Pathological findings associated with Dipetalonema spp. (Spirurida, Onchocercidae) infection in two species of Neotropical monkeys from Brazil.

Among vector-borne helminths, filarioids of the genus Dipetalonema (Spirurida: Onchocercidae) localize in several tissues and body cavities of several animal species, causing mild to moderate lesions. The pathological findings associated with Dipetalonema spp. infection in Neotropical monkeys from southern Brazil are herein described, along with a fatal case due to filarial polyserositis and entrapment of an intestinal segment. At necropsy, nematodes were observed in abdominal and thoracic cavities, or in the pericardium of 37 (31.3%) out of the 118 individuals examined (i.e., 35 Alouatta guariba clamitans and two Sapajus nigritus). In addition, at histology, 27.0% of positive animals presented microfilarie (inside blood vessels of lung, spleen, liver, and brain) and 8.1% presented adult nematodes in the heart, lung, and liver. In two cases, cross-sections of filarioids were associated with areas of epicardial thickening with intense fibrosis and pyogranulomatous inflammation in the brain, heart, liver, lungs, or spleen. The DNA fragment was amplify using the cox1 gene, sequenced and analyzed to identify the nematode species collected; presence of Wolbachia was assessed in the filarioids using the 16S rRNA gene. At BLAST analysis of the cox1 gene, 10 sequences showed 91.7% nucleotide identity with Dipetalonema gracile, and two with D. gracile (98.5%) and Dipetalonema graciliformis (98.3%). Phylogenetic analyses clustered sequences of the cox1 obtained in this study in two clades corresponding with the host species. Wolbachia sp. endosymbiont was detected in four samples. Data herein reported provide a description of pathological lesions associated with the infection by Dipetalonema spp., suggesting that they may cause disease in Neotropical monkeys. In addition, a better understanding of diversity and biology of Dipetalonema spp. in South America is needed to assess the impact they may cause in native non-human primates from Brazil.

Taxonomic assessment of the genus Procamallanus (Nematoda) in Middle American cichlids (Osteichthyes) with molecular data, and the description of a new species from Nicaragua and Costa Rica.

Procamallanus is a species-rich genus of parasitic nematodes of marine, brackish, and freshwater fishes, occurring also occasionally in amphibians and reptiles. In the Neotropical region, this genus is highly diverse, with species described from a wide range of fish families. In this study, we reassess the taxonomic status of Procamallanus rebecae with molecular and morphological data and describe a new species endemic to Nicaragua and Costa Rica. We analyzed all Procamallanus isolated from fish from the Nicaraguan lakes and some rivers in Costa Rica after an exhaustive analysis of their freshwater fish endoparasite fauna. Procamallanus rebecae is a host-specific parasite of Middle American cichlids, previously reported in southern Mexico, Nicaragua, and Costa Rica. We therefore compared these Central American specimens with individuals of P. rebecae collected in cichlids from southeastern Mexico using two genomic regions (28S rDNA and mitochondrial cytochrome oxidase subunit 1, COI). We found high levels of sequence divergence between Procamallanus from the two geographical regions, with up to 9.8 and 10.5% for both genetic markers, respectively. We also analyzed their morphology and found conspicuous differences in the shape of the mouth and the structure of the female cauda. We therefore describe the specimens of Procamallanus from Central American cichlids as a new species. Both Procamallanus species occur in different cichlid species and are allopatrically distributed. The host specificity and ancient association patterns between cichlids and Procamallanus and the jointly colonization of both hosts and parasites during their northern dispersal from South America are briefly discussed.

Diomedenema dinarctos n. sp. (Spiruromorpha: Desmidocercidae), a new species of lung nematode in Fiordland crested penguins (Eudyptes pachyrhynchus; Sphenisciformes), from South Island, New Zealand.

