Update on feline calicivirus: viral evolution, pathogenesis, epidemiology, prevention and control.

Feline calicivirus (FCV) is a prevalent and impactful viral pathogen affecting domestic cats. As an RNA virus, FCV exhibits high mutability and genetic plasticity, enabling its persistence within cat populations. Viral genetic diversity is associated with a broad spectrum of clinical manifestations, ranging from asymptomatic infections and mild oral and upper respiratory tract diseases to the potential development of virulent systemic, and even fatal conditions. This diversity poses distinctive challenges in diagnosis, treatment, and prevention of diseases caused by FCV. Over the past four decades, research has significantly deepened understanding of this pathogen, with an emphasis on molecular biology, evolutionary dynamics, vaccine development, and disease management strategies. This review discusses various facets of FCV, including its genomic structure, evolution, innate immunity, pathogenesis, epidemiology, and approaches to disease management. FCV remains a complex and evolving concern in feline health, requiring continuous research to enhance understanding of its genetic diversity, to improve vaccine efficacy, and to explore novel treatment options.

Simultaneous detection of seven bacterial pathogens transmitted by flies using the reverse line blot hybridization assay.

BACKGROUND: Traditional methods for detecting insect-borne bacterial pathogens are time-consuming and require specialized laboratory facilities, limiting their applicability in areas without access to such resources. Consequently, rapid and efficient detection methods for insect-borne bacterial diseases have become a pressing need in disease prevention and control. METHODS: We aligned the ribosomal 16S rRNA sequences of seven bacterial species (Staphylococcus aureus, Shigella flexneri, Aeromonas caviae, Vibrio vulnificus, Salmonella enterica, Proteus vulgaris, and Yersinia enterocolitica) by DNASTAR Lasergene software. Using DNASTAR Lasergene and Primer Premier software, we designed universal primers RLB-F and RLB-R, two species-specific probes for each pathogen, and a universal probe (catch-all). The PCR products of seven standard strains were hybridized with specific oligonucleotide probes fixed on the membrane for specific experimental procedures. To evaluate the sensitivity of PCR-RLB, genomic DNA was serially diluted from an initial copy number of 1010 to 100 copies/µl in distilled water. These dilutions were utilized as templates for the PCR-RLB sensitivity analysis. Simultaneous detection of seven fly-borne bacterial pathogens from field samples by the established PCR-RLB method was conducted on a total of 1060 houseflies, collected from various environments in Lanzhou, China. RESULTS: The established PCR-RLB assay is capable of detecting bacterial strains of about 103 copies/µl for S. aureus, 103 copies/µl for S. flexneri, 105 copies/µl for A. caviae, 105 copies/µl for V. vulnificus, 100 copies/µl for S. enterica, 105 copies/µl for P. vulgaris, and 100 copies/µl for Y. enterocolitica. The results demonstrate that the detection rate of the established PCR-RLB method is higher (approximately 100 times) compared to conventional PCR. This method was applied to assess the bacterial carrier status of flies in various environments in Lanzhou, China. Among the seven bacterial pathogens carried by flies, S. enterica (34.57%), S. flexneri (32.1%), and Y. enterocolitica (20.37%) were found to be the predominant species. CONCLUSIONS: Overall, this research shows that the rapid and efficient PCR-RLB detection technology could be a useful for surveillance and therefore effective prevention and control the spread of insect-borne diseases. Meanwhile, the experimental results indicate that urban sanitation and vector transmission sources are important influencing factors for pathogen transmission.

Screening of low-toxic zinc oxide nanomaterials and study the apoptosis mechanism of NSC-34 cells.

