Aarón Martin-Alonso

Emerging rabbit haemorrhagic disease virus 2 (RHDV2) at the gates of the African continent

Until the beginning of this decade, the genetic characterization of rabbit haemorrhagic disease virus (RHDV) from Iberian Peninsula had revealed the existence of two genogroups, G1 and sporadically G6. In 2010, the new emerging rabbit haemorrhagic disease variant, RHDV2 or RHDVb, was described in France, from where it has rapidly spread throughout Europe, including Iberian Peninsula countries. Nevertheless, although cases of rabbit haemorrhagic disease (RHD) have been reported in the Canary Islands, a Spanish archipelago located 100km off the coast of Morocco, no genetic characterization of RHDV had been carried out. Consequently, in order to identify the circulating RHDV strains in this archipelago, liver samples of six farm rabbits and fifteen wild rabbits were collected from several areas of the largest island, Tenerife, and analyzed for the presence of RHDV by antigen capture double antibody sandwich ELISA. In case of positive ELISA result, we amplified and sequenced two fragments of the vp60 gene, which were concatenated for phylogenetic purposes. The sequences analysis revealed the presence of RHDV2 in both farm and wild rabbits from several areas of Tenerife. This result constitutes the first finding of RHDV2 in the Canary Islands. These RHDV2 strains found in Tenerife shared two exclusive SNPs that have not been observed in the rest of RHDV2 strains. The identification of RHDV2 and the absence of classic RHDV strains in this study suggest that RHDV2 may be replacing classic strains in Tenerife, as has been also proposed in Iberian Peninsula, France and Azores. Given the proximity of the Canary Islands to the African continent, this result should raise awareness about a possible dispersal of RHDV2 from the Canary Islands to the North of Africa.

Proposal for a unified classification system and nomenclature of lagoviruses

Lagoviruses belong to the Caliciviridae family. They were first recognized as highly pathogenic viruses of the European rabbit (Oryctolagus cuniculus) and European brown hare (Lepus europaeus) that emerged in the 1970-1980s, namely, rabbit haemorrhagic disease virus (RHDV) and European brown hare syndrome virus (EBHSV), according to the host species from which they had been first detected. However, the diversity of lagoviruses has recently expanded to include new related viruses with varying pathogenicity, geographic distribution and host ranges. Together with the frequent recombination observed amongst circulating viruses, there is a clear need to establish precise guidelines for classifying and naming lagovirus strains. Therefore, here we propose a new nomenclature based on phylogenetic relationships. In this new nomenclature, a single species of lagovirus would be recognized and called Lagovirus europaeus. The species would be divided into two genogroups that correspond to RHDV- and EBHSV-related viruses, respectively. Genogroups could be subdivided into genotypes, which could themselves be subdivided into phylogenetically well-supported variants. Based on available sequences, pairwise distance cutoffs have been defined, but with the accumulation of new sequences these cutoffs may need to be revised. We propose that an international working group could coordinate the nomenclature of lagoviruses and any proposals for revision.

Seroprevalence of Angiostrongylus cantonensis in wild rodents from the Canary Islands.

Background Angiostrongylus cantonensis is a lungworm of rats (Muridae) that is the causative agent of human cerebral angiostrongyliasis. The life cycle of A. cantonensis involves rats and mollusks as the definitive and intermediate hosts, respectively. This study was designed to increase the knowledge about the occurrence and distribution of A. cantonensis in its definitive host in the Canary Islands, using parasitological and serological analysis in different areas and age groups. Methodology/Principal Findings Between 2009 and 2010, 54 black rats (Rattus rattus) from Tenerife were captured from six human-inhabited areas and sera samples were obtained. The lung nematodes were identified by morphological and molecular tools as A. cantonensis. The 31-kDa glycoprotein antigen was purified from A. cantonensis adult worms by electrophoresis and electroelution. Of the 54 tested rodents, 30 showed IgG antibodies against A. cantonensis 31-kDa antigen by ELISA. Therefore, the overall seroprevalence was 55.6% (95% CI: 42.4–68). Seroprevalent rodents were found in all the 6 areas. This 31-kDa antigen was not recognized by some sera of rats infected by other helminth species (but not A. cantonensis). Seroprevalence of IgG antibodies against A. cantonensis and prevalence based on the presence of adult worms showed significant correlation (R2 = 0.954, p<0.05). Conclusions/Significance The present results could indicate a high prevalence of A. cantonensis in Tenerife and suggest the inclusion of two new zones in the distribution area of the parasite. The commonness and wide distribution of A. cantonensis in rats implies the presence of intermediate hosts, indicating that humans may be at risk of getting infected.

