A spatial ecology study in a high-diversity host community to understand blood-feeding behaviour in Phlebotomus sandfly vectors of Leishmania.

Molecular studies indicate that Phlebotomine sandflies (Diptera: Psychodidae) blood feed on many vertebrate species, of which only a few are proven parasite reservoirs. Investigating sandfly vector feeding preferences is therefore important and requires taking into account the availability and accessibility of host species. In terms of the latter, it is necessary to consider the metabolic cost to the insect of reaching the host and moving on to a suitable breeding site. The present study used statistical modelling to compare the feeding patterns of Phlebotomus perniciosus (n = 150), Phlebotomus papatasi (n = 35) and Phlebotomus ariasi (n = 7) on each of an average of 30 host species in a wildlife park in Murcia, Spain. Sandfly feeding movement costs were estimated as a function of the distance and altitude gradients saved by the insect, assuming that they displayed 'site fidelity'. Most (87%) engorged females were caught <100 m from the host on which they had fed. Although the percentage of bloodmeals was highest on fallow deer (Dama dama) (30%) and red deer (Cervus elaphus) (26%), the predicted feeding probability after considering movement cost was highest for red deer and common eland (Taurotragus oryx), and positively associated with host census. These results suggest that, under similar circumstances, sandflies prefer to feed on some host species more than on others.

Endemic species may have complex histories: within-refugium phylogeography of an endangered Iberian vole.

Glacial refugia protected and promoted biodiversity during the Pleistocene, not only at a broader scale, but also for many endemics that contracted and expanded their ranges within refugial areas. Understanding the evolutionary history of refugial endemics is especially important in the case of endangered species to recognize the origins of their genetic structure and thus produce better informed conservation practices. The Iberian Peninsula is an important European glacial refugium, rich in endemics of conservation concern, including small mammals, such as the Cabrera vole (Microtus cabrerae). This near-threatened rodent is characterized by an unusual suite of genetic, life history and ecological traits, being restricted to isolated geographic nuclei in fast-disappearing Mediterranean subhumid herbaceous habitats. To reconstruct the evolutionary history of the Cabrera vole, we studied sequence variation at mitochondrial, autosomal and sex-linked loci, using invasive and noninvasive samples. Despite low overall mitochondrial and nuclear nucleotide diversities, we observed two main well-supported mitochondrial lineages, west and east. Phylogeographic modelling in the context of the Cabrera vole's detailed fossil record supports a demographic scenario of isolation of two populations during the Last Glacial Maximum from a single focus in the southern part of the Iberian Peninsula. In addition, our data suggest subsequent divergence within the east, and secondary contact and introgression of the expanding western population, during the late Holocene. This work emphasizes that refugial endemics may have a phylogeographic history as rich as that of more widespread species, and conservation of such endemics includes the preservation of that genetic legacy.

First reported case of fatal tuberculosis in a wild African elephant with past human-wildlife contact.

Tuberculosis is emerging/re-emerging in captive elephant populations, where it causes morbidity and deaths, although no case of TB in wild African elephants has been reported. In this paper we report the first case of fatal TB in an African elephant in the wild. The infection with Mycobacterium tuberculosis was confirmed by post-mortem and histological examinations of a female sub-adult elephant aged >12 years that died in Tsavo East National Park, Kenya, while under treatment. This case is unique in that during its lifetime the elephant had contact with both humans and wild elephants. The source of the infection was unclear because the elephant could have acquired the infection in the orphanage or in the wild. However, our results show that wild elephants can maintain human TB in the wild and that the infection can be fatal.

The opportunistic Sarcoptes scabiei: a new episode from giraffe in the drought-suffering Kenya.

The ubiquitous Sarcoptes mite is unexplainable emerging and re-emerging parasite, threatening biodiversity and human health. When a new outbreak occurs, it is not clear if it is a genuine emergence resulting from a new incidence or apparent emergence resulting from increased detection. In this paper we report, for the first time to our knowledge, an outbreak of sarcoptic mange in giraffes in the wild. Three decaying carcasses and five free-ranging subadult reticulated giraffes were observed to have mange-like lesions in the drought-suffering Wajir Region in North Eastern Kenya, while apparently all sympatric wild and domestic animals were mange-free. Affected giraffes were captured and successfully treated. The possible relations between this outbreak and annual seasons, animal age-classes and sex, and spatial distribution are discussed.

Bronchopulmonary nematode infection of Capra pyrenaica in the Sierra Nevada massif, Spain.

