Molecular investigation of Neorickettsia risticii in trematodes and snails in a region with serological evidence of this agent in horses, state of Rio de Janeiro

ABSTRACT In Brazil, some studies have indicated that Neorickettsia risticii circulates in horses, but it is unclear which are the possible intermediate vectors of this bacterium in the country. The aim of this study was to use molecular techniques in order to analyze the presence of N. risticii in snails and larval stages of trematodes in farms in a region with a history of seroreactive horses towards this bacterium, in Rio de Janeiro, Brazil. Convenience sampling was used in the studied region. The collected snails were exposed to incandescent light (60W) for 2-4 hours in order to investigate trematodes in larval forms. Deoxyribonucleic acid (DNA) was extracted from snail tissue and trematode. Real-time PCR (qPCR) technique was used to investigate the presence of a 16S rRNA gene fragment of N. risticii. Snail specimens (n=410) were collected from 11 horse-breeding farms, and the following species were identified: Melanoides tuberculata, Pomacea sp., Biomphalaria tenagophila, Physa acuta, Drepanotrema anatinum and Biomphalaria straminea. Only 3.17% (n=13/410) of the collected snails were infected by trematodes. The cercariae obtained from these snails were classified as Megalourous cercariae, Pleurolophocercus cercariae and Furcocercous cercariae. There was no amplification of the target DNA of N. risticii in the snail and trematode samples tested by qPCR. Based on these data, the transmission of N. risticii by trematodes using these snail species in this region does not appear to occur or occurs at very low rates. Thus, further studies are needed in order to clarify which species of invertebrate hosts are infected by this bacterium and potentially participate in the transmission chain of equine neorickettsiosis in the state of Rio de Janeiro, Brazil.

Localization of carbohydrate determinants common to Biomphalaria glabrata as well as to sporocysts and miracidia of Schistosoma mansoni

SUMMARYThe presence of antigenic carbohydrate epitopes shared by Biomphalaria glabrata as well as by the sporocysts and miracidia representing snail-pathogenic larval stages of Schistosoma mansoni was assayed by immunohistochemical staining of paraformaldehyde-fixed tissues. To this end, both polyclonal rabbit antiserum raised against soluble egg antigens (SEA) of S. mansoni and monoclonal antibodies recognizing the carbohydrate epitopes LDN [GalNAc(β1-4)GlcNAc(β1-)], F-LDN [Fuc(α1-3)GalNAc(β1-4)GlcNAc(β1-)], LDN-F [GalNAc(β1-4)[Fuc(α1-3)]GlcNAc(β1-)], LDN-DF [GalNAc(β1-4)[Fuc(α1-2)Fuc(α1-3)]GlcNAc(β1-)] and Lewis X [Gal(β1-4)[Fuc(α1-3)]GlcNAc(β1-)] were used. Intriguingly, anti-SEA serum as well as anti-F-LDN antibodies displayed significant binding in the foot region, anterior tissue and the hepatopancreas of uninfected snails, whereas the Lewis X epitope was only weakly detectable in the latter tissue. In contrast, increased binding of antibodies recognizing LDN, LDN-F and LDN-DF was observed in infected snail tissue, in particular in regions involved in sporocystogenesis, in addition to an enhanced binding of anti-SEA serum and antibodies reacting with F-LDN. A pronounced expression of most of these carbohydrate antigens was also observed at the surface of miracidia. Hence, the detection of shared carbohydrate determinants in uninfected snail tissue, sporocysts and miracidia may support the hypothesis of carbohydrate-based molecular mimicry as a survival strategy of S. mansoni.

Description of a tandem repeated DNA sequence of Echinostoma caproni and methods for its detection in snail and plankton samples

Echinostome larval stages in the snail have a great potential as effective competitors for the control of schistosomes and adult worms can cause painful intestinal diseases in humans. Ecology and transmission of the larval stages of trematodes are poorly understood, especially because their identification in field-collected samples by microscopy is difficult. We cloned, sequenced and analysed a 192 bp tandem repreated DNA sequence of Echinostoma caproni (EcSau3A), an often discussed antagonist of Schistosoma mansoni in Biomphalaria snails. PCR primers against this sequence can detect less than 10 fg of E. caproni DNA, 2 miracidia in snails 1 day p.i., 1 metacercaria in 50 mg snail tissue and 1 cercaria in 50 mg plankton with high specificity. Methods described in this study can support the discovery of fundamental ecological principles on distribution, host specificity and epidemiology of E. caproni larvae under field conditions.

Observations on chromosomes and gametogenesis ofTransversotrema patialense(Trematoda)

SUMMARYThe karyotype ofTransversotrema patia1enconsists of 10 pairs of chromosomes (2n= 20) of which 5 pairs are metacentrics and 5 pairs are submetacentrics. The chromosomes are large and range in size from 5 to 12 μm. The total chromosome length of the diploid complement is 16783 μm. Stages of spermatogenesis including the two gonial divisions and two reduction divisions leading to production of spermatozoa occur in cercarial embryos inside snail tissue, while the maturation divisions of the ovum occur in eggs freshly liberated by the fluke. The chiasma frequency is high, being 3888/cell and 388/bivalent. Cytological data indicate an independent phylogenetic status for the family Transversotrematidae.

Electron Microscope and Histochemical Observations on the Daughter Sporocyst of Schistosoma mattheei and Schistosoma bovis

Electron-microscopical, histological and histochemical observations were carried out on the body wall of the daughter sporocyst stage of Schistosoma mattheei and S. bovis. Electron microscopy revealed that the body wall consists of a continuous outer layer of cytoplasm, collagen fibres, fibroblasts and a layer of somatic cells which are apparently continuous with the cytoplasmic layer. This layer forms numerous microvilli at the surface.Pronounced alkaline phosphatase activity was found in the sporocyst body wall, but no evidence of esterases was found in the parasite although esterase activity was readily demonstrated in the snail tissue that the schistosome parasitized.It is concluded that the passage of substances such as glucose across the surface of the sporocyst is an active process probably mediated by enzymes and that little, if any, lipid metabolism occurs in the sporocyst stage.

Development of Carmyerius exoporus Maplestone (Trematoda: Gastrothylacidae) in a snail host

The development of Carmyerius exoporus Maplestone in the snail Anisus nata-lensis Krauss is described with the emphasis laid on the redial and cercarial productivity of the rediae of the first and subsequent generations.The miracidium gives rise to one sporocyst. From 25 to 32 germinal cells present in the miracidium, only six to eleven develop into embryo balls in the sporocyst and, of these, only three to nine develop into rediae of the first generation.The redia of the first generation gives birth to daughter rediae and cercariae. No alternation in the development of redial and cercarial embryos was noted in the rediae of C. exoporus and a small number of redial embryos was observed to develop alongside the more numerous cercarial embryos throughout the life of the first generation rediae.The rediae of the second and subsequent generations also give birth to daughter rediae and cercariae.The cercariae leave their parent redia when they are still immature and continue the development in the snail tissue.