Luz Elena Velásquez

Morphological and molecular characterization of Neotropic Lymnaeidae (Gastropoda: Lymnaeoidea), vectors of fasciolosis

Lymnaeidae play a crucial role in the transmission of fasciolosis, a disease of medical and veterinary importance. In the Neotropic, a region where fasciolosis is emergent, eight Lymnaeidae species are currently considered valid. However, our knowledge of the diversity of this taxon is hindered by the fact that lymnaeids exhibit extremely homogeneous anatomical traits. Because most species are difficult to identify using classic taxonomy, it is difficult to establish an epidemiological risk map of fasciolosis in the Neotropic. In this paper, we contribute to our understanding of the diversity of lymnaeids in this region of the world. We perform conchological, anatomical and DNA-based analyses (phylogeny and barcoding) of almost all species of Lymnaeidae inhabiting the Neotropic to compare the reliability of classic taxonomy and DNA-based approaches, and to delimitate species boundaries. Our results demonstrate that while morphological traits are unable to separate phenotypically similar species, DNA-based approaches unambiguously ascribe individuals to one species or another. We demonstrate that a taxon found in Colombia and Venezuela (Galba sp.) is closely related yet sufficiently divergent from Galba truncatula, G. humilis, G. cousini, G. cubensis, G. neotropica and G. viatrix to be considered as a different species. In addition, barcode results suggest that G. cubensis, G. neotropica and G. viatrix might be conspecifics. We conclude that conchological and anatomical characters are uninformative to identify closely related species of Lymnaeidae and that DNA-based approaches should be preferred.

Paragonimiasis: a view from Columbia

Paragonimiasis is a zoonosis caused by adult trematodes of the Paragonimus genus. The infection in humans is a result of a complex transmission cycle that includes two obligate intermediate hosts, a snail and a crustacean or a crayfish, and a definitive mammalian host. It has been shown that 9 of the more than 40 species of Paragonimus described affect humans in over 39 countries in Asia, Africa and America. It is estimated that 20.7 million people have paragonimiasis and it is calculated that 195 million people are at risk of being infected. The illness usually is caused once the parasite has settled in the lung at the site of the main clinical symptoms: cough, thoracic pain and hemoptysis. The diagnosis of paragonimiasis is based on the patients history, the parasitological findings (ova in sputum and in feces), and the result of radiological and immunological tests. In severe cases, the patient may suffer from life-threatening hemoptysis or pneumothorax. Currently, praziquantel is the drug of choice.

MORPHOLOGICAL DESCRIPTION AND LIFE CYCLE OF PARAGONIMUS SP. (TREMATODA: TROGLOTREMATIDAE): CAUSAL AGENT OF HUMAN PARAGONIMIASIS IN COLOMBIA

The first morphological description is made of all stages of the life cycle of a Paragonimus species infecting humans in Colombia. Larval stages were obtained both in vitro and from field collections. Adult Paragonimus spp. are described. The aquatic snail Aroapyrgus sp. serves as an intermediate host of this species, both naturally and experimentally, yielding rediae and cercariae. Crabs (Hypolobocera bouvieri monticola and H. emberarum) were found to be the natural second intermediate hosts, and individuals of another crab species (Strengeria sp.) were also infected in the laboratory.

Epidemiology of paragonimiasis in Colombia.

Five newly discovered endemic foci for paragonimiasis in Colombia are described for the first time. The disease was diagnosed in 24 people from the Embera Indian communities located at the Colombian Pacific Coast and investigated in 1993-98. We also describe the clinical, epidemiological and treatment response aspects. In these foci an Aroapyrgus sp. snail different from A. colombiensis was found to be the first intermediate host, and the crab Hypolobocera emberarum nsp. the second intermediate host.

Morphological and polymerase chain reaction-restriction fragment lenght polymorphism characterization of Biomphalaria kuhniana and Biomphalaria amazonica from Colombia

In Colombia, five Biomphalaria planorbid species are known: B. kuhniana, B. straminea, B. peregrina, B. canonica and B. oligoza(var. B. philippiana). Among them, B. straminea is intermediate host of Schistosoma mansoni and B. peregrina has been found to be experimentally susceptible to this parasite. B. straminea is commonly confused with B. kuhniana and they have been clustered together with B. intermedia in the complex named B. straminea. The difficulties involved in the specific identification, based on morphological data, have motivated the use of new techniques as auxiliary tools in cases of inconclusive morphological identification of such planorbid. In the present study, five Biomphalaria populations from the Colombian Amazon region and from Interandian Valleys were morphologically identified and characterized by polymerase chain reaction-restriction fragment lenght polymorphism directed at the internal transcribed spacer region of the rRNA gene, followed by digestion of the generated fragment with restriction enzymes (DdeI, AluI, RsaI, MvaI and HaeIII). Known profiles of the Brazilian species B. straminea, B. peregrina, B. kuhniana, B. intermedia and B. amazonica, besides B. kuhniana from Colombia, were used for comparison. The five populations under study were morphologically and molecularly identified as B. kuhniana and B. amazonica.

Synonymy between Lymnaea bogotensis Pilsbry, 1935 and Lymnaea cousini Jousseaume, 1887 (Gastropoda: Lymnaeidae)

The lymneid snail Lymnaea bogotensis Pilsbry 1935 is synonymized with L. cousini Jousseaume 1887, based on morphological comparisons of the reproductive systems. The shell, renal tube, and reproductive system are described and illustrated from specimens collected in the type locality and the municipality of Paipa, Colombia. Bibliographical records reveal L. columella to be the commonest lymneid in the country. The latter two species can be differentiated based on the morphology of the penial complex.

