Parasites & Vectors最新文献

Background: Songling virus (SGLV) within the genus Orthonairovirus, family Nairoviridae, is an emerging tick-borne virus associated with human febrile illness. However, no rapid detection method for SGLV has been established.

Methods: In this study, four primer sets targeting the nucleocapsid protein gene of SGLV were designed for use in the LAMP assay and evaluated to identify the optimal primer set. Recombinant plasmids were constructed and utilized for assessing the sensitivity of the assay. Tacheng tick virus 1 (TcTV-1)-, Beiji nairovirus (BJNV)-, Yezo virus (YEZV)-, severe fever with thrombocytopenia syndrome virus (SFTSV)-, and tick-borne encephalitis virus (TBEV)-positive tick samples were utilized to assess the specificity. Field-collected ticks were also evaluated as biological specimens to validate the assay.

Results: A SGLV-specific LAMP assay was established with a detection limit of 1 × 10^-2 copies/μl and could be visually confirmed by a color change from purple to blue in SGLV-positive samples. No cross-reactivity was observed in the detection of TcTV-1, BJNV, YEZV, SFTSV, and TBEV using the LAMP assay. In addition to the detection of the same seven high-copy numbers of SGLV as the SYBR Green quantitative RT-PCR assay within a reduced timeframe, the developed LAMP method also effectively identified an additional sample with a low copy number in the field-collected tick samples.

Conclusions: We successfully developed a sensitive, specific, and cost-effective visual method for the rapid detection of SGLV using the LAMP assay, which can be applied in pathogenesis and epidemiological surveillance studies of SGLV.

Background: Babesia duncani is a pathogen within the phylum Apicomplexa that causes human babesiosis. It poses a significant threat to public health, as it can be transmitted not only through tick bites but also via blood transfusion. Consequently, an understanding of the gene functions of this pathogen is necessary for the development of drugs and vaccines. However, the absence of conditional gene knockdown tools has hindered the research on this pathogen. The auxin-inducible degron (AID) system is a rapid, reversible conditional knockdown system widely used in gene function studies. Thus, there is an urgent need to establish the AID system in B. duncani to study essential gene functions.

Methods: The endogenous genes of the Skp1-Cullin-F-box (SCF) complex in B. duncani were identified and confirmed through multiple sequence alignment and conserved domain analysis. The expression of the F-box protein TIR1 from Oryza sativa (OsTIR1) was achieved by constructing a transgenic parasite strain using a homologous recombination strategy. Polymerase chain reaction (PCR), western blot, and indirect immunofluorescence assay (IFA) were used to confirm the correct monoclonal parasite strain. The degradation of enhanced green fluorescent protein (eGFP) tagged with an AID degron was detected through western blot and live-cell fluorescence microscopy after treatment of indole-3-acetic acid (IAA).

Results: In this study, Skp1, Cul1, and Rbx1 of the SCF complex in B. duncani were identified through sequence alignment and domain analysis. A pure BdTIR1 strain with expression of the OsTIR1 gene was constructed through homologous recombination and confirmed. This strain showed no significant differences from the wild type (WT) in terms of growth rate and proportions of different parasite forms. The eGFP tagged with an AID degron was successfully induced for degradation using 500 μM IAA. Grayscale analysis of western blot indicated a 61.3% reduction in eGFP expression levels, while fluorescence intensity analysis showed a 77.5% decrease in fluorescence intensity. Increasing the IAA concentration to 2 mM accelerated eGFP degradation and enhanced the extent of degradation.

Conclusions: This study demonstrated the functionality of the AID system in regulating protein levels by inducing rapid degradation of eGFP using IAA, providing an important research tool for studying essential gene functions related to invasion, egress, and virulence of B. duncani. Moreover, it also offers a construction strategy for apicomplexan parasites that have not developed an AID system.

Background: Ixodes scapularis and Ixodes pacificus are important vectors of multiple pathogens in the United States. However, their role in transmission of Bartonella spp., which are commonly reported in rodents and fleas, has been debated. Our previous investigation on Bartonella spp. in host-seeking I. scapularis and I. pacificus showed Bartonella spp. were absent in the ticks, suggesting the two species are unlikely to contribute to Bartonella transmission. It is unclear whether the absence of Bartonella spp. in the host-seeking ticks was attributable to ticks not being exposed to Bartonella in nature or being exposed but unable to acquire or transstadially transmit the bacterium. To assess the likelihood of exposure and acquisition, we tested Ixodes spp. ticks collected from rodents for Bartonella infections.

Methods: Blood-fed I. scapularis ticks (n = 792; consisting of 645 larvae and 147 nymphs), I. pacificus ticks (n = 45, all larvae), and Ixodes angustus ticks (n = 16, consisting of 11 larvae and 5 nymphs) collected from rodents from Minnesota and Washington were tested for Bartonella spp. using a quadruplex polymerase chain reaction (PCR) amplicon next-generation sequencing approach that targets Bartonella-specific fragments on gltA, ssrA, rpoB, and groEL. In parallel, rodents and fleas collected from the same field studies were investigated to compare the differences of Bartonella distribution among the ticks, fleas, and rodents.

