Rapid visual detection of Giardia duodenalis in faecal samples using an RPA-CRISPR/Cas12a system.

Giardiasis is a common intestinal infection caused by Giardia duodenalis, which is a major economic and health burden for humans and livestock. Currently, a convenient and effective detection method is urgently needed. CRISPR/Cas12a-based diagnostic methods have been widely used for nucleic acid-based detection of pathogens due to their high efficiency and sensitivity. In this study, a technique combining CRISPR/Cas12a and RPA was established that allows the detection of G. duodenalis in faecal samples by the naked eye with high sensitivity (10-1 copies/µL) and specificity (no cross-reactivity with nine common pathogens). In clinical evaluations, the RPA-CRISPR/Cas12a-based detection assay detected Giardia positivity in 2% (1/50) of human faecal samples and 47% (33/70) of cattle faecal samples, respectively, which was consistent with the results of nested PCR. Our study demonstrated that the RPA-CRISPR/Cas12a technique for G. duodenalis is stable, efficient, sensitive, specific and has low equipment requirements. This technique offers new opportunities for on-site detection in remote and poor areas.

Development of an LFD-RPA Assay for Rapid Detection of Pentatrichomonas hominis Infection in Dogs.

Pentatrichomonas hominis is a trichomonad protozoan that infects the cecum and colon of humans and other mammals. It is a zoonotic pathogen that causes diarrhea in both animals and humans. As companion animals, dogs infected with P. hominis pose a risk of transmitting it to humans. Current methods, such as direct smears and polymerase chain reaction (PCR), used for P. hominis detection have limitations, including low detection rates and the need for specialized equipment. Therefore, there is an urgent need to develop rapid, sensitive, and simple detection methods for clinical application. Recombinase polymerase amplification (RPA) has emerged as a technology for rapid pathogen detection. In this study, we developed a lateral flow dipstick (LFD)-RPA method based on the highly conserved SPO11-1 gene for detecting P. hominis infection by optimizing the primers, probes, and reaction conditions, and evaluating cross-reactivity with genomes of Giardia duodenalis and other parasites. The LFD-RPA method was then used to test 128 dog fecal samples collected from Changchun. The results confirmed the high specificity of the method with no cross-reactivity with the five other parasites. The lowest detection limit of the method was 102 copies/µL, and its sensitivity was 100 times higher than that of the conventional PCR method. Consistent with the positivity rate observed using nested PCR, 12 samples (out of 128) tested positive using this method (positivity rate, 9.38%). In conclusion, the LFD-RPA method developed in this study represents a simple and sensitive assay that allows for the rapid detection of P. hominis infection in dogs, especially in this field.

Trichomonas gallinae induces heterophil extracellular trap formation in pigeons.

Avian trichomonosis is a worldwide and cross-species epidemic, and the infection in pigeons is particularly severe. Although the disease causes a serious threat to poultry health resulting in significant economic losses, the relationship between Trichomonas gallinae (T. gallinae) and host innate immunity is still not clear. Extracellular traps (ETs) are an innate immunity response to parasitic infections. However, whether host cells can produce ETs after T. gallinae infection has not yet been reported. In the present study, the ability of T. gallinae to induce the production of heterophil extracellular traps (HETs) in pigeons was examined. T. gallinae-induced HETs were observed by scanning electron microscopy (SEM) and the main components of HETs were detected by fluorescence confocal microscopy. Changes in reactive oxygen species (ROS) and lactate dehydrogenase (LDH) were tested during the HETosis. A quantitative analysis of T. gallinae-induced HETs, the role of myeloperoxidase (MPO), store-operated Ca (2+) entry (SOCE), and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase in T. gallinae-induced HET formation were conducted by inhibitor assays. The results showed that T. gallinae induced ET formation in pigeon heterophils. ETs consisted of a DNA skeleton, neutrophil elastase (NE), MPO, and Histone3 (H3). T. gallinae-induced HETs formation in a dose- and time-dependent process. The release of T. gallinae-induced HETs depends on MPO, SOCE, and NADPH oxidase. Furthermore, after T. gallinae stimulated pigeon heterophils, ROS production was significantly increased, while no significant differences in the LDH activity were observed.

Unfolded protein response is involved in resistance to Neospora caninum infection via IRE1α-XBP1s-NOD2 Axis.

