Parasites & Vectors最新文献

Background: Zoonotic diseases pose a major threat to both human and animal health, contributing significantly to global morbidity and mortality. Accurate diagnosis is crucial for effective control and treatment, with microscopic examination serving as the gold standard, supplemented by highly sensitive molecular biology techniques. However, these confirmatory methods require skilled personnel and are subject to inter- and intra-rater variability. An innovative solution lies in artificial intelligence (AI)-powered automated tools, which offer a promising alternative. This study aimed to develop a self-supervised learning (SSL) approach using the Distillation with No Labels (DiNOv2) algorithm to extract features of protozoa from Giemsa-stained blood samples.

Methods: The development of self-supervised learning algorithms, including DiNOv2, was based on archived samples of clinically significant and significant veterinary microorganisms. These models were evaluated in comparison to a baseline Vision Transformer (ViT).

Results: Among the tested SSL models, the DiNOv2-Small version achieved exceptional performance, surpassing 99% accuracy and specificity while maintaining the lowest misclassification rate (0.263). It also demonstrated a high area under the curve (AUC) value of 0.990, underscoring its robust classification capability. Remarkably, even when trained on only 20% of the dataset, the SSL models retained performance levels comparable to baseline models trained on the full dataset. However, further reducing the sample size below 20% led to notable declines in evaluation metrics: accuracy dropped by 5-7%, recall decreased from 37.9% to 35.1%, precision fell from 35% to 25.2%, and the F1 score declined from 31.1% to 21.5%. Additionally, the AUC decreased by 4-11%, while the misclassification rate increased, indicating reduced robustness. A key limitation of this study was the highly imbalanced fine-tuned dataset between classes. Nevertheless, the inconsistent performance observed may be mitigated by employing the larger DiNOv2 model, which improves the F1 score and enhances the model's ability to handle imbalanced data while reducing reliance on labeled data.

Conclusions: The proposed method can assist laboratory technicians, particularly in resource-limited healthcare settings. Furthermore, the findings support the potential deployment of this AI-based tool for automated screening in both medical and veterinary applications.

Background: Anaplasma phagocytophilum is a complex of tick-borne bacteria of medical and veterinary relevance, whose eco-epidemiology is well characterized in the Northern Hemisphere but remains poorly understood in South America. Here, we report in Uruguay the detection and genetic characterization of a novel A. phagocytophilum ecotype in South America.

Methods: Questing Ixodes fuscipes, the only member of the Ixodes ricinus complex in the country, were collected in five localities in Uruguay, and the presence of Anaplasma spp. DNA was assessed using PCR to amplify fragments of the 16S ribosomal RNA (rrs), gltA and groEL genes.

Results: A total of 223 Ixodes fuscipes ticks were collected between 2017 and 2022 in five localities. PCR screening and subsequent sequencing identified Anaplasma spp. DNA in five nymphs from the Rivera and Tacuarembó departments. Phylogenetic analyses of rrs, gltA and groEL sequences of this bacteria confirmed the placement within the A. phagocytophilum clade. In particular, groEL-based phylogenies showed that Uruguayan sequences form a distinct and well-supported lineage, grouping with ecotype V (strain Patagonia) and being closely related to ecotype III. Pairwise genetic distance analyses of groEL sequences further supported the recognition of this lineage as a novel ecotype (ecotype VI). The detection of positive nymphs suggests acquisition from local vertebrate hosts, and the phylogenetic relationship among ecotypes III, V and VI, together with host records for immature and adult I. fuscipes, point to a potential role for small mammals, birds or cervids in maintaining the enzootic cycle of A. phagocytophilum strain "Uruguay." Although vector competence of I. fuscipes remains to be determined, these findings provide preliminary evidence of the potential involvement of this tick species in local transmission and represent the second characterization of an A. phagocytophilum ecotype in South America.

Conclusions: Anaplasma phagocytophilum is reported for the first time in Uruguay. The recognition of this new ecotype (VI) expands regional knowledge and underscores the influence of local host-vector assemblages in shaping A. phagocytophilum diversity. Broader host-vector surveys are needed to clarify its ecology, transmission dynamics and potential epidemiological implications in the region.

Background: Dermanyssus gallinae is a prevalent ectoparasite in the poultry farms, inflicting damage on chicken health through blood-sucking. Chemical acaricides commonly used for mite control often show reduced efficacy due to the development of resistance. Therefore, alternative control methods are needed, and vaccination is a promising strategy for controlling D. gallinae.

Methods: The mRNA expression of Deg-CYP-3 in mites at various developmental stages, as well as under fed and starved conditions, was analyzed. Subsequently, recombinant protein rCYP-3 was induced, purified, and employed for immunization. Following immunization, antibodies were analyzed and mite challenge was then conducted. Following a 12 h period of blood-feeding on chicks, the mites were collected to evaluate the acaricidal efficacy of the rCYP-3 vaccine.

