Ace and ace-like genes of invasive redlegged earth mite: copy number variation,target-site mutations,and their associations with organophosphate insensitivity

Background

Invasive Australian populations of redlegged earth mite, Halotydeus destructor (Tucker), are evolving increasing organophosphate resistance. In addition to the canonical ace gene, the target gene of organophosphates, the H. destructor genome contains many radiated ace-like genes that vary in copy number and amino acid sequence. In this work, we characterise copy number and target-site mutation variation at the canonical ace and ace-like genes and test for potential associations with organophosphate insensitivity. This was achieved through comparisons of whole-genome pool-seq data from alive and dead mites following organophosphate exposure.

Results

A combination of increased copy number and target-site mutations at the canonical ace was associated with organophosphate insensitivity in H. destructor. Resistant populations were segregating for G119S, A201S, F331Y at the canonical ace. A subset of populations also had copy numbers of canonical ace > 2, which potentially helps overexpress proteins carrying these target-site mutations. Haplotypes possessing different copy numbers and target-site mutations of the canonical ace gene may be under selection across H. destructor populations. We also detected some evidence that increases in copy number of radiated ace-like genes are associated with organophosphate insensitivity, which might suggest potential roles in sequestration or breakdown of organophosphates.

Conclusion

Different combinations of target-site mutations and (or) copy number variation in the canonical ace and ace-like genes may provide non-convergent ways for H. destructor to respond to organophosphate selection. However, these changes may only play a partial role in organophosphate insensitivity, which appears to have a polygenic architecture. © 2023 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.     

Unraveling the morphological patterns of a subantarctic eelpout: a geometric morphometric approach

Phenotypic variation in organisms depends on the genotype and the environmental constraints of the habitat that they exploit. Therefore, for marine species inhabiting contrasting aquatic conditions, it is expected to find covariation between the shape and its spatial distribution. We studied the morphology of the head and cephalic sensory canals of the eelpout Austrolycus depressiceps (4.5–22.5 cm TL) across its latitudinal distribution in South Pacific (45°S–55°S). Geometric morphometric analyses show that the shape varied from individuals with larger snout and an extended suborbital canal to individuals with shorter snouts and frontally compressed suborbital canal. There was size variation across the sampled populations, but that size does not have a clear latitudinal gradient. Only 8% of the shape variation relates to this size variation (allometry), represented by a decrease in the relative size of the eye, and a depression of the posterior margin of the head. There were spatial differences in the shape of the head, but these differences were probably caused by allometric effects. Additionally, 2 of the canals of the cephalic lateralis pores and the head shape showed modularity in its development. This study shows that the morphology of marine fish with a shallow distribution varies across distances of hundreds of kilometers (i.e., phenotypic modulation).     

Acute oral toxicity of zinc phosphide: an assessment for wild house mice (Mus musculus)

Irregular plagues of house mice, Mus musculus, incur major economic impacts on agricultural production in Australia. The efficacy of zinc phosphide (ZnP), the only registered broadacre control agent for mice, is reported as increasingly variable. Have mice become less sensitive over time or are they taking a sub-lethal dose and developing aversion? In this laboratory study, the sensitivity of mice (wild caught; outbred laboratory strain) was assessed using oral gavage of a range of ZnP concentrations. The estimated LD₅₀ values (72–79 mg ZnP/kg body weight) were similar for each mouse group but are significantly higher than previously reported. The willingness of mice to consume ZnP-coated grains was determined. ZnP-coated grains (50 g ZnP/kg grain) presented in the absence of alternative food were consumed and 94% of wild mice died. Mice provided with alternative food and ZnP-coated wheat grains (either 25 or 50 g ZnP/kg grain) consumed toxic and non-toxic grains, and mortality was lower (33–55%). If a sublethal amount of ZnP-coated grain was consumed, aversion occurred, mostly when alternative food was present. The sensitivity of wild house mice to ZnP in Australia is significantly lower than previously assumed. Under laboratory conditions, ZnP-coated grains coated with a new higher dose (50 g ZnP/kg grain) were readily consumed. Consumption of toxic grain occurred when alternative food was available but was decreased. Our unambiguous findings for house mice indicate a re-assessment of the ZnP loading for baits used for control of many rodents around the world may be warranted.     