Neospora caninum, an intracellular protozoan parasite, causes neosporosis resulting in major losses in the livestock industry worldwide. However, no effective drugs or vaccines have been developed to control neosporosis. An in-depth study on the immune response against N. caninum could help to search for effective approaches to prevent and treat neosporosis. The host unfolded protein response (UPR) functions as a double-edged sword in several protozoan parasite infections, either to initiate immune responses or to help parasite survival. In this study, the roles of the UPR in N. caninum infection in vitro and in vivo were explored, and the mechanism of the UPR in resistance to N. caninum infection was analyzed. The results revealed that N. caninum triggered the UPR in mouse macrophages, such as the activation of the IRE1 and PERK branches, but not the ATF6 branch. Inhibition of the IRE1α-XBP1s branch increased the N. caninum number both in vitro and in vivo, while inhibition of the PERK branch did not affect the parasite number. Furthermore, inhibition of the IRE1α-XBP1s branch reduced the production of cytokines by inhibiting NOD2 signalling and its downstream NF-κB and MAPK pathways. Taken together, the results of this study suggest that the UPR is involved in the resistance of N. caninum infection via the IRE1α-XBP1s branch by regulating NOD2 and its downstream NF-κB and MAPK pathways to induce the production of inflammatory cytokines, which provides a new perspective for the research and development of anti-N. caninum drugs.

Protective efficacy of Toxoplasma gondii bivalent MAG1 and SAG1 DNA vaccine against acute toxoplasmosis in BALB/c mice.

Toxoplasma gondii can infect a wide range of warm-blooded animals, causing a global toxoplasmosis zoonotic epidemic. Surface antigen 1 (SAG1) protein is expressed at the proliferative tachyzoite stage, whereas matrix antigen 1 (MAG1) is expressed at the bradyzoite and tachyzoite stages. These two proteins were found to perform protective roles in previous studies; however, their synergetic protective efficacy as a DNA vaccine against toxoplasmosis has not been clarified. In this study, we constructed recombinant pcDNA3.1( +)-TgMAG1 (pMAG1), pcDNA3.1( +)-TgSAG1 (pSAG1), and pcDNA3.1( +)-TgMAG1-TgSAG1 (pMAG1-SAG1) plasmids and administered them intramuscularly to immunize mice. The levels of anti-T. gondii IgG in serum and cytokines, such as Interleukin (IL)-4, IL-10, and Interferon (IFN)-γ, in splenocytes were measured using ELISA and the respective culture supernatants. Lethal doses of T. gondii (type I) RH strain tachyzoites were administered to immunized mice, and mortality was assessed. Conversely, mice infected with low doses of tachyzoites were monitored to determine their survival rates, and parasite burden analyses of the brains and livers were conducted. The bivalent TgMAG1 and TgSAG1 DNA vaccines exhibited excellent protective immunity against toxoplasmosis in mice, with higher serum IgG and splenocyte IFN-γ release levels, longer survival days, and reduced parasite burden in the brain and liver tissues (p < 0.05). These findings provide a new perspective for the development of T. gondii vaccines.

Giardia lamblia regulates the production of proinflammatory cytokines through activating the NOD2-Rip2-ROS signaling pathway in mouse macrophages.

Giardia lamblia is a zoonotic protozoan that causes the diarrheal illness giardiasis, with the highest prevalence reported in the tropics and subtropics. Giardia is currently the most frequently identified pathogen in waterborne outbreaks in the United States. Nucleotide oligomerization domain (NOD) 1 and NOD2, intracellular NOD-like receptors, recognize pathogens to induce proinflammatory and antimicrobial responses. However, the roles of NOD1 and NOD2 signaling in Giardia infection have not yet been investigated. In the present study, the activation of NOD1 and NOD2 signaling pathways and the production of proinflammatory cytokines, reactive oxygen species (ROS) and nitric oxide in mouse macrophages stimulated with G. lamblia or parasite excretory-secretory products (ESPs) were examined. The results showed that G. lamblia and ESPs activated NOD2 and its downstream adaptor protein kinase, Receptor-interacting protein 2 (Rip2), in mouse macrophages. Blocking NOD2-Rip2 signaling significantly reduced the production of ROS and subsequently decreased the phosphorylation of nuclear factor-κB p65 and extracellular signal-regulated kinase, which in turn inhibited the production of four proinflammatory cytokines, namely, interleukin (IL)-1ß, IL-6, IL-12p40 and tumor necrosis factor-α. In summary, our results indicate that the NOD2-Rip2 signal, which is activated by G. lamblia, contributes to the production of proinflammatory cytokines and ROS in mouse macrophages.

Delayed IL-12 production by macrophages during Toxoplasma gondii infection is regulated by miR-187.