Results: Bartonella spp. were commonly detected in rodents and fleas, with prevalence of 25.6% in rodents and 36.8% in fleas from Minnesota; 27.9% in rodents and 45.2% in fleas from Washington. Of all tested ticks, Bartonella DNA was detected by gltA in only one larval I. scapularis tick from Minnesota.

Conclusions: The high prevalence of Bartonella spp. in rodents and fleas coupled with extremely low prevalence of Bartonella spp. in blood-fed ticks suggests that although Ixodes ticks commonly encounter Bartonella in rodents, they rarely acquire the infection through blood feeding. Notably, ticks were at various stages of feeding on rodents when they were collected. Laboratory transmission studies are needed to assess acquisition rates in fully blood-fed ticks and to assess transstadial transmission efficiency if ticks acquire Bartonella infections from feeding to repletion.

Background: Sand flies, belonging to the Psychodidae family, represent small, hairy insects that serve as significant vectors in various important medical and veterinary diseases. Despite being recognized by the World Health Organization as an endemic area for leishmaniasis, Southeast Asia lacks comprehensive information on the species composition and biology of sand flies. To address this, the current study aimed to survey sand fly biodiversity.

Methods: Sand flies from six provinces in Southern Vietnam were collected using CDC light traps. Sand flies were subsequently identified morphologically and confirmed molecularly using mitochondrial cytochrome oxidase c subunit I (COI) and cytochrome b (cytb) sequences. BLASTN searches were conducted, and the species identity of sand flies was further confirmed through a Barcode of Life Database (BOLD) search utilizing COI sequences. Subsequently, nucleotide sequences were subjected to a panel of analyses including intraspecific variation, phylogenetic relationships and haplotype network. The average densities of collected sand flies (sand flies/trap/night) and species richness were also recorded.

Results: A total of 753 sand flies were collected. After excluding damaged specimens, six sand fly species, namely Phlebotomus stantoni, Sergentomyia khawi, Se. silvatica, Se. barraudi, Se. bailyi and Grassomyia indica, were identified. All conspecific sand fly sequences, including Ph. stantoni, Se. barraudi, Gr. indica, Se. bailyi, Se. khawi and Se. silvatica, clustered with their reference sequences, corroborating the results of morphology-based identification, BLASTN analysis and BOLD search. For intraspecific variation of sand flies obtained from the current study, COI diversity indices were consistently higher than those of cytb.

Conclusions: This study provides the first updates on morphological and molecular characterization of sand flies in Southern Vietnam. This acquired knowledge on sand fly species composition is essential for controlling sand fly-borne diseases in this potentially endemic region.

Background: Angiostrongylus vasorum, commonly known as the "French heartworm," is a nematode belonging to the Metastrongyloidea superfamily. This parasite was first identified in Toulouse, France in 1853 infecting the pulmonary arteries and the right side of the heart of a Pointer dog. Angiostrongylosis is an important infection due its severe clinical signs and potential for causing high morbidity and mortality in domestic dogs. This nematode has not been studied in Algeria. The aim of this study was investigate the presence of lungworms among different mammal species in a number of Algerian regions.

Methods: Between February 2022 and September 2023, 47 road-killed animals were collected from six administrative units (departments) in Algeria. All carcasses underwent a full parasitological necropsy, and lung tissues were preserved in 10% buffered formalin and concentrated ethanol for further study. All collected samples were subjected to histological and PCR (cytochrome c oxidase subunit 1 gene) analyses for lungworm identification.

Results: Histological examination revealed the presence of nematode eggs and larvae in the alveolar space and chronic obstructive vascular changes were detected in a single golden African wolf (Canis lupaster) collected from the department of Constantine. First-stage larvae were collected and morphologically identified as Angiostrongylus spp. The molecular identification confirmed the presence of A. vasorum. All other animals tested were negative for lungworms.

Conclusions: To the best of our knowledge, this is the first report of A. vasorum infection in an African golden wolf (Canis lupaster). We report a new host association, highlighting the importance of further studies to update the geographical distribution of A. vasorum and its epidemiology across Algeria.

Background: Phylogeny, combined with trait-based measures, offers insights into parasite sharing among hosts. However, the specific traits that mediate transmission and the aspects of host community diversity that most effectively explain parasite infection rates remain unclear, even for the Bartonella genus, a vector-borne bacteria that causes persistent blood infections in vertebrates.

Methods: This study investigated the association between rodent host traits and Bartonella infection, as well as how rodent community diversity affects the odds of infection in the Atlantic Forest, using generalized linear models. Additionally, we assessed how host traits and phylogenetic similarities influence Bartonella infection among mammal species in Brazil. To this end, rodents were sampled from ten municipalities in Rio de Janeiro, southeastern Brazil. Then, we calculated several diversity indices for each community, including Rényi's diversity profiles, Fisher's alpha, Rao's quadratic entropy (RaoQ), Functional Diversity (FDis), Functional Richness (FRic), and Functional Evenness (FEve). Finally, we compiled a network encompassing all known interactions between mammal species and Bartonella lineages recorded in Brazil.