The present investigation examined the prevalence and abundance of bronchopulmonary nematodes in 213 randomly hunted Iberian ibexes (Capra pyrenaica) (87 females and 126 males) in the Sierra Nevada mountain range in Spain between 2003 and 2006. Post mortem examination revealed an overall prevalence of 72% for adult nematodes (Cystocaulus ocreatus 44%, Muellerius capillaris 44%, Protostrongylus sp. 40%, and Dictyocaulus filaria 4%). The abundances were 13.45+/-3.97, 5.18+/-2.49, 6.36+/-2.16, and 2.27+/-0.46, respectively. Protostrongylid adults showed similar infection rates, which were statistically different from that of D. filaria. 20% of the examined Iberian ibexes were infected by three protostrongylid nematodes species, 24% of C. pyrenaica were affected by two protostrongylid species, while infestations with only one protostrongylid species were detected in 20% of the examined animals. The overall prevalence of larvae nematodes in the examined animals was 100%, and the overall abundance (number of the first stage larvae per gram) was 86.45+/-20.63. There was a high correlation between the two sets of data (adults and larvae). Results of the present investigation provided foundation for the effective control of bronchopulmonary nematode infection in Iberian ibex.

Sarcoptes mite from collection to DNA extraction: the lost realm of the neglected parasite.

Sarcoptes mite from collection to DNA extraction forms the cornerstone for studies on Sarcoptes scabiei. Whilst the new science era took a shy leap into the different facets of mite studies, the cornerstone was almost entirely neglected. Mite collection, cleaning, storage and DNA extraction were, basically, humble attempts to extrapolate, adapt, modify or 'pirate' those existing methods to the peculiarities of Sarcoptes research. These aspects usually constituted few lines, bashfully mentioned, in the materials and methods section of some papers, which arose in unique problems concerning cost-effectiveness, time profitability, safety and even worse, the credibility of the results, creating contradictory conclusions in some cases. This 'noisy' situation encouraged us to collect, classify and review, for the first time to our knowledge, some aspects relating to studies on Sarcoptes mite from collection to DNA extraction, which will be useful for further studies on Sarcoptes, and have implications for the effective control of the diseases Sarcoptes mite causes. Further studies are needed, especially to compare the profitability, safety, sensibility and specificity of the different methods of this neglected realm of the ubiquitous ectoparasite.

Is ITS-2 rDNA suitable marker for genetic characterization of Sarcoptes mites from different wild animals in different geographic areas?

The present study examined the relationship among individual Sarcoptes scabiei mites from 13 wild mammalian populations belonging to nine species in four European countries using the second internal transcribed spacer (ITS-2) of nuclear ribosomal DNA (rDNA) as genetic marker. The ITS-2 plus primer flanking 5.8S and 28S rDNA (ITS-2+) was amplified from individual mites by polymerase chain reaction (PCR) and the amplicons were sequenced directly. A total of 148 ITS-2+ sequences of 404bp in length were obtained and 67 variable sites were identified (16.59%). UPGMA analyses did not show any geographical or host-specific clustering, and a similar outcome was obtained using population pairwise Fst statistics. These results demonstrated that ITS-2 rDNA does not appear to be suitable for examining genetic diversity among mite populations.

Skin-scale genetic structure of Sarcoptes scabiei populations from individual hosts: empirical evidence from Iberian ibex-derived mites.

The objective of the present study was to examine the extent of genetic diversity among Sarcoptes scabiei individuals belonging to different skin subunits of the body from individual mangy hosts. Ten microsatellite primers were applied on 44 individual S. scabiei mites from three mangy Iberian ibexes from Sierra Nevada Mountain in Spain. Dendrograms of the mites from the individual Iberian ibexes, showing the proportion of shared alleles between pairs of individual mites representing three skin subpopulations (head, back, and abdomen subunits), allowed the clustering of some mite samples up to their skin subunits. This genetic diversity of S. scabiei at skin-scale did not have the same pattern in all considered hosts: for the first Iberian ibex (Cp1), only mites from the head subunit were grouped together; in the second individual (Cp2), the clustering was detected only for mites from the abdomen subunit; and for the third one (Cp3), only mites from the back subunit were clustered together. Our results suggest that the local colonization dynamics of S. scabiei would have influenced the nonrandom distribution of this ectoparasite, after a single infestation. Another presumable explanation to this skin-scale genetic structure could be the repeated infestations. To our knowledge, this is the first documentation of genetic structuring among S. scabiei at individual host skin-scale. Further studies are warranted to highlight determining factors of such trend, but the pattern underlined in the present study should be taken into account in diagnosis and monitoring protocols for studying the population genetic structure and life cycle of this neglected but important ectoparasite.

