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Abstract Background Cryptosporidium is a highly pathogenic parasite responsible for diarrhea in children worldwide. Here, the epidemiological status and genetic characteristics of Cryptosporidium in children with or without diarrhea were investigated with tracking of potential sources in Wenzhou City, China. Methods A total of 1032 children were recruited, 684 of whom had diarrhea and 348 without, from Yuying Children’s Hospital in Wenzhou, China. Samples of stool were collected from each participant, followed by extraction of DNA, genotyping, and molecular identification of Cryptosporidium species and subtypes. Results Twenty-two of the 1032 (2.1%) children were infected with Cryptosporidium spp. with 2.5% (17/684) and 1.4% (5/348) in diarrhoeic and asymptomatic children, respectively. Four Cryptosporidium species were identified, including C. parvum (68.2%; 15/22), C. felis (13.6%; 3/22), C. viatorum (9.1%; 2/22), and C. baileyi (9.1%; 2/22). Two C. parvum subtypes named IIdA19G1 (n = 14) and IInA10 (n = 1), and one each of C. felis (XIXa) and C. viatorum (XVaA3g) subtype was found as well. Conclusions This is the first research that identified Cryptosporidium in children of Wenzhou, China, using PCR. Identification of zoonotic C. parvum, C. felis, C. viatorum, and their subtypes indicate potential cross-species transmission of Cryptosporidium between children and animals. Additionally, the presence of C. baileyi in children suggests that this species has a wider host range than previously believed and that it possesses the capacity to infect humans.

IntroductionGlobally, rodents and shrew populations constitute crucial elements of diverse environments and animal communities. It is imperative to study their population dynamics to mitigate any potential negative impact on humans, as they can be involved in the transmission of critical zoonotic agents, such as Blastocystis. Therefore, this study aimed to identify the prevalence and genetic composition of Blastocystis in wild rodents and shrews residing in the Zhejiang provinces of China.MethodsA total of 652 wild rodents and and shrews were captured from three different regions in Zhejiang Province from April 1st to October 31, 2023. The DNA was isolated by collecting fresh feces from the intestines of each rodent or and shrew. Rodent and shrew species were examined by vertebrate cytochrome b (cytb) analysis and PCR amplification. Blastocystis was also found in all fecal samples using PCR analysis and sequencing of the partial small subunit of ribosomal RNA (SSU rRNA) gene.ResultsAmong all the samples, 6.6% (43/652) showed a positive result for Blastocystis. In the results, 6 species of rodent and shrew were identified with Blastocystis, including Apodemus agrarius (n = 36) (2.8%), Niviventer confucianus (n = 75) (17.3%), Rattus losea (n = 18) (5.6%), R. norvegicus (n = 155) (2.6%), R. tanezumi (n = 86) (3.5%), and Suncus murinus (n = 282) (7.4%). The existence of 6 Blastocystis subtypes, ST4 (n = 33), ST1 (4), ST7 (n = 3), ST2 (n = 1), ST3 (n = 1), and ST5 (n = 1), were confirmed by sequence analysis.DiscussionBased on the molecular data obtained, the wild rodents and shrews under investigation were found to be concurrently infected with zoonotic subtypes of Blastocystis, including ST1 to ST5 and ST7. This suggests that these animals could potentially pose a zoonotic threat to humans and other animals susceptible to Blastocystis infection.

Shrews play a crucial role as repositories for diverse pathogens linked to zoonotic infectious diseases. However, the genetic information regarding Cryptosporidium in Chinese shrews remains unexplored. The objectives of this study were twofold: to determine the occurrence rate of Cryptosporidium spp. in wild shrews residing in the southern part of Zhejiang Province, China, and to investigate their genetic characteristics. A total of 282 wild shrews were captured between April and October of 2023. The detection of Cryptosporidium in fecal samples, collected from each animal's rectum, was performed using PCR and sequencing of the partial small subunit of ribosomal RNA (SSU rRNA) gene. The 60-kDa glycoprotein (gp60) gene was utilized to further subtype the positive samples of C. viatorum and C. parvum. All animals were identified as Suncus murinus, and a positive result for Cryptosporidium was obtained in 14.2 % (40/282) of the samples. The following species and genotypes were identified: C. ratti (n = 19), C. parvum (n = 2), C. viatorum (n = 1), Cryptosporidium rat genotype IV (n = 13), and Cryptosporidium skunk genotype (n = 5). Furthermore, the subtypes IIdA15G1 and XVdA3 were detected within C. parvum and C. viatorum, respectively. Molecular evidence indicates that S. murinus is concurrently infected with rodent-adapted and zoonotic species/genotypes, actively contributing to the dissemination of cryptosporidiosis.

