Exploring the causal relationship between immune cell and all-cause heart failure: a Mendelian randomization study.

Background and objectives: Heart failure (HF) is a disease with numerous genetic and environmental factors that affect it. The results of previous studies indicated that immune phenotypes are associated with HF, but there have been inconclusive studies regarding a causal relationship. Therefore, Mendelian randomization (MR) analyses were undertaken to confirm the causal connections between immune phenotypes and HF, providing genetic evidence supporting the association of immune cell factors with HF risk. Methods: We selected instrumental variables that met the criteria based on data from the results of genome-wide association studies (GWAS) of immune phenotype and all-cause HF. An evaluation of the causal association between 731 immune cell factors and HF risk was carried out using the inverse variance weighted (IVW), MR-Egger regression (MR-Egger), and weighted median (WM) analysis methods. To determine the horizontal pleiotropy, heterogeneity, and stability of the genetic variants, the MR-Egger intercept test, Cochran's Q test, MR-PRESSO, and leave-one-out sensitivity analysis were performed. Results: MR principal method (IVW) analysis showed that a total of 38 immune cell-related factors were significantly causally associated with HF. Further analyses combining three methods (IVW, MR-Egger and WME) showed that six exposure factors significantly associated with heart failure, as shown below. The effect of Dendritic cell Absolute Count, CD62l- CD86+ myeloid Dendritic cell Absolute Count, CD62l- CD86+ myeloid Dendritic cell% Dendritic cell, CD39+ CD8+ T cell% CD8+ T cell, CD3 on Central Memory CD4+ T cell on heart failure was positive. Whereas, a reverse effect was observed for CD14+ CD16+ monocyte% monocyte. Conclusion: We investigated the causal relationship between immune phenotypes and all-cause HF. According to the results, Dendritic cell Absolute Count, CD62l- CD86+ myeloid Dendritic cell Absolute Count, CD62l- CD86+ myeloid Dendritic cell% Dendritic cell, CD39+ CD8+ T cell% CD8+ T cell, CD3 on Central Memory CD4+ T cell aggravate HF, and the risk of HF is decreased by CD14+ CD16+ monocyte% monocyte. These phenotypes may serve as new biomarkers, providing new therapeutic insights for the prevention and treatment of all-cause HF.

Under-Actuated Motion Control of Haidou-1 ARV Using Data-Driven, Model-Free Adaptive Sliding Mode Control Method.

We propose a data-driven, model-free adaptive sliding mode control (MFASMC) approach to address the Haidou-1 ARV under-actuated motion control problem with uncertainties, including external disturbances and parameter perturbations. Firstly, we analyzed the two main difficulties in the motion control of Haidou-1 ARV. Secondly, in order to address these problems, a MFASMC control method was introduced. It is combined by a model-free adaptive control (MFAC) method and a sliding mode control (SMC) method. The main advantage of the MFAC method is that it relies only on the real-time measurement data of an ARV instead of any mathematical modeling information, and the SMC method guarantees the MFAC method's fast convergence and low overshooting. The proposed MFASMC control method can maneuver Haidou-1 ARV cruising at the desired forward speed, heading, and depth, even when the dynamic parameters of the ARV vary widely and external disturbances exist. It also addresses the problem of under-actuated motion control for the Haidou-1 ARV. Finally, the simulation results, including comparisons with a PID method and the MFAC method, demonstrate the effectiveness of our proposed method.

Identification of a novel orthonairovirus from ticks and serological survey in animals near China-North korea border.

Emerging pathogenic tick-borne viruses (TBVs) have attracted a great deal of attention due to their significant impact on human and animal health. A novel orthonairovirus named Dadong virus (DDV) was isolated from Haemaphysalis concinna ticks in the Changbai Mountain region on the China-North Korea border. DDV can induce cytopathic effects in mammalian and human cell lines. Phylogenetic analysis showed that it belongs to the genus Orthonairovirus, family Nairoviridae, exhibiting 72.4%-81.3% nucleic acid identity to Tofla orthonairovirus, known to cause lethal infection in IFNAR KO mice. The first serological evidence of DDV circulating in cattle and mice was also obtained, with 4.0% (1/25) of cattle and 2.27% (1/44) of mice seropositive for DDV. Further investigations, including serological surveys using human samples, are required to assess the public health risk posed by DDV.

Hemalin vaccination modulates the host immune response and reproductive cycle of Haemaphysalis longicornis.