Fiordland crested penguins Eudyptes pachyrhynchus Gray are thick-billed, crested penguins endemic to New Zealand that breed in small colonies in inaccessible places. They are assessed as Vulnerable by the IUCN. This is the first report of helminth parasites from this penguin species. Herein a new species of Diomedenema is described (Spiruromorpha, Desmidocercidae) from the lungs of Fiordland crested penguins. Diomedenema dinarctos n. sp. has only two congeners and is closest morphologically to D. tavaresi. It differs from D. tavaresi in overall larger size, longer buccal cavity and oesophagus, in the distance of the excretory pore and nerve ring from the anterior end, and the ratio of spicule sizes. Eggs are twice the size reported for D. tavaresi. The only other species, D. diomedeae, is far larger in body size, with a shorter buccal cavity and oesophagus and a reduced body length to oesophagus ratio. The position in the body length of the vulva and the spicule ratio also differ strongly. We provide a DNA sequence for the 18S rDNA gene. Species of Diomedenema have been reported as causing death in albatross, so any deceased Fiordland crested penguins should be examined specifically for this worm to enable evaluation of this potentially damaging parasite in the population.

A new species of Hedruris (Nematoda: Hedruridae) from freshwater turtles, its life cycle and biogeographic distribution of the genus.

We describe Hedruris dratini n. sp. (Nematoda, Hedruridae) from Hydromedusa tectifera and Phrynops hilarii in Argentina based on morphological and molecular characters. Also, we provide information about its life cycle. The new species differs from other species of the genus by possessing the excretory pore, nerve ring and deirids at equal distance from the anterior end. Additionally, H. dratini n. sp. has mammilated eggs and males possess nine pairs of caudal papillae. The subadults and adults of H. dratini n. sp and H. orestiae were characterized by sequencing the small subunit ribosomal DNA (18S). We present for the first time a life cycle of a species of Hedruris that includes an amphipod as intermediate host and a reptile as definitive host. Furthermore, we analysed the host and geographic distribution of all Hedruris species. Although the genus has a cosmopolitan distribution and parasitizes a great host diversity, the majority of species have a Gondwanian distribution, with amphibians being the preferred hosts.

An integrative taxonomic assessment of Procamallanus (Spirocamallanus) huacraensis (Nematoda: Camallanidae), infecting the freshwater catfish Trichomycterus spegazzinii (Siluriformes: Trichomycteridae) in Argentina.

Procamallanus (Spirocamallanus) huacraensis infecting the catfish Trichomycterus spegazzinii from Escoipe River, Salta province (Argentina), is redescribed and genetically characterised for the first time, based on three genetic markers (nuclear 18S and 28S rRNA; cytochrome c oxidase subunit I [cox1] mtDNA). The phylogeny of Camallanidae was also discussed. Morphological evaluation of P. (S.) huacraensis using light and scanning electron microscopy revealed the previously undescribed features: location of deirids, accurate morphology of larvae (L1) and ovijector in females, as well as phasmids in males. Differences were found comparing the newly collected material and the type specimens, probably because the original description lacked detailing. Unfortunately, type specimens of P. (S.) huacraensis were no available for loan. The results of morphological and genetic analyses supported the validity of P. (S.) huacraensis. Inconsistencies regarding the taxonomic identification of species of Camallanidae in GenBank database were noted. Based on the current genetic database of Camallanidae, phylogenetic reconstructions using the 18S rRNA sequences were most consistent, due to the inclusion of higher number of taxa. Procamallanus (S.) huacraensis appeared as sister group of P. (S.) rarus, also isolated from a catfish in a neighbouring region. The order and habitat of hosts were also similar within some well-supported parasite lineages, but without common geographic origin. However, it is still premature to make definitive affirmations regarding the role of such features in the phylogenetic patterns of Camallanidae, given the scarcity of genetic data. The phylogenetic reconstructions also confirmed the artificiality of the morphology-based systematics of the family.

Antechiniella septentrionalis n. sp. (Spirurida: Acuariidae), a new intestinal nematode parasite of the tundra vole Microtus oeconomus (Pallas) (Rodentia: Muridae) in the north-east of Russia.

Antechiniella septentrionalis n. sp. (Spirurida: Acuariidae) is described from the duodenum of a tundra vole, Microtus oeconomus (Pallas), collected in the Magadan region in the north-east part of Russia. It differs from A. suffodiax (Beveridge & Barker, 1975) and A. sertatum Smales, 1991 mainly in having a larger number of postcloacal papillae (5-6 pairs vs 4 pairs), a differently shaped left spicule, the disposition of precloacal papillae in two rows vs one, and oblong vs oval eggs. Other differences include the different disposition of ovaries in A. septentrionalis n. sp and A. suffodiax and the different structure of deirids in A. septentrionalis n. sp. and A. sertatum. The new species was characterized molecularly (partial sequences for 18S rRNA, 28S rRNA and cox1 mtDNA). The phylogenetic analyses performed showed the affinity of the new species to the members of the Acuariidae and other spirurid nematodes.

