Redescription and First Nucleotide Sequences of Rhinobatonchocotyle pacifica Oliva & Luque, 1995 (Monogenea: Hexabothriidae), a Parasite of Pseudobatos planiceps (Garman, 1880) (Rhinopristiformes: Rhinobatidae) from Peru Abstract Introduction Specimens of Rhinobatonchocotyle pacifica Oliva & Luque, 1995 (Hexabothriidae) were collected from the gill filaments of Pacific guitarfish, Pseudobatos planiceps (Garman, 1880) (Rhinopristiformes: Rhinobatidae), from off the coast of Chorrillos, Lima, Peru. Since the original description of R. pacifica was incomplete, we herein redescribe R. pacifica based on re-examination of the holotype, paratypes, and newly collected voucher specimens. Nucleotide sequences (18S, 28S) for R. pacifica are presented for the first time, and a phylogenetic analysis conducted. Materials and Methods Monogeneans were examined with light microscopy and partial sequences of 18S and 28S rDNA genes were obtained. Results The following taxonomically important morphological features are described: presence of seminal receptacle, spiniform papillae within oral cavity, haptoral sucker sclerites and anchors, hook surface striations, muscular pad associated with each haptoral sucker sclerite (except sclerite 1), muscular bulbs in appendix suckers, and oötype lacking longitudinal rows of large cells (oötype not côtelé). The genital complex is detailed. The diagnosis of Rhinobatonchocotyle is emended to include species with or without seminal receptacle. Representative nucleotide sequences of partial 18S and 28S rDNA genes of R. pacifica were obtained for the first time and used for reconstructing the phylogeny of Hexabothriidae. Conclusion The phylogenetic reconstruction confirmed the species within the Hexabothriidae.

The Morphological and Molecular Description of Acanthogyrus ( Acanthosentis ) fusiformis n. sp. (Acanthocephala: Quadrigyridae) from the Catfish Arius sp. (Ariidae) in the Pacific Ocean off Vietnam, with Notes on Zoogeography Abstract Background Most (82%) of the 46 recognized species of Acanthogyrus (Acanthosentis) Verma and Datta, 1929 are known from Asian freshwater fishes. Only three species of Acanthosentis are known from marine or brackish water fishes from India and Pakistan. We have discovered another marine species of Acanthosentis in the Pacific Ocean, off Vietnam. Purpose The purpose is to describe the new species morphologically and molecularly and provide new information of its evolutionally relationships with other species of the subgenus. Methods Standard methods of collection and examination of marine hosts, processing and illustrating of specimens, and taxonomic identification of parasites using the extensive collection of the lead author were used. Specimens were further studied using energy-dispersive X-ray analysis and ion sectioning of hooks, SEM analysis, and molecular sequencing. Type specimens were deposited at the Harold W. Manter Lab. collection, Lincoln, Nebraska. Results Acanthogyrus (Acanthosentis) fusiformis n. sp. is described from the catfish, Arius sp. (Ariidae: Siluriformes) off the Pacific Coast of Vietnam at Bac Lieu in the Gulf of Thailand. The three other marine Indian species include A. (A.) arii Bilqees, 1971 which is also described from a similar catfish, Arius serratus Day off the Karachi coast in the Arabian Sea, Indian Ocean. Our new species from Vietnam is distinguished from the other 46 species by a combination of characters including a small fusiform trunk, complete circles of small hollow spines covering the entire trunk, prominent double apical organs often extending posteriorly past posterior hooks, middle and posterior hooks of equal size slightly smaller than anterior hooks, large neck continuous with the outline of the proboscis without distinct separation, big drop-shaped cephalic ganglion, extension of the proboscis receptacle anteriorly past the base of the proboscis up to the insertion point of the posterior hooks, presence of two para-receptacle structures (PRSs), free unattached thick lemnisci, short female reproductive system with filamentous attachment of the distal end of the uterine bell to the ventral body wall, and small narrowly ellipsoid eggs with thickened polar ends. Partial sequences of the 18S and internal transcribed spacers (ITS1-5.8S-ITS2) of ribosomal RNA were generated and used for phylogenetic analyses to confirm the taxonomic identity of Acanthogyrus (Acanthosentis) fusiformis n. sp. Conclusions We describe unique morphological features of A. fusiformis never before known in the subgenus Acanthosentis. The uniqueness of A. fusiformis is further demonstrated by its EDXA fingerprint characterized by high levels of calcium and phosphorous in hooks. The zoogeography of species of Acanthosentis is elucidated in the Indian subcontinent, the Caribbean, China, and Africa. Molecular data have been available only in few species of Acanthogyrus (Acanthosentis) to date on GenBank database. For 18S, only two sequences from unknown Acanthosentis sp. from India are available, while for the ITS1-5.8S-ITS2 region, only sequences of A. cheni from China and of two unidentified species from Malaysia are available. Additional studies of species of Acanthosentis based on morphological and molecular genetic data will be needed to reconstruct the evolutionary history and phylogenetic affinities of this group of acanthocephalans.