Toxoplasma gondii is an important zoonotic protozoan worldwide which infects most of warm-blooded mammals and birds, including human, and cause toxoplasmosis. As an intracellular parasite, T. gondii must evade host immune surveillance, such as IL-12 and IFN-γ, in order to survive and multiply in macrophages and other host cells. By delaying IL-12 secretion of host macrophages within 24 h after infection, T. gondii ensures not only self-survival but also the establishment of chronic infection of host cells. MicroRNA plays an important role in regulating gene transcription and translation. The mechanisms of IL-12 production during T. gondii infection are still unknown. Thus, understanding how the parasites manipulate IL-12 production by host macrophage is critical for the effective prevention and therapy of T. gondii infection. In the present study, regulation of delayed macrophage IL-12 production during T. gondii infection was explored. We found that the production of IL-12 after T. gondii infection was inhibited during the first 24 h and then resumed. The expression pattern of miR-187 production was consistent with the production pattern of IL-12 during T. gondii infection. The downregulation of miR-187 promoted Akt and P65 phosphorylation and delayed IL-12 production at late stage (after 24 h) of T. gondii infection. Dual-luciferase reporter assay indicated that MiR-187 targeted the NFKBIZ gene. Our results suggested that the delayed IL-12 production in mouse macrophages during T. gondii infection was regulated by miR-187.

Identification of GdRFC1 as a novel regulator of telomerase in Giardia duodenalis.

Telomerase plays a crucial role in ageing and tumourigenesis. However, the regulatory network of its activity is complicated and not fully understood. In the present study, a yeast two-hybrid screen identified a homologue of human replication factor C subunit 1 (RFC1) as a novel interacting protein of Giardia duodenalis GdTRBD (Giardia duodenalis telomerase ribonucleoprotein complex RNA binding domain GdTRBD). This interaction was further verified via GST pull-down in vitro and co-immunoprecipitation (Co-IP) and bimolecular fluorescence complementation (BiFC) in vivo. We also found that GdRFC1 (Giardia duodenalis replication factor C subunit 1) only interacted with GdTRBD in one nucleus in Giardia duodenalis via a proximity ligation assay (PLA). We reasoned that the two nuclei might have significant heterogeneity in their functional activities during the trophozoite stage and that the two molecules might be involved in other unidentified functions in addition to telomerase activity. In addition, knockdown of GdRFC1 decreased telomerase activity. Collectively, our results indicate that GdRFC1 is a novel binding partner and positive regulator of telomerase in Giardia duodenalis.

A Novel Anti-Microbial Peptide from Pseudomonas, REDLK Induced Growth Inhibition of Leishmania tarentolae Promastigote In Vitro.

Leishmaniasis is a prevalent cause of death and animal morbidity in underdeveloped countries of endemic area. However, there is few vaccine and effective drugs. Antimicrobial peptides are involved in the innate immune response in many organisms and are being developed as novel drugs against parasitic infections. In the present study, we synthesized a 5-amino acid peptide REDLK, which mutated the C-terminus of Pseudomonas exotoxin, to identify its effect on the Leishmania tarentolae. Promastigotes were incubated with different concentration of REDLK peptide, and the viability of parasite was assessed using MTT and Trypan blue dye. Morphologic damage of Leishmania was analyzed by light and electron microscopy. Cellular apoptosis was observed using the annexin V-FITC/PI apoptosis detection kit, mitochondrial membrane potential assay kit and flow cytometry. Our results showed that Leishmania tarentolae was susceptible to REDLK in a dose-dependent manner, disrupt the surface membrane integrity and caused parasite apoptosis. In our study, we demonstrated the leishmanicidal activity of an antimicrobial peptide REDLK from Pseudomonas aeruginosa against Leishmania tarentolae in vitro and present a foundation for further research of anti-leishmanial drugs.

Identification and characterization of Letm1 gene in Toxoplasma gondii.

Toxoplasma gondii is an obligate intracellular protozoan that causes toxoplasmosis. Previous studies have shown that the perturbation of mitochondrial metabolism in T. gondii results in growth deficiency in host cells and lack of virulence in animals. Members of this Letm1 protein family are inner mitochondrial membrane proteins which play a role in potassium and hydrogen ion exchange. Letm1 has not been characterized in T. gondii. In this study, a potential TgLetm1 gene (TgGT1_288400) with Letm1-like protein domain coding sequence was identified in T. gondii. Indirect immunofluorescence assays suggested that TgLetm1 localized to the mitochondria in tachyzoites, as indicated by the colocalization with mitochondrial marker Mitotracker. TgLetm1 was found in the membrane fraction by western blot analysis. To investigate the role of TgLetm1 in T. gondii, we generated a tetracycline-inducible TgLetm1-knock-down mutant. The conditional deletion of TgLetm1 resulted in mitochondrial swelling. Functional studies showed that the conditional deletion of TgLetm1 resulted in growth inhibition, deficiency in invasion and replication, and lack of virulence in mice.

