Evaluation of diagnostic techniques for early detection of heartworm in experimentally infected dogs: identification of Dirofilaria immitis-derived microRNA in the initial 28 weeks post-inoculation.

BACKGROUND: Dirofilaria immitis, commonly known as heartworm (HW), is a parasitic nematode transmitted by various mosquito species, leading to heartworm disease (HWD) in dogs. Diagnosis of HW typically involves antigen or microfilariae detection, or visualization of adult worms through imaging or post mortem examination. Polymerase chain reaction (PCR) and micro RNA (miRNA) detection have been explored for HW diagnosis. METHODS: Three dogs, previously experimentally infected with HW, underwent blood sampling every 4 weeks for 7 months. Samples were assessed for antigen presence after heat treatment, PCR amplification, and microfilaria examination using Giemsa-stained thick smears. Additionally, whole blood aliquots underwent miRNA deep sequencing and bioinformatic analysis. RESULTS: Heartworm antigen was detectable after heat treatment at 20 weeks post-inoculation and via PCR at 24 weeks, with microfilariae observed in peripheral blood smears at 28 weeks. However, deep miRNA sequencing revealed that the miRNA candidate sequences are not consistently expressed before 28 weeks of infection. CONCLUSIONS: While ancillary molecular methods such as PCR and miRNA sequencing may be less effective than antigen detection for detecting immature larval stages in an early stage of infection, our experimental findings demonstrate that circulating miRNAs can still be detected in 28 weeks post-infection.

The Domestic Dog as a Laboratory Host for Brugia malayi.

Of the three nematodes responsible for lymphatic filariasis in humans, only Brugia malayi is actively maintained in research settings owing to its viability in small animal hosts, principal among which is the domestic cat. While the microfilaremic feline host is necessary for propagation of parasites on any significant scale, this system is plagued by a number of challenges not as pronounced in canine filarial models. For this reason, we investigated the capacity in which dogs may serve as competent laboratory hosts for B. malayi. We infected a total of 20 dogs by subcutaneous injection of 500 B. malayi third-stage larvae (L3) in either a single (n = 10) or repeated infection events (125 L3 per week for four weeks; n = 10). Within each group, half of the individuals were injected in the inguinal region and half in the dorsum of the hind paw. To track the course of microfilaremia in this host, blood samples were examined by microscopy biweekly for two years following infection. Additionally, to identify cellular responses with potential value as predictors of patency, we measured peripheral blood leukocyte counts for the first year of infection. A total of 10 of 20 dogs developed detectable microfilaremia. Peak microfilaria density varied but attained levels useful for parasite propagation (median = 1933 mL-1; range: 33-9950 mL-1). Nine of these dogs remained patent at 104 weeks. A two-way ANOVA revealed no significant differences between infection groups in lifetime microfilaria production (p = 0.42), nor did regression analysis reveal any likely predictive relationships to leukocyte values. The results of this study demonstrate the competence of the dog as a host for B. malayi and its potential to serve in the laboratory role currently provided by the cat, while also clarifying the potential for zoonosis in filariasis-endemic regions.

Prenatal exposure to bisphenols affects pregnancy outcomes and offspring development in rats.

The objective of this study was to evaluate the effects of gestational exposure to low doses of bisphenol A (BPA), bisphenol S (BPS), and bisphenol F (BPF) on pregnancy outcomes and offspring development. Pregnant Sprague-Dawley rats were orally dosed with vehicle, 5 µg/kg body weight (BW)/day of BPA, BPS and BPF, or 1 µg/kg BW/day of BPF on gestational days 6-21. Pregnancy and gestational outcomes, including number of abortions and stillbirths, were monitored. Male and female offspring were subjected to morphometry at birth, followed by pre- and post-weaning body weights, post-weaning food and water intakes, and adult organ weights. Ovarian follicular counts were also obtained from adult female offspring. We observed spontaneous abortions in over 80% of dams exposed to 5 µg/kg of BPF. BPA exposure increased Graafian follicles in female offspring, while BPS and BPF exposure decreased the number of corpora lutea, suggesting reduced ovulation rates. Moreover, BPA exposure increased male kidney and prostate gland weights, BPF decreased epididymal adipose tissue weights, and BPS had modest effects on male abdominal adipose tissue weights. Prenatal BPS exposure reduced anogenital distance (AGD) in male offspring, suggesting possible feminization, whereas both BPS and BPA induced oxidative stress in the testes. These results indicate that prenatal exposure to BPF affects pregnancy outcomes, BPS alters male AGD, and all three bisphenols alter certain organ weights in male offspring and ovarian function in female offspring. Altogether, it appears that prenatal exposure to BPA or its analogues can induce reproductive toxicity even at low doses.

Independent and combined effects of Bisphenol A and Diethylhexyl Phthalate on gestational outcomes and offspring development in Sprague-Dawley rats.

Bisphenol A (BPA) and Diethylhexyl Phthalate (DEHP) are well-studied endocrine disrupting chemicals (EDCs), however, the effects of mixtures of these EDCs are not. To assess the consequences of prenatal exposure to a mixture of these EDCs, dams were orally administered either saline (control), BPA (5 µg/kg BW/day), high dose DEHP (HD-D; 7.5 mg/kg BW/day), or a combination of BPA with HD-D in experiment 1; saline, BPA (5 µg/kg BW/day), low-dose DEHP (LD-D; 5 µg/kg BW/day) or a combination of BPA with LD-D in experiment 2. Gestational weights, number of abortions, litter size and weights, number of live births and stillbirths were recorded. Morphometric measures were obtained at birth and body weight, food and water intake were monitored weekly from postnatal weeks 3-12. Offspring were sacrificed at 16-24 weeks of age and organ weights were measured. The abortion rate of dams exposed to HD-D and the mixtures, BPA + LD-D and BPA + HD-D were higher at 9, 14 and 27% respectively. Prenatal exposure to BPA or HD-D significantly decreased relative thymus weights in male but not female offspring. Apoptotic cells were detected in thymus sections of both male and female offspring prenatally exposed to DEHP. Relative heart weights increased in BPA + HD-D exposed male offspring compared to the other groups. The results indicate that a mixture of BPA and DEHP, produced a pronounced effect on pregnancy outcomes. Male offspring appear to be more susceptible to the programming effects of these EDCs or their mixture suggesting a need to reconsider the possible additive, antagonistic or synergistic effects of EDC mixtures.