With the increasing application of ZnO nanomaterials (ZnO-NMts) in the biomedical field, it is crucial to assess their potential risks to humans and the environment. Therefore, this study aimed to screen for ZnO-NMts with low toxicity and establish safe exposure limits, and investigate their mechanisms of action. The study synthesized 0D ZnO nanoparticles (ZnO NPs) and 3D ZnO nanoflowers (ZnO Nfs) with different morphologies using a hydrothermal approach for comparative research. The ZnO-NMts were characterized using X-ray diffraction (XRD), scanning electron microscopy (SEM), and transmission electron microscopy (TEM). Mouse brain neuronal cells (NSC-34) were incubated with ZnO NMts for 6, 12, and 24 h, and the cell morphology was observed using TEM. The toxic effects of ZnO Nfs on NSC-34 cells were studied using CCK-8 cell viability detection, reactive oxygen species (ROS) measurement, caspase-3 activity detection, Annexin V-FITC/PI apoptosis assay, and mitochondrial membrane potential (Δφm) measurement. The results of the research showed that ZnO-NMts caused cytoplasmic vacuolization and nuclear pyknosis. After incubating cells with 12.5 µg mL-1 ZnO-NMts for 12 h, ZnO NRfs exhibited the least toxicity and ROS levels. Additionally, there was a significant increase in caspase-3 activity, depolarization of mitochondrial membrane potential (Δφm), and the highest rate of early apoptosis.This study successfully identified ZnO NRfs with the lowest toxicity and determined the safe exposure limit to be < 12.5 µg mL-1 (12 h). These findings will contribute to the clinical use of ZnO NRfs with low toxicity and provide a foundation for further research on their potential applications in brain disease treatment.

Prevalence and Biological Characteristics of Listeria Species Isolated from Livestock and Poultry Meat in Gansu Province, China.

Listeria monocytogenes is a widespread foodborne pathogen contaminating foods during their production or processing stages. Fresh meat is susceptible to such contamination if it is not properly preserved. Our study was conducted to reveal the level of contamination and prevalence of Listeria spp. present in livestock and poultry meat from Gansu province. A total of 1,387 samples were collected from five cities in Gansu Province according to standard sampling procedures, of which 174 samples (12.5%) were positive for Listeria species. Among them, 14 isolates of L. monocytogenes (1.0%), 150 isolates of Listeria innocua (10.8%), and ten isolates of Listeria welshimeri (0.7%) were identified by conventional bacteriological and molecular identification methods. All isolates were subjected to serological assays, antimicrobial susceptibility tests, growth curve assays, determination of biofilm-forming capacity, and cluster analysis of the 16S rRNA gene sequences. Four predominant serotypes of L. monocytogenes were identified, including 1/2a (35.7%, 5/14), 1/2b (14.3%, 2/14), 1/2c (42.9%, 6/14), and 4b (7.1%, 1/14). All L. monocytogenes isolates were resistant to tetracycline and cefoxitin. Most L. innocua isolates (63.6%, 14/22) and L. welshimeri (40%, 4/10) were resistant to tetracycline. The high biofilm-forming ability was observed among 1/2c and 1/2a serotype isolates. The cluster analysis of the 16S rRNA gene sequences revealed a close genetic relationship between the three Listeria species. This study fills the gap in the knowledge of livestock and poultry meat that carry Listeria in slaughterhouses and markets in Gansu Province.

Transcriptomic analysis of Listeria monocytogenes biofilm formation at different times.

Biofilm (BF) formation is a considerable obstacle to the effective control of Listeria monocytogenes (LM). In this study, we used transcriptomics to analyze LM BF and planktonic bacteria at different stages of BF formation and growth to compare differential gene expression between the 2. We identified 1588, 1517, and 1462 differentially expressed genes (DEGs) when early formation BF and planktonic bacteria were compared at 12, 24, and 48 h, respectively. Among these, 1123 DEGs were shared across the 3 data pool. Gene Ontology functional enrichment and Kyoto Encyclopedia of Genes and Genomes pathway analyses demonstrated significant changes associated with the phosphotransferase system, the microbial metabolism in diverse environments, the flagella assembly, the bacterial chemotaxis, the bacterial secretion, the quorum sensing, and the 2-component system. The top 5 upregulated DEGs were lmo0024, lmo0374, lmo0544, hly, and lmo2434. The top 5 downregulated DEGs were lmo2192, lmo1211, cheY, lmo0689, and secY. After real-time quantitative polymerase chain reaction, the expression of these 10 DEGs were consistent with the results of the transcriptomic sequence. This research lays the foundation for further studies on mechanisms regulating BF formation and will help to identify BF inhibitors to reduce the risk of LM infection.

