Strain-level variations of Dirofilaria immitis microfilariae in two biochemical assays

Background: The increase in reports of resistance to macrocyclic lactones in the canine heartworm, Dirofilaria immitis is alarming. While DNA based tests have been well-validated, they can be expensive. In a previous study, we showed that two biochemical tests adapted to a 96- well plate format and read in a spectrophotometer could detect differences among lab validated D. immitis isolates. The two tests- Resazurin reduction and Hoechst 33342 efflux—detect metabolism and P-glycoprotein activity respectively in microfilariae isolated from infected dog blood. Methods: Our objective was to optimize the two assays further by testing various assay parameters in D. immitis isolates not tested previously. We tested microfilarial seeding density, incubation time and the effect of in vitro treatment with ivermectin and doxycycline in five other D. immitis isolates—JYD-34, Big Head, Berkeley, Georgia III and LOL. All assays were performed in 3 technical replicates and 2–4 biological replicates. To understand the molecular basis of the assays, we also performed qPCR for selected drug metabolism and elimination associated genes of the ABC transporter and cytochrome P450 gene families. Results: Metabolism and ABC transporter activity as detected by these assays varied between strains. Anthelmintic status (resistant or susceptible) did not correlate with metabolism or P-gp efflux. Basal transcriptional variations were found between strains in ABC transporter and cytochrome P450 genes. Conclusions: These assays provide a greater understanding of the biochemical variation among isolates of D. immitis, which can be exploited in the future to develop in vitro diagnostic tests capable of differentiating susceptible and resistant isolates.This article is published as Hampton, Naomi, Vicki Smith, Matthew T. Brewer, and Jeba RJ Jesudoss Chelladurai. "Strain-level variations of Dirofilaria immitis microfilariae in two biochemical assays." PloS one 19, no. 7 (2024): e0307261. doi: https://doi.org/10.1371/journal.pone.0307261. Copyright: © 2024 Hampton et al. This is an open access article distributed under the terms of the (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited

Naturally Acquired Sarcoptes scabiei Infestation in a Captive Southern Tamandua (Tamandua tetradactyla) and a Capybara (Hydrochoeris hydrochaeris)

A privately-owned southern tamandua (Tamandua tetradactyla) and a capybara (Hydrochoeris hydrochaeris) were presented for severe pruritus. Superficial skin scrapings revealed numerous Sarcoptes scabiei mites. Morphological examination and mitochondrial DNA sequencing revealed that both patients were infested with Sarcoptes scabiei. The animals were treated with selamectin (9 mg/kg) topically once, and the owner was instructed to repeat treatment in two weeks. The tamandua died of unknown causes three weeks after initial examination before reevaluation could occur. Clinical signs and skin lesions in the capybara resolved after two treatments with selamectin. This is the first documented report of sarcoptic mange in a southern tamandua. In addition, this is the first documented case of natural transmission of Sarcoptes mites between a tamandua and capybara in captivity. Finally, this is the first described use of topical selamectin for the treatment and resolution of sarcoptic mange in a capybara

Perineal Urethrostomy Enables Susceptibility of Bull Calves as a Natural Host Model for Bovine Trichomonosis

Tritrichomonas foetus is a sexually transmitted protozoan that causes early embryonic death in cattle. A challenge in trichomonosis research is that in vivo studies of treatments, diagnostic strategies, and vaccines are severely hampered by the logistical challenge and cost of maintaining adult bulls. Since natural infections are diagnosed in postpubescent animals, the paradigm is that only mature breeding bulls can be infected. In this study, we hypothesized that prepubescent bull calves could be artificially infected with T. foetus trophozoites for the purpose of conducting research trials. Initial attempts to directly infect bull calves with two different parasite isolates resulted in the sporadic and transient detection of parasite DNA but not culturable trophozoites. In vitro and in vivo studies suggested that urine directly inhibited trophozoites, likely by osmotic damage and mechanical flushing action. Studies utilizing a perineal urethrostomy to remove urine flow from the prepuce resulted in the ability to colonize the prepuce, with live organisms being cultured for as long as 15 days post-inoculation. Future studies optimizing this technique have the potential to accelerate the pace of bovine trichomonosis research and may have applications in the study of human trichomoniasis.This article is published as Martin, Katy A., Jenna E. Bayne, Krystal Chinchilla-Vargas, Sara L. Reece, Jeba RJ Chelladurai, Tyler A. Harm, Jodi D. Smith, Courtney N. Blake, Douglas E. Jones, and Matthew T. Brewer. "Perineal Urethrostomy Enables Susceptibility of Bull Calves as a Natural Host Model for Bovine Trichomonosis." Microorganisms (2025). doi: https://doi.org/10.3390/microorganisms13051070

