Epidemiological and phylogenetic investigation of Babesia spp. in dogs of Hainan Province/Island, China.

Babesiosis is a significant tick-borne disease, which is globally prevalent. Many previous research studies have discussed the presence of Babesia gibsoni, Babesia vogeli, and Babesia canis in dogs in China. In the present study, we have used distinct molecular approaches to detect the presence of Babesia spp. in dogs of Hainan Province/Island, China. A total of 1106 dog blood samples were collected from the Island, of which 61 dog samples were found to be positive for Babesia vogeli. The highest infection rate was 56.7% (17/30) detected from Tunchang, followed by 25.0% (3/12) from Baisha and 10.4% (5/48) from Wenchang. There was only one positive case of Babesia gibsoni, and the infection rate was found to be 0.1% (1/1106). The sequencing results showed that the subjected sample sequences were identical and resembled the Babesia vogeli and Babesia gibsoni sequences available in the database. The results derived from this study will be helpful for planning effective strategies for the treatment, control, and prevention of babesiosis in dogs of Hainan Province/Island.

Molecular identification and functional analysis of Niemann-Pick type C2 proteins，carriers for semiochemicals and other hydrophobic compounds in the brown dog tick, Rhipicephalus linnaei.

Ticks are important vectors of many pathogens with tremendous impact on human and animal health. Studies of semiochemical interactions and mechanisms underlying chemoreception can provide important tools in tick management. Niemann-Pick type C2 (NPC2) proteins have been proposed as one type of chemoreceptor in arthropods. Here, we cloned two NPC2 genes in the brown dog tick, Rhipicephalus linnaei, the tropical lineage previously named R. sanguineus sensu lato and characterized them functionally. R.linNPC2a and R.linNPC2b genes were found to be expressed at each developmental stage with the highest level in adult males. By using quantitative real-time PCR we revealed expression in multiple tissues, including midgut, ovary, salivary glands and legs. Ligand binding analysis revealed that R.linNPC2b bound a wide spectrum of compounds, with ß-ionone, α-amylcinnamaldehyde, 2-nitrophenol and benzaldehyde displaying the strongest binding affinity (Ki < 10 µM), whereas R.linNPC2a showed a more narrow ligand binding range, with intermediate binding affinity to α-amylcinnamaldehyde and 2-nitrophenol (Ki < 20 µM). Molecular docking indicated that the amino acid residue Phe89, Leu77 and Val131 of R.linNPC2a and Phe70, Leu132 and Phe73 of R.linNPC2b could bind multiple ligands. These residues might thus play a key role in the identification of the volatiles. Our results contribute to the understanding of olfactory mechanisms of R. linnaei and can offer new pathways towards new management strategies.

An innovative and user-friendly smartphone-assisted molecular diagnostic approach for rapid detection of canine vector-borne diseases.

Present-day diagnostic tools and technologies for canine diseases and other vector-borne parasitic diseases hardly meet the requirements of an efficient and rapid diagnostic tool, which can be suitable for use at the point-of-care in resource-limited settings. Loop-mediated isothermal amplification (LAMP) technique has been always a method of choice in the development and validation of quick, precise, and sensitive diagnostic assays for pathogen detection and to reorganize point-of-care (POC) molecular diagnostics. In this study, we have demonstrated an efficient detection system for parasitic vector-borne pathogens like Ehrlichia canis and Hepatozoon canis by linking the LAMP assay to a smartphone via a simple, inexpensive, and a portable "LAMP box," All the components of the LAMP box were connected to each other wirelessly. This LAMP box was made up of an isothermal heating pad mounted below an aluminum base which served as a platform for the reaction tubes and LAMP assay. The entire setup could be connected to a smartphone via an inbuilt Wi-Fi that allowed the user to establish the connection to control the LAMP box. A 5 V USB power source was used as a power supply. The sensitivity of the LAMP assay was estimated to be up to 10-6 dilution limit using the amplified, purified, and quantified specific DNA templates. It can also serve as an efficient diagnostic platform for many other veterinary infectious or parasitic diseases of zoonotic origin majorly towards field-based diagnostics.

The prevalence of canine dirofilariasis in China: a systematic review and meta-analysis.

