Guilherme G. Verocai

Whence river blindness? The domestication of mammals and host-parasite co-evolution in the nematode genus Onchocerca☆

The genus Onchocerca includes 34 described species and represents one of the largest genera of the filarial nematodes within the family Onchocercidae. Representative members of this genus are mainly parasites of ungulates, with some exceptions such as Onchocerca lupi and Onchocerca volvulus, infecting carnivores and/or humans. For a long time, the evolutionary relationships amongst onchocercids remained poorly studied, as the systematics of this genus was impaired by the high morphological variability of species included in the taxon. Although some molecular phylogenies were developed, these studies were mainly focused on bovine Onchocerca spp. and O. volvulus, including assessments of Wolbachia endosymbionts. In the present study, we analysed 13 Onchocerca spp. from a larger host spectrum using a panel of seven different genes. Analysis of the coxI marker supports its usefulness for the identification of species within the genus. The evolutionary history of the genus has been herein revised by multi-gene phylogenies, presenting three strongly supported clades of Onchocerca spp. Analyses of co-evolutionary scenarios between Onchocerca and their vertebrate hosts underline the effect of domestication on Onchocerca speciation. Our study indicates that a host switch event occurred between Bovidae, Canidae and humans. Cophylogenetic analyses between Onchocerca and the endosymbiotic bacterium Wolbachia indicate the strongest co-evolutionary pattern ever registered within the filarial nematodes. Finally, this dataset indicates that the clade composed by O. lupi, Onchocerca gutturosa, Onchocerca lienalis, Onchocerca ochengi and O. volvulus derived from recent speciation.

Varestrongylus (Nematoda: Protostrongylidae), lungworms of ungulates: a phylogenetic framework based on comparative morphology

Varestrongylus Bhalerao, 1932 comprises ten valid lungworm species infecting wild and domestic ungulates from Eurasia and North America. Here, we present a phylogenetic hypothesis for the genus based on morphological characters in a broader context for the family Protostrongylidae and discuss species relationships and aspects of character evolution. Phylogenetic analysis of 25 structural attributes, including binary and multistate characters, among the 10 species of Varestrongylus resulted in one fully resolved most parsimonious tree (61 steps; consistency index = 0.672, retention index = 0.722, and consistency index excluding uninformative characters = 0.667). Varestrongylus forms a monophyletic clade and is the sister of Pneumostrongylus, supporting recognition of the subfamily Varestrongylinae. Monophyly for Varestrongylus is diagnosed by six unequivocal synapomorphies, all associated with structural characters of the copulatory system of males. Varestrongylus pneumonicus is basal, and sister to all other species within the genus, which form two subclades. The subclade I contains V. sagittatus + V. tuvae and V. qinghaiensis + V. longispiculatus. Subclade II contains V. alpenae, V. capricola, V. capreoli, and V. eleguneniensis + V. alces. Both subclades are diagnosed by two unambiguous synapomorphies. Highlighted is the continuing importance of phylogenetic assessments based on comparative morphology as a foundation to explore the structure of the biosphere across space and time.

Comparison between McMaster and Mini-FLOTAC methods for the enumeration of Eimeria maxima oocysts in poultry excreta

Monitoring Eimeria shedding has become more important due to the recent restrictions to the use of antibiotics within the poultry industry. Therefore, there is a need for the implementation of more precise and accurate quantitative diagnostic techniques. The objective of this study was to compare the precision and accuracy between the Mini-FLOTAC and the McMaster techniques for quantitative diagnosis of Eimeria maxima oocyst in poultry. Twelve pools of excreta samples of broiler chickens experimentally infected with E. maxima were analyzed for the comparison between Mini-FLOTAC and McMaster technique using, the detection limits (dl) of 23 and 25, respectively. Additionally, six excreta samples were used to compare the precision of different dl (5, 10, 23, and 46) using the Mini-FLOTAC technique. For precision comparisons, five technical replicates of each sample (five replicate slides on one excreta slurry) were read for calculating the mean oocyst per gram of excreta (OPG) count, standard deviation (SD), coefficient of variation (CV), and precision of both aforementioned comparisons. To compare accuracy between the methods (McMaster, and Mini-FLOTAC dl 5 and 23), excreta from uninfected chickens was spiked with 100, 500, 1,000, 5,000, or 10,000 OPG; additional samples remained unspiked (negative control). For each spiking level, three samples were read in triplicate, totaling nine reads per spiking level per technique. Data were transformed using log10 to obtain normality and homogeneity of variances. A significant correlation (R = 0.74; p = 0.006) was observed between the mean OPG of the McMaster dl 25 and the Mini-FLOTAC dl 23. Mean OPG, CV, SD, and precision were not statistically different between the McMaster dl 25 and Mini-FLOTAC dl 23. Despite the absence of statistical difference (p > 0.05), Mini-FLOTAC dl 5 showed a numerically lower SD and CV than Mini-FLOTAC dl 23. The Pearson correlation coefficient revealed significant and positive correlation among the four dl (p ≤ 0.05). In the accuracy study, it was observed that the Mini-FLOTAC dl 5 and 23 were more accurate than the McMaster for 100 OPG, and the Mini-FLOTAC dl 23 had the highest accuracy for 500 OPG. The McMaster and Mini-FLOTAC dl 23 techniques were more accurate than the Mini-FLOTAC dl 5 for 5,000 OPG, and both dl of the Mini-FLOTAC were less accurate for 10,000 OPG counts than the McMaster technique. However, the overall accuracy of the Mini-FLOTAC dl 23 was higher than the McMaster and Mini-FLOTAC dl 5 techniques.

