Molecular cloning, identification, transcriptional analysis, and silencing of enolase on the life cycle of Haemaphysalis longicornis (Acari, Ixodidae) tick.

Ticks, blood-sucking ectoparasites, spread diseases to humans and animals. Haemaphysalis longicornis is a significant vector for tick-borne diseases in medical and veterinary contexts. Identifying protective antigens in H. longicornis for an anti-tick vaccine is a key tick control strategy. Enolase, a multifunctional protein, significantly converts D-2-phosphoglycerate and phosphoenolpyruvate in glycolysis and gluconeogenesis in cell cytoplasm. This study cloned a complete open reading frame (ORF) of enolase from the H. longicornis tick and characterized its transcriptional and silencing effect. We amplified the full-length cDNA of the enolase gene using rapid amplification of cDNA ends. The complete cDNA, with an ORF of 1,297 nucleotides, encoded a 432-amino acid polypeptide. Enolase of the Jeju strain H. longicornis exhibited the highest sequence similarity with H. flava (98%), followed by Dermacentor silvarum (82%). The enolase motifs identified included N-terminal and C-terminal regions, magnesium binding sites, and several phosphorylation sites. Reverse transcription-polymerase chain reaction (RT-PCR) analysis indicated that enolase mRNA transcripts were expressed across all developmental stages of ticks and organs such as salivary gland and midgut. RT-PCR showed higher transcript levels in syn-ganglia, suggesting that synganglion nerves influence enolase,s role in tick salivary glands. We injected enolase double-stranded RNA into adult unfed female ticks, after which they were subsequently fed with normal unfed males until they spontaneously dropped off. RNA interference significantly (P<0.05) reduced feeding and reproduction, along with abnormalities in eggs (no embryos) and hatching. These findings suggest enolase is a promising target for future tick control strategies.

First Evidence of Fowl Adenovirus Induced Inclusion Body Hepatitis in Chicken in Bangladesh.

Background: The livestock sector contributes 1.90% to the GDP in Bangladesh during 2021-22. Poultry is one of the important subsectors struggling with diseases. Fowl adenoviruses (FAdVs) cause numerous diseases resulting in economic losses to the poultry industry worldwide. Several FAdV serotypes cause inclusion body hepatitis in chicken. Although FAdV infection was suspected, there was no confirmatory report from Bangladesh. The study was conducted to investigate the FAdV infection and antibodies in chicken. Methods: A total of 50 samples, each composed of liver and spleen, were collected from different chickens of Gazipur, Dinajpur, and Panchagarh district. Each location belongs to A, B, and C poultry zones of Bangladesh, respectively. Viruses were detected by real-time PCR and conventional PCR. Blood samples (n = 303) were collected at the beginning and after the recovery from infection and tested by indirect ELISA. Sequencing of PCR products was done for serotyping and phylogenetic analysis. Results: Clinical signs were observed including anorexia, drowsiness, ruffled feathers, reduced body weight, lack of uniformity, and high mortality (15-25%). Enlarged friable liver with yellow to tan color mottled with the focal soft area, fluid in pericardial sac, swollen and hemorrhagic kidneys, enlarged congested spleen and pancreas, etc. were found on postmortem examination. FAdVs were detected in 90% of the flocks except commercial layer flock from Dinajpur. Three serotypes, namely, 8b (70%), 11 (10%), and 5 (10%) were detected. Anti-FAdV antibody was detected in 80% flocks at the beginning of infection and in 90% of the flocks after recovery from infection. The antibody titer increases significantly (p < 0.05) after recovery from infection. Phylogenetic analysis revealed that the Bangladeshi FAdVs have close identity with viruses from Asia, Europe, and South and North America. Conclusions: These findings suggested that several introductions of FAdVs were taken place in Bangladesh. To combat the disease, vaccination along with maintenance of biosecurity is essential.

The pharmacological and biological roles of eriodictyol.

Eriodictyol is a flavonoid in the flavanones subclass. It is abundantly present in a wide range of medicinal plants, citrus fruits, and vegetables that are considered to have potential health importance. Having the considerable medicinal properties, eriodictyol has been predicted to clarify the mode of action in various cellular and molecular pathways. Evidence for the existing therapeutic roles of eriodictyol includes antioxidant, anti-inflammatory, anti-cancer, neuroprotective, cardioprotective, anti-diabetic, anti-obesity, hepatoprotective, and miscellaneous. Therefore, this review aims to present the recent evidence regarding the mechanisms of action of eriodictyol in different signaling pathways in a specific disease condition. In view of the immense therapeutic effects, eriodictyol may serve as a potential drug source to enhance community health standards.

Molecular cloning, expression and impact of ribosomal protein S-27 silencing in Haemaphysalis longicornis (Acari: Ixodidae).