Morphologic Observations and Novel 18S rRNA Sequences of Abbreviata hastaspicula and Abbreviata antarctica from Varanus spp. Lizards in Australia.

The nematodes Abbreviata antarctica von Linstow, 1899, and Abbreviata hastaspicula Jones, 1979 , are predominant spirurid nematodes in species of Varanus lizards in Australia. However, genetic knowledge of these two species of nematode is lacking. In this study, nematodes removed from Varanus gouldii were examined using integrated morphologic and molecular methods. We extracted DNA from A. hastaspicula and A. antarctica for PCR and sequencing. Specific 18S small subunit ribosomal RNA (rRNA) primers were designed on the basis of existing 18S rRNA sequences of Physalopterinae strains. Species of Abbreviata, which are closely similar morphologically, may be misidentified, especially the larvae of different species of Abbreviata that cannot be differentiated. The findings of this study will improve the accuracy in identification of A. hastaspicula and A. antarctica, both morphologically and molecularly.

Seasonal variation of Mastophorus muris (Nematoda: Spirurida) in the water vole Arvicola amphibius from southern Sweden.

This study focused on the spirurid nematode Mastophorus muris in water voles (Arvicola amphibius) trapped in three regions in southern Sweden during spring and fall 2013. The collection of water voles formed part of a larger project (EMIRO) on the cestode Echinococcus multilocularis in rodents. The voles' stomach contents were examined for the presence of M. muris. Prevalence, mean abundance and mean intensity of infection were calculated. A generalized linear model model was used to examine the effects of sex, functional group, season and region on the number of M. muris individuals in each vole. Forty-seven of 181 (26%) voles were infected with M. muris, with up to 74 worms each. The overall mean intensity (worms per infected vole) was 15 (95% CI 10-21), and abundance (mean number of worms in all voles) was 4 (95% CI 2-6). Model output indicated a significant effect of season and region with respect to abundance of nematode infection, which was independent of sex and functional group of the investigated host.

Molecular evidence of infection with air sac nematodes in the great tit (Parus major) and the captive-bred gyrfalcon (Falco rusticolus).

Serratospiculiasis is a parasitic disease caused by filariid nematodes of the genus Serratospiculum that parasitise the air sacs of various species of falcons, bald eagles and Cooper's hawks around the world. An infection with Serratospiculum was recently confirmed in a nonspecific host, the great tit, in Slovakia. Parasitic material from this host was fixed for molecular analysis. Nematode found in the air sacs from a captive-bred gyrfalcon was also stored. Analysis of small subunit (18S) ribosomal DNA (18S rDNA) gene indicated that sequences from Serratospiculum sp. and Serratospiculoides amaculata were closely related to a reference sequence from Serratospiculum tendo, in agreement with morphology. This study is the first to generate molecular data and infer the phylogenetic position of S. amaculata as the first representative of the genus Serratospiculoides.

Filarial nematode (order: Spirurida) surveillance in urban habitats, in the city of Pécs (Hungary).

As part of the seasonal mosquito control activities in the city of Pécs (Baranya County, Hungary), a total of 1123 adult female mosquitoes belonging to 18 species (including the invasive species Aedes koreicus) were collected from human-inhabited areas, using CO2-baited traps, during two consecutive years. To survey the presence and prevalence of filarial parasites in these mosquitoes, we performed a molecular survey for filarial DNA, attempted by PCR using generic primers (COI), and followed by DNA sequencing. Filaroid nematode DNA was detected in 4% of investigated mosquito pools. Out of 410 pools, 9 pools of mosquitoes were positive for Dirofilaria repens (Aedes vexans, Aedes koreicus, Coquillettidia richiardii), and/or Dirofilaria immitis (Ae. vexans, Cq. richiardii), and further 8 pools were positive for Setaria tundra (Ae. vexans, Cq. richiardii). Our study provides novel insight for prevalence of filaroid nematodes in mosquitoes occurring in close proximity to humans, thereby highlights the possible human and veterinary health importance of these mosquito species, including the recently introduced invasive mosquito Ae. koreicus.