In vitro leishmanicidal activity of antimicrobial peptide KDEL against Leishmania tarentolae.

Leishmaniasis, caused by the intracellular protozoan parasite Leishmania, remains an important neglected tropical infectious disease. Infection may be lethal if untreated. Currently, the available drugs for the disease are limited by high toxicity and drug resistance. There is an urgent need to develop novel anti-leishmanial strategies. Antimicrobial peptides (AMPs) have been described as the first-line immune defense against pathogenic microbes and are being developed as emerging anti-parasitic therapies. In the present study, we showed the anti-leishmanial activity of the synthetic 4-amino acid peptide lysine, aspartic acid, glutamic acid, and leucine (KDEL), the endoplasmic reticulum retention sequence, against Leishmania tarentolae promastigote and amastigote. Different concentrations of KDEL peptides were incubated with promastigotes, MTT viability assay, and promastigote assay were carried out. Macrophages infected with GFP-transfected L. tarentolae promastigotes were incubated with KDEL peptides, and the anti-amastigote activity of the KDEL peptides was measured by fluorescence microscopy. The damage of L. tarentolae was observed by light microscopy and electron microscopy. The cell apoptosis was analyzed using the Annexin V-FITC/PI apoptosis detection kit and mitochondrial membrane potential assay kit and by flow cytometry. Results showed that L. tarentolae was susceptible to KDEL peptides in a dose-dependent manner, and KDEL peptides disrupted the surface membrane integrity and caused cell apoptosis. In our study, we found for the first time an AMP KDEL from Pseudomonas aeruginosa and proved its significant therapeutic potential as a novel anti-leishmanial drug.

A novel detection method of Cryptosporidium parvum infection in cattle based on Cryptosporidium parvum virus 1.

Cryptosporidium parvum is an important zoonotic parasite that causes significant economic loss in the animal husbandry industry, especially the cattle industry. As there is no specific vaccine or drug against Cryptosporidium, a rapid and accurate method for the detection of C. parvum is of great significance. In this study, colloidal gold strips were developed based on Cryptosporidium parvum virus 1 (CSpV1) for the detection of C. parvum infection in cattle fecal samples. The colloidal gold solution was prepared by reducing trisodium citrate and the CSpV1 #5 monoclonal antibody was labeled with colloidal gold. A polyclonal antibody against the CSpV1 capsid protein and an anti-mouse IgG antibody were coated on the colloidal gold strips for use in the test and control lines, respectively. Our results showed that the detection sensitivity in fecal samples was up to a 1:64 dilution. There was no cross-reaction with Cryptosporidium andersoni or Giardia in the fecal samples. The different preservation conditions (room temperature, 4°C, and 37°C) and preservation time (7, 30, 60, and 90 days) were analyzed. The data showed that the strips could be preserved for 90 days at 4°C and for 60 days at room temperature or 37°C. The colloidal gold strips were used to detect the samples of 120 clinical fecal in Changchun, China. The results indicated that the rate of a positive test was 5% (6/120). This study provides a rapid and accurate method for detecting C. parvum infection in cattle and humans.

An Eimeria acervulina OTU protease exhibits linkage-specific deubiquitinase activity.