Results: The Deg-CYP-3 gene was expressed across all life stages and maintained stable expression levels under both fed and starved conditions. The recombinant protein rCYP-3 was successfully expressed in Escherichia coli and efficiently secreted into the culture supernatant. Immunization with rCYP-3 induced a specific IgY immune response in chicks, as confirmed by ELISA. Moreover, anti-rCYP-3 serum specifically recognized P450 proteins extracted from D. gallinae, as demonstrated by Western blot analysis. Immunization resulted in an 8.1% reduction in adult mite survival (P > 0.05), whereas nymph survival decreased significantly by 22.4% (P < 0.01). In addition, oviposition rate, hatching rate, and fecundity were reduced by 2.8%, 2.2%, and 22.0%, respectively, in the immunized group. Overall, vaccine efficacy was calculated to be 30.6% in immunized birds. Furthermore, the expression level of Deg-CYP-3 in mites fed on immunized hosts was significantly lower than that in mites from the unimmunized control group.

Conclusions: Our findings demonstrated that the Deg-CYP-3 gene exhibits high transcriptional activity during both the adult and nymph stages of D. gallinae. Moreover, its expression remains consistent regardless of the feeding status of adult mites. Immunization with rCYP-3 effectively reduced mite survival, reproductive capacity, and gene expression levels, demonstrating its potential as a preventive and control strategy against D. gallinae.

Background: Tsetse flies (Diptera: Glossinidae) are vectors of human and animal trypanosomes. The Glossina palpalis gambiensis Burkina Faso (BKF) colony, established in 1972 and rejuvenated once in 1981, is a long-standing closed colony used extensively for research and vector control. While its performance and competitiveness for sterile insect technique (SIT) programs are regularly monitored, its vector competence (VC) data are outdated. This study aimed to update the VC data of this 47-year-old colony (from the onset of experiment in 2019) against Trypanosoma congolense and T. brucei brucei in laboratory conditions using trypanosome clone and tsetse fly individuals from the BKF colony.

Methods: Vector competence was studied by infecting rats with T. congolense IL1180 and T. b. brucei BE8P2P2, on which tsetse flies received their first blood meal. Dissections were subsequently performed at different time intervals.

Results: Following experimental infections with T. congolense IL1180, 10.58% (20/189) of G. p. gambiensis developed mature infections (trypanosomes in the proboscis), resulting in an average VC index of 0.106. For T. b. brucei BE8P2P2, 4.21% (11/261) of flies developed mature infections (trypanosomes in the salivary glands), yielding an average VC index of 0.042.

Conclusions: The VC for T. b. brucei aligned with previous findings from 21 years ago, though a different trypanosome isolate was used at that time. However, using the same trypanosome strain, the observed competence for T. congolense was 4.8 times higher than previously reported. These results raise questions about the long-term effects of insectary rearing on VC, particularly in the absence of prolonged parasite exposure.

Background: For decades, insecticides have been central to controlling the yellow fever mosquito, Aedes aegypti (L.), but extensive use has driven resistance development. This study investigates resistance of Ae. aegypti to pyrethroids (permethrin, deltamethrin) and organophosphates (malathion, pirimiphos-methyl) and their underlying mechanisms across Malaysia, Thailand, Indonesia, and the USA.

Methods: Adult female Ae. aegypti (3-5 days old, non-blood-fed) were subjected to World Health Organization (WHO) tube bioassays using 0.4% permethrin, 0.03% deltamethrin, 5% malathion, and 60 mg/m² pirimiphos-methyl. Each assay included four replicates of 25 mosquitoes, with mortality assessed at 24 h post-exposure. Genomic DNA was extracted from 10 resistant individuals per population, and two coding regions of the voltage-gated sodium channel (VGSC) gene (domains II and III) were amplified and sequenced to detect known and novel kdr mutations. For biochemical analysis, 40 newly emerged, non-blood-fed females per strain were individually homogenized to quantify mixed-function oxidase (MFO), esterase (α- and β-EST), glutathione S-transferase (GST), and acetylcholinesterase (AChE) activity.

