High Prevalence of Microsporidia in the North African Hedgehog (Atelerix algirus) in the Canary Islands, Spain.

Microsporidia are unicellular eukaryotic obligate intracellular parasites with a wide range of hosts reported worldwide; however, little is known about the epidemiological data on microsporidia infection in animals from the Canary Islands. Since data on microsporidia infection in hedgehog species are scarce, the aim of this study was to analyze the presence and identity of microsporidia in a group of North African hedgehogs (Atelerix algirus) using microscopic and molecular methods. From December 2020 to September 2021, a total of 36 fecal samples were collected from naturally deceased hedgehogs from Tenerife and Gran Canaria. All samples showed spore-compatible structures (100%; 36/36) under microscopic analysis, of which 61.1% (22/36) were amplified via the nested-polymerase chain reaction (PCR) targeting the partial sequence of the 16S rRNA gene, the internal transcribed spacer (ITS) region, and the partial sequence of the 5.8S rRNA gene. After Sanger sequencing and ITS analysis, Enterocytozoon bieneusi was detected in 47.2% (17/36) of the samples, identifying two novel genotypes (AAE1 and AAE2), followed by the detection of an undetermined species in 8.3% (3/36) and Encephalitozoon cuniculi genotype I in 5.6% (2/36) of the samples. This study constitutes the first report of microsporidia species in Atelerix algirus worldwide, highlighting the high prevalence of zoonotic species.

Study of Zoonotic Pathogens in Alien Population of Veiled Chameleons (Chamaeleo calyptratus) in the Canary Islands (Spain).

Veiled chameleons (Chamaeleo calyptratus) are native to the Arabian Peninsula that have been introduced as pets in many regions around the world, such as the Canary Islands (Spain). In this work, the gastrointestinal content from veiled chameleons of Gran Canaria island (Canary Islands) has been analyzed to determine the presence of zoonotic bacteria. Forty animals were analyzed using different selective culture media and PCR. The most isolated bacteria were Yersinia enterocolitica (52.4%), followed by Salmonella spp. (40.0%), with positive isolates for Salmonella Tyhpi and Salmonella Typhimurium. Pseudomonas spp. was found in 32.5% of the chameleons. More than half were positive for Pseudomonas aeruginosa. Antibiotic-resistant Staphylococcus spp. was detected in six animals plus one isolate of non-resistant Staphylococcus hominis. Multiple mycobacteria species belonging to both tuberculous and non-tuberculous complexes were identified as well as Escherichia coli carrying the stx1 and eae virulence genes with 12.5% and 7.5% prevalence, respectively. Listeria monocytogenes, Campylobacter spp., and Vibrio spp. were found in lower proportion (<5%). The results obtained indicate that veiled chameleons in Gran Canaria could be playing a role in the maintenance and dissemination of the pathogens detected, harming public health and biodiversity.

Data on New Intermediate and Accidental Hosts Naturally Infected with Angiostrongylus cantonensis in La Gomera and Gran Canaria (Canary Islands, Spain).

Angiostrongylus cantonensis is a metastrongyloid nematode and the etiologic agent of angiostrongyliasis, a disease characterized by eosinophilic meningitis. This emerging zoonotic parasite has undergone great expansion, including in some regions of Europe and America. In the Canary Islands, the parasite was first discovered parasitizing Rattus rattus on the island of Tenerife in 2010. To date, the distribution of this parasite in the Canary Islands has been restricted to the northern zone and the main cities of Tenerife. Using molecular tools for the sentinel species present in the Canary Islands, this study confirmed the presence of the nematode on two other islands in the Canary Archipelago: La Gomera and Gran Canaria. Furthermore, this emerging parasite was detected, besides in the common definitive host R. rattus, in wild Mus musculus and Felis catus and in four terrestrial gastropod species, Limacus flavus, Milax gagates, Insulivitrina emmersoni, and Insulivitrina oromii, two of them endemic to La Gomera, for the first time, increasing the number of non-definitive host species. This study reinforces the expansion character of A. cantonensis and highlights the importance of knowledge about sentinel species for identifying new transmission locations that help prevent and control the transmission of the parasite and, thus, prevent public health problems.

Study of tick-borne zoonotic pathogens in questing and feeding ticks in Tenerife, Canary Islands, Spain.