Fractionated oral dosing and its effect on cyclophosphamide pharmacokinetics in dogs with high-grade multicentric lymphoma

Cyclophosphamide (CP) is an alkylating agent commonly included in multi-drug treatment protocols for canine cancer. As a prodrug, CP requires hepatic metabolism for activation to the intermediate compound 4-hydroxycyclophosphamide (4-OHCP) which then spontaneously forms alkylating phosphoramide mustard. CP is frequently administered in a fractionated manner, with the total dose given over multiple days. CP is reported to cause auto-induction of metabolism in humans, with faster CP clearance and relatively increased 4-OHCP formation following fractionated versus bolus dosing, however canine pharmacokinetic studies of CP dose fractionation are lacking. The study objective was to evaluate the pharmacokinetics of fractionated oral CP dosing at a dose of 200–250 mg/m² over 3 to 4 days in a prospectively identified population of cancer-bearing dogs. Plasma concentrations of CP and 4-OHCP were measured by ultra-high performance liquid chromatography tandem-mass spectrometry in eight dogs following the first and last doses to assess for auto-induction of CP metabolism. No significant difference in the rate of CP elimination between first and last doses were detected (0.73 ± 0.46 vs. 1.22 ± 0.5 h⁻¹; p = .125). Additionally, no significant difference in dose-normalized 4-OHCP exposure was identified between first and last doses (5.9 ± 2.1 vs. 7.9 ± 6.4 h × ng/ml; p = .936). These results suggest that fractionated dosing may not increase exposure to the active metabolite of CP in dogs as it does in humans. As such, standard bolus dosing and fractionated dosing may be equivalent in terms of bio-activation of CP in dogs administered a dose of 200–250 mg/m².     

Extracellular heat shock protein 70 levels in tumour-bearing dogs and cats treated with radiation therapy and hyperthermia

Hyperthermia is a form of a cancer treatment which is frequently applied in combination with radiotherapy (RT) to improve therapy responses and radiosensitivity. The mode of action of hyperthermia is multifactorial; the one hand by altering the amount of the blood circulation in the treated tissue, on the other hand by modulating molecular pathways involved in cell survival processes and immunogenic interactions. One of the most dominant proteins induced by hyperthermia is the major stress-inducible heat shock protein 70 (Hsp70). Hsp70 can be found in the blood either as a free-protein (free HSP70) derived from necrotic cells, or lipid-bound (liposomal Hsp70) when it is actively released in extracellular vesicles (EVs) by living cells. The aim of the study was to evaluate the levels of free and liposomal Hsp70 before and after treatment with RT alone or hyperthermia combined with radiotherapy (HTRT) in dogs and cats to evaluate therapy responses. Peripheral blood was collected from feline and canine patients before and at 2, 4, 6 and 24 h after treatment with RT or HTRT. Hsp70 enzyme-linked immunosorbent assays (ELISAs) were performed to determine the free and liposomal Hsp70 concentrations in the serum. The levels were analysed after the first fraction of radiation to study immediate effects and after all applied fractions to study cumulative effects. The levels of free and liposomal Hsp70 levels in the circulation were not affected by the first singular treatment and cumulative effects of RT in cats however, after finalizing all treatment cycles with HTRT free and liposomal Hsp70 levels significantly increased. In dogs, HTRT, but not treatment with RT alone, significantly affected liposomal Hsp70 levels during the first fraction. Free Hsp70 levels were significantly increased after RT, but not HTRT, during the first fraction in dogs. In dogs, on the other hand, RT alone resulted in a significant increase in liposomal Hsp70, but HTRT did not significantly affect the liposomal Hsp70 when cumulative effects were analysed. Free Hsp70 was significantly induced in dogs after both, RT and HTRT when cumulative effects were analysed. RT and HTRT treatments differentially affect the levels of free and liposomal Hsp70 in dogs and cats. Both forms of Hsp70 could potentially be further investigated as potential liquid biopsy markers to study responses to RT and HTRT treatment in companion animals.     

Evaluation of mechlorethamine,vinblastine, procarbazine,and prednisone for the treatment of resistant multicentric canine lymphoma

Multi-agent chemotherapy successfully induces remission in most naïve, high-grade canine lymphoma patients; however, disease recurrence is common. MOPP (mechlorethamine, vincristine, procarbazine, and prednisone) is an effective rescue protocol used to re-induce remission, but is associated with gastrointestinal toxicity and can be a less desirable option for patients that previously failed vincristine-containing protocols. Therefore, alternative members of the vinca alkaloid family, such as vinblastine, could be potentially advantageous as substitutes for vincristine to reduce gastrointestinal toxicity and chemoresistance. The objective of this study was to report the clinical outcomes and toxicity of 36 dogs with relapsed or refractory multicentric lymphoma treated with a modified MOPP protocol whereby vincristine was replaced with vinblastine (MVPP). The overall response rate to MVPP was 25% with a median progression free survival of 15 days and a median overall survival of 45 days. MVPP at the prescribed doses resulted in modest and transient clinical benefit, but was well tolerated with no treatment delays or hospitalizations secondary to side effects. Given the minimal toxicity, dose intensification could be considered to improve clinical responses.     