Efficacy of spinosad on the treatment of myiasis caused by Cochliomyia hominivorax (Diptera: Calliphoridae) in dogs

The purpose of this study was to evaluate the overall efficacy, larval expulsion and larvicidal effect of spinosad on the treatment of myiasis caused by New World screwworm Cochliomyia hominivorax in naturally infested dogs. Six Beagle dogs presenting with myiasis were treated once with spinosad 560 mg, with dosages ranging from 43 to 577 mg/kg. Dogs were observed with 15 min post treatment, and with intervals of 15 min within the first hour followed by hourly evaluations up to six hours post-treatment. After this period, the remaining larvae were mechanically removed. Spontaneous expulsion of larvae was variable among dogs. All dogs received support treatment and were followed up for healing of the myiasis-associated lesion. On average, the highest larval expulsion occurred four hours after treatment. A single spinosad treatment had an overall efficacy of 79.7% (56.7-100%). The average larval expulsion rate was 73.3% (56.7-100%), reached its maximum within 6 h post-treatment, and the average larvicidal effect was only 23.7% (0-58.3%). No animal needed a second dosage 24 h post-treatment, lesions were completely healed within 8 days post-treatment. Despite not reaching the desirable 100% efficacy, spinosad treatment is an option in managing NSW myiasis, in conjunction with mechanical removal of maggots that remain in the lesion and support treatment.

A cryptic species of Onchocerca (Nematoda: Onchocercidae) in blackflies ( Simulium spp.) from southern California, USA

BackgroundEntomological surveillance for pathogens based on molecular screening of putative arthropod vectors such as blackflies (Diptera: Simuliidae) is becoming increasingly important. Surveillance provides a means to understand host and geographical patterns of underestimated biodiversity among North American species of Onchocerca and a pathway to identify and track expanding emergence of the zoonotic Onchocerca lupi. Herein, we have screened two blackfly species, Simulium tescorum and Simulium vittatum (s.l.), from Los Angeles County, southern California, USA for DNA of filarioid nematodes to better understand species richness and limits within the genus Onchocerca.MethodsA total of 1056 and 378 female blackflies was collected using CO2-baited mosquito traps from March to November of 2015 and 2016, respectively. All blackflies during 2015 were individually processed for DNA extraction and PCR targeting of the cytochrome c oxidase subunit 1 (cox1) of the mitochondrial DNA (mtDNA). Specimens of S. tescorum collected in 2016 were processed individually with heads and bodies extracted separately, whereas those of S. vittatum (s.l.) were processed in pooled samples with heads and bodies extracted separately. A subset of filarioid-positive samples from 2015 and all samples from 2016 were screened using a PCR targeting the NADH dehydrogenase subunit 5 (nad5) gene (mtDNA).ResultsIn 2015, 356 S. tescorum (33.7%) and 683 S. vittatum (s.l.) (64.7%) were collected, and an additional 17 specimens were not assessed morphologically. In 2016, a total of 378 blackflies was collected. Of these, 43 (11.6%) were S. tescorum and 327 (88.4%) were S. vittatum (s.l.), and an additional 8 specimens were not assessed morphologically. In 2015, Onchocerca sequences were detected in 4.8% (n = 17) of S. tescorum samples, and only one S. vittatum (0.15%). In 2016, only a single S. vittatum pool was positive for the same cryptic Onchocerca species. In phylogenetic comparisons based on nad5, the Onchocerca sequences from California formed a clade with those isolates in white-tailed deer from upstate New York, suggesting these belong to a single widespread cryptic species.ConclusionsAn uncharacterized species of Onchocerca associated with cervid hosts was found in blackflies from southern California. Sequence data demonstrated it is likely conspecific with an unnamed species of Onchocerca previously found in white-tailed deer from upstate New York. Current data support recognition of a broad geographical distribution across North America for an apparently cryptic species of Onchocerca that is discrete from O. cervipedis, considered to be a typical filarioid among cervids. Our data suggest that this cryptic species of Onchocerca may infect subspecies of white-tailed deer (Odocoileus virginianus), and mule and black-tailed deer (Odocoileus hemionus) at temporal latitudes. The blackflies Simulium tescorum and S. vittatum (s.l.) (presumably, S. tribulatum) are putative vectors. Discovery of a cryptic complex indicates that species diversity and putative associations for definitive hosts and vectors of Onchocerca species in North America must be reassessed.