Results: We found no significant relationship between diversity indices and the odds of Bartonella infection in rodent communities. Furthermore, there was no statistical support for the influence of individual-level traits (e.g., body length, sex, and age) or species-level ecological traits (e.g., locomotor habitat, dietary guild, and activity period) on Bartonella infection in rodents. A country-scale analysis, considering all mammal species, revealed no effect of host traits or phylogeny on Bartonella infection.

Conclusions: This study highlighted wild mammals that share Bartonella lineages with livestock, synanthropic, and domestic animals, underscoring the complexity of their maintenance cycle within the One Health framework. A key question arising from our findings is whether molecular host-cell interactions outweigh host body mass and ecological traits in influencing Bartonella infection, potentially opening new avenues for understanding host-parasite relationships and infection ecology.

Background: Rhipicephalus sanguineus sensu lato (s.l.) and Ctenocephalides felis are among the most important year-round ectoparasites of dogs. The persistent efficacy of one treatment with fluralaner injectable suspension (Bravecto^® 150 mg/ml powder and solvent for suspension for dogs, referred to as Bravecto^® injectable) was investigated in a negative-controlled, randomised, partially blinded 12-month laboratory study.

Methods: A total of 20 dogs were randomly allocated to two equal groups (treatment and control). Treatment-group dogs were injected subcutaneously on study day 0 with the investigational veterinary product at the recommended dose of 15 mg fluralaner/kg body weight (0.1 mL/kg), whereas the control group dogs received saline solution (0.1 mL/kg). Each dog was infested with 50 (25 female, 25 male) adult R. sanguineus s.l. and 100 adult C. felis 2 days before treatment, 5 and 28 days after treatment, and then once monthly for a 12-month period. Live tick and flea counts were performed 48 h after treatment or subsequent infestation, respectively. Efficacy was determined by comparing arithmetic means of the treatment group tick and flea counts with those of the control group. Infestation was considered adequate if at least 25.0% of ticks and 40.0% of fleas were recovered from at least six dogs in the control group at the respective assessment times.

Results: Adequate R. sanguineus s.l. and C. felis infestations of control group dogs were observed at each time point. Arithmetic mean treatment group values were significantly lower than those of the control group at all time points. The immediate efficacy when treating existing infestations of R. sanguineus s.l. and C. felis (infestation 2 days before treatment), was 49.7% and 89.7%, respectively. The persistent efficacy against post-treatment re-infestations was 94.4-100% against R. sanguineus s.l. and 92.2-100% against C. felis. Seven dogs in the control group developed flea allergy dermatitis due to the repeated re-infestations over the study period, whereas no dogs in the treatment group were affected. No clinically relevant side effects were observed over the entire study period.

Conclusions: The fluralaner injectable suspension (Bravecto^® injectable) provides 1 year of efficacy against R. sanguineus s.l. and C. felis infestations in dogs following a single treatment, allowing once-yearly treatment, which can significantly improve owner compliance with year-round protection of dogs.

Background: Cryptosporidium spp. are important zoonotic parasites that can cause moderate to severe diarrhea in humans and animals. Among the three Cryptosporidium species infecting the intestines of calves, Cryptosporidium parvum has a broad host range and causes severe diarrhea in calves, while Cryptosporidium bovis and Cryptosporidium ryanae mainly infect calves without obvious clinical symptoms. Comparative genomic analysis revealed differences in the copy number of genes encoding the nonfinancial disclosure quality (NFDQ) secretory protein family among the three species, suggesting that this protein family may be associated with the host range or pathogenicity of Cryptosporidium spp. To understand the function of cgd8_10 encoded NFDQ1, tagged and knockout strains were constructed and characterized in this study.

Methods: To determine the localization of NFDQ1, we used clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) technology to tag the C-terminus of NFDQ1 with three hemagglutinin epitopes (3 × HA). The tagged strain was constructed, and the genomic insertion was confirmed by polymerase chain reaction (PCR). Immunofluorescence assays were performed to observe the localization of NFDQ1 both in extracellular sporozoites and at various intracellular developmental stages. Immunoelectron microscopy was used to study the ultrastructural localization of NFDQ1. Then, the ΔNFDQ1 strain was generated by CRISPR/Cas9 and the in vitro growth assay on HCT-8 cells was used to analyze of phenotypic changes after knockout NFDQ1 in parasites.

Results: The NFDQ1 tagging and knockout stains were successfully constructed by CRISPR/Cas9 technology and the insertions of transgenic strains were validated by PCR. The expression of NFDQ1 was validated in parasite by western blot. Immunofluorescence and immune-electron microscopy assay showed that NFDQ1 expressed in both asexual and sexual stages of C. parvum, where it was localized to the cytoplasm of the parasite. Upon ablation of NFDQ1, the ΔNFDQ1 strain showed an apparent growth retardation during sexual replication in vitro.

Conclusions: NFDQ1 is a cytoplasmic protein without specific localization to secretory organelles, and it may participate in C. parvum growth during sexual reproduction. Future study should determine the role of NFDQ1 following C. parvum infection in vivo.