Evaluation of hemostatic derangements associated with canine anaphylaxis and the relationship to syndrome severity

Objective

To describe hemostatic derangements associated with canine anaphylaxis and to assess for association with syndrome severity.

Design

Prospective observational study.

Setting

University teaching hospital.

Animals

Twenty-seven client-owned dogs, recruited from November 2018 to January 2022, diagnosed with anaphylaxis of varying severity were included. Study inclusion required presentation <6 hours after initiation of clinical signs, no medications or history of illness within the prior 2 weeks, lack of comorbidities expected to affect hemostasis, and lack of a disease state that could alternatively explain the clinical presentation.

Interventions

Blood samples were collected within the first hour of presentation for CBC, serum biochemistry, prothrombin time (PT), activated partial thromboplastin time (aPTT), and viscoelastic coagulation testing for use with a cartridge-based point-of-care device.

Measurements and main results

Clotting time and clot formation time were prolonged, alpha angle and maximum clot firmness were decreased, PT and aPTT were prolonged, and platelet counts were lower in severe cases compared to mild and moderate cases. There were no differences for any parameter between mild and moderate cases. The presence or absence of abdominal effusion was not associated with hemostatic status.

Conclusions

Global hemostatic derangements consistent with hypocoagulability are a prominent feature of severe anaphylaxis in dogs and should be considered for routine evaluation.     

Comprehensive transcriptional profiling and functional study of lymphangiogenic growth factor in placentome of early-stage pregnant water buffalo

The aim of the present study was to evaluate the expression and localization of lymphangiogenic factors (VEGF-C and VEGF-D), their receptor (VEGFR3) and lymphatic endothelial marker (LYVE1) in buffalo placenta during early pregnancy [EP], and to investigate the functional role of lymphangiogenic growth factors in placental lymphangiogenesis. The mRNA and protein expression of VEGF-C, VEGF-D, their receptor VEGFR3 and LYVE1 showed significant expression in EP1 (29–42 days) and EP2 stages (51–82 days) both in caruncle (maternal part) and cotyledon (foetal part) of the buffalo placenta. Immunoreactivity of VEGF-C, VEGF-D and LYVE1 was observed around the endometrial gland, in lymphatics and trophoblast cells, whereas VEGFR3 mainly localized in lymphatics of the caruncle and cotyledons. Cultured trophoblast cells were treated with VEGF-C/VEGF-D (50, 100 and 150 ng/ml) and combined doses of VEGF-C and VEGF-D (150 ng/ml) each for different time durations (24, 48 and 72 h). The mRNA expression of LYVE1 and PCNA was significantly (p < .001) upregulated with VEGF-C and VEGF-D and combined treatment (@150 ng/ml), as well as significantly downregulating Caspase-3 at 48 and 72 h. Thus, the present study provides evidence that lymphangiogenic factors are expressed in buffalo placental compartments and they may play a significant role in the regulation of placental function in water buffaloes.     

Onchocerca sp. in an imported Zangersheide gelding causing suspensory ligament desmitis

A 5-year-old imported Zangersheide gelding was evaluated for SC swellings over both forelimbs and lameness localized to the distal metacarpus. Ultrasound examination of the SC masses was compatible with verminous granulomas. Linear hyperechoic foci were present within the suspensory ligament branches of both forelimbs, suggestive of ligamentous parasitic infiltrates. A diagnosis of onchocerciasis was confirmed on biopsy of a SC mass. The gelding was treated with ivermectin and a tapering course of PO dexamethasone but was eventually euthanized. Necropsy confirmed the presence of SC eosinophilic granulomas and degenerative suspensory ligament desmitis, both with intralesional nematodes. Given the location and appearance of the nematode, a diagnosis of Onchocerca sp., most likely O. reticulata, was made. Onchocerciasis should be included as a differential diagnosis for multifocal suspensory ligament desmitis with these sonographic characteristics when paired with SC masses in imported European Warmbloods.