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Onchocerca volvulus is a filarial parasite that is a major cause of dermatitis and blindness in endemic regions primarily in sub-Saharan Africa. Widespread efforts to control the disease caused by O. volvulus infection (onchocerciasis) began in 1974 and in recent years, following successful elimination of transmission in much of the Americas, the focus of efforts in Africa has moved from control to the more challenging goal of elimination of transmission in all endemic countries. Mass drug administration (MDA) with ivermectin has reached more than 150 million people and elimination of transmission has been confirmed in four South American countries, with at least two African countries having now stopped MDA as they approach verification of elimination. It is essential that accurate data for active transmission are used to assist in making the critical decision to stop MDA, since missing low levels of transmission and infection can lead to continued spread or recrudescence of the disease. Current World Health Organization guidelines for MDA stopping decisions and post-treatment surveillance include screening pools of the Simulium blackfly vector for the presence of O. volvulus larvae using a PCR-ELISA-based molecular technique. In this study, we address the potential of an updated, practical, standardized molecular diagnostic tool with increased sensitivity and species-specificity by comparing several candidate qPCR assays. When paired with heat-stable reagents, a qPCR assay with a mitochondrial DNA target (OvND5) was found to be more sensitive and species-specific than an O150 qPCR, which targets a non-protein coding repetitive DNA sequence. The OvND5 assay detected 19/20 pools of 100 blackfly heads spiked with a single L3, compared to 16/20 for the O150 qPCR assay. Given the improved sensitivity, species-specificity and resistance to PCR inhibitors, we identified OvND5 as the optimal target for field sample detection. All reagents for this assay can be shipped at room temperature with no loss of activity. The qPCR protocol we propose is also simpler, faster, and more cost-effective than the current end-point molecular assays.

The search for new macrofilaricidal drugs against onchocerciasis that can be administered in shorter regimens than required for doxycycline (DOX, 200mg/d given for 4-6 weeks), identified minocycline (MIN) with superior efficacy to DOX. Further reduction in the treatment regimen may be achieved with co-administration with standard anti-filarial drugs. Therefore a randomized, open-label, pilot trial was carried out in an area in Ghana endemic for onchocerciasis, comprising 5 different regimens: the standard regimen DOX 200mg/d for 4 weeks (DOX 4w, N = 33), the experimental regimens MIN 200mg/d for 3 weeks (MIN 3w; N = 30), DOX 200mg/d for 3 weeks plus albendazole (ALB) 800mg/d for 3 days (DOX 3w + ALB 3d, N = 32), DOX 200mg/d for 3 weeks (DOX 3w, N = 31) and ALB 800mg for 3 days (ALB 3d, N = 30). Out of 158 randomized participants, 116 (74.4%) were present for the follow-up at 6 months of whom 99 participants (63.5%) followed the treatment per protocol and underwent surgery. Histological analysis of the adult worms in the extirpated nodules revealed absence of Wolbachia in 98.8% (DOX 4w), 81.4% (DOX 3w + ALB 3d), 72.7% (MIN 3w), 64.1% (DOX 3w) and 35.2% (ALB 3d) of the female worms. All 4 treatment regimens showed superiority to ALB 3d (p < 0.001, p < 0.001, p = 0.002, p = 0.008, respectively), which was confirmed by real-time PCR. Additionally, DOX 4w showed superiority to all other treatment arms. Furthermore DOX 4w and DOX 3w + ALB 3d showed a higher amount of female worms with degenerated embryogenesis compared to ALB 3d (p = 0.028, p = 0.042, respectively). These results confirm earlier studies that DOX 4w is sufficient for Wolbachia depletion and the desired parasitological effects. The data further suggest that there is an additive effect of ALB (3 days) on top of that of DOX alone, and that MIN shows a trend for stronger potency than DOX. These latter two results are preliminary and need confirmation in a fully randomized controlled phase 2 trial. ClinicalTrials.gov #06010453.