Haemaphysalis longicornis can transmit high varieties of tick-borne pathogens (TBPs), and a primary strategy for preventing the transmission of those TBPs is to control ticks. Hemalin, a thrombin inhibitor of the Kunitz-type family and a crucial component in H. longicornis feeding process has been isolated from parthenogentic ticks. This study aimed to evaluate the validity of a recombinant Hemalin (rHlHemalin) vaccination as an anti-tick vaccine against H. longicornis in rabbits to find a new candidate for an effective tick control. In this study, mouse splenocytes were isolated and used to investigate immune responses after rHlHemalin stimulation. The rabbits were vaccinated with the rHlHemalin protein. After tick challenges, body weight at engorgement, egg mass, and the reproductive cycle of H. longicornis were evaluated. To confirm the vaccination, the passive immunization tests of α-rHlHemalin sera were performed. The results showed that the rHlHemalin protein could stimulate cytokine production in mouse splenocytes. Vaccination assay revealed that the periods from tick infestations to egg-hatch in the vaccination group were significantly longer than those in the phosphate buffer saline (PBS) group (P = 0.0003). In addition, the tick body weight at engorgement (P = 0.0019) and egg mass at 10 days after oviposition (P = 0.0232) were higher than those in the PBS group. These findings were consistent with the current passive immunization results and suggest rHlHemalin vaccination extended the reproductive cycle in H. longicornis but did not decrease the body weight at engorgement or weight of egg mass. Therefore, it is debatable whether Hemalin vaccination is highly-effective anti-tick vaccine or not. However, due to the importance of thrombin inhibitors in tick blood feeding and blood digestion, additional inhibitor-based vaccines should be developed aiming to find an effective and environmentally friendly biological strategy to combat ticks.

First description of Blastocystis sp. and Entamoeba sp. infecting zoo animals in the Qinghai-Tibetan plateau area, China.

Protozoan parasites are a well-known threat to human health, particularly for people working at or visiting zoos, and potentially cause zoonotic diseases in humans. Captive wildlife may be potential reservoirs for human infection with protozoan parasites. Therefore, focusing on zoonotic protozoan infections in zoo animals is critical. However, there is no report on this topic in the Qinghai-Tibetan Plateau region. In this study, a total of 167 and 103 fecal samples were collected from 12 animal species from Qinghai-Tibet Plateau Wildlife Park in winter and summer, respectively, to detection the prevalence of infections and subtype distribution with Entamoeba sp., Cryptosporidium sp., Giardia duodenalis, Enteromicrosporidia bieneusi sp., Blastocystis sp. by PCR assay. The results showed that a total of 21 fecal samples collected in winter, including from 2 white-lipped deer, 8 Sika deer, 6 blue sheep, 2 wolves and 3 bears, were positive for Entamoeba, with a 12.6% (21/167) positive rate. However, 4.9% (5/103) of animals in summer were positive for Entamoeba, including 1 snow leopard, 1 tiger, 1 Tibetan argali and 2 mouflon. Moreover, 1 white-lipped deer and 1 bear were found to be positive for Blastocystis sp., one zoonotic STs (ST10) was identified and found in white-lipped deer. We found no effect on season on Blastocystis sp. and Entamoeba sp. colonization. To the best of our knowledge, this study is the first description of Blastocystis sp. and Entamoeba sp. infecting zoo animals in the plateau area. The findings provide the latest data on Entamoeba sp. and Blastocystis sp. in zoo animals in China.

Novel Orthonairovirus Isolated from Ticks near China-North Korea Border.

We isolated a new orthonairovirus from Dermacentor silvarum ticks near the China-North Korea border. Phylogenetic analysis showed 71.9%-73.0% nucleic acid identity to the recently discovered Songling orthonairovirus, which causes febrile illness in humans. We recommend enhanced surveillance for infection by this new virus among humans and livestock.

The life cycle of Dermacentor nuttalli from the Qinghai-Tibetan Plateau under laboratory conditions and detection of spotted fever group Rickettsia spp.