Infection of prepubertal heifer calves as a natural host model for Tritrichomonas foetus

IntroductionTritrichomonas foetus is a sexually transmitted flagellate that causes economic loss in the cattle industry throughout the world. In the United States, there are no approved treatments for the parasite. Owing to its transmission strategy, T. foetus typically infects cattle of breeding age. However, in vivo studies of treatment, diagnostic strategies, and vaccination are severely hampered by the maintenance and cost of maintaining adult cattle in research settings. In this study, we investigated the utility of infecting pre-pubescent heifer calves with T. foetus.MethodsFour independent cohorts of cross-bred prepubertal heifer calves were vaginally inoculated with T. foetus trophozoites previously derived from a naturally-infected bull. Infections were assessed by culture, PCR, DNA sequencing, histopathology, gross pathology, and lesion scoring. In addition, reproductive tract tissue was assessed for the presence of galectin-1, a putative receptor for T. foetus trophozoite adhesion.ResultsOur experiments revealed that despite being in anestrus, heifer calves were amenable to infection with trophozoites for as long as 42 days post-infection as determined by PCR and culture of the organism. Histopathology revealed inflammation throughout the reproductive tract of infected calves. Infection resulted in endometritis with lymphoplasmacytic infiltration and demonstrated that trophozoites could pass through the cervix even during anestrus in prepubescent heifers. In addition, immunohistochemistry of the vagina, cervix, and uterus demonstrated robust expression of galectin-1.ConclusionOur experiments demonstrated that prepubertal heifer calves are a suitable natural host model for bovine trichomonosis. This is a significant breakthrough in the field and also has potential for advancing the human trichomoniasis research agenda

Genomic differences and species delimitation: a case for two species in the zoonotic cestode<i>Dipylidium caninum</i>

AbstractDipylidium caninum(Linnaeus, 1758) is a common zoonotic cestode of dogs and cats worldwide. Previous studies have demonstrated the existence of largely host associated canine and feline genotypes based on infection studies, genetic differences at the nuclear 28S rDNA gene and complete mitochondrial genomes. There have been no comparative studies at a genome-wide scale. Here, we sequenced the genomes of a dog and cat isolate ofDipylidium caninumfrom the United States using the Illumina platform and conducted comparative analyses with the reference draft genome. Complete mitochondrial genomes were used to confirm the genotypes of the isolates.D. caninumcanine and feline genomes generated in this study had mean coverage depths of 45x and 26x and an average identity of 98% and 89% respectively when compared to the reference genome. SNPs were 20 times higher in the feline isolate. Comparison and species delimitation using universally conserved orthologs and protein coding mitochondrial genes revealed that the canine and feline isolates are different species. Data from this study builds a base for future integrative taxonomy. Further genomic studies from geographically diverse populations are necessary to understand implications for taxonomy, epidemiology, veterinary clinical medicine, and anthelmintic resistance.</jats:p

Comparative Genomic Analysis and Species Delimitation: A Case for Two Species in the Zoonotic Cestode Dipylidium caninum

Dipylidium caninum (Linnaeus, 1758) is a common zoonotic cestode of dogs and cats worldwide. Previous studies have demonstrated the existence of largely host-associated canine and feline genotypes based on infection studies, differences at the 28S rDNA gene, and complete mitochondrial genomes. There have been no comparative genome-wide studies. Here, we sequenced the genomes of a dog and cat isolate of Dipylidium caninum from the United States using the Illumina platform at mean coverage depths of 45&times; and 26&times; and conducted comparative analyses with the reference draft genome. Complete mitochondrial genomes were used to confirm the genotypes of the isolates. Genomes of D. caninum canine and feline genotypes generated in this study, had an average identity of 98% and 89%, respectively, when compared to the reference genome. SNPs were 20 times higher in the feline isolate. Comparison and species delimitation using universally conserved orthologs and protein-coding mitochondrial genes revealed that the canine and feline isolates are different species. Data from this study build a base for future integrative taxonomy. Further genomic studies from geographically diverse populations are necessary to understand implications for taxonomy, epidemiology, veterinary clinical medicine, and anthelmintic resistance