Identification of a novel assemblage G subgenotype and a zoonotic assemblage B in rodent isolates of Giardia duodenalis in the Canary Islands, Spain

The flagellated parasite Giardia duodenalis is known as one of the most common causes of protozoal diarrhoea in both humans and animals worldwide. The aim of the present work was to perform the first study of G. duodenalis in rodents in the Canary Islands (Spain) and analyse the level of genetic variation and the potential zoonotic role of the isolates. Stool samples were collected from 284 wild rodents and Giardia cysts were detected by light microscopy. The overall prevalence of giardiasis was 25 · 4% and ranged from 19 · 4% in El Hierro to 34% in Gran Canaria. Positive samples were further characterized by PCR and nucleotide sequencing of the triose phosphate isomerase (TPI), β-giardin (BG) and glutamate dehydrogenase (GDH) genes. Our study revealed assemblage G as the most frequent genotype and identified two rodent-infecting G. duodenalis haplotypes of this assemblage, HI and HII. Phylogenetic analysis supported the monophyly of haplotype HI, which we suggest to be considered as a novel G. duodenalis sub-assemblage GII, due to the high genetic distances among this sub-genotype and assemblage G. Furthermore, G. duodenalis assemblage B was detected in an inhabited area in La Palma, a fact that may pose a potential risk of G. duodenalis transmission from rodents to humans.

Intermediate Hosts of Angiostrongylus cantonensis in Tenerife, Spain

The nematode Angiostrongylus cantonensis is the causative agent of human angiostrongyliasis, the main clinical manifestation of which is eosinophilic meningitis. Although this parasite has been found recently in its definitive rat host in Tenerife (Canary Islands, Spain), showing a widespread distribution over the north-east part of the island, there are no available data regarding which snail and/or slug species are acting as intermediate hosts on this island. Consequently, the objective of this work was to determine the possible role of three mollusc species, Plutonia lamarckii, Cornu aspersum and Theba pisana, as intermediate hosts of A. cantonensis in Tenerife. Between 2011 and 2014, 233 molluscs were collected from five biotopes where rats had been found previously to harbor either adult worms or antibodies against A. cantonensis, and the identification was carried out on the basis of morphological features and a LAMP technique. The prevalence of A. cantonensis larvae in the mollusc samples, based on morphological identification, was 19.3%, whereas 59 out of the 98 individuals (60.2%) analyzed by LAMP were positive. Positive results were obtained for the three mollusc species analyzed and two of the positive samples, both obtained from P. lamarckii, were confirmed as positive by 18S rRNA and ITS1 PCR. Sequence analysis of 18S rRNA PCR products showed 100% similarity with previously published A. cantonensis sequences. These results may be relevant from a public health point of view, since all the biotopes from which the samples were obtained were in inhabited areas or areas with human activity, but it is also important from the perspective of a possible transmission to other accidental hosts, such as dogs and horses, animals that are present in some of the areas analyzed.

On the biology of Spiruroidea parasites of murine rodents on El Hierro (Canary Islands, Spain) with molecular characterization of Streptopharagus greenbergi Wertheim, 1993.

This paper reports the role of darkling beetles Pimelia laevigata costipennis and Hegeter amaroides (Tenebrionidae) as intermediate hosts of spiruroid nematodes parasites of the black rat and house mouse of El Hierro (Canary Islands). Larvae of spiruroid species were found in the two tenebrionids (18.1% in P. l. costipennis, 7.8% in H. amaroides), Streptopharagus greenbergi being predominant in both (16.1% and 7.1%, respectively), ahead of Mastophorus muris and Gongylonema type larva. The larval stages of S. greenbergi are described for the first time, and adult worms were obtained experimentally from an infected laboratory rat, allowing the identification of the species. Morphometric measurements of experimental adults match those of adults detected in naturally infected rats on the island. Molecular data for S. greenbergi, and the ITS nucleotide sequence of the genus Streptopharagus are also provided for the first time. After the isolation of S. greenbergi DNA and amplification of the ITS region, the ITS1 of this spirocercid was sequenced and deposited in the GenBank database.

Full genome sequences are key to disclose RHDV2 emergence in the Macaronesian islands

A recent publication by Carvalho et al. in “Virus Genes” (June 2017) reported the presence of the new variant of rabbit hemorrhagic disease virus (RHDV2) in the two larger islands of the archipelago of Madeira. Based on the capsid protein sequence, the authors suggested that the high sequence identity, along with the short time spanning between outbreaks, points to dissemination from Porto Santo to Madeira. By including information of the full RHDV2 genome of strains from Azores, Madeira, and the Canary Islands, we confirm the results obtained by Carvalho et al., but further show that several subtypes of RHDV2 circulate in these islands: non-recombinant RHDV2 in the Canary Islands, G1/RHDV2 in Azores, Porto Santo and Madeira, and NP/RHDV2 also in Madeira. Here we conclude that RHDV2 has been independently introduced in these archipelagos, and that in Madeira at least two independent introductions must have occurred. We provide additional information on the dynamics of RHDV2 in the Macaronesian archipelagos of Azores, Madeira, and the Canary Islands and highlight the importance of analyzing RHDV2 complete genome.

