Surveillance of phlebotomine fauna and Didelphis marsupialis (Didelphimorphia: Didelphidae) infection in an area highly endemic for visceral leishmaniasis in Colombia. / Vigilancia de la fauna flebotomínea e infección de Didelphis marsupialis (Didelphimorphia: Didelphidae) en un área de alta endemia de leishmaniasis visceral en Colombia

INTRODUCTION: The study of the interaction between the parasite, the vector and the mammalian hosts, including man, allows to understand the behavior of the leishmaniases. OBJECTIVE: To determine the presence of Lutzomyia species and to detect the Leishmania infection in Didelphis marsupialis in an endemic area for visceral leishmaniasis. MATERIALS AND METHODS: Phlebotomine fauna and individuals of D. marsupialis were collected with CDC and Tomahawk™ traps, respectively. The species of Lutzomyia were identified using the Young and Duncan key (1994). Ear and tail biopsies and blood samples from D. marsupialis were taken to identify the Leishmania species by amplifying a fragment of the gene associated with the 70 kD heat shock protein. RESULTS: Seven Lutzomyia species were identified: Lu. evansi, Lu. gomezi, Lu. panamensis, Lu. dubitans, Lu. cayennensis cayennensis, Lu. rangeliana and Lu. trinidadensis. The first three species have epidemiological importance in Colombia because of their implications in the transmission of the Leishmania parasite. Sixty-five tissue samples from 19 D. marsupialis individuals were negative for Leishmania spp. CONCLUSIONS: The presence of the Lutzomyia species that have been identified as vectors for Leishmania inside and around houses in the village of El Bledo, in El Carmen de Bolívar represents a risk of infection. Furthermore, the presence of Lu. panamensis is reported for first time in El Carmen de Bolívar in Colombia. Although the lack of detection of Leishmania spp. in D. marsupialis samples may suggest that D. marsupialis does not play an important role in the transmission cycle of Leishmania in this region, it is necessary to carry out further longitudinal studies to confirm this hypothesis.

Introducción. El estudio de la interacción entre el parásito, el vector y los huéspedes mamíferos, incluido el hombre, permite entender el comportamiento de la leishmaniasis. Objetivo. Determinar la presencia de especies del género Lutzomyia y detectar la infección por Leishmania spp. en Didelphis marsupialis en un área endémica de leishmaniasis visceral. Materiales y métodos. Se recolectaron flebotomíneos y D. marsupialis con trampas CDC y Tomahawk™, respectivamente. Las especies de Lutzomyia se identificaron usando la clave de Young y Duncan, 1994. Se tomaron biopsias de oreja, cola y muestras de sangre de D. marsupialis para diagnosticar Leishmania spp. mediante la amplificación de un fragmento del gen de la proteína de choque térmico de 70 kD. Resultados. Se identificaron siete especies de Lutzomyia: Lu. evansi, Lu. gomezi, Lu. panamensis, Lu. dubitans, Lu. cayennensis cayennensis, Lu. rangeliana y Lu. trinidadensis. Las tres primeras especies son reconocidas como vectores en el país por estar implicadas en la transmisión de Leishmania spp. En total, 65 muestras de tejidos de oreja, cola y de sangre provenientes de 19 D. marsupialis fueron negativas para Leishmania spp. en la PCR-HSP70. Conclusiones. La presencia de flebotomíneos con importancia epidemiológica en la zona evaluada representa un riesgo de transmisión. Asimismo, Lu. panamensis es reportada por primera vez en El Bledo (Carmen de Bolívar). La ausencia de Leishmania spp. en D. marsupialis podría sugerir que esta especie no tiene un papel importante en el ciclo de transmisión de Leishmania en la vereda El Bledo, por lo que es necesario profundizar en estudios longitudinales para corroborar esta hipótesis.