Dermacentor nuttalli has been a focus of study because tick-borne pathogens have been widely identified in this tick from northern and southwestern China. The aim of this study was to characterize the life cycle of D. nuttalli under laboratory conditions and to detect spotted fever group (SFG) Rickettsia in the midgut and salivary glands of both field-collected and first laboratory generation adults. D. nuttalli ticks were collected in the field on the Qinghai-Tibetan Plateau from March to April 2021 and their life cycle was studied under laboratory conditions. Tick identify was molecularly confirmed, and SFG Rickettsia were detected in the midgut and salivary glands of males and females by PCR targeting different rickettsial genes. The results showed that the life cycle of D. nuttalli under laboratory conditions was completed in an average of 86.1 days. High positivity of Rickettsia spp. was detected in the midgut and salivary glands of both males (92.0%) and females (93.0%) of field-collected D. nuttalli ticks. However, a relatively lower positivity (4.0-6.0%) was detected in first laboratory generation adults. Furthermore, sequencing analysis showed that the Rickettsia sequences obtained in this study shared 98.6 to 100% nucleotide identity with Rickettsia slovaca and Rickettsia raoultii isolated from Dermacentor spp. in China. Phylogenetic analysis of Rickettsia spp. based on the gltA, ompA, ompB and sca4 genes revealed that the Rickettsia sequences obtained could be classified as belonging to R. slovaca and R. raoultii clades. This study described for the first time the life cycle of D. nuttalli from the Qinghai-Tibetan Plateau under laboratory conditions. Two species of SFG Rickettsia were detected in the midgut and salivary glands of males and females in both field-collected and first laboratory-generation adults of D. nuttalli. Our study provides new insights into pathogen detection in ticks in the Qinghai-Tibet Plateau, and the relationships among hosts, ticks, and pathogens.

The diurnal salivary glands transcriptome of Dermacentor nuttalli from the first four days of blood feeding.

The ixodid tick Dermacentor nuttalli is distributed from southern Siberia to North China and is a vector of many pathogens. This species can have severe impacts on animal husbandry and human health. To date, the control of D. nuttalli is limited to the use of acaricides such as organophosphorus, synthetic pyrethroids and amidine pesticides. There are no environmentally friendly or reliable prevention and control measures, and little is known regarding key antigens involved in blood feeding. Salivary glands are major tissues involved in the blood feeding and pathogen transmission of ticks. Therefore, this study focused on salivary glands tissue to identify the dominant antigens of D. nuttalli involved in tick feeding. For this, high-throughput RNA sequencing (RNA-seq) was used for analysis. The transcriptome of female D. nuttalli ticks was assembled and characterized, and differentially expressed genes (DEGs) were identified in the salivary glands of ticks that had not fed (0 h) and of ticks after 24, 48, 72 and 96 h of feeding. There were 22,802,784, 22,275,013, 26,629,453, 24,982,389, and 22,596,230 high-quality clean reads obtained from salivary glands tissues at the five different blood feeding time points. The total number of annotated unigenes was 100,347. The differences in gene expression between different time points were compared, and functional enrichment was performed. Quantitative reverse transcription PCR (RT‒qPCR) was used to validate the RNA-seq results, the results of which showed that the differences in expressed transcripts presented similar trends. Among the identified DEGs, the most numerous were those with catalytic and binding activities and those involved in diverse metabolic pathways and cellular processes. The expression patterns of homologous and family-member proteins throughout the blood feeding period exhibited significant differences, strongly suggesting that the transcriptome composition is highly dynamic and likely subjected to important variation throughout the life cycle. Studies of gene sequences in D. nuttalli will greatly increase the information on tick protective antigens, which could potentially function as effective vaccine candidates or drug targets for the development of environmentally friendly acaricides.

Establishment of a Real-Time Recombinase Polymerase Amplification for Rapid Detection of Pathogenic Yersinia enterocolitica.

Yersinia enterocolitica is a zoonotic proto-microbe that is widespread throughout the world, causes self-limiting diseases in humans or animals and even leads to sepsis and death in patients with severe cases. In this study, a real-time recombinase polymerase amplification (RPA) assay for pathogenic Y. enterocolitica was established based on the ail gene. The results showed that the RPA detection for Y. enterocolitica could be completed within 20 min at an isothermal temperature of 38 °C by optimizing the conditions in the primers and Exo probe. Moreover, the sensitivity of the current RT-RPA was 10-4 ng/µL, and the study found that the assay was negative in the application of the genomic DNA of other pathogens. These suggest the establishment of a rapid and sensitive real-time RPA method for the detection of pathogenic Y. enterocolitica, which can provide new understandings for the early diagnosis of the pathogens.

De novo assembled transcriptomics assisted label-free quantitative proteomics analysis reveals sex-specific proteins in the intestinal tissue of Haemaphysalis qinghaiensis.