La formation de biofilm (BF) est un obstacle considérable à la maîtrise efficace de Listeria monocytogenes (LM). Dans cette étude, nous avons utilisé la transcriptomique pour analyser le BF et les bactéries planctoniques de LM à différents stades de la formation et de la croissance du BF afin de comparer l'expression différentielle des gènes entre les deux. Nous avons identifié 1588, 1517 et 1462 gènes exprimés de manière différentielle (DEGs) lors de la formation précoce du BF et les bactéries planctoniques ont été comparées à 12, 24 et 48 h, respectivement. Parmi ceux-ci, 1123 DEGs ont été partagés entre les trois pools de données. L'enrichissement fonctionnel de l'ontologie génique et les analyses des voies de l'Encyclopédie des gènes et des génomes de Kyoto ont démontré des changements significatifs associés au système de phosphotransférase, au métabolisme microbien dans divers environnements, à l'assemblage des flagelles, à la chimiotaxie bactérienne, à la sécrétion bactérienne, à la détection du quorum et au système à deux composants. Les cinq principaux DEGs régulés à la hausse étaient lmo0024, lmo0374, lmo0544, hly et l mo2434. Les 5 principaux DEGs régulés à la baisse étaient lmo2192, lmo1211, cheY, lmo0689 et secY. Après réaction d'amplification en chaîne par la polymérase quantitative en temps réel, l'expression de ces dix DEGs était cohérente avec les résultats du séquence transcriptomique. Cette recherche jette les bases d'études ultérieures sur les mécanismes régulant la formation de BF et aidera à identifier les inhibiteurs de BF pour réduire le risque d'infection LM.(Traduit par Docteur Serge Messier).

Effect of matrine in MAC-T cells and their transcriptome analysis: A basic study.

Matrine, an alkaloid derived from herbal medicine, has a wide range of biological activities, including antibacterial. Matrine was toxic to multiple cells at high concentrations. Bovine mammary epithelial cells (MAC-T) could be used as model cells for cow breast. Matrine was a feasible option to replace antibiotics in the prevention or treatment of mastitis against the background of prohibiting antibiotics, but the safe concentration of matrine on MAC-T cells and the mechanism of action for matrine at different concentrations were still unclear. In this study, different concentrations of matrine (0.5, 1, 1.5, 2, 2.5 and 3 mg/mL) were used to treat MAC-T cells for various time periods (4, 8, 12, 16 and 24 h) and measure their lactic dehydrogenase (LDH). And then the optimal doses (2 mg/mL) were chosen to detect the apoptosis at various time periods by flow cytometry and transcriptome analysis was performed between the control and 2 mg/mL matrine-treated MAC-T cells for 8 hours. The results showed that matrine was not cytotoxic at 0.5 mg/mL, but it was cytotoxic at 1~3 mg/mL. In addition, matrine induced apoptosis in MAC-T cells at 2 mg/mL and the proportion of apoptosis cells increases with time by flow cytometry. RNA-seq analysis identified 1645 DEGs, 676 of which were expressed up-regulated and 969 were expressed down-regulated. The Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis indicated the following pathways were linked to matrine-induced toxicity and apoptosis, including cytokine-cytokine receptor interaction pathway, viral protein interaction with cytokine and cytokine receptor, P53 and PPAR pathway. We found 7 DEGs associated with matrine toxicity and apoptosis. This study would provide a basis for the safety of matrine in the prevention or treatment of mastitis.

Characterization and Preliminary Application of Phage Isolated From Listeria monocytogenes.