Ticks, obligatory blood-feeding arthropods, are a major pathogen vector in humans and animals worldwide. Anti-tick vaccines are an exciting alternative to chemical acaricides for controlling these disease-transmitting vectors. However, identification of protective antigens for anti-tick vaccine development is challenging. Different ribosomal proteins play multifunctional roles in tick survival and feeding. Here, we first report the cloning and molecular characterization of ribosomal protein S27 (RPS-27) from the hard tick Haemaphysalis longicornis. We identified a complete open reading frame (ORF) of RPS-27: a 255-bp (base pair) cDNA encoding a mature protein of 84 amino-acid residues with a 9.4-kDa predicted molecular mass. Amino-acid sequence analysis revealed that RPS-27 was highly conserved among different tick and vertebrate animals with identity ranges of 97-98% and 60-85%, respectively. Phylogenetic tree analysis showed that RPS-27 from different tick species clustered together. Reverse transcription-polymerase chain reaction (RT-PCR) analysis showed that the RPS-27 mRNA transcript was expressed in all life stages. At the tissue level, it was more highly expressed in the salivary gland than in the midgut for both the fed and unfed conditions, which indicates a role for RPS-27 in tick feeding. In vitro analysis showed that recombinant RPS-27 (10-RPS-27) was successfully expressed in a pGEMEX-2 vector with an estimated 45-kDa molecular mass. The functional importance of RPS-27 was determined by gene silencing through RNA interference (RNAi). RPS-27 silencing showed a significant (P < 0.05) reduction of feeding abilityand engorgement weight after the blood meal in both nymph and adult female ticks and also significantly (P < 0.05) reduced molting rate in nymph. In addition, RPS-27 silencing in eggs led to abnormalities in shape and hatching. Taken together, our results suggest that RPS-27 is an important molecule that plays multiple roles in the tick life cycle including in both feeding and reproduction. Therefore, RPS-27 is an exciting target for future tick control strategies.

Molecular cloning and transcriptional and functional analysis of glycogen synthase kinase-3ß in Haemaphysalis longicornis (Acari, Ixodidae).

Glycogen synthase kinase 3 (GSK-3), which belongs to the serine/threonine kinase family, regulates glycogen metabolism, Wnt signaling, hormonal regulation, and embryonic development in many eukaryotes. Here, we cloned a complete open reading frame (ORF) of glycogen synthase kinase 3ß (GSK-3ß) from Haemaphysalis longicornis and characterized its transcriptional and functional status. The ORF of GSK-3ß possesses 1242 nucleotides encoding a mature protein of 413 amino acid residues. GSK-3ß nucleotide and protein sequences are highly conserved among different vertebrate and invertebrate animals, with identity between 47.8-100% and 63.2-88.7%, respectively. Sequence comparison showed one signature domain between the residues of 51 and 335 amino acids, which was identified as a protein kinase (serine/threonine). RT-PCR showed GSK-3ß mRNA present in all developmental stages of H. longicornis. Interestingly, a higher transcript level was observed in nymph and 7-day-old eggs compared with others by real-time PCR, indicating a role of GSK-3ß in the early stages of life. The functional status of GSK-3ß was characterized by RNA interference (RNAi) and caused significant (p < 0.05) reduction in feeding and reproduction, as well as an abnormality in eggs and hatching. Taken together, our results suggest that GSK-3ß may be an important candidate for a multiple antigen vaccine for controlling the tick population.

Impact of Subolesin and Cystatin Knockdown by RNA Interference in Adult Female Haemaphysalis longicornis (Acari: Ixodidae) on Blood Engorgement and Reproduction.

Currently, multi-antigenic vaccine use is the method of choice for the strategic control of ticks. Therefore, determining the efficacy of combined antigens is a promising avenue of research in the development of anti-tick vaccines. The antigen responsible for blood intake and reproduction has proven suitable as a vaccine antigen. It has been shown to silence Haemaphysalis longicornis salivary cystatin (HlSC-1) and subolesin by RNA interference. Adult unfed female ticks were injected with double-stranded RNA of (A) subolesin, (B) cystatin, (C) subolesin plus cystatin, and (D) injection buffer, then fed alongside normal unfed males up to spontaneous drop-down. The percentage of knockdowns was determined by real-time polymerase chain reaction. Sixty-three percent and 53% knockdown rates were observed in subolesin and cystatin double-stranded RNA-injected ticks respectively, while 32 and 26% knockdown rates of subolesin and cystatin transcript were observed in subolesin plus cystatin double-stranded RNA-injected ticks. Subolesin and/or cystatin knockdown causes a significant (p < 0.05) reduction in tick engorgement, egg mass weight, and egg conversion ratio. Most importantly, combined silencing did not act synergistically, but caused a similarly significant (p < 0.05) reduction in tick engorgement, egg mass weight, and egg conversion ratio. Therefore, the elucidation of multiple antigens may be helpful in the future of vaccines.