Infectivity and Screening of Anti-piperaquine Genes in Mice Infected with Piperaquine-Sensitive and Piperaquine-Resistant Plasmodium berghei Abstract Background Piperaquine (PQ) is one of the major components of artemisinin-based combination therapy for malaria. However, the mechanism of PQ resistance has remained unclear. Methods In this study, we infected mice with PQ-resistant Plasmodium berghei ANKA strain line (PbPQR) or PQ-sensitive P. berghei ANKA strain line (PbPQS) and their survival rates, parasitemia, and spleen sizes were compared. In addition, we constructed genomic DNA subtractive library of spleens from the infected mice, and screened the potential PQ-resistant related genes from genomic DNA of PbPQR line using the representational difference analysis (RDA) method. Clones of the subtractive library were screened by PCR, and related genes were sequenced and analyzed using BLAST software of NCBI. Results Compared to PbPQS-infected mice, PbPQR-infected mice survived significantly longer, and had significantly lowered parasitemia rate and significantly increased splenomegaly. Among the total of 502 clones picked, 494 were sequenced and 96 unique PCR fragments were obtained; in which 24 DNA fragments were homologous to chromosomes related to immune function of mice. ORF Finder blasting showed that at the protein level, 26 encoded proteins were homologous to 18 hypothetical PbANKA proteins and 13 encoded proteins were homologous to “ferlin-like protein” family of PbANKA. In addition, there were more immune-related DNA molecules, ubiquitous PbANKA homology at the ORF fragment level, and enriched ferlin-like protein families identified from PbPQR-infected mice than those from PbPQS-infected mice. Conclusion These findings suggest that PbPQR may induce stronger protective immune response than that of PbPQS in infected mice.

Pravastatin and Simvastatin Pretreatment in Combination with Pyrimethamine and Sulfadiazine Reduces Infection Process of Toxoplasma gondii Tachyzoites (RH Strain) in HeLa Cells Abstract Purpose Toxoplasma gondii is a protozoan from phylum Apicomplexa, which causes the toxoplasmosis infection; this one exhibits an apicoplast organelle which assists in the metabolism of isoprenoids and other pivotal mediators for the parasite survival. Statins are drugs that inhibit cholesterol synthesis, blocking the conversion of the substrate HMG-CoA to mevalonate, thus preventing the initial processes of the biosynthesis of these precursors, both in humans and parasite. Our goal was to verify whether the Toxoplasma gondii (RH strain) tachyzoites form pretreated with pravastatin and simvastatin in association with pyrimethamine and sulfadiazine at low concentrations could affect the infection processes, suggesting direct action on protozoa intracellular proliferation through the inhibition of isoprenoids in the parasite’s apicoplast. Methods To have the adhesion, infection, and parasite proliferation during experimental infection investigated, HeLa cells (105) were subjected to a 24-hour infection by T. gondii tachyzoites forms of RH strain (5 × 105) pretreated for 30 min with pravastatin and/or simvastatin combined or not with pyrimethamine and sulfadiazine. Results Combined with conventional drugs at low concentrations pravastatin and simvastatin inhibit the adhesion, invasion, and intracellular proliferation of T. gondii in HeLa cells which are similar to the positive control. Conclusion Pravastatin and simvastatin in association with pyrimethamine and sulfadiazine at low concentrations can be regarded as a promising, effective alternative to toxoplasmosis treatment with reduced side effects.