Results: High resistance levels were recorded in Malaysian and US Ae. aegypti strains, with low mortality ranging between 9% and 22% for pyrethroids. New mutations T1520I (8-15%) and I1011M (10-15%) were identified in Malaysian populations, the first detection of T1520I in the country, while V1016I (10%) was newly detected in Indonesian strains. Malaysian mosquitoes had multiple kdr mutations (S989P, V1016G, F1534C, and T1520I) in triple- and quadruple-haplotype combinations. The US Riverside strain showed a nine- to 10-fold increase in β-EST and three- to fivefold increase in MFO and GST activity compared to the VCRU susceptible strain, indicating strong metabolic resistance. In contrast, the highly resistant Malaysian Hamna strain exhibited no significant upregulation (P > 0.05) in detoxifying enzymes, suggesting resistance was driven primarily by kdr mutations. Thai strains lacked kdr mutations but exhibited altered AChE (20-35% remaining activity) and elevated GST (2-3 times higher than control).

Conclusions: The detection of novel kdr mutations and diverse resistance mechanisms underscores the adaptability of Ae. aegypti to insecticide pressure and highlights the urgent need for continuous monitoring and integrated resistance management strategies.

Background: Trypanosomoses are parasitic diseases caused by Trypanosoma protozoa transmitted by tsetse flies (Glossina spp.) to humans and animals. These diseases cause major health and economic disruptions in sub-Saharan Africa. Despite the development and wide implementation of control strategies, the disease burden remains high and complementary tools are needed. Ivermectin is an endectocide toxic to arthropods, including Glossina. The aim of this study was to test the efficacy of different doses of ivermectin administered to cattle on the survival and fecundity of Glossina palpalis gambiensis Vanderplank, 1949 in Burkina Faso.

Methods: This study compared the survival and fecundity of tsetse flies exposed to cattle treated with ivermectin-clorsulon (onefold veterinary therapeutic dose [TD; 0.2 mg/kg], twofold TD [2TD; 0.4 mg/kg], and fourfold TD [4TD; 0.8 mg/kg]) with those of flies exposed to control cattle (no treatment). Direct-skin blood-feeding experiments were performed at different days post-injection (DPI) (DPI: 1, 8, 15, 22, 29, and 36). The 30-day fly survival was analyzed using Kaplan-Meier curves and Cox proportional hazards models. Fecundity parameters were compared among treatments using generalized linear modeling (GLM). Time to first pupation was also measured.

Results: Fly mortality differed significantly between treatments (χ² = 353.63, df = 3, P < 0.001), with 30-day mortality rates at 1 DPI of 24.0%, 59.8%, 88.9%, and 90.4% in the control, TD, 2TD and 4TD groups, respectively. Treatments also significantly affected pupal production (χ² = 353.63, df = 3, P < 0.001), with a decrease of 43.6-100% relative to control at 1 DPI. In addition, in tsetse flies exposed to the treatment, the deposition of the first larva occurred 9-10 days later than in nonexposed flies, in both the 2TD and 4TD groups at 1 DPI. In the 4TD group, toxic effects lasted until 15 DPI (survival) and 8 DPI (fecundity parameters).

Conclusions: In our experiment, blood meals from cattle treated with an ivermectin-clorsulon formulation significantly reduced tsetse fly survival and fecundity, two key traits influencing vectorial capacity. Thus, treatment of domestic animals with the formulation has the potential to reduce trypanosomes transmission and improve both human and animal health in sub-Saharan Africa.

Background: This research aimed to investigate the prevalence and diversity of Bartonella in small mammals and their ectoparasites from the Central Highlands of Madagascar and to refine existing information on potential associated zoonotic diseases.

Methods: A retrospective analysis was performed on mammals and their ectoparasites collected in the Fandriana and Ankazobe districts, including 253 spleen samples from seven small mammal species and 183 individual ectoparasites (132 fleas and 51 ticks). Genomic DNA was extracted and amplified by polymerase chain reaction (PCR) targeting the nuoG gene (346 bp). Sanger sequencing of the PCR products was performed to assess Bartonella diversity using phylogenetic analysis.

Results: In total, 60.1% (152/253) of small mammals and 15.9% (21/132) of fleas tested positive for Bartonella, with Rattus rattus (69.1%, 137/198) and the associated flea Synopsyllus fonquerniei (21.2%, 14/66) having the highest infection rates. At the same sampled locations, adult R. rattus were more frequently infected with Bartonella than juveniles. Phylogenetic analysis revealed five associated clades of Bartonella with two clades recognized as a potential zoonotic species (B. elizabethae and B. kosoyi).

Conclusions: Using molecular tools, we report a high prevalence of Bartonella in small mammals and their fleas in the Central Highlands of Madagascar. Two potential Bartonella zoonotic species were identified in R. rattus and their fleas. As these bacteria are generally vector-borne, they could have a significant impact on public health in the vicinity of our study areas and, in general, in Madagascar, and merit further investigation.

