Veterinary parasitology最新文献

Monogenean parasites are harmful pathogens in aquaculture systems. Current treatment strategies for monogenean infections are unsatisfactory, making the discovery of new drugs urgent. Thymoquinone (TQ), a natural monoterpene isolated from Nigella sativa L., has demonstrated its potential as lead structure against the monogenean parasites. In the present study, the anthelmintic activity of 26 selected TQ analogues was investigated against the monogenean parasite Gyrodactylus kobayashii in the goldfish (Carassius auratus). The results indicated that 18 TQ analogues displayed the EC₅₀ value below 1.0 mg/L, 14 of them showed more potent anthelmintic activity than TQ (EC₅₀=0.303 mg/L). Particularly, compounds 9 and 23 exhibited the best efficacies with the EC₅₀ values of 0.058 and 0.034 mg/L, outperforming the standard drug praziquantel as well. Structure-activity relationship analysis revealed that large electron-donating substituents at the 2-position of the quinone ring contribute to enhanced in vivo anthelmintic activity. Additionally, four analogues (9, 12, 23 and 25) displayed low fish toxicity, with the therapeutic index (TI, LC₅₀/EC₅₀) above 9.23, and were selected for further in silico drug‑likeness assessment. The results demonstrated an excellent drug-likeness profile and an adherence to major pharmaceutical companies' filters for compounds 9 and 23. Besides, TQ analogue 9 was chosen for an in vivo study in large-scale trial based on its potency, low fish toxicity, and excellent drug-likeness profiles. Treatment of G. kobayashii infected goldfish for 24 h at 0.1 and 0.2 mg/L was effective, reducing the infection prevalence from 100 % to 0 % post-treatment. The experimental fish exhibited normal behavior, despite minor modifications were displayed in the gills and liver according to histopathological results. Collectively, this study suggested that TQ analogues are excellent scaffolds in the discovery of novel anthelmintic agents against monogenean infections.

Medicinal plants are considered promising candidates for controlling parasitic pathogen in aquaculture. Our previous study demonstrated that the crude extracts of Pseudolarix amabilis exhibit promising anti-Dactylogyrus intermedius activity. However, the specific compounds responsible for the antiparasitic effects of these crude extracts remain elusive. In this study, the bioactive compounds from the ethyl acetate extract of P. amabilis were isolated by the multi-column chromatography and in vivo bioassay-guided methods. Two crystalline compounds were identified as (+)-catechin through the nuclear magnetic resonance spectroscopy and specific rotation analysis. (+)-Catechin showed 98.1 % antiparasitic activity at 20 mg/L with the median effective concentration (EC₅₀) of 4.3 mg/L. The 96 h median lethal concentration (LC₅₀) of (+)-catechin for zebrafish larvae and goldfish was determined to be 32.9 and 152.8 mg/L, respectively. The therapeutic index (TI) of (+)-catechin was 6.8 and 35.5, indicating a potential for safe application in aquaculture. These findings suggest that (+)-catechin could be further developed as a viable therapeutic agent against D. intermedius.

Toxoplasmosis is an important public health concern. Cats play a crucial role in increasing the risk of toxoplasmosis transmission to humans. Early diagnosis in cats is essential for the prevention and control of toxoplasmosis. In this study, we found that T. gondii aldolase (ALD) could be an effective diagnostic antigen, and then the recombinant ALD protein was expressed using the pET SUMO protein expression system, the mouse monoclonal antibody (MoAb) and rabbit polyclonal antibody (PoAb) of ALD were successfully produced, respectively. Furthermore, a reliable sandwich enzyme-linked immunosorbent assay (sELISA) was developed to detect circulating ALD in the sera of experimentally and naturally infected cats. rALD sELISA could detect T. gondii infection from 7DPI (post-infection day) to 14DPI with 100 % sensitivity and specificity, but could not detect T. gondii infection after 21DPI, indicating that it is a good early diagnosis tool. The detection limit was 7.8 ng/ml, the coefficients of variation (CV) of repeated tests within batches and between batches were confirmed to be less than 10 %. The results of 70 cat clinical serum samples detected by rALD sELISA were in almost perfect agreement beyond chance with those of a commercial ELISA kit (Cohen's kappa coefficient = 0.883). This sandwich ELISA method has high accuracy and can be used for early diagnosis of toxoplasmosis in cats.

