AP2XI-2 is a key negative regulator of Toxoplasma gondii merogony.

Sexual development in Toxoplasma gondii is a multistep process that culminates in the production of oocysts, constituting approximately 50% of human infections. However, the molecular mechanisms governing sexual commitment in this parasite remain poorly understood. Here, we demonstrate that the transcription factors AP2XI-2 and AP2XII-1 act as negative regulators, suppressing merozoite-primed pre-sexual commitment during asexual development. Depletion of AP2XI-2 in type II Pru strain induces merogony and production of mature merozoites in an alkaline medium but not in a neutral medium. In contrast, AP2XII-1-depleted Pru strain undergoes several rounds of merogony and produces merozoites in a neutral medium, with more pronounced effects observed under alkaline conditions. Additionally, we identified two additional AP2XI-2-interacting proteins involved in repressing merozoite programming. These findings underscore the intricate regulation of pre-sexual commitment by a network of factors and suggest that AP2XI-2 or AP2XII-1-depleted Pru parasites can serve as a model for studying merogony in vitro.

A newly characterized dense granule protein (GRA76) is important for the growth and virulence of Toxoplasma gondii.

Pathogenicity of the zoonotic pathogen Toxoplasma gondii largely depends on the secretion of effector proteins into the extracellular milieu and host cell cytosol, including the dense granule proteins (GRAs). The protein-encoding gene TGME49_299780 was previously identified as a contributor to parasite fitness. However, its involvement in parasite growth, virulence and infectivity in vitro and in vivo remains unknown. Here, we comprehensively examined the role of this new protein, termed GRA76, in parasite pathogenicity. Subcellular localization revealed high expression of GRA76 in tachyzoites inside the parasitophorous vacuole (PV). However, its expression was significantly decreased in bradyzoites. A CRISPR-Cas9 approach was used to knock out the gra76 gene in the T. gondii type I RH strain and type II Pru strain. The in vitro plaque assays and intracellular replication showed the involvement of GRA76 in replication of RH and Pru strains. Deletion of the gra76 gene significantly decreased parasite virulence, and reduced the brain cyst burden in mice. Using RNA sequencing, we detected a significant increase in the expression of bradyzoite-associated genes such as BAG1 and LDH2 in the PruΔgra76 strain compared with the wild-type Pru strain. Using an in vitro bradyzoite differentiation assay, we showed that loss of GRA76 significantly increased the propensity for parasites to form bradyzoites. Immunization with PruΔgra76 conferred partial protection against acute and chronic infection in mice. These findings show the important role of GRA76 in the pathogenesis of T. gondii and highlight the potential of PruΔgra76 as a candidate for a live-attenuated vaccine.

Black-box attacks on dynamic graphs via adversarial topology perturbations.

Research and analysis of attacks on dynamic graph is beneficial for information systems to investigate vulnerabilities and strength abilities in resisting malicious attacks. Existing attacks on dynamic graphs mainly focus on rewiring original graph structures, which are often infeasible in real-world scenarios. To address this issue, we adopt a novel strategy by injecting both fake nodes and links to attack dynamic graphs. Based on that, we present the first study on attacking dynamic graphs via adversarial topology perturbations in a restricted black-box setting, in which downstream graph learning tasks are unknown. Specifically, we first divide dynamic graph structure perturbations into three sub-tasks and transform them as a sequential decision making process. Then, we propose a hierarchical reinforcement learning based black-box attack (HRBBA) framework to model three sub-tasks as attack policies. In addition, an imperceptible perturbation constraint to guarantee the concealment of attacks is incorporated into HRBBA. Finally, HRBBA is optimized based on the actor-critic process. Extensive experiments on four real-world dynamic graphs show that the performance of diverse dynamic graph learning methods (victim methods) on tasks like link prediction, node classification and network clustering can be substantially degraded under HRBBA attack.

The splicing factor SR2 is an important virulence factor of Toxoplasma gondii.