Background: Ongoing antimalarial drug resistance surveillance is essential to guide effective treatment strategies. Historically, resistance to chloroquine and sulfadoxine-pyrimethamine (SP) has been associated with well-characterized mutations in the chloroquine resistance transporter (Pfcrt; K76T) and antifolate pathway genes, including dihydrofolate reductase (Pfdhfr; N51I, C59R, S108N) and dihydropteroate synthase (Pfdhps; A437G, K540E, A581G). Since the introduction of artemisinin-based combination therapies (ACTs), 13 mutations in the kelch 13 (PfK13) propeller domain have emerged as World Health Organization (WHO) validated markers of partial artemisinin resistance. This study aimed to characterize temporal trends in both established, Pfcrt and Pfk13, and less well-described potential markers, cysteine desulfurase (Pfnfs) and Pfcoronin, using febrile malaria samples collected across diverse regions of Kenya between 2013 and 2022.

Methods: The temporal trend of these markers of resistance were assessed by screening archived P. falciparum-positive dried blood spots (DBS). A total of 1750 DBS samples were collected from therapeutic efficacy studies (TES) conducted across distinct malaria transmission settings in Kenya, including coastal Kenya (Kwale 2013, n = 350; 2018, n = 150), the lake endemic region of Western Kenya (Kisumu 2015, n = 314; Busia 2016, n = 334), and the highland epidemic region of western Kenya (Kisii 2017, n = 314). Additional samples were obtained from an hrp2 study conducted in Kisii in 2022, (n = 288). Parasite genomic DNA was extracted using the Chelex-saponin method and confirmed by a Pf18S real-time polymerase chain reaction (RT-PCR). Pfk13, Pfcrt, Pfnfs, and Pfcoronin PCR amplicons were sequenced using capillary electrophoresis, Illumina Miseq or Oxford Nanopore (GridION) platforms.

Results: The prevalence of Pfcrt mutations declined over time and no WHO-validated Pfk13 mutations associated with artemisinin resistance were detected. However, synonymous substitutions at WHO-validated codons C469C and P553P were identified. In the Pfcoronin gene, nonsynonymous mutations distinct from those reported in West Africa were observed at high frequencies (> 75%). Notably, the Pfnfs-K65Q mutation, previously associated with reduced lumefantrine sensitivity in West Africa, was detected in more than 80% of the samples.

Conclusions: Our findings reveal no WHO-validated k13 mutations up until 2022 and confirm previous findings of a reduction in the Pfcrt resistance genotypes over time. This study underscores the importance of continued molecular surveillance and suggests that resistance may evolve through different pathways in East compared with West Africa and Southeast Asia.

Background: The poultry red mite, Dermanyssus gallinae, is a haematophagous ectoparasite causing significant economic losses in the commercial egg-laying sector. Blood meal digestion by D. gallinae is required for nutrient acquisition, with acidic lysosomal proteinases such as cathepsin L and cathepsin D playing a critical role in haemoglobin digestion. This study investigated the role of a cathepsin D-like aspartyl proteinase, Dg-CatD-1, in the haemoglobin digestion cascade.

Methods: Haemoglobin processing was investigated by RNA interference (RNAi)-mediated silencing of Dg-CatD-1 and assessing the impact on haemoglobin digestion. RNAi-mediated knockdown of Dg-CatD-1 was achieved by feeding a target-specific double-stranded RNA (dsRNA) to D. gallinae in a blood meal. The minimum length and concentration of Dg-CatD-1 dsRNA for effective knockdown was determined. In addition, the effect of Dg-CatD-1 knockdown on mite digestive physiology, haemoglobin digestion, and egg-laying by adult female mites was assessed.

Results: Feeding Dg-CatD-1 dsRNAs via a blood meal to adult female D. gallinae mites resulted in a substantial knockdown of target gene expression. The minimum length and concentration of dsRNA required for effective Dg-CatD-1 knockdown were 25 base pairs (bp, at 200 ng/μl) (61% knockdown) and 25 ng/μl (at 485  bp) (42% knockdown), respectively. When Dg-CatD-1 dsRNA was delivered as a single feed it resulted in up to 91% reduction in Dg-CatD-1 expression, although no observable effect on blood digestion was observed. The phenotypic impact of Dg-CatD-1 knockdown was demonstrated following two consecutive rounds of Dg-CatD-1 dsRNA feeding where knockdown reduced the ability of mites to process and clear their blood meal relative to control non-specific dsRNA-fed mites.

Conclusions: This work highlights the importance of Dg-CatD-1 as an essential enzyme in the haemoglobin digestion pathway of D. gallinae. These findings open avenues for the development of targeted control strategies aimed at disrupting the digestive processes of D. gallinae. Furthermore, this research suggests that reductions in gene expression via RNAi do not always lead to corresponding decreases in protein levels or observable phenotypes. Repeated exposure to dsRNA may be necessary to reveal phenotypic effects of gene knockdown.