Surveillance of phlebotomine fauna and Didelphis marsupialis (Didelphimorphia: Didelphidae) infection in an area highly endemic for visceral leishmaniasis in Colombia / Vigilancia de la fauna flebotomínea e infección de Didelphis marsupialis (Didelphimorphia: Didelphidae) en un área de alta endemia de leishmaniasis visceral en Colombia

Abstract Introduction: The study of the interaction between the parasite, the vector and the mammalian hosts, including man, allows to understand the behavior of the leishmaniases. Objective: To determine the presence of Lutzomyia species and to detect the Leishmania infection in Didelphis marsupialis in an endemic area for visceral leishmaniasis. Materials and methods: Phlebotomine fauna and individuals of D. marsupialis were collected with CDC and Tomahawk™ traps, respectively. The species of Lutzomyia were identified using the Young and Duncan key (1994). Ear and tail biopsies and blood samples from D. marsupialis were taken to identify the Leishmania species by amplifying a fragment of the gene associated with the 70 kD heat shock protein. Results: Seven Lutzomyia species were identified: Lu. evansi, Lu. gomezi, Lu. panamensis, Lu. dubitans, Lu. cayennensis cayennensis, Lu. rangeliana and Lu. trinidadensis. The first three species have epidemiological importance in Colombia because of their implications in the transmission of the Leishmania parasite. Sixty-five tissue samples from 19 D. marsupialis individuals were negative for Leishmania spp. Conclusions: The presence of the Lutzomyia species that have been identified as vectors for Leishmania inside and around houses in the village of El Bledo, in El Carmen de Bolívar represents a risk of infection. Furthermore, the presence of Lu. panamensis is reported for first time in El Carmen de Bolívar in Colombia. Although the lack of detection of Leishmania spp. in D. marsupialis samples may suggest that D. marsupialis does not play an important role in the transmission cycle of Leishmania in this region, it is necessary to carry out further longitudinal studies to confirm this hypothesis.

Resumen Introducción. El estudio de la interacción entre el parásito, el vector y los huéspedes mamíferos, incluido el hombre, permite entender el comportamiento de la leishmaniasis. Objetivo. Determinar la presencia de especies del género Lutzomyia y detectar la infección por Leishmania spp. en Didelphis marsupialis en un área endémica de leishmaniasis visceral. Materiales y métodos. Se recolectaron flebotomíneos y D. marsupialis con trampas CDC y Tomahawk™, respectivamente. Las especies de Lutzomyia se identificaron usando la clave de Young y Duncan, 1994. Se tomaron biopsias de oreja, cola y muestras de sangre de D. marsupialis para diagnosticar Leishmania spp. mediante la amplificación de un fragmento del gen de la proteína de choque térmico de 70 kD. Resultados. Se identificaron siete especies de Lutzomyia: Lu. evansi, Lu. gomezi, Lu. panamensis, Lu. dubitans, Lu. cayennensis cayennensis, Lu. rangeliana y Lu. trinidadensis. Las tres primeras especies son reconocidas como vectores en el país por estar implicadas en la transmisión de Leishmania spp. En total, 65 muestras de tejidos de oreja, cola y de sangre provenientes de 19 D. marsupialis fueron negativas para Leishmania spp. en la PCR-HSP70. Conclusiones. La presencia de flebotomíneos con importancia epidemiológica en la zona evaluada representa un riesgo de transmisión. Asimismo, Lu. panamensis es reportada por primera vez en El Bledo (Carmen de Bolívar). La ausencia de Leishmania spp. en D. marsupialis podría sugerir que esta especie no tiene un papel importante en el ciclo de transmisión de Leishmania en la vereda El Bledo, por lo que es necesario profundizar en estudios longitudinales para corroborar esta hipótesis.

Transcriptomic variation of locally-infected skin of Epinephelus coioides reveals the mucosal immune mechanism against Cryptocaryon irritans.