Within the research conducted in the years 2016–2022 in the area of Volovsky Mountains in Slovakia, 63,950 biting midges were collected during 74 trapping sessions. The aim of the study was to identify species composition of biting midges, their host preference and potential transmission of parasites by these insects under natural conditions. The collected biting midges fell into 29 species and the most common were the Culicoides ( C. obsoletus / C. scoticus / C. montanus ) that accounted for 47.9% of the collected biting midges. Identification of species was based on the morphology of biting midges and the use of molecular methods. We confirmed positive suckling results on red deer in three samples namely C. montanus , C. scoticus and C. deltus . We examined these samples for the presence of Onchocerca worm DNA. We confirmed the presence of O. flexuosa DNA in one C. deltus sample. The host preference of biting midges was identified by molecular technique that involved sequencing a 350-bp sequence of the mitochondrial cytochrome b gene (cyt b). The presence of Onchocerca flexuosa DNA in C. deltus was confirmed by sequencing of fragments of mitochondrial genes cox1. The sequences matched the previously published sequences for O. flexuosa . Data on high prevalence of infections caused by Onchocerca worms in red deer in Slovakia have already been published and indicated favourable conditions for the vectors and a suitable environment for parasite circulation in Slovakia. According to the authors’ knowledge, this was the first ever detection of O. flexuosa in C. deltus in wild nature.

BACKGROUND: microRNAs (miRNAs), a class of short, non-coding RNA can be found in a highly stable, cell-free form in mammalian body fluids. Specific miRNAs are secreted by parasitic nematodes in exosomes and have been detected in the serum of murine and dog hosts infected with the filarial nematodes Litomosoides sigmodontis and Dirofilaria immitis, respectively. Here we identify extracellular, parasite-derived small RNAs associated with Onchocerca species infecting cattle and humans. METHODS: Small RNA libraries were prepared from total RNA extracted from the nodule fluid of cattle infected with Onchocerca ochengi as well as serum and plasma from humans infected with Onchocerca volvulus in Cameroon and Ghana. Parasite-derived miRNAs were identified based on the criteria that sequences unambiguously map to hairpin structures in Onchocerca genomes, do not align to the human genome and are not present in European control serum. RESULTS: A total of 62 mature miRNAs from 52 distinct pre-miRNA candidates were identified in nodule fluid from cattle infected with O. ochengi of which 59 are identical in the genome of the human parasite O. volvulus. Six of the extracellular miRNAs were also identified in sequencing analyses of serum and plasma from humans infected with O. volvulus. Based on sequencing analysis the abundance levels of the parasite miRNAs in serum or plasma range from 5 to 127 reads/per million total host miRNA reads identified, comparable to our previous analyses of Schistosoma mansoni and L. sigmodontis miRNAs in serum. All six of the O. volvulus miRNAs identified have orthologs in other filarial nematodes and four were identified in the serum of mice infected with L. sigmodontis. CONCLUSIONS: We have identified parasite-derived miRNAs associated with onchocerciasis in cattle and humans. Our results confirm the conserved nature of RNA secretion by diverse nematodes. Additional species-specific small RNAs from O. volvulus may be present in serum based on the novel miRNA sequences identified in the nodule fluid. In our analyses comparison to European control serum illuminates the scope for false-positives, warranting caution in criteria that should be applied to identification of biomarkers of infection.

Long-standing reports of open sores on the hind legs of moose ( Alces alces ) have been recorded in Alaska (as well as Canada, Europe, and Michigan), eliciting concerns about causes and infection. We used histological and genomic methods to investigate the sores from 20 adult moose on the Kenai Peninsula, Alaska. We paired this with thermal imagery and molt scoring of adult moose to further describe sore formation and understand its timing. Severe, ulcerative and eosinophilic dermatitis was found in all moose with sores present, and microfilariae within intraepidermal pustules were additionally found in four samples. Genetic analysis of sores from moose revealed a previously unknown genetic lineage of Onchocerca . Adult moose molt and lose their barrier of protection against flies in June and July during peak fly activity, leaving them vulnerable and allowing the development of sores. In summary, our results indicate that the cause for the sores on the hindleg of moose is a previously unknown genetic lineage of Onchocerca , probably transmitted by black flies, in timing with the molt cycle of adult moose. These sores leave moose exposed to pathogens, making them vulnerable, and challenging their health and fitness.