Listeria monocytogenes (LM) is one of the four major foodborne bacteria that cause bacteremia and meningitis. To explore the control of listeriosis with natural phages, we used the double-layer agar plate method to isolate LM from slaughterhouse sewage and designated LP8. The result of electron microscopy indicated that the phage belonged to the family of Myoviridae. Whole-genome sequencing indicated that the genome size of LP8 is 87,038 bp and contains 120 genes. Mice were infected with LM and treated with penicillin G sodium, LP8, and the combination of these two. From the levels of lymphocyte subsets (CD4+, CD8+), the expression of cytokines (TNF-α, IL1ß, IL-10, and IFN-γ), observation of pathological changes in organs (heart, liver, spleen, kidney, and brain), and the bacterial load of the spleen, we concluded the therapeutic effect of LP8 against listeriosis and demonstrate the feasibility of a combined therapy to reduce the use of antibiotics. This provides a new avenue for the treatment of listeriosis.

Effect of Two Different Drug-Resistant Staphylococcus aureus Strains on the Physiological Properties of MAC-T Cells and Their Transcriptome Analysis.

Staphylococcus aureus (S. aureus) is one of the main pathogens causing mastitis in dairy cows. The current work mainly focuses on the pathway of apoptosis induction in MAC-T cells caused by S. aureus infection or other factors. However, the physiological characteristics of S. aureus infected MAC-T cells and the resulting mRNA expression profile remain unknown particularly in the case of diverse drug resistant strains. Methicillin-resistant S. aureus (MRSA) and methicillin-susceptible S. aureus (MSSA) strains were used to infect MAC-T cells to investigate this issue. The adhesion, invasion and apoptosis ability of MRSA-infected group and MSSA-infected group was assessed over time (2, 4, 6, 8, and 12 h). After 8 h, the RNA sequencing was conducted on the MRSA-infected and the MSSA-infected with uninfected MAC-T cells as controls. The results showed that the adhesion and invasion ability of MRSA-infected and MSSA-infected to MAC-T cells increased and then decreased with infection time, peaking at 8 h. The adhesion and invasion rates of the MSSA-infected were substantially lower than those of the MRSA-infected, and the invasion rate of the MSSA-infected group was nearly non-existent. Then the apoptosis rate of MAC-T cells increased as the infection time increased. The transcriptome analysis revealed 549 differentially expressed mRNAs and 390 differentially expressed mRNAs in MRSA-infected and MSSA-infected MAC-T cells, respectively, compared to the uninfected MAC-T cells. According to GO analysis, these differentially expressed genes were involved in immune response, inflammation, apoptosis, and other processes. The Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis indicated the following pathways were linked to adhesion, invasion inflammation and apoptosis, including AMPK, FOXO, HIF-1, IL-17, JAK-STAT, MAPK, mTOR, NF-κB, p53, PI3K-Akt, TNF, Toll-like receptor, Rap1, RAS, prion disease, the bacterial invasion of epithelial cells pathway. We found 86 DEGs from 41 KEGG-enriched pathways associated with adhesion, invasion, apoptosis, and inflammation, all of which were implicated in MAC-T cells resistance to MRSA and MSSA infection. This study offers helpful data toward understanding the effect of different drug-resistant S. aureus on dairy cow mammary epithelial cells and aid in the prevention of mastitis in the dairy industry.

The Characteristics and Function of Internalin G in Listeria monocytogenes.

In order to clarified characteristics and function of internalin G (inlG) in Listeria monocytogenes ATCC®19111 (1/2a) (LM), the immune protection of the inlG was evaluated in mice, the homologous recombination was used to construct inlG deletion strains, and their biological characteristics were studied by the transcriptomics analysis. As a result, the immunization of mice with the purified protein achieved a protective effect against bacterial infection. The deletion strain LM-AinlG was successfully constructed with genetic stability. The mouse infection test showed that the virulence of LM was decreased after the deletion of the inlG gene. The deletion strain showed enhanced adhesion to and invasion of Caco-2 cells. Compared to the wild strain, 18 genes were up-regulated, and 24 genes were down-regulated in the LM-AinlG. This study has laid a foundation for further research on the function of inlG and the pathogenesis of LM. In this study, immunization of mice with the purified inlG protein achieved a protective effect against Listeria monocytogenes infection. The virulence of LM-ΔinlG was decreased by mouse infection. However, the adhesion and invasion ability to Caco-2 cell were enhanced. Compared to the wild strain, 18 genes were up-regulated, and 24 genes were down-regulated in the LM-ΔinlG. This study has laid a foundation for further study of the function of the inlG and the listeriosis.