Fish skin is the largest immunologically active mucosal organ, providing first-line defense against external pathogens. However, the skin-associated immune mechanisms of fish are still unclear. Cryptocaryon irritans is an obligate ectoparasitic ciliated protozoan that infects almost all marine fish, and is believed to be an excellent pathogen model to study fish mucosal immunity. In this study, a de novo transcriptome assembly of Epinephelus coioides skin post C. irritans tail-infection was performed for the first time using the Illumina HiSeq™ 2500 system. Comparative analyses of infected skin (group Isk) and uninfected skin (group Nsk) from the same challenged fish and control skin (group C) from uninfected control fish were conducted. As a result, a total of 91,082 unigenes with an average length of 2880 base pairs were obtained and among them, 38,704 and 48,617 unigenes were annotated based on homology with matches in the non-redundant and zebrafish database, respectively. Pairwise comparison resulted in 10,115 differentially-expressed genes (DEGs) in the Isk/C group comparison (4,983 up-regulated and 5,132 down-regulated), 2,275 DEGs in the Isk/Nsk group comparison (1,319 up-regulated and 956 down-regulated) and 4,566 DEGs in the Nsk/C group comparison (1,534 up-regulated and 3,032 down-regulated). Seven immune-related categories including 91 differentially-expressed immune genes (86 up-regulated and 5 down-regulated) were scrutinized. Both DEGs and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis and immune-related gene expression analysis were used, and both analyses showed that the genes were more significantly altered in the locally-infected skin than in the uninfected skin of the same challenged fish. This suggests the skin's local immune response is important for host defense against this ectoparasite infection. Innate immune molecules, including hepcidin, C-type lectin, transferrin, transferrin receptor protein, serum amyloid A, cathepsin and complement components were significantly up-regulated (fold-change ranged from 3.3 to 12,944) in infected skin compared with control skin. The up-regulation of chemokines and chemokine receptors and activation of the leukocyte transendothelial migration pathway suggested that leucocytes intensively migrated to the local infected sites to mount a local immune defense. Toll-like receptors (TLRs) 1, 2, 5 and 5S were most significantly up-regulated in the infected skin, suggesting that these TLRs may be involved in parasite pathogen-associated molecular pattern (PAMPs) recognition. Up-regulation of the dendritic cell markers CD209 and CD83 and other antigen presentation pathway molecules provided evidence for skin local antigen presentation. Up-regulation of the T cell markers CD4 and CD48, B cell markers CD22 and CD81 and B cell receptor signaling kinase Lyn, showed the presence and population expansion of T/B cells at locally-infected sites, which suggested possible activation of a local specific immune response in the skin. Our results will facilitate in-depth understanding of local immune defense mechanisms in fish skin against ectoparasite infection.

Alaria mesocercariae in the tails of red-sided garter snakes: evidence for parasite-mediated caudectomy.

Trematodes of the genus Alaria develop into an arrested stage, known as mesocercariae, within their amphibian second intermediate host. The mesocercariae are frequently transmitted to a non-obligate paratenic host before reaching a definitive host where further development and reproduction can occur. Snakes are common paratenic hosts for Alaria spp. with the mesocercariae often aggregating in the host's tail. In the current study, we used morphological examination and molecular analyses based on partial sequences of nuclear large ribosomal subunit gene and mitochondrial cytochrome C oxidase subunit 1 gene to identify larvae in the tails of red-sided garter snakes (Thamnophis sirtalis parietalis) as mesocercariae of Alaria marcianae, Alaria mustelae, and Alaria sp. as well as metacercariae of Diplostomidae sp. of unknown generic affiliation. We assessed infection prevalence, absolute and relative intensity, and associated pathological changes in these snakes. Infection prevalence was 100 % for both male and female snakes. Infection intensity ranged from 11 to more than 2000 mesocercariae per snake tail but did not differ between the sexes. Gross pathological changes included tail swelling while histopathological changes included mild inflammation and the presence of mucus-filled pseudocysts surrounding mesocercariae, as well as the compression and degeneration of muscle fibers. Our results indicate that mesocercariae can lead to extensive muscle damage and loss in both sexes which likely increases the fragility of the tail making it more prone to breakage. As tail loss in garter snakes can affect both survival and reproduction, infection by Alaria mesocercariae clearly has serious fitness implications for these snakes.

Identification of different Bartonella species in the cattle tail louse (Haematopinus quadripertusus) and in cattle blood.