The hard tick Haemaphysalis qinghaiensis is the vector of a wide variety of infectious agents, such as spirochetes and other bacteria as well as viruses in the western plateau of China. Tick midgut is the key tissue involved in the host-pathogen-vector interface. Multiple midgut proteins are related to key functions in blood digestion, tick survival, and tick-borne pathogen transmission. However, information on the sex-specific proteins expressed in the midgut tissue of H. qinghaiensis for which the genome has not been sequenced is limited. Hence, we assembled and characterized the transcriptome of the H. qinghaiensis midgut and identified the differentially expressed genes (DEGs) in female and male ticks. The sequencing of the mRNA for this nonmodel species is essential for producing a protein database for mass spectrometry-based identification. Here, we combined high-throughput parallel sequencing and label-free quantitative proteomics analysis to extensively characterize the tick midgut using massive RNA sequencing and mass spectrometry, which allowed the detection of genes and proteins. A total of 279,186 transcripts were annotated into 125,790 coding sequences (CDSs), which were manually curated into 96 different gene families. A total of 12,837 DEGs between the two sexes were found by RNA-seq analysis. Of these, 5401 were upregulated genes, while 7436 were downregulated genes. The most common molecular functions were those related to the endocrine system, translation, signal transduction, transport, and catabolism. Meanwhile, the most common biological processes were related to cellular processes, metabolic processes, cellular anatomical entities, and cargo receptor activities. An analysis of the label-free protein quantitation dataset showed 272 upregulated proteins and 46 downregulated proteins when the fold-change was >2.0 (LC-MS/MS). Association analysis of the transcriptome and proteome with GO functional enrichment showed that the majority of the genes (proteins) were those related to catalytic activity, binding, cellular processes, metabolic processes, and responses to stimuli. This study aims to elucidate the digestive physiology of H. qinghaiensis as well as its physiological sexual dimorphism. This will allow the identification of protein candidates with physiological importance that could be used as targets to control the vector as well as the transmission of tick-borne pathogens to humans and animals.

Serological Analysis of IgG and IgM Antibodies against Anaplasma spp. in Various Animal Species of the Qinghai-Tibetan Plateau.

Anaplasma genus infects the blood cells of humans and animals by biting, causing zoonotic anaplasmosis. However, limited data are available on carrier animals for Anaplasma spp. antibodies in the Qinghai−Tibetan Plateau Area. Therefore, a serological indirect ELISA diagnostic method based on the major surface protein 5 (MSP5), derived from Anaplasma phagocytophilum, was developed in this study to analyze both IgG and IgM antibodies of Anaplasma spp. in a total of 3952 animals from the Qinghai−Tibetan Plateau, including yaks (Bos grunniens), cows (Bos taurus), cattle (Bos taurus domesticus), Tibetan sheep (Ovis aries), horses (Equus ferus caballus), pigs (Sus domesticus), chickens (Gallus gallus domesticus), donkeys (Equus asinus), stray dogs (Canis sp.), and stray cats (Felis sp.). The results showed that recombinant MSP5 protein was expressed and was successfully used to establish the indirect ELISA methods. The overall positivity for Anaplasma IgG and IgM antibodies was 14.6% (578/3952) and 7.9% (312/3952), respectively, and a total of 123 animals (3.1%) were both IgG- and IgM-positive. Moreover, the most prevalent Anaplasma IgG positivity was exhibited by donkeys (82.5%), followed by stray dogs, Tibetan sheep, pigs, chickens, horses, yaks, cows, cattle, and stray cats. The analysis for IgM antibody positivity revealed that IgM positivity was the most prevalent in the stray dogs (30.1%), followed by horses, yaks, Tibetan sheep, cows, stray cats, and cattle. Moreover, the results revealed significant differences (p < 0.05) at different altitudes in Anaplasma-specific IgG in the yaks, Tibetan sheep, and horses, and in IgM in the yaks and Tibetan sheep. In conclusion, this study is the first to demonstrate that yaks, cows, cattle, Tibetan sheep, horses, donkeys, stray dogs, stray cats, pigs, and chickens living in the Qinghai−Tibet Plateau are carrier animals for Anaplasma spp. IgG or IgM antibodies. The current findings provide valuable current data on the seroepidemiology of anaplasmosis in China and for plateau areas of the world.

Seroepidemiology of Neosporosis in Various Animals in the Qinghai-Tibetan Plateau.