Further study on Procamallanus (Spirocamallanus) pintoi (Kohn et Fernandes, 1988) (Nematoda: Camallanidae) in Corydoras paleatus and Corydoras micracanthus (Siluriformes: Callichthyidae) from Salta, Argentina, with a key to congeneric species from Neotropical Realm.

This study describes the morphology of Procamallanus (Spirocamallanus) pintoi (Kohn et Fernandes, 1988), isolated from the intestine of Corydoras micracanthus Regan, 1912 and C. paleatus (Jenyns, 1842) (both Callichthyidae, Siluriformes) from Bermejo and Juramento River basins, respectively, province of Salta, Argentina. This is the first record of P. (S.) pintoi in C. micracanthus from Northwestern Argentina. The light and scanning electron microscopy observations of P. (S.) pintoi, revealed a few previously unreported morphological details of the cephalic region, vulva and caudal region of males. In addition, morphometric variability was observed in, mainly body size, glandular oesophagus length and position of the vulva between present specimens and those described in previous studies. A key to the species of Procamallanus (Spirocamallanus) from the Neotropical Realm is proposed.

Evaluation of a spot-on imidacloprid-moxidectin formulation (Advocate®) for the treatment of naturally occurring esophageal spirocercosis in dogs: a double-blinded, placebo-controlled study.

BACKGROUND: Dogs are the definitive hosts of Spirocerca lupi. Spirocercosis is treated by prolonged avermectin administration by injection or daily oral doses. In this prospective, double-blinded, placebo-controlled, clinical trial, the efficacy of imidacloprid and moxidectin spot-on formulation (Advocate®) was compared to injectable doramectin (Dectomax®). Dogs diagnosed with benign esophageal spirocercosis were divided randomly into doramectin (400 µg/kg IM) or moxidectin and imidacloprid spot-on (2.5-6.25 mg/kg and 10-25 mg/kg, respectively) groups and treated weekly for 12 consecutive weeks. Dogs were followed for 20 weeks by physical examination, owners' questionnaire, blood work, fecal floatation, PCR and endoscopy. RESULTS: All the doramectin group dogs (n = 10) completed the treatment and follow-up, and the disease had completely resolved in all by week 12. Of the Advocate® group (n = 10), four had complete resolution at week 12, four had partial resolution, one dog did not respond to treatment, and one dog was switched to the doramectin protocol on week 5 due to persistent severe clinical signs. PCR analysis was more sensitive in detecting S. lupi eggs compared to fecal floatation. Discrepancies were detected on 22 occasions, of which on 20 occasions, the PCR was positive while fecal floatation was negative, and only on two occasions the PCR results were negative while fecal flotation was positive. CONCLUSIONS: The present results indicate that weekly Advocate® spot-on administration may be effective for treating benign esophageal spirocercosis, but is less effective than the currently used injectable doramectin therapy at the dose and duration used herein.

A new species of Procyrnea (Nematoda: Habronematidae) parasitic in Rhea pennata (Aves: Rheidae) from Patagonia, Argentina, with a key to species of the genus.

Procyrnea choique n. sp. is described from the lesser rhea, Rhea pennata d´Orbigny (Aves: Rheidae), from the Protected Natural Area Península Valdés, Chubut province, Argentina. The new species resembles P. ficheuri, P. murrayi, P. excisiformis, P. dollfusi, P. haliasturi, P. anterovulvata, P. graculae, P. brevicaudata, P. uncinipenis, P. javaensis, P. ameerae, P. ornata, P. aegotheles, P. spiralis, P. ruschii and P. aptera, mainly in the absence of lateral alae; but differs from its congeners by having a left spicule without barbs, the position of the vulva which is post-equatorial, the absence of lateral ridges, absent median precloacal papilla, asymmetrical caudal alae, males more than 6 mm long, left spicule 1 mm long and spicule ratio 1:3. Our results extend the taxonomy of Procyrnea Chabaud, 1958 and comprise the first report of a habronematid from R. pennata. A key to species of Procyrnea is presented.