Bartonella spp. are worldwide-distributed facultative intracellular bacteria that exhibit an immense genomic diversity across mammal and arthropod hosts. The occurrence of cattle-associated Bartonella species was investigated in the cattle tail louse Haematopinus quadripertusus and in dairy cattle blood from Israel. Lice were collected from cattle from two dairy farms during summer 2011, and both lice and cow blood samples were collected from additional seven farms during the successive winter. The lice were identified morphologically and molecularly using 18S rRNA sequencing. Thereafter, they were screened for Bartonella DNA by conventional and real-time PCR assays using four partial genetic loci (gltA, rpoB, ssrA, and internal transcribed spacer [ITS]). A potentially novel Bartonella variant, closely related to other ruminant bartonellae, was identified in 11 of 13 louse pools collected in summer. In the cattle blood, the prevalence of Bartonella infection was 38%, identified as B. bovis and B. henselae (24 and 12%, respectively). A third genotype, closely related to Bartonella melophagi and Bartonella chomelii (based on the ssrA gene) and to B. bovis (based on the ITS sequence) was identified in a single cow. The relatively high prevalence of these Bartonella species in cattle and the occurrence of phylogenetically diverse Bartonella variants in both cattle and their lice suggest the potential role of this animal system in the generation of Bartonella species diversity.

Evaluation of continuous 4-day exposure to peracetic acid as a treatment for Ichthyophthirius multifiliis.

The parasitic ciliate Ichthyophthirius multifiliis infests all species of freshwater fish and can cause severe economic losses in fish breeding. The most effective treatment, malachite green, has been banned in Europe and North America for use in food fish production. Peracetic acid (PAA) was found to be toxic to I. multifiliis theronts at low concentrations. I. multifiliis-infested carp were exposed to 1 mg/l PAA in a dynamic exposure by means of peristaltic pumps. Five days after infestation, gills, tail fins, and skin below the dorsal fin were observed microscopically for I. multifiliis abundance. After PAA exposure, PAA-treated fish showed lower infestation of I. multifiliis in all investigated tissues than the unexposed control fish. The infestation increased in the control group whereas the infestation in the PAA-exposed groups significantly decreased (p = 0.0083, Bonferroni correction). The fish in the two exposure groups showed a slight reinfestation with I. multifiliis. This might be caused by a peroxide degradation (hydrolysis) and/or reduction of the delivered PAA concentration. Thus, PAA concentrations were possibly too low to be effective on the released trophonts and/or the infective theronts. This hypothesis is corroborated by the fact that the I. multifiliis in the gills, skin, and fins of the PAA-exposed carp were in an early developmental stage.

Occurrence of Trichodina heterodentata (Ciliophora: Trichodinidae) infesting tadpoles of Rhinella pombali (Anura: Bufonidae) in the Neotropical area.

Data on trichodinid ciliophorans in Brazil are scarce and generally do not use the modern silver-impregnation technique, which is essential to the taxonomy of the group. The present study reports not only on the first occurrence of Trichodina heterodentata in Brazil but also on the first record of this trichodinid infesting tadpoles of the species Rhinella pombali. This study also constitutes a second report on T. heterodentata from tadpoles. In September 2007, tadpoles were collected from a stream in a small farm in the agricultural area of the city of Juiz de Fora, southeastern Brazil. The ciliophorans found on the tadpoles' bodies and tails were submitted to techniques such as silver impregnation and scanning electron microscopy. Our biometric data of T. heterodentata population infesting Rhinella pombali were compared to five other South-African populations of T. heterodentata infesting tadpoles of Xenopus laevis laevis. Results were discussed in terms of low host specificity as well as the wide geographical distribution of this trichodinid.

Morphology and 18S rDNA of Henneguya gurlei (Myxosporea) from Ameiurus nebulosus (Siluriformes) in North Carolina.

Henneguya gurlei was isolated from Ameiurus nebulosus captured in North Carolina and redescribed using critical morphological features and 18S small-subunit ribosomal RNA (SSU rDNA) gene sequence. Plasmodia are white, spherical, or subspherical, occur in clusters, measure up to 1.8 mm in length, and are located on the dorsal, pectoral, and anal fins. Histologically, plasmodia are located in the dermis and subdermally, and the larger cysts disrupt the melanocyte pigment layer. The spore body is lanceolate, 18.2 +/- 0.3 microm (range 15.7-20.3) in length, and 5.4 +/- 0.1 microm (range 3.8-6.1) in width in valvular view. The caudal appendages are 41.1 +/- 1.1 microm (range 34.0-49.7) in length. Polar capsules are pyriform and of unequal size. The longer polar capsule measures 6.2 +/- 0.1 microm (range 5.48-7.06), while the shorter is 5.7 +/- 0.1 microm (range 4.8-6.4) in length. Polar capsule width is 1.2 +/- 0.03 microm (range 1.0-1.54). The total length of the spore is 60.9 +/- 1.2 microm (range 48.7-68.5). Morphologically, this species is similar to other species of Henneguya that are known to infect ictalurids. Based on SSU rDNA sequences, this species is most closely related to H. exilis and H. ictaluri, which infect Ictalurus punctatus.