Serological survey of antibodies to Toxoplasma gondii and Coxiella burnetii in rodents in north-western African islands (Canary Islands and Cape Verde)

Abstract Coxiella burnetii and Toxoplasma gondii are intracellular parasites that cause important reproductive disorders in animals and humans worldwide, resulting in high economic losses. The aim of the present study was to analyse the possible role of peridomestic small mammals in the maintenance and transmission of C. burnetii and T. gondii in the north-western African archipelagos of the Canary Islands and Cape Verde, where these species are commonly found affecting humans and farm animals. Between 2009 and 2013, 108 black rats (Rattus rattus) and 77 mice (Mus musculus) were analysed for the presence of Coxiella and Toxoplasma antibodies by enzyme-linked immunosorbent assay (ELISA) and indirect immunofluorescence (IFA), respectively. Our results showed a wide distribution of C. burnetii and T. gondii, except for T. gondii in Cape Verde, in both rodent species. The overall seroprevalence of C. burnetii antibodies was 12.4%; 21.1% for Cape Verde and 10.2% for the Canary Islands. With respect to T. gondii, seropositive rodents were only observed in the Canary Islands, with an overall seroprevalence of 15%. Considering the fact that both pathogens can infect a large range of hosts, including livestock and humans, the results are of public health and veterinary importance and could be used by governmental entities to manage risk factors and to prevent future cases of Q fever and toxoplasmosis.Coxiella burnetii and Toxoplasma gondii are intracellular parasites that cause important reproductive disorders in animals and humans worldwide, resulting in high economic losses. The aim of the present study was to analyse the possible role of peridomestic small mammals in the maintenance and transmission of C. burnetii and T. gondii in the north-western African archipelagos of the Canary Islands and Cape Verde, where these species are commonly found affecting humans and farm animals. Between 2009 and 2013, 108 black rats (Rattus rattus) and 77 mice (Mus musculus) were analysed for the presence of Coxiella and Toxoplasma antibodies by enzyme-linked immunosorbent assay (ELISA) and indirect immunofluorescence (IFA), respectively. Our results showed a wide distribution of C. burnetii and T. gondii, except for T. gondii in Cape Verde, in both rodent species. The overall seroprevalence of C. burnetii antibodies was 12.4%; 21.1% for Cape Verde and 10.2% for the Canary Islands. With respect to T. gondii, seropositive rodents were only observed in the Canary Islands, with an overall seroprevalence of 15%. Considering the fact that both pathogens can infect a large range of hosts, including livestock and humans, the results are of public health and veterinary importance and could be used by governmental entities to manage risk factors and to prevent future cases of Q fever and toxoplasmosis.

First ocular report of Gurltia paralysans (Wolffhügel, 1933) in cat

Abstract A two-year-old cat from Tenerife, Canary Islands (Spain), presented with two months history of uveitis in the left eye. It had been treated for two months but still showed an active uveitis. After new treatment, eye examination showed a mobile worm in the anterior chamber. Following surgical removal, the worm was obtained. Morphological study revealed that it was a male metastrongyloid nematode (Nematoda: Metastrongyloidea) with caudal bursa and two similar spicules. Molecular tools based on the 18Sr RNA gene sequence identified the parasite as Gurltia paralysans, which is a neurotropic nematode previously found in South America. Therefore, this article describes the first report of ophthalmic case of parasitism by G. paralysans, and cites for first time this species out of South America.

Differentially expressed microRNAs in experimental cerebral malaria and their involvement in endocytosis, adherens junctions, FoxO and TGF-β signalling pathways

Cerebral malaria (CM) is the most severe manifestation of infection with Plasmodium, however its pathogenesis is still not completely understood. microRNA (miRNA) have been an area of focus in infectious disease research, due to their ability to affect normal biological processes, and have been shown to play roles in various viral, bacterial and parasitic infections, including malaria. The expression of miRNA was studied following infection of CBA mice with either Plasmodium berghei ANKA (causing CM), or Plasmodium yoelii (causing severe but non-cerebral malaria (NCM)). Using microarray analysis, miRNA expression was compared in the brains of non-infected (NI), NCM and CM mice. Six miRNA were significantly dysregulated between NCM and CM mice, and four of these, miR-19a-3p, miR-19b-3p, miR-142-3p and miR-223-3p, were further validated by qPCR assays. These miRNA are significantly involved in several pathways relevant to CM, including the TGF-β and endocytosis pathways. Dysregulation of these miRNA during CM specifically compared with NCM suggests that these miRNA, through their regulation of downstream targets, may be vitally involved in the neurological syndrome. Our data implies that, at least in the mouse model, miRNA may play a regulatory role in CM pathogenesis.