BACKGROUND: Dirofilariasis, the disease caused by Dirofilaria spp., and in particular by Dirofilaria immitis and Dirofilaria repens in canines, occurs frequently in canids and felids, and occasionally in humans, in temperate, sub-tropical and tropical regions globally. Although highly effective, safe and convenient preventive medicines have been available for the treatment of dirofilariasis for the past three decades, the disease remains a major veterinary and public health concern in endemic areas. The insect vectors, host-parasite relationships and interactions of Dirofilaria spp. have received little attention in China, and there is very little information in English regarding the prevalence of dirofilariasis in animals and humans in the country. The aim of this systematic review and meta-analysis is to evaluate the status of canine dirofilariasis in China based on the available literature in English and in Chinese. METHODS: We systematically searched five databases for epidemiologic studies on the prevalence of canine dirofilariasis in China and finally selected 42 studies eligible for inclusion in the systematic review and meta-analysis. The meta-analysis was performed using the random effects model in the meta package in R v4.2.1. RESULTS: The random effects model gave a pooled and weighted prevalence of Dirofilaria infection among dogs in China in the past 100 years of 13.8% (2896/51,313, 95% confidence interval 8.2-20.4%) with a high level of heterogeneity (I2 =  99.5%). CONCLUSIONS: Our analyses indicated that the prevalence of canine dirofilariasis in China has gradually declined, but that the range of Dirofilaria spp. has expanded. Older and outdoor dogs presented a higher rate of positive infection. The findings indicated that more attention should be paid to host factors for the effective control and management of this disease.

Molecular Detection and Phylogenetic Characterization of Anaplasma spp. in Dogs from Hainan Province/Island, China.

Anaplasmosis is a serious infection which is transmitted by ticks and mosquitos. There are very few reports and studies that have been carried out to understand the prevalence, distribution, and epidemiological profile of Anaplasma spp. infection in dogs in Hainan province/island. In the present study, we have tried to understand the prevalence, distribution, and occurrence of Anaplasma spp. infections in dogs (n = 1051) in Hainan Island/Province to establish a surveillance-based study. The confirmed positive samples by Polymerase chain reaction (PCR) were subjected to capillary sequencing for further strain-specific confirmation, followed by the construction of phylogenetic trees to determine their genetic relations. Various statistical tools were used to analyze related risk factors. There were three species of Anaplasma detected from the Hainan region; namely, A. phagocytophilum, A. bovis, and A. platys. The overall prevalence of Anaplasma is 9.7% (102/1051). A. phagocytopihum was prevalent in 1.0% of dogs (11/1051), A. bovis was found in 2.7% of dogs (28/1051), and A. platys in 6.0% of dogs (63/1051). Our surveillance-based study conducted to understand the occurrence and distribution pattern of Anaplasma spp. in Hainan will help in designing effective control measures along with management strategies so as to treat and control the infection in the area.

Genomic epidemiology of SARS-CoV-2 from Uttar Pradesh, India.

The various strains and mutations of SARS-CoV-2 have been tracked using several forms of genomic classification systems. The present study reports high-throughput sequencing and analysis of 99 SARS-CoV-2 specimens from Western Uttar Pradesh using sequences obtained from the GISAID database, followed by phylogeny and clade classification. Phylogenetic analysis revealed that Omicron lineages BA-2-like (55.55%) followed by Delta lineage-B.1.617.2 (45.5%) were predominantly circulating in this area Signature substitution at positions S: N501Y, S: D614G, S: T478K, S: K417N, S: E484A, S: P681H, and S: S477N were commonly detected in the Omicron variant-BA-2-like, however S: D614G, S: L452R, S: P681R and S: D950N were confined to Delta variant-B.1.617.2. We have also identified three escape variants in the S gene at codon position 19 (T19I/R), 484 (E484A/Q), and 681 (P681R/H) during the fourth and fifth waves in India. Based on the phylogenetic diversification studies and similar changes in other lineages, our analysis revealed indications of convergent evolution as the virus adjusts to the shifting immunological profile of its human host. To the best of our knowledge, this study is an approach to comprehensively map the circulating SARS-CoV-2 strains from Western Uttar Pradesh using an integrated approach of whole genome sequencing and phylogenetic analysis. These findings will be extremely valuable in developing a structured approach toward pandemic preparedness and evidence-based intervention plans in the future.

Evolutionary genomics analysis reveals gene expansion and functional diversity of arylalkylamine N-acetyltransferases in the Culicinae subfamily of mosquitoes.

Arylalkylamine N-acetyltransferase (aaNAT), considered a potential new insecticide target, catalyzes the acetylation of arylalkylamine substrates such as serotonin and dopamine and, hence, mediates diverse functions in insects. However, the origin of insect aaNATs (iaaNATs) and the evolutionary process that generates multiple aaNATs in mosquitoes remain largely unknown. Here, we have analyzed the genomes of 33 species to explore and expand our understanding of the molecular evolution of this gene family in detail. We show that aaNAT orthologs are present in Bacteria, Cephalochordata, Chondrichthyes, Cnidaria, Crustacea, Mammalia, Placozoa, and Teleoste, as well as those from a number of insects, but are absent in some species of Annelida, Echinozoa, and Mollusca as well as Arachnida. Particularly, more than 10 aaNATs were detected in the Culicinae subfamily of mosquitoes. Molecular evolutionary analysis of aaNAT/aaNAT-like genes in mosquitoes reveals that tandem duplication events led to gene expansion in the Culicinae subfamily of mosquitoes more than 190 million years ago. Further selection analysis demonstrates that mosquito aaNATs evolved under strongly positive pressures that generated functional diversity following gene duplication events. Overall, this study may provide novel insights into the molecular evolution of the aaNAT family in mosquitoes.