Neosporosis is a worldwide infectious disease caused by intracellular parasite Neospora caninum that is a major pathogen of abortion in cattle and neurological disorders in other hosts. However, limited data are available on animals exposed to N. caninum in the Qinghai-Tibetan Plateau Area (QTPA), and little is known about whether animals in the plateau area play an important role in the epidemiology of N. caninum. Therefore, indirect ELISAs based on a combination of NcSAG1 and NcGRA7 antigens were developed to examine both N. caninum-specific IgG and IgM antibodies in Tibetan sheep, yak, cow, pig, cattle, horse, chicken, camel, and donkey from the QTPA in this study. The results showed that all current species present- IgG and IgM-positive animals, and that the overall seroprevalence of N. caninum were 18.6 (703/3,782) and 48.1% (1,820/3,782) for the IgG and IgM antibodies, respectively. Further analysis found significant differences from different altitudes in IgG in Tibetan sheep and IgM in the yak. Hence, the present serological results indicate that the tested animal populations in the QTPA are suffering from N. caninum infections or have become carriers of N. caninum antibodies. To the best of our knowledge, this is the first report on current N. caninum-infected animals in the QTPA, the first epidemiology of neosporosis in cow and camel in China, and the first record of N. caninum IgM antibodies in all the surveyed animals in China. This study provides the latest valuable data on the epidemiology of neosporosis in China and in plateau areas of the world.

Survey of tick species and molecular detection of selected tick-borne pathogens in Yanbian, China.

Ticks and tick-borne diseases pose a significant threat to public health. In this study, we aimed to determine the tick species distribution and pathogens carried by ticks in Yanbian, China. A total of 2673 questing ticks were collected from eight counties and cities in Yanbian and were morphologically identified. The presence of Candidatus Rickettsia tarasevichiae (CRT), spotted fever group Rickettsia (SFGR), severe fever thrombocytopenia syndrome virus (SFTSV), Theileria, and other pathogens was confirmed using polymerase chain reaction (PCR) and real-time quantitative PCR assays, followed by phylogenetic and genotypic analyses. According to the morphological identification, the tick species in Yanbian consisted of Haemaphysalis longicornis, Ixodes persulcatus, Dermacentor silvarum, H. japonica, and H. concinna. In H. longicornis, CRT, SFGR, SFTSV and Theileria orientalis were detected, while CRT, SFGR, and SFTSV were detected in I. persulcatus, H. japonica, and D. silvarum. Only SFTSV was detected in H. concinna. Mixed infection with CRT and SFTSV was observed in I. persulcatus and H. japonica. The gene sequences of all tested pathogens exhibited 95.7%-100% identity with the corresponding sequences deposited in GenBank. Phylogenetic analysis showed that different SFGR and SFTSV genotypes were closely related to the Korean strains. This study is the first to describe the genetic diversity of SFGR Candidatus Rickettsia longicornii in H. longicornis in Yanbian, China, using the ompA, ompB, sca4, and rrs genes. These results provide epidemiological data to support the prevention and control of ticks and tick-borne diseases in the border areas of China, North Korea, and Russia.

Title: Enquête sur les espèces de tiques et détection moléculaire de certains agents pathogènes transmis par les tiques à Yanbian, en Chine. Abstract: Les tiques et les maladies transmises par les tiques constituent une menace importante pour la santé publique. Dans cette étude, nous avons cherché à déterminer la distribution des espèces et les agents pathogènes portés par les tiques à Yanbian, en Chine. Un total de 2 673 tiques errantes ont été collectées dans huit comtés et villes de Yanbian et identifiées morphologiquement. La présence de Candidatus Rickettsia tarasevichiae (CRT), de Rickettsia du groupe de la fièvre boutonneuse (SFGR), du virus du syndrome de la fièvre thrombocytopénique sévère (SFTSV), de Theileria et d'autres agents pathogènes a été confirmée à l'aide d'une réaction en chaîne par polymérase (PCR) et de PCR quantitative en temps réel, suivies par des analyses phylogénétiques et génotypiques. Selon leur identification morphologique, les espèces de tiques à Yanbian se composaient de Haemaphysalis longicornis, Ixodes persulcatus, Dermacentor silvarum, H. japonica et H. concinna. Chez H. longicornis, CRT, SFGR, SFTSV et Theileria orientalis ont été détectés, tandis que CRT, SFGR et SFTSV ont été détectés chez I. persulcatus, H. japonica et D. silvarum. Seul le SFTSV a été détecté chez H. concinna. Une infection mixte par CRT et SFTSV a été observée chez I. persulcatus et H. japonica. Les séquences des gènes de tous les agents pathogènes testés présentaient une identité de 95,7 % à 100 % avec les séquences correspondantes déposées dans GenBank. L'analyse phylogénétique a montré que différents génotypes SFGR et SFTSV étaient étroitement liés aux souches coréennes. Cette étude est la première à décrire la diversité génétique de SFGR Candidatus Rickettsia longicornii chez H. longicornis à Yanbian, en Chine, en utilisant les gènes ompA, ompB, sca4 et rrs. Ces résultats fournissent des données épidémiologiques pour soutenir la prévention et le contrôle des tiques et des maladies transmises par les tiques dans les zones frontalières de la Chine, de la Corée du Nord et de la Russie.