Serine/arginine-rich (SR) proteins are key factors with important roles in constitutive and alternative splicing (AS) of pre-mRNAs. However, the role of SR splicing factors in the pathogenicity of T. gondii remains largely unexplored. Here, we investigated the role of splicing factor SR2, a homolog of Plasmodium falciparum SR1, in the pathogenicity of T. gondii. We functionally characterized the predicted SR2 in T. gondii by gene knockout and studied its subcellular localization by endogenous protein HA tagging using CRISPR-Cas9 gene editing. The results showed that SR2 was localized in the nucleus and expressed in the tachyzoite and bradyzoite stages. In vitro studies including plaque formation, invasion, intracellular replication, egress and bradyzoite differentiation assays showed that deletion of SR2 in type I RH strain and type II Pru strains had no significant effect on the parasite growth and bradyzoite differentiation (p > 0.05). Interestingly, the disruption of SR2 in RH type I (p < 0.0001) and Pru type II (p < 0.05) strains resulted in varying degrees of attenuated virulence. In addition, disruption of SR2 in type II Pru strain significantly reduced brain cyst burden by ~80% (p < 0.0001). Collectively, these results suggest that splicing factor SR2 is important for the pathogenicity of T. gondii, providing a new target for the control and treatment of toxoplasmosis.

The Toxoplasma protein phosphatase 6 catalytic subunit (TgPP6C) is essential for cell cycle progression and virulence.

Protein phosphatases are post-translational regulators of Toxoplasma gondii proliferation, tachyzoite-bradyzoite differentiation and pathogenesis. Here, we identify the putative protein phosphatase 6 (TgPP6) subunits of T. gondii and elucidate their role in the parasite lytic cycle. The putative catalytic subunit TgPP6C and regulatory subunit TgPP6R likely form a complex whereas the predicted structural subunit TgPP6S, with low homology to the human PP6 structural subunit, does not coassemble with TgPP6C and TgPP6R. Functional studies showed that TgPP6C and TgPP6R are essential for parasite growth and replication. The ablation of TgPP6C significantly reduced the synchronous division of the parasite's daughter cells during endodyogeny, resulting in disordered rosettes. Moreover, the six conserved motifs of TgPP6C were required for efficient endodyogeny. Phosphoproteomic analysis revealed that ablation of TgPP6C predominately altered the phosphorylation status of proteins involved in the regulation of the parasite cell cycle. Deletion of TgPP6C significantly attenuated the parasite virulence in mice. Immunization of mice with TgPP6C-deficient type I RH strain induced protective immunity against challenge with a lethal dose of RH or PYS tachyzoites and Pru cysts. Taken together, the results show that TgPP6C contributes to the cell division, replication and pathogenicity in T. gondii.

Prevalence and Multilocus Genotyping of Giardia duodenalis in Donkeys in Shanxi Province, North China.

Giardia duodenalis is a ubiquitous flagellated protozoan, causing significant economic losses to animal husbandry and posing threats to public health. China ranks the world's sixth largest major producer of donkeys, rearing approximately 2.6 million donkeys in 2019, but limited investigation of G. duodenalis prevalence has been conducted in the past, and it is yet to be known whether donkeys in Shanxi Province are infected with G. duodenalis. In the present study, a total of 815 fecal samples collected from donkeys in representative regions of Shanxi Province, North China, were examined for G. duodenalis using nested PCR. Then, the assemblages and multilocus genotypes (MLGs) were examined based on three established loci: namely, ß-giardin (bg), triosephosphate isomerase (tpi), and glutamate dehydrogenase (gdh). The overall prevalence of G. duodenalis in donkeys in Shanxi Province was 16.81% (137/815). The region was identified as the main risk factor for the observed difference in G. duodenalis prevalence in donkeys among the three study areas (χ2 = 21.611, p < 0.001). Assemblages A, E, and B were identified, with the latter as the predominant assemblage. Three MLGs (MLG-novel-1 to 3) were formed based on sequence variation among the three loci. The present study reveals the presence of G. duodenalis in donkeys in Shanxi Province, North China, for the first time, which not only enriches the data on the distribution of G. duodenalis in donkeys in China but also provides useful baseline data for planning control strategies against G. duodenalis infection in the sampled areas.

Counting manatee aggregations using deep neural networks and Anisotropic Gaussian Kernel.

Manatees are aquatic mammals with voracious appetites. They rely on sea grass as the main food source, and often spend up to eight hours a day grazing. They move slow and frequently stay in groups (i.e. aggregations) in shallow water to search for food, making them vulnerable to environment change and other risks. Accurate counting manatee aggregations within a region is not only biologically meaningful in observing their habit, but also crucial for designing safety rules for boaters, divers, etc., as well as scheduling nursing, intervention, and other plans. In this paper, we propose a deep learning based crowd counting approach to automatically count number of manatees within a region, by using low quality images as input. Because manatees have unique shape and they often stay in shallow water in groups, water surface reflection, occlusion, camouflage etc. making it difficult to accurately count manatee numbers. To address the challenges, we propose to use Anisotropic Gaussian Kernel (AGK), with tunable rotation and variances, to ensure that density functions can maximally capture shapes of individual manatees in different aggregations. After that, we apply AGK kernel to different types of deep neural networks primarily designed for crowd counting, including VGG, SANet, Congested Scene Recognition network (CSRNet), MARUNet etc. to learn manatee densities and calculate number of manatees in the scene. By using generic low quality images extracted from surveillance videos, our experiment results and comparison show that AGK kernel based manatee counting achieves minimum Mean Absolute Error (MAE) and Root Mean Square Error (RMSE). The proposed method works particularly well for counting manatee aggregations in environments with complex background.