Functional analysis reveals ionotropic GABA receptor subunit RDL is a target site of ivermectin and fluralaner in the yellow fever mosquito, Aedes aegypti.

BACKGROUND: Ionotropic γ-aminobutyric acid (iGABA) receptors are involved in various physiological activities in insects, including sleep, olfactory memory, movement, and resistance to viruses. Ivermectin and fluralaner can disturb the insect nervous system by binding to iGABA receptors, and are therefore an effective means for controlling insect pests. However, the molecular mechanisms underlying the insecticidal effect of both the compounds on Aedes. aegypti remain unexplored. RESULTS: In this study, we investigated the spatiotemporal expression profile of Ae. aegypti RDL (Ae-RDL), a subunit of iGABA receptor. RDL dsRNA suppressed the expression of Ae-RDL mRNA in Ae. aegypti larvae and adult by 60% and 50.67%,  resepectly. However, the physiology of Ae. aegypti larvae was not significantly affected. The mortality of Ae. aegypti larvae and adult females subjected to Ae-RDL knockdown significantly decreased after exposure to ivermectin and fluralaner. Additionally, Ae-RDL was cloned into Xenopus laevis oocytes and characterized using the two-electrode voltage-clamp method. The inward current was induced by GABA binding to the functional Ae-RDL homomeric receptors at a median effective concentration (EC50 ) of 100.4 ± 59.95 µM (n > 3). The significant inhibitory effect of ivermectin and fluralaner on inward current indicated that both insecticides exerted a significant antagonistic effect on Ae-RDL. However, ivermectin also showed strong agonistic as well as weak activation effects on Ae-RDL. These contrasting effects of ivermectin on Ae-RDL depended on ivermectin concentration. CONCLUSION: Our study revealed that Ae-RDL subunit is a target of ivermectin and fluralaner, providing new insights into the insecticidal mechanism of both compounds in Ae. aegypti. © 2022 Society of Chemical Industry.

Identification of Theileria spp. and investigation of hematological profiles of their infections in goats in Hainan Island, China.

Theileria spp. are a group of parasites primarily transmitted by ticks and can pose a significant threat to domestic and wild animals globally. The main objective of this study was to understand the epidemiology of Theileria spp. in goats of Hainan Island/province, which is the only tropical region of China, and to study their hematological profiles in naturally infected goats. A total of 464 blood samples were collected from randomly selected local adult goats (Capra hircus, local domestic breed with black hair), from six cities and eight counties of Hainan, from November 2017 to October 2020. Blood smear microscopy of the sample and a nested polymerase chain reaction (nPCR) targeting the 18S rRNA gene combined with DNA sequencing were used to detect piroplasm infections in goats. Data analysis of the obtained sequences revealed that all the sequences were highly similar to the Theileria luwenshuni 18S rRNA gene sequence from the database. This result is consistent with the microscopic examination. In the hematological test, hematocrit, mean corpuscular volume, and mean corpuscular hemoglobin of the goats naturally infected with T. luwenshuni significantly increased, while mean corpuscular hemoglobin concentration and red blood cell distribution width (RDW) were significantly decreased. Results showed that T. luwenshuni could cause macrocytic, hypochromic anemia in goats. This study provides reliable and comprehensive information about the epidemiology of the parasite infections and hematological profile of the infected goats in Hainan, which encourages further investigations to develop practical control strategies for Theileria spp. infections in tropical areas.