Ticks are vectors of many zoonotic pathogens of clinical relevance, including Anaplasma and Rickettsia species. Since few data about these tick-borne pathogens are available in the Canary Islands, the aim of the present study was to screen their presence in questing and feeding ticks on the island of Tenerife. A total of 81 ticks was removed from six hedgehogs, and eight ticks were collected from the grass. PCR assays for tick species identification based on the LSU rRNA gene, and detection of Anaplasma spp. and Rickettsia spp. by targeting the 16s rRNA and gltA gene, respectively, were carried out. Rhipicephalus sanguineus and Haemaphysalis formosensis tick species were identified, with Rickettsia hoogstraalii detected in H. formosensis. In addition, Rickettsia helvetica and one unidentified Rickettsia species were detected. The overall prevalence of Rickettsia in ticks was 12.2%, and none harbored Anaplasma sp. This work constitutes the first identification in the Canary Islands of pathogenic R. helvetica and the species of unknown pathogenicity R. hoogstraalii in questing ticks. The clinical relevance of the pathogens identified highlights the need for studies with increased sample size and locations, including potential hosts, as well as warning the population about the relevance of ticks as vectors.

Zoonotic Bacteria in Anolis sp., an Invasive Species Introduced to the Canary Islands (Spain).

Lizards belonging to the genus Anolis are native to America and have been introduced in many parts of the world. In this work, a gastrointestinal microbiological analysis from Anolis sp. introduced to Tenerife, Canary Island, was carried out. A total of 74 individuals were analyzed by culture and molecular tools. Pseudomonas spp. was the most prevalent bacteria isolated (64.3%), followed by enteropathogenic Escherichia coli with at least one of the investigated virulent genes (stx1, stx2, and eae) (44.6%). The stx2 gene was more prevalent which differs to that reported in other reptiles, probably due to wastewater transmission. Campylobacter spp. was detected in 32.4% of the animals, highlighting the detection of C. jejuni and C. fetus by their relevance to public health. The zoonotic Staphylococcus lugdunensis, found in 14.9% of the animals, was firstly detected in reptiles. Vibrio sp. which is more associated with aquatic environments was found in 10.8% of the lizards in this study, with Vibrio cholerae being found in two of the animals. The prevalence of Salmonella sp. (5.4%) was low, compared with other studies carried out in reptiles. These results indicate that Anolis sp. in Tenerife could be playing a role in the maintenance and spread of the pathogens detected, being a possible risk factor for public health and biodiversity conservation.

Molecular Detection of Microsporidia in Rabbits (Oryctolagus cuniculus) in Tenerife, Canary Islands, Spain.

Enterocytozoon bieneusi and Encephalitozoon spp. are microsporidia with zoonotic potential that have been identified in humans, as well as in a large group of wild and domestic animals. Several wildlife species have been studied as reservoirs of zoonotic microsporidia in mainland Spain, including the European rabbit (Oryctolagus cuniculus). Due to a lack of data on microsporidia infection in wildlife on the Canary Islands, the aim of this work was to analyze the prevalence and identify the species of microsporidia in rabbits in Tenerife. Between 2015 and 2017, a total of 50 fecal samples were collected from rabbits in eight municipalities of Tenerife, Canary Islands, Spain. Seven of the fifty samples (14%) were amplified using nested polymerase chain reaction (PCR) targeting the partial sequence of the 16S rRNA gene, the internal transcribed spacer (ITS) region, and the partial sequence of the 5.8S rRNA gene. Sanger sequencing reveals the presence of Encephalitozoon cuniculi genotype I in two samples (4%), and undescribed microsporidia species in five samples (10%). This study constitutes the first molecular detection and genotyping of E. cuniculi in rabbits in Spain.

Morphological and Molecular Identification of Anisakis spp. (Nematoda: Anisakidae) in Commercial Fish from the Canary Islands Coast (Spain): Epidemiological Data.

The study aimed to perform the molecular identification of Anisakis larvae in commercial fish from the coast of the Canary Islands and to provide data on their infection level for the host and the species of this nematode parasite that we could find in several species of commercial interest in the Canary Archipelago. Fish specimens (n = 172) from the Canary coasts were examined for parasites. In total, 495 larvae were identified; PCR was carried out for the entire ITS rDNA and cox2 mtDNA region, obtaining sixteen sequences for the entire ITS rDNA region and fifteen for the cox2 mtDNA, this being the first contribution of nucleotide sequences of Anisakis species of fish caught from the Canary Islands. An overall prevalence of 25% was obtained in the fish analyzed, and five species of Anisakis were identified, these being Anisakis simplex (s.s.), Anisakis pegreffii, Anisakis physeteris, Anisakis nascettii and Anisakis typica and the hybrid Anisakis simplex x Anisakis pegreffii. The results obtained in this study have relevance for public health, since the pathology will depend on the species of Anisakis, so it is important to know the health status of fish in the waters of the Canary Islands to assure a safer consumption and take adequate measures, in addition to the provision of epidemiological data.