During the intestinal stage of Trichinella spiralis (T. spiralis) infection, it can stimulate host's intestinal peristalsis to expulse worms. 5-hydroxytryptamine (5-HT) is a neurotransmitter which can regulate the contraction of intestinal smooth muscle. IL-33 specifically binds to ST2 receptor to promote the secretion of 5-HT by intestinal enterochromaffin cells. However, it remains unclear whether the host is able to modulate the secretion of 5-HT to expulse worms through the IL-33-ST2 signaling pathway during the intestinal stage of T. spiralis infection. Therefore, ST2 inhibitor iST2 was used in a T. spiralis infected mouse model and MODE-K cells to analyze the role of IL-33-ST2 signaling pathway in the secretion of 5-HT during the intestinal phase of T. spiralis infection. The results indicated that the expression of ST2, IL-33, and TPH1(tryptophan hydroxylase 1, the rate-limiting enzyme in the biosynthesis of 5-HT) in the small intestine were increased during the intestinal phase of T. spiralis infection. Meanwhile the levels of secretory 5-HT and IL-33 in the small intestine were significantly increased. After iST2 treatment, the level of 5-HT was significantly decreased, resulting in diminished worms expulsion capability. The decrease of 5-HT was observed in MODE-K cells treated with excretory-secretory products of T. spiralis post iST2 treatment. The above results demonstrated that IL-33-ST2 signaling pathway might play a crucial role in promoting the secretion of 5-HT which enhancing the ability of the intestine to expulse worms during the intestinal stage of T. spiralis.

The complex life cycle of the rumen fluke Calicophoron daubneyi is similar to that of the liver fluke Fasciola hepatica. Interestingly, C. daubneyi and F. hepatica share the same intermediate host, Galba truncatula. However, in contrast to its relative, experimental production of metacercariae is a major challenge for C. daubneyi, hampering a detailed analysis of its life cycle, especially in the definitive host. G. truncatula snails collected from natural habitats were bred in glass Petri dishes and fed dried organic lettuce leaves. C. daubneyi eggs were obtained from feces of naturally infected cattle and incubated until miracidia were hatching. Subsequently, these miracidia were allowed to infect snails, which were kept under specific laboratory conditions to monitor the shedding of metacercariae. In total, 177 G. truncatula snails were exposed to C. daubneyi miracidia during eleven snail infection trials. Sixty-eight of these snails survived for longer than 30 days post-infection (p.i.). From day 35 p.i., seven snails from five trials started shedding an average number of 106 metacercariae (range: 38-186) per snail. Three ewe lambs (aged 7-10 months) were inoculated orally with 150 metacercariae each. A different batch of metacercariae (obtained from three different snail trials) was used for each lamb. Another two lambs served as controls. All animals were regularly examined clinically, hematologically and coproscopically, using sedimentation techniques for the detection of trematode eggs. Low numbers of C. daubneyi eggs were detected in fecal samples of two of the three inoculated lambs on day 86 post-inoculation (yielding ≤ 2 epg), but only one lamb continued to shed eggs (up to 6 epg) until the end of the experiment (day 104 post-inoculation). None of the animals showed any abnormal clinical findings or blood parameters throughout the course of the study. Production of C. daubneyi metacercariae under laboratory conditions is reported, followed by experimental infection of the definitive host, thus completing the full life cycle of this parasite under experimental conditions. However, neither the survival rate of the snails nor the amount of metacercariae produced were comparable to previously published experiments using F. hepatica, necessitating further optimization of the laboratory protocols. Nevertheless, the results can serve as a starting point for more in-depth studies of this increasingly important trematode.