Thubunaea eleodori sp. nov. (Nematoda: Physalopteridae) from Liolaemus eleodori (Sauria: Liolaemidae) from Argentina.

Thubunaea eleodori sp. nov. is described from the stomach of Liolaemus eleodori (Sauria: Liolaemidae) from San Guillermo National Park, Province of San Juan, Argentina. T. eleodori is most similar to those species lacking spicules, T. cnemidophorus, T. fitsimonsi, T. parkeri, T. schukurovi, and T. smogorzhewskii. T. eleodori is separated from these species based on the papilla pattern. T. eleodori has 12 pedunculate papillae and 14 sessile papillae, T. smogorzhewskii lacks pedunculate papillae, T. fitsimonsi and T. parkeri lack sessile papillae, and T. cnemidophorus has14-16 pedunculate papillae and 12 sessile papillae. T. eleodori represents the first member of the genus to be reported from Argentina.

A Quantitative PCR Protocol for Detection of Oxyspirura petrowi in Northern Bobwhites (Colinus virginianus).

Oxyspirura petrowi is a parasitic nematode that infects wild birds. This parasite has a broad host range, but has recently been reported in high prevalences from native Galliformes species in the United States. In order to better understand the impact O. petrowi has on wild bird populations, we developed a quantitative PCR protocol to detect infections in wild northern bobwhites (Colinus virginianus). We used paired fecal and cloacal swab samples from wild caught and experimentally infected northern bobwhites and matching fecal float data from experimentally infected birds to validate our assay. Overall we detected more positive birds from fecal samples than the paired cloacal swabs and there was strong agreement between the qPCR results from fecal samples and from fecal flotation (84%; κ = 0.69 [0.53-0.84 95% CI]). We also detected O. petrowi DNA in ten replicates of samples spiked with one O. petrowi egg. This qPCR assay is an effective assay to detect O. petrowi infections in wild birds. Our results suggest that fecal samples are the most appropriate sample for detecting infections; although, cloacal swabs can be useful for determining if O. petrowi is circulating in a population.

Differential Evolutionary Selection and Natural Evolvability Observed in ALT Proteins of Human Filarial Parasites.

The abundant larval transcript (ALT-2) protein is present in all members of the Filarioidea, and has been reported as a potential candidate antigen for a subunit vaccine against lymphatic filariasis. To assess the potential for vaccine escape or heterologous protection, we examined the evolutionary selection acting on ALT-2. The ratios of nonsynonymous (K(a)) to synonymous (K(s)) mutation frequencies (ω) were calculated for the alt-2 genes of the lymphatic filariasis agents Brugia malayi and Wuchereria bancrofti and the agents of river blindness and African eyeworm disease Onchocerca volvulus and Loa loa. Two distinct Bayesian models of sequence evolution showed that ALT-2 of W. bancrofti and L. loa were under significant (P<0.05; P < 0.001) diversifying selection, while ALT-2 of B. malayi and O. volvulus were under neutral to stabilizing selection. Diversifying selection as measured by ω values was notably strongest on the region of ALT-2 encoding the signal peptide of L. loa and was elevated in the variable acidic domain of L. loa and W. bancrofti. Phylogenetic analysis indicated that the ALT-2 consensus sequences formed three clades: the first consisting of B. malayi, the second consisting of W. bancrofti, and the third containing both O. volvulus and L. loa. ALT-2 selection was therefore not predictable by phylogeny or pathology, as the two species parasitizing the eye were selected differently, as were the two species parasitizing the lymphatic system. The most immunogenic regions of L. loa and W. bancrofti ALT-2 sequence as modeled by antigenicity prediction analysis did not correspond with elevated levels of diversifying selection, and were not selected differently than predicted antigenic epitopes in B. malayi and O. volvulus. Measurements of ALT-2 evolvability made by χ2 analysis between alleles that were stable (O. volvulus and B. malayi) and those that were under diversifying selection (W. bancrofti and L. loa) indicated significant (P<0.01) deviations from a normal distribution for both W. bancrofti and L. loa. The relationship between evolvability and selection in L. loa followed a second order polynomial distribution (R2 = 0.89), indicating that the two factors relate to one another in accordance with an additional unknown factor. Taken together, these findings indicate discrete evolutionary drivers acting on ALT-2 of the four organisms examined, and the described variation has implications for design of novel vaccines and diagnostic reagents. Additionally, this represents the first mathematical description of evolvability in a naturally occurring setting.