A Peculiar Distribution of the Emerging Nematode Angiostrongylus cantonensis in the Canary Islands (Spain): Recent Introduction or Isolation Effect?

Angiostrongylus cantonensis is an emerging zoonotic nematode recognized as the leading cause of eosinophilic meningitis in the word. After its discovery in China, it was recorded in 30 countries worldwide. Recently, it has expanded to new areas such as South America and it has been recently found in the Atlantic island of Tenerife (Canary Islands). In order to characterize the distribution of A. cantonensis in the Canary Islands, the lungs of 1462 rodents were sampled in eight islands of the archipelago over 13 years and were then analyzed for A. cantonensis. Remarkably, the parasite was detected only in Tenerife, in Rattus rattus (19.7%) and Rattus norvegicus (7.14%). They were concretely in the northern part of the island, which had a warmer and more humid climate than the south and main cities. The absence of this nematode in other islands with similar environmental conditions could be explained by an isolation effect or by a recent introduction of the parasite in the islands. Besides, the presence in Tenerife of the most invasive lineage of A. cantonensis reinforced the hypothesis of a recent introduction on this island. This study highlights the need to implement control measures to prevent the expansion to other areas in order to avoid the transmission to humans and other animals.

Bartonella species and haplotypes in rodents and their fleas in Lanzarote and El Hierro in the Canary Islands, Spain.

Because isolated ecosystems contribute to species variability, especially oceanic island ecosystems, the present work focused on the study of the Bartonella species and haplotypes in Lanzarote and El Hierro, two Canary islands with evident bioclimatic differences between them. A total of 123 rodents and 110 fleas from two islands were screened for the presence of Bartonella by PCR analysis of the gltA and nuoG genes. The overall prevalence was 5.7% in rodents and 20.4% in fleas. A total of seven gltA-haplotypes was found in both rodents and fleas, belonging to the species Bartonella mastomydis and Bartonella tribocorum in Lanzarote, and to Bartonella rochalimae and Bartonella elizabethae in El Hierro, as well as recently described species Bartonella kosoyi in both islands. Besides, potential co-infections were detected based on the nuoG analysis. Further, Xenopsylla cheopis was the only flea species identified. Our study shows that isolated ecosystems such as the Canary Islands lead to the appearance of new Bartonella haplotypes along different biotopes, with diverse flea species involved in the spreading of the pathogen being of great relevance due to the zoonotic potential of the species found.

Molecular detection of Bartonella henselae, Bartonella clarridgeiae and Rickettsia felis in cat and dog fleas in Tenerife, Canary Islands, Spain.

The cat flea Ctenocephalides felis is the main vector of Bartonella henselae and Bartonella clarridgeiae, the causative agents of cat-scratch disease (CSD) and the spotted-fever agent Rickettsia felis. In spite of their worldwide distribution, there are no data on the occurrence of CSD-causing Bartonella species or the prevalence of Rickettsia species in the Canary Islands, Spain. Therefore, the aim of our study was to screen cat and dog fleas for both pathogens. A total of 128 C. felis from cats and dogs were screened for Bartonella and Rickettsia by PCR. Bartonella henselae (2.3%) and B. clarridgeiae (3.9%) were found in fleas infesting cats, whereas R. felis was identified in both cat (36.6%) and dog (40.7%) fleas. Further, co-infections were observed. This work constitutes the first finding of CSD-causing Bartonella species and the first study on the prevalence of R. felis in fleas from domestic animals in the Canary Islands. These results indicate public health importance, as associated infections could be misdiagnosed in the Archipelago despite their clinical relevance. Establishing human and animal routine diagnosis procedures for these pathogens along with improving vector control in shelters is necessary in order to prevent the spread of the infections among animals.

Evaluating Different Methodologies for Bioprospecting Actinomycetes in Canary Islands Soils.

Actinomycetes are a wide group of Gram positive prokaryotes, the soil being their most characteristic habitat, where they play important ecological functions. Their immense biotechnological potential as producers of bioactive molecules of great commercial and industrial interest is exemplified by most antibiotics for clinical use being derived from this group of bacteria. In this work several methodologies and culture media were tested for the isolation of actinomycetes in soils from three different edafoclimatic areas of Tenerife (Canary Islands, Spain): an arid zone of the southeast coast of the island, a humid area in the laurel forest and the Canary high mountain. The results of this work evidenced that just an air drying of the sample during 7 days (pretreatment) produces high counts of actinomycetes versus non-actinomycetes in the three soils and in any of the culture media studied, making unnecessary post physical and chemical treatments. However, the only method that produced an exclusive isolation of actinomycetes was the use of 0.22-µm filters as a physical barrier, and hence being a tool of unquestionable utility to estimate actinomycetes populations in soil.The analysis by BOX rep-PCR determined that the richness and genetic diversity of the isolates of these localities are very high. The greatest similarities were, with one exception, found between strains from the same locations. Izaña was one that obtained the highest diversity. Regarding the biotecnology potential as antibiotic produces isolates from Güimar soils are the most promising as 7 out of 10 isolates showed some antibiotic activity.