Onchocerca lupi is a filarial nematode that causes ocular onchocercosis in canines globally including North America and areas of Europe, North Africa, and the Middle East. Reported incidence of this parasite in canines has continued to steadily escalate since the early 21 st century and was more recently documented in humans. Whole genome sequencing (WGS) of this parasite can provide insight into gene content, provide novel surveillance targets, and elucidate the origin and range expansion. However, past attempts of whole genome sequencing of other Onchocerca species reported a substantial portion of their data unusable due to the variable over-abundance of host DNA in samples. Here, we have developed a method to determine the host-to-parasite DNA ratio using a quantitative PCR (qPCR) approach that relies on two standard plasmids each of which contains a single copy gene specific to the parasite genus Onchocerca (major body wall myosin gene, myosin) or a single copy gene specific to the canine host (polycystin-1 precursor, pkd1 ). These plasmid standards were used to determine the copy number of the myosin and pkd1 genes within a sample to calculate the ratio of parasite and host DNA. Furthermore, whole genome sequence (WGS) data for three O . lupi isolates were consistent with our host-to-parasite DNA ratio results. Our study demonstrates, despite unified DNA extraction methods, variable quantities of host DNA within any one sample which will likely affect downstream WGS applications. Our quantification assay of host-to-parasite genome copy number provides a robust and accurate method of assessing canine host DNA load in an O . lupi specimen that will allow informed sample selection for WGS. This study has also provided the first whole genome draft sequence for this species. This approach is also useful for future focused WGS studies of other parasites.

This case-series describes the 6 human infections with Onchocerca lupi, a parasite known to infect cats and dogs, that have been identified in the United States since 2013. Unlike cases reported outside the country, the American patients have not had subconjunctival nodules but have manifested more invasive disease (eg, spinal, orbital, and subdermal nodules). Diagnosis remains challenging in the absence of a serologic test. Treatment should be guided by what is done for Onchocerca volvulus as there are no data for O. lupi. Available evidence suggests that there may be transmission in southwestern United States, but the risk of transmission to humans is not known. Research is needed to better define the burden of disease in the United States and develop appropriately-targeted prevention strategies.

Background Onchocerca lupi is a zoonotic, vector-borne filarioid nematode that mainly infects wild and domestic canids in the Southwestern USA, Europe, Asia, and Africa. Clinical canine infections are associated with ocular disease, characterized by the presence of nodules and conjunctivitis. Subclinical cases can be challenging to diagnose, even with evaluation of cutaneous tissues for microfilariae. Current diagnostic tests include conventional polymerase chain reaction (cPCR) to detect O. lupi DNA, and, alternatively, real-time PCR (qPCR), which provides more rapid results and higher throughput. The objectives of this study were to: I) optimize a novel qPCR assay that detects O. lupi and II) to assess the prevalence of O. lupi in shelter dogs from Albuquerque, NM, USA. Methods This probe-based qPCR was optimized with a detection threshold of 0.33 pg for DNA of an adult female O. lupi . We further optimized the assay by performing a dynamic range test to determine the ideal dilution factor and inclusion of an internal positive control. We collected skin snips from the interscapular region of 404 dogs between January and September 2023. Demographics were recorded, including age, sex, American Kennel Club breed groups, and coat color. Dogs were separated into age groups, including juveniles ≤ 1 year old ( n =  120; 29.7%), adults > 1–7 years old ( n =  260; 64.3%), and seniors > 7 years old ( n =  24; 5.9%). Of those, 194 were female, and 210 were male. We also had nine different American Kennel Club breed groups represented, as well as two coat colors: single (33.0%) and mixed (67.0%). Genomic DNA was subjected to cPCR followed by Sanger sequencing and our probe-based qPCR. Both PCRs targeted a fragment of the cytochrome oxidase c subunit 1 ( cox1 ) of the mitochondrial DNA. We performed statistical analysis to assess any association between exposure factors, such as age, sex, breed, and coat color and the outcome, whether O. lupi was present. Results Overall, eight (1.9%; 95% confidence interval (CI) 0.8–3.8%) dogs tested O. lupi -positive via qPCR and five (1.2%; 95% CI 0.4–2.8%) via cPCR. Of the qPCR-positive dogs, six were adults and two were juveniles. Age ( P  = 0.704), sex ( P  = 0.910), breed groups ( P  = 0.217), and coat color ( P  = 0.781) were not statistically associated with a qPCR-positive result with a cutoff of P  < 0.2. In addition, 20 dogs tested positive for Cercopithifilaria bainae via cPCR and sequencing, but these did not cross-react with our qPCR. Conclusions This is the first epidemiological study on O. lupi in a canine population from an urban center within an endemic area in North America. Active surveillance using reliable diagnostic tools can better elucidate the epidemiology of this zoonotic parasite and enable the implementation of strategies for control and prevention. Graphical Abstract