TITLE: Identification de Theileria spp. et enquête sur les profils hématologiques de leurs infections chez les chèvres de l'île de Hainan, en Chine. ABSTRACT: Les Theileria spp. sont un groupe de parasites principalement transmis par les tiques qui peuvent constituer une menace importante pour les animaux domestiques et sauvages dans le monde. L'objectif principal de cette étude était de comprendre l'épidémiologie de Theileria spp. chez les chèvres de l'île/province de Hainan, qui est la seule région tropicale de Chine et étudier les profils hématologiques des chèvres naturellement infectées. 464 échantillons de sang ont été prélevés sur des chèvres adultes locales sélectionnées au hasard (Capra hircus, race domestique locale à poils noirs), dans 6 villes et 8 comtés de Hainan, de novembre 2017 à octobre 2020. L'étude microscopique du frottis sanguin de l'échantillon et la réaction en chaîne par polymérase nichée (nPCR) ciblant le gène de l'ARNr 18S combinée au séquençage de l'ADN ont été utilisées pour détecter les infections à piroplasmes chez les chèvres. L'analyse des séquences obtenues a révélé que toutes les séquences étaient très similaires à la séquence du gène de l'ARNr 18S de T. luwenshuni de la base de données. Le résultat est cohérent avec l'examen microscopique. Dans le test hématologique, l'hématocrite, le volume corpusculaire moyen et l'hémoglobine corpusculaire moyenne des chèvres naturellement infectées par T. luwenshuni ont augmenté de manière significative, tandis que la concentration moyenne d'hémoglobine corpusculaire et la largeur de distribution des globules rouges (RDW) ont été significativement diminuées. Les résultats ont montré que T. luwenshuni pouvait provoquer une anémie macrocytaire et une anémie hypochrome chez les chèvres. Cette étude fournit des informations fiables et complètes sur l'épidémiologie des infections parasitaires et le profil hématologique des chèvres infectées à Hainan, ce qui encourage des investigations supplémentaires pour développer des stratégies pratiques de contrôle des infections par Theileria spp. dans les zones tropicales.

Structure and Functions of Bacterial Outer Membrane Protein A, A Potential Therapeutic Target for Bacterial Infection.

Outer membrane protein A (OmpA) is a unique outer membrane protein which is abundantly present in the outer membrane of Gram-negative bacteria. OmpA is a transmembrane structural protein with a conserved amino acid sequence among different bacteria. This protein is involved in a number of functions like adhesion, toxicity, invasiveness, and biofilm formation in Gram-negative bacteria. Many studies have proposed that OmpA could be a therapeutic target for bacterial infection. Our review focusses on the studies involving recent development in the structure and functions of OmpA and further discussing its potential as a therapeutic target for bacterial infection.

ZnO Nanolower-Based NanoPCR as an Efficient Diagnostic Tool for Quick Diagnosis of Canine Vector-Borne Pathogens.

: Polymerase chain reaction (PCR) is a unique technique in molecular biology and biotechnology for amplifying target DNA strands, and is also considered as a gold standard for the diagnosis of many canine diseases as well as many other infectious diseases. However, PCR still faces many challenges and issues related to its sensitivity, specificity, efficiency, and turnaround time. To address these issues, we described the use of unique ZnO nanoflowers in PCR reaction and an efficient ZnO nanoflower-based PCR (nanoPCR) for the molecular diagnosis of canine vector-borne diseases (CVBDs). A total of 1 mM of an aqueous solution of ZnO nanoflowers incorporated in PCR showed a significant enhancement of the PCR assay with respect to its sensitivity and specificity for the diagnosis of two important CVBDs, Babesia canis vogeli and Hepatozoon canis. Interestingly, it drastically reduced the turnaround time of the PCR assay without compromising the yield of the amplified DNA, which can be of benefit for veterinary practitioners for the improved management of diseases. This can be attributed to the favorable adsorption of ZnO nanoflowers to the DNA and thermal conductivity of ZnO nanoflowers. The unique ZnO nanoflower-assisted nanoPCR greatly improved the yield, purity, and quality of the amplified products, but the mechanism behind these properties and the effects and changes due to the different concentrations of ZnO nanoflowers in the PCR system needs to be further studied.

Identification and immunotherapeutic targeting of antigens induced by chemotherapy.

Cancer cells differ from normal cells in their response to chemotherapy. We exploited this dissimilarity by identifying and targeting tumor-specific, cell-surface proteins whose expression is induced by the chemotherapeutic irinotecan (CPT-11; Camptosar). A cytotoxin-armed antibody reactive with one of these drug-induced surface proteins, the LY6D/E48 antigen, originally identified as the target of a monoclonal antibody reactive with squamous cell carcinomas, caused complete regression of colorectal tumor xenografts in mice treated with CPT-11, whereas either agent alone was less effective. These results suggest that a positive therapeutic index may be generated for other drug combinations by immunotherapeutic targeting of chemotherapy-induced antigens.

3,4-Dihydroxyphenylacetaldehyde synthase and cuticle formation in insects.

Cuticle is the most important structure that protects mosquitoes and other insect species from adverse environmental conditions and infections of microorganism. The physiology and biochemistry of insect cuticle formation have been studied for many years and our understanding of cuticle formation and hardening has increased considerably. This is especially true for flexible cuticle. The recent discovery of a novel enzyme that catalyzes the production of 3,4-dihydroxyphenylacetaldehyde (DOPAL) in insects provides intriguing insights concerning the flexible cuticle formation in insects. For convenience, the enzyme that catalyzes the production DOPAL from l-dopa is named DOPAL synthase. In this mini-review, we summarize the biochemical pathways of cuticle formation and hardening in general and discuss DOPAL synthase-mediated protein crosslinking in insect flexible cuticle in particular.