Vertebrate Ticks Distribution and Their Role as Vectors in Relation to Road Edges and Underpasses.

Roads fragment vertebrate populations and affect the dynamics and dispersal patterns of vertebrate parasites. We evaluated how vertebrate ticks distribute near roads and road underpasses in human-caused road-fragmented landscapes in Seville, SW Spain. We sampled 49 stations with 93 individualized sampling points and assessed tick abundance. We explored the relationship between tick presence and abundance and distance to the nearest road and underpass (drainage culverts and other passages used by vertebrates), and landscape features through categorical regression and nonparametric statistics. The presence of the tick-borne pathogens Borrelia sp. and Bartonella sp. was also analyzed by PCR. We found preliminary evidence of high relative tick abundance next to roads and in the vicinity of road underpasses. Plant cover type was related to tick presence in this road context. Implications of road permeability and edge effect in patterns of vertebrate-tick relationships in road fragmentation contexts are discussed. Both Borrelia sp. and Bartonella sp. were detected in the ticks analyzed. This is the first report of these bacteria in ticks from Seville. The results confirm the potential risk of acquiring Lyme disease and bartonellosis in this area.

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), ß-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.

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.

Parasite fauna of rodents (Murinae) from El Hierro (Canary Islands, Spain): a multidisciplinary approach.

The parasite fauna (protozoa, helminths and insects) of the two most widespread Murinae rodents in El Hierro (Canary Islands, Spain), the black rat (Rattus rattus) and the house mouse (Mus musculus domesticus) was studied. Faunistic, ecological, ecotoxicological data, as well as information on the biology of some nematode parasites of R. rattus are provided. The present work is unprecedented in the Canary Islands, and provides the first data on the parasite biodiversity in Murinae from the archipelago. Concerning to parasitofaunas stands out: a) impoverishment of biodiversity of helminths respect of which have the same hosts in other islands; b) increasing the number of species of Siphonaptera, even compared with flea species that parasitize the same hosts from continental biotopes.

Finding of Parastrongylus cantonensis (Chen, 1935) in Rattus rattus in Tenerife, Canary Islands (Spain).

Parastrongylus cantonensis is a parasite of murid rodents that can infect humans and cause health problems as eosinophilic meningitis. Although it is endemic in south Asia, the Pacific islands, Australia, USA, and a few Caribbean islands, it has been extended to new geographical regions. In the Canary Islands (Spain) a survey of helminths of Rattus rattus, Rattus norvegicus and Mus musculus domesticus was carried out. Furthermore, five species of molluscs were examined for nematode larvae to determine whether they are potential intermediate hosts of P. cantonensis. Nematodes were found in the lungs of 15% of 67 R. rattus examined in Tenerife, one of the four studied islands, with a prevalence of 20% in the highest focus of infection. Based on morphological and molecular analysis, with the complete internal transcribed spacer 2 (ITS-2) and a fragment of the small subunit ribosomal RNA (SSU rRNA) nucleotide sequences, nematodes were identified as P. cantonensis. Larval nematodes found from snails and slugs were identified as third-stage (L(3)) Metastrongyloidea, but the molecular study showed that they did not belong to P. cantonensis. This is the first finding of angiostrongyliasis in rats in the Canary Islands (Spain). New molecular data for this species and Parastrongylus dujardini are reported. The presence of P. cantonensis in Tenerife could be of importance from the public health point of view. Further studies are required in order to look for other potential foci of infections in the Canary Islands.

HLA class II alleles in patients with multiple sclerosis in the Biscay province (Basque Country, Spain).