Navigation within host tissues: cues for orientation of Diplostomum spathaceum (Trematoda) in fish towards veins, head and eye.

Cercariae of Diplostomum spathaceum penetrate the skin of fish, and then migrate along blood vessels and tissues towards the head and the eye-lens. We studied their orientation behaviour in tail fins of guppies and in chemical concentration gradients within agar-filled choice chambers. In fins, they entered veins and orientated cranially, independent of the blood flow and living cells. In choice chambers, they were attracted by a small molecular fraction of fish serum, D-glucose (at 1, 10, and 1000 microM), D-mannose, D-maltotriose and Cl-ions, whereas D-glucosamine repelled them (even at 1.0 nM). Amino acids were not attractive, but arginine in tetrapeptides attracted at concentrations as low as 1 microm and melatonin at 0.4-4.3 PM. We suggest a preliminary model for the behaviour of diplostomula in fish fins and attracting (+) or repelling (-) host cues: (1) migration towards deeper skin layers and avoidance of skin surface, cues: Cl-ions (+ and -), glucose (+), glucosamine (-), light radiation (-); (2) orientation in cranial direction, cue: Cl-ions (+); (3) localization of blood vessels, cues: glucose (+), arginine-residues (+); (4) localization of the retina, cue: melatonin (+). A comparison with the navigation mechanisms of tissue-migrating schistosomules and hookworm larvae reveals an enormous diversity of strategies.

The site of Leishmania major infection determines disease severity and immune responses.

Inbred strains of mice infected with Leishmania major have been classified as genetically resistant or susceptible on the basis of their ability to cure their lesions, the parasite burden in the draining lymph nodes, and their type of T helper cell immune responses to the parasite. Using the intradermal infection at the base of the tail and the ear pinna, we compared for the first time the above-mentioned parameters in six strains of mice infected with metacyclic promastigotes, and we show that the severity of disease depends greatly on the site of infection. Although the well-documented pattern of disease susceptibility of BALB/c and C57BL/6 mice described for the footpad and base-of-the-tail models of leishmaniasis were confirmed, C3H/HeN and DBA/2 mice, which are intermediate and susceptible, respectively, in the tail and other models, were resistant to ear infection. Moreover, in the CBA/H, C3H/HeN, C57BL/6J, and DBA/2 mouse strains, there was little correlation between the pattern of cytokines produced and the disease phenotype observed at the ear and tail sites. We conclude that the definition of susceptibility and the immune mechanisms leading to susceptibility or resistance to infection may differ substantially depending on the route of infection.

Demodicosis in an American bison.

An 18-month-old, male American bison (Bison bison) was presented with 7- to 9-mm size nodules periorbital, perineal, and on the ventral surface of the tail. Demodex spp. were identified from the exudate by microscopic examination. Examination 6 mo later revealed that the infestation had nearly cleared without treatment.

Isolation of Leishmania (L.) mexicana from wild rodents and their possible role in the transmission of localized cutaneous leishmaniasis in the state of Campeche, Mexico.

Leishmania parasites were isolated from four of seven small rodent species captured in the State of Campeche, Mexico. Signs and/or symptoms of leishmanial infection were restricted to the tail of the mice. Peromyscus yucatanicus endemic to the peninsula of Yucatan and Oryzomys melanotis were added to the list of carriers of Leishmania. Leishmania (L.) mexicana, characterized by an indirect immunofluorescence antibody method using monoclonal antibodies, was identified in one Sigmodon hispidus and one Or. melanotis. The possible role of the different species as primary reservoirs of Leishmania in the peninsula of Yucatan is discussed.

Morphology and cytology study of Foleyella agamae complex (Nematoda: Filarinae) infecting the agamid lizard Agama agama in Nigeria.