Morphological, molecular and phylogenetic analyses of the spirurid nematode Stegophorus macronectes (Johnston & Mawson, 1942).

Stegophorus macronectes (Johnston & Mawson, 1942) is a gastrointestinal parasite found in Antarctic seabirds. The original description of the species, which was based only on females, is poor and fragmented with some unclear diagnostic characters. This study provides new morphometric and molecular data on this previously poorly described parasite. Nuclear rDNA sequences (18S, 5.8S, 28S and internal transcribed spacer (ITS) regions) were isolated from S. macronectes specimens collected from the chinstrap penguin Pygoscelis antarctica Forster on Deception Island, Antarctica. Using 18S rDNA sequences, phylogenetic analyses (maximum likelihood, maximum parsimony and Bayesian inference) of the order Spirurida were performed to determine the phylogenetic location of this species. Primer pairs of the ITS regions were designed for genus-level identification of specimens, regardless of their cycle, as an alternative to coprological methods. The utility of this molecular method for identification of morphologically altered specimens is also discussed.

Stammerinema hyalinum n. comb. for Filaria hyalina von Linstow, 1890 and its recognition as a senior synonym of Stammerinema rhopalocephalum (Soltys, 1952) (Spirurida: Acuariidae), a parasite of shrews.

Based on a re-examination of type-specimens of Filaria hyalina von Linstow, 1890 from Sorex araneus L. (Mammalia: Soricidae) and morphological studies (light and scanning electron microscopy) of specimens collected from the same host species in Bulgaria and previously identified as Stammerinema rhopalocephalum (Soltys, 1952), both these forms are considered to be conspecific. Accordingly, F. hyalina is transferred to the genus Stammerinema Osche, 1955 as Stammerinema hyalinum n. comb. and the species originally described as Synhimantus rhopalocephalus Soltys, 1952 is considered its junior synonym.

Morphological and molecular observations on the status of Crassicauda magna, a parasite of the subcutaneous tissues of the pygmy sperm whale, with a re-evaluation of the systematic relationships of the genus Crassicauda.

Members of the genus Crassicauda (Nematoda: Spirurida) are parasites of the body tissues of whales and dolphins. Owing to the large size of worms and difficulties in the recovery of entire nematodes from the tissues of hosts, limited information is available on morphological descriptions of both male and female worms. Furthermore, there are currently no available sequence data for this genus to assist with such identifications. This paper describes for the first time features of the anterior extremity and the male tail of Crassicauda magna, suggesting that Crassicauda duguyi may be a synonym of this species. In addition, molecular data are presented for the genus for the first time suggesting that the genus belongs within the superfamily Acuarioidea rather than within the Habronematoidea, in which it is currently placed.

Morphology, ultrastructure and molecular characterisation of Spiroxys japonica Morishita, 1926 (Spirurida: Gnathostomatidae) from Pelophylax nigromaculatus (Hallowell) (Amphibia: Ranidae).

Gnathostomatid nematodes identified morphologically as Spiroxys japonica Morishita, 1926 were collected from the dark-spotted frog Pelophylax nigromaculatus (Hallowell) (Amphibia: Ranidae) in China. Light and scanning electron microscopy were used to study the morphology of this species in detail. Previously unreported morphological features are revealed and others corrected. In addition, adult nematodes of S. japonica collected from P. nigromaculatus and Spiroxys hanzaki Hasegawa, Miyata & Doi, 1998 collected from Andrias japonicus (Temminck) (Caudata: Cryptobranchidae) in China and Japan, respectively, and the third-stage larva of S. japonica collected from Lithobates catesbeianus (Shaw) (Anura: Ranidae) in Japan, were characterised using molecular methods by sequencing and analysing ribosomal [large ribosomal DNA (18S) and internal transcribed space] and mitochondrial [cytochrome c oxidase subunit 1] target regions, respectively. The new morphological and genetic data contributes to a more accurate diagnosis of this hitherto little known nematode genus.