Heartworm infection caused by Dirofilaria immitis induces a devastating disease that greatly affects the global canine population. The mechanism leading to heartworm pathology has been attributed to be mostly by mechanical damage of the worm to the dog´s vascular system and immune-mediated, but the latter processes are not completely understood. Autoantibodies targeting host molecules such as lipids and nucleic acids have been described with pathological roles during malaria and COVID-19 and mediating anemia and thrombocytopenia. We hypothesized that autoantibodies could be present and have a pathological role during canine heartworm disease caused by D. immitis. In this study, we analyzed the levels of autoantibodies (IgM and IgG) against membrane lipid phosphatidylserine (PS) and DNA in the serum of 169 canine samples based on D. immitis infection. First, our results found significant levels of anti-PS IgM and IgG autoantibodies that were associated with D. immitis-positive when compared to D. immitis-negative samples. Second, we found that autoantibodies, particularly anti-PS, are correlated with hematological parameters such as low platelet count suggesting an association with pathologies such as thrombocytopenia. Altogether, these findings elucidate the understudied presence and pathological role of autoantibodies during canine heartworm disease by D. immitis with implications as biomarkers of disease.

Based on an exhaustive review, a theory was proposed with an immunoendocrine perspective on the reactivation of dormant Toxocara canis larvae in pregnant bitches and their transmission to fetuses through the placenta, or milk and colostrum to puppies. A historical review was carried out on the reactivation of dormant larvae of T. canis in pregnant bitches which suggested that larval reactivation of T. canis is not solely the effect of a single hormone (prolactin) but is the result of a series of events triggered by progesterone, prolactin, and estrogens. In the first third of gestation, progesterone is capable of directly stimulating the reactivation of larvae through hormonal receptors, indirectly downregulating the granulomatous proinflammatory response around dormant T. canis larvae, and directing the response to a Th2 profile with increased levels of antibodies and blood eosinophils. After a time, when progesterone levels decrease, prolactin and estrogen maintain larval stimulation through hormonal receptors and downregulation of Th1 and the granulomatous proinflammatory response. Collectively, these hormones play major roles in the reactivation of T. canis larvae in pregnant bitches. The series of complex events that occur during larval reactivation is a clear example of transregulation, in which host hormones regulate the vital functions of the parasite to positively influence its establishment and/or proliferation. Understanding larval reactivation from an immunoendocrine perspective helps us to comprehensively understand the complex parasite-host relationship of T. canis.

Dairy heifers with gastrointestinal nematodes have reduced growth rates, and delayed age at puberty and milk production onset related to late mammary gland development. IGF1 and Notch signaling systems are important in this process, and an altered profile of serum IGF1 has been associated with the detrimental effect of the nematodes on parenchymal development. In this context, we aimed to study the molecular mechanisms involved in bovine mammary gland development around pre and postpuberty, focusing on proliferative and angiogenic processes that involve the Notch and IGF1 pathways. We used mammary tissue samples from pre and pubertal heifers, treated or untreated with anthelmintics, and MAC-T bovine mammary epithelial cells in vitro. Anthelminthic treatment effectively lowered EPG in feces. Mammary glands from treated heifers had increased proliferation rate (measured by PCNA) and angiogenic marker expression (VEGF and CD34), as well as increased αSMA area compared to age-matched control parasitized heifers. These changes were preceded by increased expression of Notch targets at 20 wk of age (HES1, HEY2, and HEY1), indicating a possible interaction. Similarly, IGF1R expression was increased at 30 weeks of age. To study the crosstalk between systems, bovine MAC-T cells were treated with DAPT (50 μM) to inhibit Notch signaling. DAPT decreased the proliferation of cells as evidenced by a decrease in PCNA, pERK, CYCYLIN D1; and the wound healing capacity of HMEC cells was impaired in the presence of the supernatants of DAPT-treated cells. Furthermore, DAPT decreased IGF1 and increased IGF1R mRNA levels in MAC-T cells. On the other hand, cells treated with 10 ng/mL IGF1 Increased their proliferation (MTS assay), and induced a strong tendency to increase Notch target genes (HEY1, and HES1). Furthermore, IGF1 treatment tampered the decrease in the proliferation rate induced by DAPT. Finally, a positive correlation between the IGF1R and Notch target genes (HEY1, and HES1) further suggested a relation between these two signaling systems in the bovine mammary gland. In conclusion, pubertal delay related to parasitosis is counteracted by anthelminthic treatments, which increase serum IGF1, mammary cell proliferation, and angiogenesis. We postulate the Notch pathway, mainly through the HEY1 target gene, which is modulated by the IGF1 system, may regulate both proliferative and angiogenic processes favoring normal development of the bovine mammary gland during puberty. In addition, we demonstrate that the interaction between the Notch and the IGF1 pathways may affect cell proliferation.