Three cytotypes of Foleyella agamae were isolated from naturally infected wild-caught Agama agama in Nigeria. Cytotype A (2n = 8) has a body length/oesophageal length ratio (L/O) of 87.6 +/- 14.2 in female worms and a large somatic size (length, 63.8 +/- 12.7 mm in female worms and 25.5 +/- 3.4 mm in males). Cytotype B (2n = 6) has an L/O of 43.4 +/- 6.6 in females and a small to average somatic size (length, 36.7 +/- 11.9 mm in females and 19.5 +/- 2.3 mm in males). Cytotype C (2n = 4) has an L/O of 23.8 +/- 8.2 in female worms and a small somatic size (length, 18.8 +/- 3.8 mm in females and 14.1 +/- 3.0 mm in males). No cytotype with odd numbers of chromosomes was observed. Comparisons with similar types of speciation in medically important filarial parasites were drawn. Introgressive hydridisation in the speciation of filarial parasites is postulated.

Influence of a menhaden oil diet on cercarial penetration of Schistosoma mansoni.

The ability of a menhaden oil (MO) diet to influence cercarial penetration into mouse tail skin was evaluated. Male CD-1 mice 4-6 wk old (15.2 g average weight) were fed a 0, 10%, or 20% MO-supplemented diet for 2 wk. After this time mice were infected with either 65 +/- 3 or 145 +/- 3 [35S]methionine/cysteine-labeled cercariae for 1 hr by tail immersion. Twenty-four hours and 7 days later groups of mice were killed and their tail skin removed and autoradiographed. At 24 hr postinfection, mice fed a 20% MO diet had significantly higher cercarial penetration than controls and 10% MO diets (56% +/- 5.2 vs. 44% +/- 2.9, P = 0.02, 1-tailed t-test). After 7 days mice fed a 20% MO diet retained more radioactive foci than controls or 10% MO diets (21% +/- 2.0 vs. 15% +/- 1.3, P = 0.01, 1-tailed t-test).

Observations on the New World screwworm fly in Libya and the risk of its entrance into Egypt.

Data on traumatic myiasis caused by Cochliomyia hominivorax in Libya were reported from August 1988 until February 1989. A total of 468 cases of screwworm myiasis were recorded in seven species of livestock and 229 humans, mainly children, were also found to be infested. Cattle and sheep were the most common hosts in livestock. In cattle, the principal infestation site was the umbilicus of neonates; in sheep, it was mainly the fatty tail. Animal myiasis reached its peak in October and November, but disappeared abruptly in winter on the onset of cold weather. Screwworm distribution was confined to the northwest of Libya. The potential dispersion of the fly from this area and the possibility of infestation of other countries, particularly Egypt, are discussed. Food and Agriculture Organization (FAO) efforts to confront the New World screwworm problem in Africa are mentioned.

Epidemiology of sheep myiases in São Paulo State, Brazil.

A survey of Diptera species causing cutaneous myiases on sheep in Botucatu, São Paulo, Brazil was made to determine seasonal incidence, predilection sites and the factors predisposing to infestation. Sheep were checked daily for myiases for one year. At two week intervals larvae from wounds were collected for identification. Only larvae of Dermatobia hominis and Cochliomyia hominivorax were found. Myiases due to C. hominivorax were observed during the whole year with high incidence from January to April. The feet, vulva, tail and scrotum were most frequently infested. Wounds were the commonest predisposing factor.

Experimental infection routes of Angiostrongylus cantonensis in mice.

Stomach intubation is the most common method used in the experimental infection of animals with Angiostrongylus cantonensis. In order to compare the effectiveness of other possible transmission methods, groups of BALB/c mice were given infective third-stage larvae of A. cantonensis by different routes including intraperitoneal or subcutaneous injections, and penetration of anal mucosa, vaginal mucosa, conjunctival mucosa, lacerated skin, unabraded skin, foot pad and tail skin, while stomach intubation was used as control. Recovery of fifth-stage larvae was higher in mice inoculated with third-stage larvae subcutaneously. Successful infections were established through all experimental transmission routes except tail skin penetration. This study suggests that oral infection may not be the only route for the transmission of human angiostrongyliasis, and subcutaneous infection may be a better method for experimental infection.