Multimethodological Approach to Gastrointestinal Microsporidiosis in HIV-Infected Patients Abstract Purpose Microsporidiosis is an opportunistic infection that produces chronic diarrhoea and cholangiopathy in patients with AIDS, mainly caused by two species of microsporidia, Enterocytozoon bieneusi and Encephalitozon intestinalis. The aim of this work was to develop an integral system for the diagnosis of microsporidiosis of the intestine and biliary tract in HIV-infected patients, comprising microscopic and molecular techniques. Methods The study population comprised 143 adult patients of both sexes with diagnosis of HIV infection, with chronic diarrhoea, and with or without HIV-associated cholangiopathy. Stool studies for microsporidia identification of spores were performed on each patient. A video esofagogastroduodenoscopy with biopsy collection was also carried out for routine histology and semi-thin sections stained with Azure II. Species identification was carried out by transmission electron microscopy and/or polymerase chain reaction for the species E. bieneusi and E. intestinalis. Results Out of the 143 patients a total of 12.6% (n = 18) were infected with microsporidia. Microsporidia species identified in most cases was E. bieneusi (16/18 cases), followed by E. intestinalis (4/18), all of these last ones in coinfection with E. bieneusi. Conclusions Clinical, imaging, microscopic and molecular analyses, when applied in a systematic and integrated approach, allow diagnosis and identification of microsporidia at species level in AIDS patients with chronic diarrhoea, and with or without HIV-associated cholangiopathy.

Seroprevalences of Rickettsia conorii , Ehrlichia canis and Coxiella burnetii in Dogs from Montenegro Abstract Purpose The incidence of vector-borne zoonoses has been increasing in Europe as a result of global climate change, and rickettsioses are a significant etiologic entity among these infections. The objective of this study was to investigate the seroprevalences of Rickettsia conorii, Ehrlichia canis and Coxiella burnetii in dogs in Montenegro. Methods The seroepidemiological study covered 259 dogs, of which 155 were owned dogs suspected of infection with agents of rickettsial aetiology from the continental (Podgorica) or five coastal municipalities of Montenegro (Budva, Herceg Novi, Kotor, Ulcinj and Bar), and 104 dogs were from a public shelter in Podgorica. The presence of specific IgG antibodies against R. conorii was analysed using a commercial indirect enzyme-linked immunosorbent assay (ELISA) test, and the presence of antibodies against E. canis and C. burnetii was analysed using commercial indirect immunofluorescence tests (IFAT). Results Seroprevalences of 73.36, 19.3 and 1.16% were determined for R. conorii, E. canis and C. burnetii, respectively. R. conorii was significantly more prevalent (χ2 = 14.53; p < 0.001) in owned dogs (81.93%) than in dogs from the public shelter (60.6%), while E. canis was more prevalent (χ2 = 12.31; p < 0.001) in dogs from the public shelter (29.81%) than in owned dogs (12.26%). Coinfection with two pathogens was determined in 40 (15.44%) dogs, and the prevalence of R. conorii/E. canis (χ2 = 4.23; p < 0.05) was greater in dogs from the public shelter (20.19%) than in owned dogs (10.97%). Conclusion The prevailing evidence from this study shows that dogs from Montenegro are exposed to pathogens of veterinary and public health importance. This calls for the One Health approach to sensitise the public on the risks of zoonoses from dogs and to formulate policies and strategies to mitigate their spread and safeguard public health.

Spatial and Seasonal Distribution of Holochilus sciureus with Schistosoma mansoni Infection in an Endemic Area for Schistosomiasis in Brazil Abstract Introduction The occurrence of wild rodents in the Schistosoma mansoni life cycle may increase the transmission of schistosomiasis and understanding the spatial and seasonal distribution of the natural infection of these reservoirs is essential for the development of more effective control strategies. Material and methods In this study, Holochilus sciureus rodents were captured from the flooded fields of São Bento city (an endemic area for schistosomiasis in Northeast Brazil), during the dry and rainy seasons. All these sites were georeferenced and the prevalence of S. mansoniwas verified by the presence of adult worms in the mesenteric branches and livers of each animal. Results It was demonstrated that almost half of the animals collected were naturally infected with S. mansoni (45.45%) and this infection was not related to the distance from urban areas. Furthermore, the temperature, precipitation, sex, and weight did not interfere with the H. sciureus infection rates. Interestingly, we found, for the first time, the natural infection of H. sciureus in another endemic city of S. mansoni in Northeast Brazil. Conclusions Therefore, the H. sciureus rodent shows a high natural infection rate for S. mansoni in the flooded fields of São Bento, regardless of the weather station, capture distance, and biological aspects. Additionally, we have demonstrated that H. sciureus naturally parasitized with S. mansoni may inhabit a much more extensive area that is currently described in the literature.