The study aimed to conduct a survey on the occurrence of benzimidazole (BZ) resistance in strongyles by in vitro egg hatch test (EHT) and larval development test (LDT) and to identify the effective indicators of early resistance detection on horse farms with associated risk factors analysis appraisal. In total, 203 horses from 8 farms underwent the fecal sampling of which 77 horses were selected for in vitro testing. Simultaneously, 18 horses were chosen to analyse the results of in vitro tests compared to the in vivo fecal egg count reduction test (FECRT). The EHT indicated the presence of resistant strongyles in all farms with an exceeded threshold of 0.1 μg/ml thiabendazole (TBZ) in 43 horses (55.84 %). The percent egg hatch at 0.1 μg/ml TBZ in "resistant parasites" varies from 39.0 ± 6.0 % to 91.0 ± 1.0 %. The hatching range between 8.5 ± 1.5 % and 50.0 ± 6.0 % at a concentration of 0.1 μg/ml TBZ was detected wherein the recommended threshold was not exceeded. The analysis of the EHT results demonstrated that egg hatching at the 0.1 µg/ml TBZ concentration was identified as the most important predictor of the early detection of BZ resistance. In the LDT, the mean TBZ concentration which interrupted the development of 50 % and 99 % larvae to the infective stage (L3) from all horses was 0.0753 ± 0.0454 and 0.6798 ± 1.9144 μg/ml TBZ, respectively. Only cyathostomin L3 were found at TBZ concentrations ≥ 0.08 μg/ml and only in samples from 81.8 % of horses (36/44). A comparison of LDT results did not show a statistically significant agreement with EHT and FECRT. In conclusion, the in vitro EHT could be implemented as an indicator for early BZ resistance detection and showed that monitoring of hatching at selected concentrations could detect presence and estimate the proportion of the resistant parasite population on the horse farms.

Trichinellosis is a serious foodborne and zoonotic parasitic disease caused by Trichinella family. At present, the main on-site detection method for Trichinella spiralis (T. spiralis) infection is the lateral flow assay (LFA). Other diagnostic techniques for this parasite cannot be applied to on-site testing due to their reliance on special instruments. Here, we established an ELISA smartphone-based method for detecting anti-T. spiralis antibodies in pig serum. The use of horseradish peroxidase-labeled goat anti-pig IgG-modified gold nanoparticle (AuNPs@HRP-IgG) effectively increased the sensitivity of the method. The entire reaction was carried out at room temperature without the need for special instruments. A low-cost and portable device for imaging and processing experimental data was also developed. Validation analysis revealed that the specificity of the test was 98.89 %, while its sensitivity was 100.00 %. T. spiralis antibodies could be detected in pig serum beginning at 25 dpi after infection with the muscle larvae. This visual immunosensor facilitates on-site detection of T. spiralis, especially in regions lacking specialized laboratory equipment.