Ubiquitination is an important post-translational modification process that regulates many cellular processes. Proteins can be modified at single or multiple lysine residues by a single ubiquitin protein or by ubiquitin oligomers. It is important to note that the type of ubiquitin chains determines the functional outcome of the modification. Ubiquitin or ubiquitin chains can be removed by deubiquitinases (DUBs). In our previous study, the Eimeria tenella ovarian tumour (Et-OTU) DUB was shown to regulate the telomerase activity of E. tenella and affect E. tenella proliferation. The amino acid sequences of Et-OTU (GenBank: XP_013229759.1) and Eimeria acervulina (E. acervulina) ovarian tumour (Ea-OTUD3) DUB (XP_013250378.1) are 74% identical. Although Et-OTU may regulate E. tenella telomerase activity, whether Ea-OTUD3 affects E. acervulina growth and reproduction remains unclear. We show here that Ea-OTUD3 belongs to the OTU domain class of cysteine protease deubiquitinating enzymes. Ea-OTUD3 is highly linkage-specific, cleaving K48 (Lys48)-, K63-, and K6-linked diubiquitin but not K29-, K33-, and K11-linked diubiquitin. The precise linkage preference of Ea-OTUD3 among these three nonlinear diubiquitin chains is K6 > K48 > K63. Recombinant Ea-OTUD3, but not its catalytic-site mutant Ea-OTUD3 (C247A), exhibits activity against diubiquitin. Ea-OTUD3 removes ubiquitin from the K48-, but to a lesser extent from the K63-linked ubiquitinated E. acervulina proteins of the modified target protein, thereby exhibiting the characteristics of deubiquitinase. This study reveals that the Ea-OTUD3 is a novel functional deubiquitinating enzyme. Furthermore, the Ea-OTUD3 protein may regulate the stability of some K48-linked ubiquitinated E. acervulina proteins.

Morphological variability, molecular phylogeny, and biological characteristics of the nematophagous fungus Duddingtonia flagrans.

This study aims to investigate the molecular phylogenetic analysis, morphological variability, nematode-capturing ability, and other biological properties of Chinese Duddingtonia flagrans isolates. We isolated 13 isolates of D. flagrans and found features that have never been reported before, such as two to three septa incluing club-shaped conidia. Meanwhile, we conducted molecular phylogenetic analysis of the seven isolates and tested the radical growth of the isolates under different pH values, temperatures, and media. The capturing ability against infective larvae (L3) of Cooperia spp. in yak was detected in vitro. Finally, one isolate was selected for scanning electron microscopy (SEM) to investigate the trap formation process. The fungal sequence was obtained and submitted to GenBank (Accession no. KY288614.1, KU881774.1, KP257593.1, KY419119.1, MF488979.1, MF488980.1, and MF488981.1), and the tested isolates were identified as D. flagrans. Except for three isolates, the radial growth of the other isolates on 2% corn meal agar and 2% water agar exhibited faster growth than on other media. The fungus could not grow at 10 and 40°C but grew within 11 to 30°C. Moreover, it did not grow at pH 1-3 and 13-14, but instead at pH 4-12. In the in vitro experimental, L3s were reduced by 94.36%, 88.15%, and 91.04% for SDH035, DH055, and F088, respectively. SEM results showed that at 8 hr post addition of nematodes, some of the latter were captured. In the later stages of the interaction of the fungus with nematodes, a large number of chlamydospores were produced, especially on the predation trap. Results of the present study provided information about the molecular phylogenetic analysis, morphological variability, nematode-capturing ability, and other biological properties of Chinese Arthrobotrys flagrans isolates before administering them for biocontrol.

Peroxisome proliferator-activated receptor-γ-mediated polarization of macrophages in Neospora caninum infection.

BACKGROUND: Neospora caninum is an apicomplexan parasite closely related Toxoplasma gondii, which causes neurological disease and abortion in multiple animal species. Macrophage polarization plays an important role in host immune responses to parasites infection, such as Toxoplasma gondii, Leishmania, Trypanosoma cruzi. However, the dynamics of macrophage polarization, as well as the possible mechanism that regulate macrophage polarization, during N. caninum infection remains unclear. METHODS: The M1 and M2-phenotypic markers of peritoneal macrophages from mice infected with tachyzoites of Nc-1 were analyzed by flow cytometry (FCM) analysis. Then J774A.1 cells were respectively treated with GW9662 and RGZ, and stimulated by tachyzoites of Nc-1. M1 and M2-phenotypic markers were determined by FCM and ELISA. And the activations of PPAR-γ and NF-κB were determined by Western blotting. RESULTS: In this study, our data showed that macrophages were preferentially differentiated into the M1 type during the acute stage of N. caninum infection, while the level of M2 macrophages significantly increased during the chronic stage of infection. In vitro study, compared with the GW9662 group and RGZ group, N. caninum can promote M2-polarized phenotype through up-regulate the activity of PPAR-γ and inhibting NF-κB activation. CONCLUSION: In conclusion, this study demonstrated that macrophages are plastic since M1 differentiated macrophages can express M2 markers with N. caninum infection through up-regulating the activity of PPAR-γ and inhibting NF-κB activation and may be providing new insights for the prevention and treatment of N. caninum infection.

Identification and characterization of GRA6/GRA7 of Neospora caninum in MDBK cells.