Toxoplasma gondii and Neospora caninum Infections in Stray Cats and Dogs in the Qinghai-Tibetan Plateau Area, China.

Toxoplasma gondii and Neospora caninum belong to the Apicomplexan protozoa which is an obligate intracellular parasite, causing toxoplasmosis and neosporosis throughout the world. Cats and dogs are the definitive hosts of these two parasites. However, information on the epidemiology of toxoplasmosis and neosporosis in stray cats and dogs in the Qinghai-Tibetan Plateau Area (QTPA) is limited, and little is known about the diversity of the diseases. Therefore, the aim of this study was to perform indirect ELISA tests based on recombinant TgSAG1, TgGRA1, NcSAG1 and NcGRA7 proteins to establish a detailed record of the seroprevalence of T. gondii and N. caninum-specific IgG and IgM antibodies in serum samples and to develop qPCR amplification based on TgB1 and NcNc5 genes to conduct molecular epidemiology in feces from stray cats and dogs in the QTPA. In the current study, a total of 128 cat serum samples were analyzed through serological tests in which 53 (41.4%) and 57 (44.5%) samples were found positive for T. gondii specific-IgG and IgM antibodies, and 2 (1.6%) and 74 (57.8%) samples were confirmed positive for N. caninum specific-IgG and IgM antibodies, respectively. Out of 224 stray dog sera, 59.8% and 58.9% were recorded as positive against anti-Toxoplasma IgG and IgM antibodies, 17.9% and 64.7% were detected positive against Neospora IgG and IgM. On the other hand, 1 of 18 cat fecal samples was successfully amplified within the Ct value of 10 to 30 while no cat was positive for neosporosis. Moreover, a higher prevalence of toxoplasmosis in stray dogs (14.5%, 16/110) than of neosporosis (5.5%, 6/110) with different parasite numbers were found. Further analysis showed that no significant sex differences were found nor between the overall infection rates of T. gondii and N. caninum in this study. This study suggests that stray cats and dogs play key roles in the transmission and prevalence of T. gondii and N. caninum in the plateau area.

The Cross-Species Immunity During Acute Babesia Co-Infection in Mice.

Babesiosis causes high morbidity and mortality in immunocompromised individuals. An earlier study suggested that lethal Babesia rodhaini infection in murine can be evaded by Babesia microti primary infection via activated macrophage-based immune response during the chronic stage of infection. However, whether the same immune dynamics occur during acute B. microti co-infection is not known. Hence, we used the mouse model to investigate the host immunity during simultaneous acute disease caused by two Babesia species of different pathogenicity. Results showed that B. microti primary infection attenuated parasitemia and conferred immunity in challenge-infected mice as early as day 4 post-primary infection. Likewise, acute Babesia co-infection undermined the splenic immune response, characterized by the significant decrease in splenic B and T cells leading to the reduction in antibody levels and decline in humoral immunity. Interestingly, increased macrophage and natural killer splenic cell populations were observed, depicting their subtle role in the protection. Pro-inflammatory cytokines (i.e. IFN-γ, TNF-α) were downregulated, while the anti-inflammatory cytokine IL-10 was upregulated in mouse sera during the acute phase of Babesia co-infection. Herein, the major cytokines implicated in the lethality caused by B. rodhaini infection were IFN- γ and IL-10. Surprisingly, significant differences in the levels of serum IFN- γ and IL-10 between co-infected survival groups (day 4 and 6 challenge) indicated that even a two-day delay in challenge infection was crucial for the resulting pathology. Additionally, oxidative stress in the form of reactive oxygen species contributed to the severity of pathology during acute babesiosis. Histopathological examination of the spleen showed that the erosion of the marginal zone was more pronounced during B. rodhaini infection, while the loss of cellularity of the marginal zone was less evident during co-infection. Future research warrants investigation of the roles of various immune cell subtypes in the mechanism involved in the protection of Babesia co-infected hosts.

Inhibitory effect of naphthoquine phosphate on Babesia gibsoni in vitro and Babesia rodhaini in vivo.