Onchocerca lupi is a parasitic filarioid and the causative agent of canine ocular onchocercosis, a zoonotic disease of domestic dogs with sporadic reports in humans. A 13-year-old dog with no travel history outside of Israel was presented to an ophthalmology veterinary clinic in Israel with severe right ocular and periocular disease. After surgical exploration, thin helminths were removed from the dorsal sclera of the eye and identified as Onchocerca lupi by polymerase chain reaction according to the cytochrome c oxidase subunit I (cox1), reduced nicotinamide adenine dinucleotide dehydrogenase subunit 5 (nad5) and 12S rRNA genes. Phylogenetic trees and haplotype networks of the cox1 and nad5 genes confirmed the circulation of two genotypes: genotype 1 with worms from dogs, cats and humans from both the Old and New Worlds, and genotype 2 with specimens from Portugal and Spain. The Israeli sequences clustered in genotype 1 and were identical to O. lupi from the USA. Evidence of two genotypes separated geographically sheds light on the phylogeography and evolution of this zoonotic pathogen, and suggests a diverse pathology observed in different regions of the world.

The impact of large scale Mass Drug Adminstration (MDA) of ivermectin on active onchocerciasis transmission by Simulium damnosum, which transmits the parasite O. volvulus is of great importance for onchocerciasis control programmes. We investigated in the Mbam river system area, the impact of MDA of ivermectin on entomological indices and also verify if there are river system factors that could have favoured the transmission of onchocerciasis in this area and contribute to the persistence of disease. We compared three independent techniques to detect Onchocerca larvae in blackflies and also analyzed the river system within 9 months post-MDA of ivermectin. Simulium flies were captured before and after 1, 3, 6 and 9months of ivermectin-MDA. The biting rate was determined and 41% of the flies dissected while the rest were grouped into pools of 100 flies for DNA extraction. The extracted DNA was then subjected to O-150 LAMP and real-time PCR for the detection of infection by Onchocerca species using pool screening. The river system was analysed and the water discharge compared between rainy and dry seasons. We used human landing collection method (previously called human bait) to collect 22,274 adult female Simulium flies from Mbam River System. Of this number, 9,134 were dissected while 129 pools constituted for molecular screening. Overall biting and parous rates of 1113 flies/man/day and 24.7%, respectively, were observed. All diagnostic techniques detected similar rates of O. volvulus infection (P = 0.9252) and infectivity (P = 0.4825) at all monitoring time points. Onchocerca ochengi larvae were only detected in 2 of the 129 pools. Analysis of the river drainage revealed two hydroelectric dams constructed on the tributaries of the Mbam river were the key contributing factor to the high-water discharge during both rainy and dry seasons. Results from fly dissection (Microscopy), real-time PCR and LAMP revealed the same trends pre- and post-MDA. The infection rate with animal Onchocerca sp was exceptionally low. The dense river system generate important breeding sites that govern the abundance of Simulium during both dry and rainy seasons.