Neospora caninum, an apicomplexan parasite, is recognized as a major bovine abortifacient. Dense granule antigens (GRAs) play important roles in the formation and modification of parasitophorous vacuoles (PVs) in Toxoplasma gondii. However, a few studies investigating GRAs have been reported in N. caninum. The aim of the present study was to characterize the dense GRA6/GRA7 of N. caninum in PVs using MDBK cells as a host cell model. Neospora caninum was inoculated into MDBK cells, and changes were observed using a transmission electron microscope (TEM). Neospora caninum GRA6/GRA7 were identified and characterized using bioinformatics, cell fractionation, and immunofluorescence. The TEM results revealed that integrated PVs were present in MDBK cells after N. caninum infection. Bioinformatics analysis showed that NcGRA6/NcGRA7 shared 28.76% and 29.66% homology with T. gondii GRA6/GRA7 (TgGRA6/TgGRA7) but had similar signal peptides, transmembrane domains, and motifs. Cell fractionation and subcellular localization analyses both showed that NcGRA6 was distributed in the lumen and intravacuolar network in soluble and transmembrane forms. The transmembrane form of NcGRA7 was observed in the PV membrane. These data lay a foundation for further study on bovine neosporosis and NcGRA6/NcGRA7 function during PV formation.

The p38 MAPK inhibitor, SB203580, inhibits cell invasion by Neospora caninum.

The Apicomplexan parasite Neospora caninum is an obligate intracellular parasitic protozoan. It can cause severe diseases in a number of animals throughout the world. Infection with N. caninum leads to abortions in pregnant animals and neuromuscular disorders of newborns which cause great economic losses to animal husbandry. However, the mechanism of cell invasion by N. caninum is still unclear. This paper aims to investigate the impact of SB203580, a p38 MAPK inhibitor, on host cell invasion by N. caninum. The results suggested the presence of putative p38 MAPK homologues in N. caninum, and incubation of N. caninum with SB203580 markedly reduced the tachyzoite motility and microneme exocytosis (NcMIC2, 3, and 6). Furthermore, treatment or pretreatment of MDBK cells with SB203580 effectively reduced cell invasion by N. caninum. Therefore, SB203580 affected both, parasites and host cells, resulting in inhibition of cell invasion by N. caninum.

Seroprevalence of Dirofilaria immitis in Cats from Liaoning Province, Northeastern China.

The present study was performed to investigate the seroprevalence and risk factors for Dirofilaria immitis infection in cats from Liaoning province, northeastern China. From October 2014 to September 2016, sera of 651 cats, including 364 domestic cats and 287 feral cats (332 females and 319 males) were assessed. They were tested for the presence of D. immitis antigen using SNAP Heartworm RT test kit. In this population, the average prevalence was 4.5%. Age and rearing conditions (feral or domestic) were found to be associated with the prevalence of D. immitis. The prevalence was significantly higher in feral cats compared with domestic cats (8.4% vs 1.4%, P<0.01). There was no significant difference between males and females (4.7% vs 4.2%, P>0.05), but older cats (≥3 years old) showed a statistically higher prevalence compared with younger cats (<3 years old) in feral populations (16.8 vs 2.4%, P<0.01), while the difference between the age groups was not statistically significant in domestic cats (2.4% vs 0.51%, P>0.05), all these results suggest that outdoor exposure time may be one of the most important factors for D. immitis prevalence in cats. Results reveal that D. immitis are prevalence in domestic and feral cats in northeastern China, which indicates that appropriate preventive measures should be taken to decrease the incidence of feline heartworm disease in Liaoning province, northeastern China.

Simple and nonradioactive detection of microRNAs using digoxigenin (DIG)-labeled probes with high sensitivity.

The discovery of microRNAs (miRNAs), which are ∼21-23 nucleotides that can regulate targeted mRNA by transcript cleavage or protein translation suppression, has changed the landscape of biomedical field greatly. At present, Northern blot analysis based on radioisotopes is still the most popular method on the detection of miRNAs for its high sensitivity. However, radioisotopes have been known for certain disadvantages, such as instability, expense, and safety; thus, developing a nonradioactive and highly sensitive method is needed. Here, we report a simple, nonradioactive, and sensitive method for miRNAs detection based on 5'-phos-3'-DIG-labeled probes prepared through splinted ligation and EDC cross-linking (DSLE). The method was more sensitive than traditional Northern blots with a DIG-labeled DNA probe and can detect as low as 2 fmol of miRNAs. The whole procedure can be completed within 6-8 h. DSLE method is very convenient, cost-effective, time-saving, and highly sensitive.