BACKGROUND: Drug resistance and toxic side effects are major challenges in the treatment of babesiosis. As such, new drugs are needed to combat the emergence of drug resistance in Babesia parasites and to develop alternative treatment strategies. A combination of naphthoquine (NQ) and artemisinin is an antimalarial therapy in pharmaceutical markets. The present study repurposed NQ as a drug for the treatment of babesiosis by evaluating the anti-Babesia activity of naphthoquine phosphate (NQP) alone. METHODS: An in vitro growth inhibition assay of NQP was tested on Babesia gibsoni cultures using a SYBR Green I-based fluorescence assay. In addition, the in vivo growth inhibitory effect of NQP was evaluated using BALB/c mice infected with Babesia rodhaini. The parasitemia level and hematocrit values were monitored to determine the therapeutic efficacy of NQP and the clinical improvements in NQP-treated mice. RESULTS: The half maximal inhibitory concentration of NQP against B. gibsoni in vitro was 3.3 ± 0.5 µM. Oral administration of NQP for 5 consecutive days at a dose of 40 mg/kg of body weight resulted in significant inhibition of B. rodhaini growth in mice as compared with that of the control group. All NQP-treated mice survived, whereas the mice in the control group died between days 6 and 9 post-infection. CONCLUSION: This is the first study to evaluate the anti-Babesia activity of NQP in vitro and in vivo. Our findings suggest that NQP is a promising drug for treating Babesia infections, and drug repurposing may provide new treatment strategies for babesiosis.

Application of Toxoplasma gondii-specific SAG1, GRA7 and BAG1 proteins in serodiagnosis of animal toxoplasmosis.

Toxoplasmosis is a zoonotic disease caused by the obligate intracellular protozoan parasite T. gondii which is widely prevalent in humans and animals worldwide. The diagnosis of toxoplasmosis and distinguishing acute or chronic T. gondii infections have utmost importance for humans and animals. The TgSAG1, TgGRA7, and TgBAG1 proteins were used in the present study to develop the serological rSAG1-ELISA, rGRA7-ELISA and rBAG1-ELISA methods for the testing of T. gondii specific IgG and IgM antibodies and differentiating acute or chronic toxoplasmosis in 3733 animals, including Tibetan sheep, yaks, pigs, cows, cattle, horses, chickens, camels and donkeys from the Qinghai-Tibetan Plateau. The ELISA tests showed that the overall positivity of IgG antibody was 21.1% (786/3733), 15.3% (570/3733) and 18.2% (680/3733) for rSAG1-, rGRA7- and rBAG1-ELISA, respectively, and the positivity of IgM antibody was 11.8% (439/3733), 13.0% (486/3733) and 11.8% (442/3733) for rSAG1-, rGRA7- and rBAG1-ELISA, respectively. A total of 241 animals (6.5%) positive for all rSAG1-, rGRA7- and rBAG1-IgG were found in this study, and the 141 animals (3.8%) tested were anti-T. gondii IgM positive in all three ELISAs. Moreover, the 338, 284 and 377 animals were IgG positive in rSAG1 + rGRA7-, rBAG1 + rGRA7- and rSAG1 + rBAG1- ELISAs respectively, and the 346, 178 and 166 animals in rSAG1 + rGRA7-, rBAG1 + rGRA7- and rSAG1 + rBAG1-ELISAs were IgM positive respectively. The results confirmed that the application of SAG1, GRA7, and BAG1 recombinant antigens could successfully be used in the detection of specific IgG and IgM antibodies for distinguishing between acute or chronic T. gondii infections. It is inferred that the forms in which current animal species in the plateau area were infected with T. gondii, and the period of infection or the clinical manifestations of the current infections may be different. The present study provides substantial clinical evidence for the differential diagnosis of toxoplasmosis, and the classification of acute and chronic T. gondii infections.

Discovering the Potent Inhibitors Against Babesia bovis in vitro and Babesia microti in vivo by Repurposing the Natural Product Compounds.