Globally, Enterocytozoon bieneusi has been detected in humans and various animal hosts. Wild rats and shrews have the potential to act as carriers of E. bieneusi, facilitating the parasite’s transmission to humans and domestic animals. We aimed to investigate the prevalence of E. bieneusi in 652 wild rats and shrews from Zhejiang Province, China, by amplifying the internal transcribed spacer (ITS) region of rDNA through polymerase chain reaction (PCR). To determine animal species, we amplified the Cytochrome b (Cyt-b) gene in their fecal DNA using PCR. Furthermore, we determined the genotype of E. bieneusi by amplifying the ITS region of rDNA through PCR. Genetic traits and zoonotic potential were evaluated using similarity and phylogenetic analyses. Suncus murinus (n = 282) and five rat species, Rattus losea (n = 18), Apodemus agrarius (n = 36), Rattus tanezumi (n = 86), Rattus norvegicus (n = 155), and Niviventer niviventer (n = 75), were identified. The average infection rate of E. bieneusi was 14.1% (92/652) with 18.1% (51/282) in S. murinus and 11.1% (41/370) in rats (27.8% in R. losea, 22.2% in A. agrarius, 10.5% in R. tanezumi, 8.4% in R. norvegicus, and 8.0% in N. niviventer). Thirty-three genotypes were identified, including 16 known genotypes. The most commonly known genotypes were HNR-VI (n = 47) and Peru11 (n = 6). Type IV, KIN-1, SHW7, and HNPL-II were each found in two samples, while Macaque4, CH5, K, Henan-III, Henan-V, HNP-II, HNPL-I, HNPL-III, HNHZ-II, and HNHZ-III were each found in one sample. Additionally, 17 novel genotypes were discovered: WZR-VIII (n = 5), WZR-I to WZR-VII, WZR-IX to WZR-XII, and WZSH-I to WZSH-V (n = 1 each). Those 33 genotypes were divided into three groups: Group 1 (n = 25), Group 2 (n = 3), and Group 13 (n = 5). The initial report underscores the extensive occurrence and notable genetic diversity of E. bieneusi in wild rats and shrews from Zhejiang province, China. These results suggest that these animals play a pivotal role in the transmission of E. bieneusi. Furthermore, animals carrying the zoonotic genotypes of E. bieneusi pose a serious threat to residents.

Abstract Background Rodents, globally overpopulated, are an important source for zoonotic disease transmission to humans, including Enterocytozoon bieneusi (one of the most prevalent zoonotic pathogens). Here, we studied the prevalence and performed genetic analyses of E. bieneusi in rodents from the Hainan Province of China. Methods A total of 603 fresh fecal samples were gathered from 369 wild rats, 117 bamboo rats, 93 Asiatic brush-tailed porcupine and 24 red-bellied squirrels. The wild rats were identified to the species level by amplification of a 421-bp region of the cytb gene from fecal DNA using PCR. Genotype analysis was performed by amplification of the internal transcribed spacer (ITS) region of rDNA of E. bieneusi using PCR. Results Seven wild rat species were identified. The average rate of infection with E. bieneusi was 15.8% (95/603) with 18.7% (69/369) in wild rats, 11.9% (25/210) in farmed rodents and 4.2% (1/24) in red-bellied squirrels. Sixteen E. bieneusi genotypes were identified, including 9 known genotypes (D, Type IV, PigEBITS7, Peru8, Peru11, ESH02, S7, EbpA and CHG5), and 7 novel genotypes (HNR-I to HNR-VII). Genotype D (44.2%, 42/95) predominated, followed by PigEBITS7 (20.0%, 19/95), HNR-VII (15.8%, 15/95), Type IV (5.3%, 5/95), HNR-III (2.1%, 2/95), HNR-VI (2.1%, 2/95) and each of the remaining 10 genotypes (1.1%, 1/95). The phylogenetic analysis of the ITS region of E. bieneusi divided the identified genotypes into the following four groups: Group 1 (n = 13), Group 2 (n = 1), Group 12 (n = 1), and the novel Group 13 (n = 1). Conclusions To our knowledge, this is the first report on the identification of E. bieneusi in rodents from Hainan, China. The zoonotic potential of the identified E. bieneusi genotypes suggested that the rodents poses a serious threat to the local inhabitants. Thus, measures need to be taken to control the population of wild rats in the areas investigated in this study, along with identification of safe methods for disposal of farmed rodent feces. Additionally, the local people should be made aware of the risk of disease transmission from rodents to humans.