Molecular Characterization of Hard Ticks by Cytochrome c Oxidase Subunit 1 Sequences.

Although widely studied, the natural diversity of the hard tick is not well known. In this study, we collected 194 sequences from 67 species, covering 7 genera of hard tick. The 5' region of the mitochondrial cytochrome c oxidase subunit 1 region (586 bp) has been used to investigate intra- and inter-species variation and the phylogenetic tree of neighbor joining method has been used for assessment. As a result, by comparing the K2P-distance of intra- and interspecies, 30 samples (15.2%) shown that interspecies distance was larger than the minimum interspecfic distance. From the phylogenetic analysis, 86.8% (49) of the species were identified correctly at the genus level. On deeper analysis on these species suggested the possibility of presence cryptic species. Therefore, further work is required to delineate species boundaries and to develop a more complete understanding of hard tick diversity over larger scale.

Identification and characterization of Tu88, an antigenic gene from Theileria uilenbergi.

Theileria uilenbergi is a pathogen that causes ovine theileriosis. Prevention and control of theileriosis relies on its diagnosis at early stages of occurrence and requires understanding of proteins with antigenic properties from the pathogen. Despite its prevalence in China, only a few molecules with antigenic properties have been characterized from T. uilenbergi. In this study, we identified a cDNA named Tu88 by immunoscreening a T. uilenbergi merozoite cDNA library with T. uilenbergi-positive sera from infected sheep. Recombinant Tu88 (rTu88) expressed in bacteria reacted strongly with the positive sera of T. uilenbergi in western blot analysis indicating its potential as an antigen. Southern blot analysis showed that it is a single copy gene. Protein localization by immunostaining blood smears from an infected sheep demonstrated the presence of native Tu88 in merozoites. These findings suggest that Tu88 is a potential candidate antigen for the development of a sero-diagnostic tool.

Phylogenetic analysis of ruminant Theileria spp. from China based on 28S ribosomal RNA gene.

Species identification using DNA sequences is the basis for DNA taxonomy. In this study, we sequenced the ribosomal large-subunit RNA gene sequences (3,037-3,061 bp) in length of 13 Chinese Theileria stocks that were infective to cattle and sheep. The complete 28S rRNA gene is relatively difficult to amplify and its conserved region is not important for phylogenetic study. Therefore, we selected the D2-D3 region from the complete 28S rRNA sequences for phylogenetic analysis. Our analyses of 28S rRNA gene sequences showed that the 28S rRNA was useful as a phylogenetic marker for analyzing the relationships among Theileria spp. in ruminants. In addition, the D2-D3 region was a short segment that could be used instead of the whole 28S rRNA sequence during the phylogenetic analysis of Theileria, and it may be an ideal DNA barcode.

Coevolutionary analyses of the relationships between piroplasmids and their hard tick hosts.

Host-parasite coevolution is a key driver of biological diversity. To examine the evolutionary relationships between piroplasmids and their hard tick hosts, we calculated the molecular clock and conducted phylogenetic analyses of both groups. Based on our results, we conclude that the divergence time of piroplasmids (∼56 Mya) is later than divergence time of their hard tick hosts (∼86 Mya). From analyses of the evolution of both piroplasmid and vector lineages and their association, we know that hard ticks transmit piroplasmids with high genus specificity and low species specificity.

The life cycle of Haemaphysalis qinghaiensis (Acari: Ixodidae) ticks under laboratory conditions.