In the present study, we screened 502 natural product compounds against the in vitro growth of Babesia (B.) bovis. Then, the novel and potent identified compounds were further evaluated for their in vitro efficacies using viability and cytotoxicity assays. The in vivo inhibitory effects of the selected compounds were evaluated using B. microti "rodent strain" in mice model. Three potent compounds, namely, Rottlerin (RL), Narasin (NR), Lasalocid acid (LA), exhibited the lowest IC50 (half-maximal inhibitory concentration) as follows: 5.45 ± 1.20 µM for RL, 1.86 ± 0.66 µM for NR, and 3.56 ± 1.41 µM for LA. The viability result revealed the ability of RL and LA to prevent the regrowth of treated parasite at 4 × IC50 and 2 × IC50, respectively, while 4 × IC50 of NR was sufficient to stop the regrowth of parasite. The hematology parameters of B. microti in vivo were different in the NR-treated groups as compared to the infected/untreated group. Interestingly, intraperitoneal administration of NR exhibiting inhibition in the growth of B. microti in mice was similar to that observed after administration of the commonly used antibabesial drug, diminazene aceturate (DA) (76.57% for DA, 74.73% for NR). Our findings indicate the richness of natural product compounds by novel potent antibabesial candidates, and the identified potent compounds, especially NR, might be used for the treatment of animal babesiosis.

Molecular detection of Anaplasma spp., Babesia spp. and Theileria spp. in yaks (Bos grunniens) and Tibetan sheep (Ovis aries) on the Qinghai-Tibetan Plateau, China.

BACKGROUND: Anaplasma, Babesia and Theileria are tick-borne pathogens (TBPs) that affect livestock worldwide. However, information on these pathogens in yaks (Bos grunniens) and Tibetan sheep (Ovis aries) on the Qinghai-Tibet Plateau (QTP), China, is limited. In this study, Anaplasma spp., Babesia spp. and Theileria spp. infections were assessed in yaks and Tibetan sheep from Qinghai Province. METHODS: A total of 734 blood samples were collected from 425 yaks and 309 Tibetan sheep at nine sampling sites. Standard or nested polymerase chain reaction was employed to screen all the blood samples using species- or genus-specific primers. RESULTS: The results showed that 14.1% (60/425) of yaks and 79.9% (247/309) of Tibetan sheep were infected with at least one pathogen. Anaplasma ovis, Anaplasma bovis, Anaplasma capra, Anaplasma phagocytophilum, Babesia bovis and Theileria spp. were detected in this study, with total infection rates for all the assessed animals of 22.1% (162/734), 16.3% (120/734), 23.6% (173/734), 8.2% (60/734), 2.7% (20/734) and 19.3% (142/734), respectively. For yaks, the infection rate of A. bovis was 6.4% (27/425), that of B. bovis was 4.7% (20/425) and that of Theileria spp. was 3.3% (14/425). Moreover, 52.4% (162/309) of the Tibetan sheep samples were infected with A. ovis, 30.1% (93/309) with A. bovis, 56.0% (173/309) with A. capra, 19.4% (60/309) with A. phagocytophilum and 41.4% (128/309) with Theileria spp. CONCLUSIONS: This study revealed the prevalence of Anaplasma spp., Babesia spp. and Theileria spp. in yaks and Tibetan sheep in Qinghai Province, China, and provides new data for a better understanding of the epidemiology of TBPs in these animals in this area of the QTP, China.

In vitro screening of novel anti-Babesia gibsoni drugs from natural products.

Babesia gibsoni is a tick-transmitted intraerythrocytic apicomplexan parasite that causes babesiosis in dogs. Due to the strong side effects and lack of efficacy of current drugs, novel drugs against B. gibsoni are urgently needed. Natural products as a source for new drugs is a good choice for screening drugs against B. gibsoni. The current study focuses on identifying novel potential drugs from natural products against B. gibsoniin vitro. Parasite inhibition was verified using a SYBR green I-based fluorescence assay. A total of 502 natural product compounds were screened for anti-B. gibsoni activity in vitro. Twenty-four compounds showed high growth inhibition (>80%) on B. gibsoni and 5 plant-derived compounds were selected for further study. The half-maximal inhibitory concentration (IC50) values of lycorine (LY), vincristine sulfate (VS), emetine·2HCl (EME), harringtonine (HT) and cephaeline·HBr (CEP) were 784.4 ± 3.3, 643.0 ± 2.8, 253.1 ± 1.4, 23.4 ± 1.2, and 108.1 ± 4.3 nM, respectively. The Madin-Darby canine kidney (MDCK) cell line was used to assess cytotoxicity of hit compounds. All compounds showed minimal toxicity to the MDCK cells. The effects of hit compounds combined with diminazene aceturate (DA) on B. gibsoni were further evaluated in vitro. VS, EME, HT or CEP combined with DA showed synergistic effects against B. gibsoni, whereas LY combined with DA showed an antagonistic effect against B. gibsoni. The results obtained in this study indicate that LY, VS, EME, HT and CEP are promising compounds for B. gibsoni treatment.