Abstract Background Angiostrongylus cantonensis can cause severe symptoms of central nervous system infections. In the host, this parasite localizes in the blood and cerebrospinal fluid, and its secreted components can impact immune responses. Our previous study demonstrated that immune responses were inhibited in A. cantonensis-infected mice immunized with Ac-Galectin-1 (AcGal-1). However, the mechanisms by which AcGal-1 regulates the immune responses remain unclear. Macrophages are innate immune cells that rapidly respond to infection. The direct impact of AcGal-1 on macrophages may affect the immune responses. Methods AcGal-1 protein was purified by nickel ion affinity chromatography. The effect of AcGal-1 on the apoptosis of macrophages was detected using CCK-8 assay, flow cytometry and western blot. Macrophage membrane proteins bound to AcGal-1 were obtained using the His-tag-based pull-down assay and identified via mass spectrometry. Co-localization of AcGal-1 and the macrophage membrane protein Annexin A2 was observed by immunofluorescence microscopy, and their interaction was validated by co-immunoprecipitation experiments. SiRNA-mediated knockdown of Annexin A2 was used to determine if AcGal-1-induced macrophage apoptosis required interaction with Annexin A2. The phosphorylation level of apoptotic signal pathway protein was detected by phospho-antibody microarray and western blot. Results Our study showed that AcGal-1 caused apoptosis of the macrophages. AcGal-1 increased the expression of apoptosis proteins caspase-3, caspase-9, Bax, but reduced the expression of anti-apoptosis protein Bcl-2. AcGal-1 interacted with the membrane protein Annexin A2, and knockdown of Annexin A2 expression increased Bcl-2 but decreased Bax levels in AcGal-1-treated cells. Moreover, AcGal-1 increased JNK phosphorylation and the inhibition of JNK phosphorylation in AcGal-1-treated cells decreased the expression of caspase-3, -9, Bax and almost restored Bcl-2 to the level observed in control cells. Conclusions AcGal-1 can induce the apoptosis of macrophages by binding to Annexin A2 and activating JNK downstream the apoptotic signaling pathway.

Enterocytozoon bieneusi is a microsporidian and zoonotic species. This study investigated the prevalence and distribution of E. bieneusi genotypes in farmed masked palm civets using nested PCR, as well as assessed their zoonotic potential by phylogenetic analysis of the ITS region of the rRNA region. Here, we collected 251 fecal specimens from farmed masked palm civets (Paguma larvata) from the Hainan Island, China. In total, 128 of 251 samples were positive for E. bieneusi, with an average infection rate of 51.0%. Seventeen genotypes were identified including 12 known genotypes—HNR-VI (n = 56), SHR1 (n = 45), SHW7 (n = 6), KIN-1 (n = 3), D (n = 3), New1 (n = 3), EbpC (n = 2), CHC5 (n = 1), CHG19 (n = 1), CHN4 (n = 1), EbpA (n = 1), and Henan-III (n = 1)—and five novel genotypes (HNPL-I to HNPL-II; one each). Phylogenetic analysis categorized these genotypes into two groups. Thirteen of them were members of the zoonotic group 1, and the remaining four genotypes were in group 12. This study has shown that the infection rates of E. bieneusi in masked palm civets from Hainan were relatively high and provide baseline data to control and prevent microsporidiosis in farm-related communities. Therefore, infections in masked palm civets with zoonotic genotypes D, EbpC, CHN4, EbpA, KIN-1, and Henan-III should be considered potential threats to public health.

Cryptosporidiosis is a major public health problem in humans and animals. Information on the prevalence and molecular diversity of Cryptosporidium in farmed deer in northeastern China is limited. In this study, the prevalence of these parasites was investigated in four farmed deer species, including 125 reindeer, 109 red deer, 86 sika deer, and 18 Siberian roe deer by nested PCR amplification of the partial small subunit of ribosomal RNA (SSU rRNA) gene. C. ubiquitum isolates were subtyped using nested PCR and sequence analysis of the 60-kDa glycoprotein (gp60) gene. The overall prevalence of Cryptosporidium was 7.1%, with 15.1% for sika deer, 4.0% for reindeer, 4.6% for red deer, and 5.6% for roe deer. C. ubiquitum (n = 4), C. xiaoi (n = 2), and Cryptosporidium deer genotype (n = 18) were identified. All four C. ubiquitum isolates belonged to the XIIa subtype (n = 4). This study confirms that Cryptosporidium deer genotype is widely occurring in deer in the investigated areas. Presence of zoonotic C. ubiquitum XIIa subtype indicates that farmed deer represent potential source of zoonotic cryptosporidia and might pose a threat to human health.