The developmental stages in the life cycle of Haemaphysalis qinghaiensis were investigated under laboratory conditions. The larval, nymphal and adult ticks were fed on sheep at 25-27 °C, 50 % relative humidity (RH) and exposed to daylight. All free-living stages were maintained in an incubator (28 °C with 90 % RH and a 12-h photoperiod). The whole life cycle of H. qinghaiensis was completed in an average of 176 days (range 118-247 days). The average developmental periods were 34.44 days for egg incubation; 5.83, 4.20 and 33.70 days for larval pre-feeding, feeding and pre-molting; and 3.88, 5.30 and 46.50 days for nymphal pre-feeding, feeding and pre-molting. The average times for pre-feeding, feeding, pre-oviposition and oviposition of female adult ticks were 2.60, 11.40, 8.50, and 19.35 days, respectively. The results confirmed the positive correlation between the weight of the engorged female and the egg mass laid (r = 0.557, P < 0.05). The reproductive efficiency index and reproductive fitness index in females were 5.49 and 4.98, respectively. Engorged nymphs moulting to females (4.53 ± 0.16 mg) were significantly heavier (P < 0.001) than those moulting to males (3.45 ± 0.19 mg). The overall sex ratio of the adult ticks was 1:1.1 (M:F).

Rapid identification and differentiation of Theileria sergenti and Theileria sinensis using a loop-mediated isothermal amplification (LAMP) assay.

The present study developed and validated a species-specific loop-mediated isothermal amplification (LAMP) assay for the rapid detection and discrimination of two benign bovine Theileria species -T. sergenti and T. sinensis. The LAMP assay is inexpensive and easy to perform and involves a rapid reaction-the amplification can be performed in 55 min or 50 min under isothermal conditions of 61°C or 63°C, respectively, by employing a set of four species-specific primer mixtures. The results can be checked using agarose gels. The optimal assay conditions, under which the assay exhibited with no cross-reaction with other closely related tick-borne parasites (T. annulata, Babesia bovis, B. bigemina, B. major, B. ovata, B. U. sp., Anaplasma marginale) or between the two Theileria species of interest, was established. The assay is approximately 10-fold more sensitive than the conventional specific PCR assay. The LAMP assay was validated using DNA from 6 standard stocks in the laboratory and was evaluated for its diagnostic utility using blood samples collected from experimentally and naturally infection cattle or yaks in China. These findings indicate that this Theileria species-specific LAMP assay may have potential clinical applications for the detection and differentiation of two benign bovine Theileria species -T. sergenti and T. sinensis, especially in endemic countries.

Phylogenetic analysis based on 28S rRNA of Babesia spp. in ruminants in China.

Molecular phylogenetic analyses are mainly based on the small ribosomal RNA subunit (18S rRNA), internal transcribed spacer regions, and other molecular markers. We compared the phylogenetic relationships of Babesia spp. using large subunit ribosomal RNA, i.e., 28S rRNA, and the united 28S + 18S rRNA sequence fragments from 11 isolates of Babesia spp. collected in China. Due to sequence length and variability, the 28S rRNA gene contained more information than the 18S rRNA gene and could be used to elucidate the phlyogenetic relationships of B. motasi, B. major, and B. bovis. Thus, 28S rRNA is another candidate marker that can be used for the phylogenetic analysis of Babesia spp. However, the united fragment (28S + 18S) analysis provided better supported phylogenetic relationships than single genes for Babesia spp. in China.

Loop-mediated isothermal amplification (LAMP) method based on two species-specific primer sets for the rapid identification of Chinese Babesia bovis and B. bigemina.

Bovine babesiosis is a tick-transmitted hemoprotozoan disease that is mainly caused by Babesia bovis and/or Babesia bigemina and is characterized by significant morbidity and mortality worldwide. This disease is widespread in most parts of China. However, it is difficult to rapidly discriminate between the B. bovis and B. bigemina species. To detect and distinguish these species, a loop-mediated isothermal amplification (LAMP) platform that targets specific sequences of the internal transcribed spacer (ITS) genes was developed. Specificity testing revealed that there was no cross-reaction with the other tick-borne parasites B. ovate, B. major, unnamed bovine Babesia, Theileria annulata, Theileria sinensis, Theileria sergenti, and Anaplasma marginale, or with bovine white blood cells. The sensitivity of the LAMP method was 0.1 pg DNA for both B. bovis and B. bigemina, which was superior to that of the classical PCR methods. This assay was evaluated for its diagnostic utility using blood samples collected from experimentally and naturally infected cattle in China. These findings indicate that the Babesia species-specific LAMP assay may have potential clinical application in the detection and differentiation of Babesia species, particularly in countries in which babesiosis is endemic.