Molecular Identification of Onchocerca spp. Larvae in Simulium damnosum sensu lato Collected in Northern Uganda

Previous studies have demonstrated that the presence of larvae of other filarial species in Simulium damnosum sensu lato can distort estimates of transmission potential for Onchocerca volvulus in West Africa. However, studies conducted in foci of onchocerciasis in West Central Uganda indicated that larvae other thanO. volvuluswere not common in vectors collected there. Recent data collected in Northern Uganda revealed a striking discordance between estimates of the prevalence of flies carrying O. volvulus infective larvae obtained from molecular pool screening and dissection methods. To resolve this discrepancy, sequences from three mitochondrially encoded genes were analyzed from the larvae collected by dissection. All larvae analyzed were Onchocerca ochengi v. Siisa, a parasite of cattle, or Onchocerca ramachandrini, a parasite of warthogs. These results suggest that nonhuman parasite larvae are common in vectors in Northern Uganda, underscoring the necessity for molecular identification methods to accurately estimate O. volvulus transmission. In 2007, Uganda announced a program to eliminate onchocerciasis in all 17 foci in the country, becoming the first country in Africa to make complete elimination of onchocerciasis a national goal. The strategic plan of the Uganda Onchocerciasis Elimination Program was based on twice per year mass drug administration of Mectizan (ivermectin) to the afflicted communities supplemented with localized vector control measures where appropriate. In 2008, the Uganda Ministry of Health established an advisory committee, known as the Uganda Onchocerciasis Elimination Expert Advisory Committee (UOEEAC) to provide technical guidance to the elimination program. One of the first actions of the UOEEAC was to consult with the Ugandan Ministry of Health to develop a set of guidelines for the program to use for verifying the suppression and eventual elimination of transmission of Onchocerca volvulus. These guidelines were based on the guidelines for the elimination of onchocerciasis published by theWorld Health Organization in 2001 and updated in 2016. The guidelines rely on a combination of serological indicators of transmission (development of antibodies against the parasite specific antigen Ov16 in children) and entomological indicators (presence of infective stage larvae in the black fly vectors of the parasite) to confirm suppression of transmission. Detection of infective larvae in the vector black flies has advantageswhen used tomonitor transmission ofO. volvulus. Most importantly, it provides the most timely and accurate measure of transmission. Traditionally, detection of infective larvae in vectors has been accomplished through dissection of vector black flies. However, there are some disadvantages associated with the method. First, in the face of an effective control or elimination program, flies carrying infective larvae become increasingly rare. This means that large numbers of flies need to be examined to detect the rare infectious fly, which is laborious and expensive. Second, in West Africa, it has long been known that Simulium damnosum s.l., the most important vector throughout Africa also serves as the vector for a number of Onchocerca species associated with domestic or wild ungulate hosts (e.g., bovids, suids). These larvae are difficult or impossible to distinguish from O. volvulus morphologically, confounding accurate measurement of the prevalence of flies carrying infective larvae of the human parasite. Molecular methods involving polymerase chain reaction (PCR) amplification of O. volvulus specific DNA sequences in DNA prepared from pools of flies or from individual larvae have been developed to overcome these difficulties. When molecular identifications were performed on infective larvae identified by dissection inWest Africa, it was found that about half of the larvae provisionally identified as O. volvulus by field dissection teams were ungulate-associated Onchocerca. By contrast, one report applying molecular identificationmethods to larvae collected from Kabarole and Kasese districts in West Central Uganda suggested that all were O. volvulus, suggesting that ungulate-associatedOnchocerca might pose less of a problem in East Africa. Onchocerciasis elimination efforts inNorthernUgandawere hindered by political unrest in the area for several years. However, after the restoration of peace to this area, elimination efforts accelerated in 2012. These efforts included both serosurveys of children to measure exposure to the parasite, aswell as entomological surveys, inwhich a portion of the flies collected were screened in pools using molecular methods, whereas a portion were screened using traditional dissection. In 2015, significantly larger numbers of flies carrying infective larvae were detected by the field dissection teams than were found by PCR pool screening. To further investigate the reason for this discrepancy, the larvae collected by the dissection teams were subjected to a detailed molecular analysis. Simulium damnosum females were collected using standard methods from several sites in the Madi-Mid North onchocerciasis focus of Northern Uganda (Figure 1). Flies were collected from the districts of Nwoya, Amuru (Elegu), and Moyo, as part of routine surveillance activities conducted by *Address correspondence to Thomas R. Unnasch, Global Health Infectious Disease Research, 3720 Spectrum Boulevard Suite 304, Tampa, FL 33612. E-mail: tunnasch@health.usf.edu