Anthelminthic potential of the Ficus insipida latex on monogeneans of Colossoma macropomum (Serrasalmidae), a medicinal plant from the Amazon Abstract Introduction Herbal therapy is a potentially beneficial alternative for fish aquaculture, since it may be cheaper and more effective than chemotherapy. The aim of this study was to investigate the in vitro efficacy of Ficus insipida latex on monogeneans of Colossoma macropomum gills. Materials and methods To evaluate the anthelmintic activity, four concentrations of F. insipida latex (250, 500, 750 and 1000 µL/L) and exposure time to cause immobilization on monogeneans were used. In addition, two control groups, being one with water from the breeding tank and the other with breeding water from the tank + 70% ethyl alcohol were used. Results At the concentration of 250 µL/L of F. insipida latex, the immobilization of the monogeneans occurred after 4 h of exposure, while at the concentration of 500 µL/L, it occurred after 2 h. At the concentrations of 750 and 1000 µL/L of F. insipida latex, the immobilization of the monogeneans occurred after 1 h and 30 min, respectively. After exposure to 250 µL/L of F. insipida latex, 100% of immobilization of monogeneans was observed within 4 h, to 500 and 750 µL/L, 100% immobilization occurred within 4 h and to 1000 µL/L, 100% mortality occurred after 2 h. Conclusions Thus, we recommended the use of 1000 μL/L of F. insipida latex for therapeutic baths in of C. macropomum against monogeneans, after previous test of toxicity.

First Report on the Genus Gyrinicola Yamaguti, 1938 from India with a Description of a New Species Abstract Introduction Gyrinicola dehradunensis sp. nov. from the intestine of tadpoles of the small paa frog, Nanorana minica, from Dehradun (Uttarakhand), India is described and illustrated. Gyrinicola dehradunensis is the first species of the genus recorded from India and the second species recorded from Asia. Methods Light microscopy was used for the identification of nematodes using BX53 DIC/BF Olympus research microscope with an attached DP27 digital camera. Drawings for the description of the new species were made from photomicrographs. For molecular studies, DNA was isolated using Qiagen, DNeasy® Blood and Tissue Kit and amplified using r-DNA ITS-18S, and ITS-1.58S primers. Results The new species is differentiated from the known species by the absence of larvated eggs and the presence of a tooth-like projection in the buccal region. N. minica is a new host record for the genus. Four r-DNA ITS1 sequences of the new species have been submitted to the NCBI Genbank.

Monogenoidea (Dactylogyridae, Gyrodactylidae) from Red River Pupfish, Cyprinodon rubrofluviatilis (Cyprinodontiformes: Cyprinodontidae), from Kansas and Texas, USA Abstract Background The gills of Red River Pupfish (Cyprinodon rubrofluviatilis) collected from Kansas and Texas, U.S.A., were found to be infected with three monogenoideans, Fundulotrema prolongis, Gyrodactylus rubrofluviatilae n. sp., and a species of Salsuginus. Results Gyrodactylus rubrofluviatilae n. sp. appears to be a member of a group of six closely related species possessing hamuli with mesially folded roots and a linguiform ventral bar shield, and parasitizing closely related species of Cyprinodon of the North American Gulf Coast and inland waters of the Southwestern United States and northern México. Gyrodactylus rubrofluviatilae n. sp. differs from its close congeners in the morphology of the hamuli, ventral bar, ventral bar shield, and marginal hooks. Because the morphology of the male copulatory complex was not determined of what we believe will eventually be a new species of Salsuginus, a species description is deferred. This is the first report of any parasite from this host. Conclusions Many North American species currently recognized within the Cyprinodontiformes have not yet been surveyed for species of Fundulotrema, Gyrodactylus, or Salsuginus. This, coupled with the high host specificity generally recognized for these monogeneans, portends there are likely additional new species yet to be discovered. When possible, to help augment morphological data, further studies should employ comprehensive intra- and interspecies molecular analyses to help resolve host–parasite phylogenies.