Functional Characterization of Eight Zinc Finger Motif-Containing Proteins in Toxoplasma gondii Type I RH Strain Using the CRISPR-Cas9 System.

The Zinc finger protein (ZFP) family is widely distributed in eukaryotes and interacts with DNA, RNA, and various proteins to participate in many molecular processes. In the present study, the biological functions of eight ZFP genes in the lytic cycle and the pathogenicity of Toxoplasma gondii were examined using the CRISPR-Cas9 system. Immunofluorescence showed that four ZFPs (RH248270-HA, RH255310-HA, RH309200-HA, and RH236640-HA) were localized in the cytoplasm, and one ZFP (RH273150-HA) was located in the nucleus, while the expression level of RH285190-HA, RH260870-HA, and RH248450-HA was undetectable. No significant differences were detected between seven RHΔzfp strains (RHΔ285190, RHΔ248270, RHΔ260870, RHΔ255310, RHΔ309200, RHΔ248450, and RHΔ236640) and the wild-type (WT) strain in the T. gondii lytic cycle, including plaque formation, invasion, intracellular replication, and egress, as well as in vitro virulence (p > 0.05). However, the RHΔ273150 strain exhibited significantly lower replication efficiency compared to the other seven RHΔzfp strains and the WT strain, while in vivo virulence in mice was not significantly affected. Comparative expression analysis of the eight zfp genes indicates that certain genes may have essential functions in the sexual reproductive stage of T. gondii. Taken together, these findings expand our current understanding of the roles of ZFPs in T. gondii.

Transcriptomic, 16S ribosomal ribonucleic acid and network pharmacology analyses shed light on the anticoccidial mechanism of green tea polyphenols against Eimeria tenella infection in Wuliangshan black-boned chickens.

BACKGROUND: Eimeria tenella is an obligate intracellular parasitic protozoan that invades the chicken cecum and causes coccidiosis, which induces acute lesions and weight loss. Elucidating the anticoccidial mechanism of action of green tea polyphenols could aid the development of anticoccidial drugs and resolve the problem of drug resistance in E. tenella. METHODS: We constructed a model of E. tenella infection in Wuliangshan black-boned chickens, an indigenous breed of Yunnan Province, China, to study the efficacy of green tea polyphenols against the infection. Alterations in gene expression and in the microbial flora in the cecum were analyzed by ribonucleic acid (RNA) sequencing and 16S ribosomal RNA (rRNA) sequencing. Quantitative real-time polymerase chain reaction was used to verify the host gene expression data obtained by RNA sequencing. Network pharmacology and molecular docking were used to clarify the interactions between the component green tea polyphenols and the targeted proteins; potential anticoccidial herbs were also analyzed. RESULTS: Treatment with the green tea polyphenols led to a reduction in the lesion score and weight loss of the chickens induced by E. tenella infection. The expression of matrix metalloproteinase 7 (MMP7), MMP1, nitric oxide synthase 2 and ephrin type-A receptor 2 was significantly altered in the E. tenella infection plus green tea polyphenol-treated group and in the E. tenella infection group compared with the control group; these genes were also predicted targets of tea polyphenols. Furthermore, the tea polyphenol (-)-epigallocatechin gallate acted on most of the targets, and the molecular docking analysis showed that it has good affinity with interferon induced with helicase C domain 1 protein. 16S ribosomal RNA sequencing showed that the green tea polyphenols had a regulatory effect on changes in the fecal microbiota induced by E. tenella infection. In total, 171 herbs were predicted to act on two or three targets in MMP7, MMP1, nitric oxide synthase 2 and ephrin type-A receptor 2. CONCLUSIONS: Green tea polyphenols can directly or indirectly regulate host gene expression and alter the growth of microbiota. The results presented here shed light on the mechanism of action of green tea polyphenols against E. tenella infection in chickens, and have implications for the development of novel anticoccidial products.