Efficacy of ponazuril in weaned feeder lambs with naturally acquired coccidiosis

Coccidiosis is a common and economically significant parasitic disease that causes diarrhea, weight loss, and death in lambs. Coccidiostats currently labeled in the US for ovine coccidiosis are challenging to administer at effective doses in sick lambs and clinically ineffectual when used late in the disease process. Newer triazinone-class coccidiocidial drugs, such as toltrazuril (Baycox®, Bayer Animal Health) are effective across a greater range of Eimeria's life cycle, resulting in improved clinical response and treatment compliance. Ponazuril (toltrazuril sulfone), an active metabolite of toltrazuril, is labeled in the United States for treatment of equine protozoal myeloencephalitis and is being used extra-label in sheep and goats for treatment of coccidiosis despite a lack of published data regarding its clinical effectiveness in sheep. The objective of this study is to determine the efficacy (fecal oocyst shedding, fecal consistency, and lamb growth performance) of a single low (2.27 mg/lb; 5 mg/kg) or high (9.1 mg/lb; 20 mg/kg) oral dose of ponazuril for treatment of naturally-acquired coccidiosis (Eimeria spp) in commercial feeder lambs.</jats:p

Assessment of in vitro efficacy for common surface disinfectants and antiseptics against Tritrichomonas foetus trophozoites

The protozoan Tritrichomonas foetus causes early embryonic death in cattle, there are no legal options for treating this parasite in the United States, and there are few developed protocols for cleaning veterinary and obstetrical equipment that may have been contaminated with trophozoites. In this study, we evaluated bleach, ethanol, acetic acid, chlorhexidine gluconate, and hydrogen peroxide solutions for the ability to kill trophozoites in vitro. Our findings suggested that ethanol and bleach could adequately disinfect tools and equipment. Acetic acid, chlorhexidine, and hydrogen peroxide had applications as surface disinfectants in addition to potential as local topical treatments due to their past uses in veterinary theriogenology. Chlorhexidine gluconate demonstrated trophocidal effects by damaging parasite cell membranes and had the lowest effective concentration 50 (EC50) of any compound tested and was in the micromolar range. These findings, in conjunction with accepted clinical uses of chlorhexidine gluconate suggest that this is a convenient agent for disinfecting equipment. In addition, topical use of chlorhexidine is relatively common, setting the stage for further investigation of this compound as a topical therapeutic option for bovine trichomonosis.This article is published as Martin, Katy A., Matt Brewer, Kris Kovach, Elizabeth Carreiro, Jeba Jesudoss Chelladurai, and Erica Moscoso. "Assessment of in vitro efficacy of common surface disinfectants and antiseptics against Tritrichomonas foetus trophozoites." Frontiers in Veterinary Science 10: 1282274. doi: https://doi.org/10.3389/fvets.2023.1282274. © 2023 Martin, Kovach, Moscoso, Carreiro, Jesudoss Chelladurai and Brewer. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY)

Room Decontamination Using Ionized Hydrogen Peroxide Fog and Mist Reduces Hatching Rates of Syphacia obvelata Ova

This study evaluated the efficacy of ionized hydrogen peroxide (iHP) fog and mist for environmental and surface decontaminationof Syphacia obvelata ova in rodent rooms. Ova were collected by perianal tape impression from S. obvelata infectedmice. In experiment 1, ova were exposed to iHP using a whole-room fogging decontamination system with a 15 min initialfog application cycle in unoccupied rodent rooms. Ova were removed from the fogged environment after a 15 min, 30 min, 90min, or 240 min iHP exposure time. In experiment 2, a second cohort of ova were exposed to iHP using the whole-room foggingdecontamination system. Ova were removed after 3, 4 or 6 continuous fog application cycles with 45 min dwelling timebetween each cycle and 15 h dwelling time for the last time point. In experiment 3, a third set of ova was exposed to an iHPsurface misting unit with 1, 2, or 3 iHP mist applications. A 7 min contact time followed each application. After exposure, ovawere incubated in a hatching medium for 6 h. Control ova were maintained at room temperature without iHP exposure beforeincubation in the hatching medium. After incubation, the number of ova hatched was assessed by microscopic examination.For experiment 1, results ranged from 46% to 57% of exposed ova hatched. For experiment 2, results ranged from 43% to 49%of ova hatched. For experiment 3, 37% to 46% of exposed ova hatched. Conversely, for the control groups above 80% of ovahatched for all 3 experiments. These data suggest that exposure to iHP fog and mist has variable effectiveness in reducingviability of S. obvelata ova at the time points tracked. Further studies are needed to identify iHP exposures that will furtherreduce or eliminate the hatching of rodent pinworm ova. </jats:p