Abstract Background Anopheles anthropophagus, one of the most important mosquito-borne disease vectors in Asia, mainly takes blood meals from humans and transmits both malaria and filariae. MicroRNAs (miRNAs) are small non-coding RNAs, and play a critical role in many cellular processes, including development, differentiation, apoptosis and innate immunity. Methods We investigated the global miRNA expression profile of male and female adults of A. anthropophagus using illumina Hiseq2000 sequencing combined with Northern blot. Results By using the miRNAs of the closely-related species Anopheles gambiae and Aedes aegypti as reference, we obtained 102 miRNAs candidates out of 12.43 million raw sequencing reads for male and 16.51 million reads for female, with 81 of them found as known miRNAs in An. gambiae and/or Ae. aegypti, and the remaining 21 miRNAs were considered as novel. By analyzing the revised read count of miRNAs in male and female, 29 known miRNAs show sexual difference expression: >2-fold in the read count of the same miRNAs in male and female. Especially for miR-989, which is highly expressed in the female mosquitoes, but shows almost no detected expression in male mosquitoes, indicating that miR-989 may be involved in the physiological activity of female mosquito adults. The expression of four miRNAs in different growth stages of mosquito were further identified by Northern blot. Several miRNAs show the stage-specific expression, of which miR-2943 only expressed in the egg stage, suggesting that miR-2943 may be associated with the development of mosquito eggs. Conclusions The present study represents the first global characterization of An. anthropophagus miRNAs in sexual differences and stage-specific functions. A better understanding of the functions of these miRNAs will offer new insights in mosquito biology and has implications for the effective control of mosquito-borne infectious diseases.

Platelets play crucial roles in hemostasis, thrombosis, wound healing, inflammation, angiogenesis, and tumor metastases. Because they are anucleated blood cells, platelets lack nuclear DNA, but they do contain mitochondrial DNA, which plays a key role in regulating apoptosis. Recent evidence has suggested that miRNAs are also involved in regulating gene expression and apoptosis in platelets. Our previous study showed that the expression of miR-326 increased visibly when apheresis platelets were stored in vitro. The antiapoptotic Bcl-2 family regulator Bcl-xL has been identified as a putative target of miR-326. In the present study, dual reporter luciferase assays were used to characterize the function of miR-326 in the regulation of the apoptosis of platelet cells. These assays demonstrated that miR-326 bound to the 3'-translated region of Bcl-xL. To directly assess the functional effects of miR-326 expression, levels of Bcl-xL and the apoptotic status of stored apheresis platelets were measured after transfection of miR-326 mimic or inhibitor. Results indicated that miR-326 inhibited Bcl-xL expression and induced apoptosis in stored platelets. Additionally, miR-326 inhibited Bcl-2 protein expression and enhanced Bak expression, possibly through an indirect mechanism, though there was no effect on the expression of Bax. The effect of miR-326 appeared to be limited to apoptosis, with no significant effect on platelet activation. These results provide new insight into the molecular mechanisms affecting differential platelet gene regulation, which may increase understanding of the role of platelet apoptosis in multiple diseases.

Introduction: Mycoplasma pneumoniae (M. pneumoniae) is the most common atypical pathogen that causes respiratory infections in humans. Laboratory tests are important in the diagnosis of M. pneumoniae because of the atypical features in clinical signs and symptoms. Nowadays, both the P1 adhesin gene and 16S ribosomal (r) RNA (rRNA) gene of M. pneumoniae have been widely detected by polymerase chain reaction (PCR). The purpose of the present study was to evaluate the most suitable target in the detection of M. pneumonia via nested PCR. Methodology: Both the P1 adhesin gene and 16S rRNA gene for nested PCR reaction conditions were optimized through an orthogonal test and single-factor experiment. Then, the sensitivity of the two sets of targets was evaluated. Finally, based on the optimal conditions, 55 clinical samples of throat swabs collected from adult patients in 2013 were examined by established nested PCR. Result: The results revealed that PCR detection of the 16S rRNA gene was more sensitive than the P1 adhesin gene because the detection limits for both the P1 gene and 16S rRNA gene were at least 100 fg and 10 fg of M. pneumoniae DNA, respectively. Furthermore, the positive rate for detection of the 16S rRNA gene (30/55; 54.5%) was higher than that of the P1 adhesin gene (25/55; 45.5%). Conclusion: Our results indicate that the 16S rRNA gene is more suitable for diagnosis of M. pneumoniae infection than the P1 adhesin gene due to its higher sensitivity and positive rate in clinical samples. [ABSTRACT FROM AUTHOR]