A DNA barcode for Piroplasmea.

Due to the difficulty in morphological identification the development of reliable molecular tools for species distinction is a priority for piroplasma. Previous studies based on 18S rRNA and other gene sequences provided a backbone for the phylogeny of piroplasma. However, it is difficult to discriminate species in a comprehensive sample. Here, the abilities of eight DNA regions including 18S rRNA, 28S rRNA, internal transcribed spacer (ITS) regions and COI genes, have been compared as candidates of DNA barcodes for piroplasma. In total, 484 sequences of piroplasma were collected from this study and GenBank. The eight proposed DNA regions were evaluated according to the criterion of Consortium for the Barcode of Life (CBOL). From this evaluation, ITS2 had 100% PCR amplification efficiency, an ideal sequence length, the largest gap between the intra- and inter-specific divergence, 98% identification efficiency at the genus level, and 92% at the species level. Thus, we propose that ITS2 is the most ideal DNA barcode based on the current database for piroplasma.

Loop-mediated isothermal amplification (LAMP) assays for the detection of Theileria annulata infection in China targeting the 18S rRNA and ITS sequences.

We have developed two loop-mediated isothermal amplification (LAMP) assays for the detection of Theileria annulata, an economically important cattle disease in China that occurs in subtropical and tropical areas. These assays target the ribosomal RNA (18S rRNA) and ITS LAMP sequences. The primer set for each gene target consists of four primers, and each set recognizes six distinct regions on the target gene to allow for the highly specific detection of T. annulata. The specific ladder bands were amplified from the autologous genomic DNA of four Chinese-laboratory-preserved standard T. annulata stocks, and there were no cross-reactions with the genomic DNA of normal bovine blood and other protozoan species. The LAMP assays were sufficiently sensitive to detect 0.1 pg/µl of genomic DNA. Furthermore, DNA extracted from blood collected from cattle experimentally infected with T. annulata (18-105 days post-infection) was amplified, demonstrating the high sensitivity of these primers. Of the 351 field samples collected from China, 24.5% were positively detected by two LAMP primers, and 18.2% were found to be positive for T. annulata infection by PCR. These results indicate that the LAMP assay could be a potential diagnostic tool for epidemiological studies of T. annulata infection in China.

Virulence of Beauveria bassiana, Metarhizium anisopliae and Paecilomyces lilacinus to the engorged female Hyalomma anatolicum anatolicum tick (Acari: Ixodidae).

The tick is a common ectoparasite of livestock and humans, and is responsible for the transmission of pathogens among hosts. Direct and indirect impacts of ticks include limiting the sustainable development of the animal husbandry industry and detrimental effects on human health. Despite these negative effects, the main method of controlling ticks remains the application of chemical acaricides, which can lead to ambient pollution and the development of tick resistance to them. The biocontrol of ticks is one of the alternative control methods that has received recent research attention. The present study used Tenebrio moliter bait methods to collect 13 species of entomopathogenic fungi from different areas in China that were then tested to observe their effects on engorged female Hyalomma anatolicum anatolicum ticks. The results showed that more than half of the isolates had some pathogenic effects on the ticks; in particular, two Beauveria bassiana strains (B.bAT01, B.bAT17) and one Metarhizium anisopliae strain (M.aAT26) were highly virulent, causing up to 90% mortality. In addition, H. anatolicum anatolicum females were treated with B. bassiana B.bAT17 using different concentrations of the fungus. Results revealed that B. bassiana B.bAT17 is highly pathogenic against engorged H. anatolicum anatolicum females. This is the first report of the pathogenic effect of entomopathogenic fungi on engorged H. anatolicum anatolicum females. However, studies of the efficiency of this fungus against ticks in the field are required before it can be used for tick management in practice.