Genetic susceptibility to multiple sclerosis (MS) is associated with genes of the major histocompatibility complex, particularly with the HLA DRB1*1501-DQA1*0102-DQB1*0602 haplotype in Caucasians. To investigate the association of DRB1, DQA1 and DQB1 alleles and haplotypes with MS in Biscay, Basque Country, northern Spain, we examined 197 patients and 200 regionally matched controls. High resolution HLA class II typing was performed by polymerase chain reaction followed by sequence-specific oligonucleotide probe hybridization. Several alleles were overrepresented in MS patients compared with those of controls: DRB1*0402, DRB1*1303, DRB1*1501, DQA1*0102, DQB1*0301, and DQB1*0602. DQB1*0602 was the only potentially predisposing allele for MS that withstood Bonferroni correction and maintained the association in a logistic regression model. On the other hand, several alleles showed lower frequencies in the MS group: DRB1*0101, DQA1*0101, DQB1*0303, and DQB1*0501, but only DRB1*0101 and DQB1*0303 maintained a negative association with the disease in the regression analysis. Three haplotypes were identified as potentially predisposing for MS in our population: DRB1*1501-DQA1*0102-DQB1*0602, DRB1*0402-DQA1*0301-DQB1*0302, and HLA-DRB1*013-DQA1*05-DQB1*0301. Additionally, three haplotypes associated with a lower risk for MS were identified, exhibiting DRB1*0101-DQA1*0101-DQB1*0501 the strongest negative association with MS [12% in controls vs. 3.8% in MS, Pc = 0.00047, OR = 0.290 (95% CI = 0.160­0.528)], and suggesting, therefore, a putative protective role for this haplotype in the population under study.

A new anoplocephalid (Cestoda: Cyclophyllidea) from Gallotia atlantica (Reptilia, Lacertidae) in the Canary Islands, Spain.

A new species of anoplocephalid cestode is described from Gallotia atlantica (Reptilia, Lacertidae) on the Canary Islands, Spain. Oochoristica feliui n. sp. belongs to the group of Oochoristica spp. having circular suckers and fewer than 25 testes in a single cluster: O. lygosomae Burt, 1933; O. lygosomatis Skinker, 1935; O. elongata Dupouy et Kechemir, 1973; O. jonnesi Bursey, McAllister and Freed, 1997; O. junkea Johri, 1950; O. macallisteri Bursey and Goldberg, 1996; O. novaezelandae Schmidt and Allison, 1985; O. parvogenitalis Dupouy and Kechemir, 1973; and O. sobolevi (Spasskii, 1948) Spaskii, 1951. Oochoristica feliui n. sp. differs from these species in a variety of characters such as the number of proglottids, size of the scolex and suckers, presence of a neck, size and shape of the ovary (divided into 5-6 lobules), ovoid shape of the vitelline gland, a spined cirrus, size of eggs, oncosphere and oncosphere hooks, and the presence of 2 osmoregulatory canals. Gallotia spp. are endemic to the Canary Islands. This is the first report of an adult cestode species in this lizard.

Occurrence and genotype characterization of Giardia duodenalis in goat kids from the Canary Islands, Spain.

Giardia duodenalis (syn. Giardia lamblia, Giardia intestinalis) is a wide-spread intestinal protozoa of both humans and animals. Although giardiosis in goat is commonly asymptomatic, young kids may bear an enteric disease associated with persistent diarrhoea and delayed weight gain. In the present study we have analysed the occurrence of Giardia in 315 young goat kids (2-6 months old) from Gran Canaria Island (Spain) through visualization of faecal cysts. The identification of genotypes of G. duodenalis among the farms was attained by nested PCR of the triophosphate isomerase (TPI) and single PCR of beta-giardin genes and subsequent sequencing. Positive samples were found in 42.2% of the animals and 95.5% of the farms. Goat faecal specimens were positive for only livestock-associated G. duodenalis assemblage E genotype for both TPI and beta-giardin genes. The genetic analysis of these two loci revealed the presence of different haplotypes among the farms included in the survey and high homology with homologous genes from cattle and sheep. Altogether, the data presented here provide additional information to the prevalence and genetic characterization of Giardia isolates. The absence of assemblages A and B in this study suggests that zoonotic transmission of Giardia from goats could be of low epidemiological significance, although these findings should be validated in studies including other geographical areas, age groups and larger number of samples.

Clinical efficacy of antiparasite treatments against intestinal helminths and haematic protozoa in Gallotia caesaris (lizards).

Intestinal helminths and blood protozoa are common parasites of lizards. In captivity they can be a serious problem, but no information on treatments is available. In this study, several antiparasitic drugs were studied in Gallotia caesaris (Lacertida) from La Gomera, the Canary Islands, Spain. A lack of efficacy of chloroquine, and a decrease in haemogregarine (Apicomplexa) infection by atovaquone-proguanil was demonstrated. Furthermore, a positive effect of fenbendazole against intestinal nematode infection was observed. In the Canary Islands, Gallotia bravoana and Gallotia simonyi (Lacertida) are included in a captive breading recovery plan. The present results are useful in order to keep these animals in good condition and to control their parasites. Moreover, these results are important for reptiles kept as pets, due to the previous lack of knowledge of parasite management.