Cultivo in vitro de larvas L3 de nematodos obtenidas del caracol gigante africano Lissachatina fulica (Mollusca: Gastropoda) en Santa Fe de Antioquia

INTRODUCTION Over 170 municipalities in Colombia have been invaded by Lissachatina fulica, an African snail that can carry larvae of nematodes of interest in human and veterinary health. Nematodes enter the host snail as larvae L1 and then change to L2 and L3, the infectious form for vertebrates. OBJECTIVE To standardize culture in vitro of L3 carried by L. fulica from Santa Fe de Antioquia. MATERIALS AND METHODS Between July and November, 2014, 10 snails were collected, killed, and conserved with HCl 0.7%. Larvae were recovered using the Baermann technique and cultured for 36 days in Schneider, DMEM and RPMI media, with and without SFB 20% and distilled water with SFB 20%. Replacements were made every 36 hours; larvae were measured with an ocular micrometer on a microscope. Summary statistics were estimated; box and whisker plots were made; the t Student test was performed in SPSS 18™. A p-value below 0.05 was assumed as significant. RESULTS Fifty per cent of the larvae survived. The highest survival and growth was 85% in supplemented DMEM. The final average length of larvae in supplemented media exceeded the initial one. There were significant differences between the average length of larvae cultured in supplemented media and the initial length. The initial width of larvae did not change. CONCLUSIONS The best medium for the culture of L3 larvae was supplemented DMEM. The length provided more information than the width for the larval growth evaluation. The larvae studied did not correspond to Angiostrongylus cantonensis, A. costaricensis or Aelurostrongylus abstrusus.

Standardization of a multiplex real-time PCR test for the identification of Angiostrongylus cantonensis, A. costaricensis and A. vasorum

INTRODUCTION Angiostrongyliasis is a disease caused by Angiostrongylus nematodes that is present worldwide. The infections with the highest impact on human and animal health are caused by A. cantonensis, A. costaricensis, and A. vasorum. Clinical forms of the disease in humans are eosinophilic meningitis and abdominal angiostrongyliasis, while the most common effect on dogs are cardiopulmonary damages. It is deemed as an emerging disease as the result of the global dissemination of the African snail Lissachatina fulica, an intermediary host of these parasites. The few diagnostic methods for Angiostrongylus spp. are unspecific, costly, and not very sensitive. It is urgent to develop a sensitive, specific and accessible diagnostic tool for the control of human and animal angiostrongyliasis. OBJECTIVE To develop a qPCR multiple test to identify the three pathogenic species of Angiostrongylus. MATERIALS AND METHODS Through a bio-informatic analysis, we selected a sequence of the ITS-2 region of the Angiostrongylus genome to guarantee the specificity of primers and probes. We extracted DNA from adult parasites as positive control, and from larvae using the DNeasy Blood&Tissue® kit. Quantitative PCR reactions were conducted on a Smartcycler Cepheid® thermocycler using a master mix QuantiTect® kit. DNA from human beings, other parasites and the African snail was used as negative control. RESULTS The threshold cycle values for positive DNA controls were: 21 for Angiostrongylus cantonensis, 22 for A. costaricensis, and 31 for A. vasorum. In negative controls, the threshold cycle was zero. qPCR showed an amplification efficiency of 2 (100%). CONCLUSIONS A multiple qPCR was standardized at the laboratory for three clinically significant species of Angiostrongylus.

The disease is another: therapeutic itinerary of bovine fascioliosis in Rionegro (Antioquia)

This article presents results of an anthropological research project that was carried out in the municipality of Rionegro in the department of Antioquia, Colombia. The disease caused by hepatic fasciola, a parasite native to Europe, and is now present on every continent and is considered to be the pathogenic disease with the greatest latitudinal, longitudinal, and altitudinal distribution. There are no specific observable symptoms in infected animals that allow a diagnosis, which must be done in veterinary laboratories, which is scarcely ever the case given that the disease rarely causes death. Through the use of ethnographic methods the researchers accessed various interlocutors linked to the pecuary field, which relayed their interpretations about bovine fasciolosis and about the common practices dealing with it ranging from lay knowledge to dialogues with expert and professional understanding. The text illustrates the therapeutic itinerary that is undertaken when certain changes in behavior and appearance are perceived, it describes the course of action that is followed in order to restore the animals’ health, and it considers the decisions that livestock farmers take when cures fail, all of which are the result of the fact that it is not mandatory to declare bovine fasciolosis. Academics in the field of national veterinary medicine who research the basic aspects related to the parasite’s life cycle are not familiar with the lay knowledge produced about this disease. An analysis of this knoweldge could contribute to the design of control strategies given that it presents in detail the doubts and uncertainties that have historically surrounded fasciolosis. The tendency to conceive it as a “new” disease, and the

Paragonimus Mexicanus (Digenea: Troglotrematidae) de Valle de Pérdidas, Urrao, Antioquia

This review establishing the specific status of Paragonimus sp. Form Valle de Perdidas by comparing morphological of metacercariae and adult stages whit Paragonimus caliensis from Valle del Cauca Colombia, and P. mexicanus from Ecuador, Mexico, Peru, and Venezuela described in the literature. Identification conflicts are due to the wide morphological variation in the reproductive system (ovary and testis) and the finding of free and encysted metacercariae in the same host (Velez et al, 2003). The digenean from Valle de Perdidas was identified as P. mexicanus based on geographical distribution, wide intraspecific variation in the shape and size of ovary and testes, and the fact that excyst metacercariae have a morphology like the described for P. mexicanus free metacercariae. This study also proposes the synonyms between P. mexicanus and P. caliensis based on intraspecific variation of adults and a possible polymorphism in the metacercariae cyst, since the morphology of the ovary and testes as well as the eventual formation of cysts in metacercariae were criteria for considering P. caliensis a new species.