HotSHOT Plus ThermalSHOCK, a new and efficient technique for preparation of PCR-quality mite genomic DNA.

The present study adapted the HotSHOT method, a technique which has been successfully applied on different kinds of tissues, to studies of Sarcoptes. Some modifications of this technique were made which allowed the quick preparation of PCR-quality Sarcoptes genomic DNA (gDNA), namely applying sodium hydroxide as a substrate for three cycles of thermal shock, followed by a short incubation and pH adjustment with a Tris solution (HotSHOT Plus ThermalSHOCK). The performance of this technique was tested by amplifying a approximately 450-bp rDNA fragment of the second internal transcribed spacer (ITS-2) and by multi-locus genotyping using ten microsatellites on 520 individual Sarcoptes samples. No difference in performance was observed between gDNA samples prepared using the HotSHOT Plus ThermalSHOCK technique and those prepared using a commercial kit utilizing proteinase K digestion. The results demonstrated that the HotSHOT Plus ThermalSHOCK technique is time-saving, economic, and easily automatable for the preparation of PCR-quality mite gDNA, which has implications for studying the molecular biology of mites with human and animal health significance. Although tested in the present study using Sarcoptes mites as a model, this technique may find broad applicability in extraction of gDNA from other parasites with small sizes and hard bodies.

Spotted fever group Rickettsia in brown dog ticks Rhipicephalus sanguineus in southwestern Spain.

A total of 2,229 adults ticks (1,428 males and 801 females) belonging to the brown dog tick, Rhipicephalus sanguineus Latreille, 1806, collected from dogs in Seville province (Andalusia), distributed in 500 lots ranging from one to eight specimens per lot, were examined for the presence of rickettsiae by molecular techniques. Specific rickettsiae DNA were detected in 90 lots (18%) of ticks tested. Sequence analysis of amplicons revealed that R. sanguineus ticks were infected exclusively with Rickettsia massiliae (including the strain Bar-29). The results of this study extend the knowledge of the geographic distribution and prevalence of these spotted fever group (SFG) rickettsiae and indicate that at least two of them, with yet uncertain pathogenicity to humans, are present in brown dog ticks in south western Spain. Although Mediterranean spotted fever (MSF) is an endemic disease in Andalusia, Rickettsia conorii was not found, whereas R. massiliae, recently described as a pathogenic species, was highly prevalent in this area. Our data suggest that in Andalusia a number of MSF or MSF-like cases attributed to R. conorii could have been actually caused by other SFG rickettsia present in R. sanguineus, particularly, R. massiliae.

Oestrid myiasis in European mouflon from Spain.

From February 1992 to March 1997, 245 European mouflon (Ovis orientalis musimon) from Sierras de Cazorla, Segura y Las Villas Natural Park (southern Spain) were surveyed for oestrid larvae in order to estimate prevalence and mean intensity of parasitism by Oestrus ovis. Over 46 percent of the animals surveyed were infected, with a mean intensity of 9.6 larvae/host parasitized. No significant differences in prevalence rates between host sexes were observed, but older mouflons were infected with more larvae than younger ones.

Hematologic parameters of the Spanish ibex (Capra pyrenaica).

Thirteen hematologic parameters were measured in 52 Spanish ibexes (Capra pyrenaica) from Sierra Nevada Natural Park, southern Spain. The animals were captured, maintained in captivity, and physically restrained for blood collection. We compared hematologic values for healthy ibexes with animals infected with sarcoptic mites and animals treated for scabies and recovered. Ibexes with scabies showed a decreased number of erythrocytes, a higher mean corpuscular volume and mean corpuscular hemoglobin, and increased numbers of band neutrophils. The number of lymphocytes was significantly higher in animals that had recovered from scabies as compared with infested ones. The only difference observed between healthy and recovered animals was in the number of monocytes, which remained significantly higher in animals that had recovered from scabies. No hemoparasites were found in this study.

Oestrosis in red deer from Spain.

A survey of naso-pharyngeal myiasis affecting red deer (Cervus elaphus) in southern Spain was conducted. The parasites involved were the larvae of Pharyngomyia picta and Cephenemyia auribarbis (Diptera:Oestridae), which coexist sympatrically within this host. Males and older animals had higher prevalences and intensities of fly larvae. Differences in behaviour and habitat use by male and female deer, and the increase of head size in older males are possibly responsible for this. There were low densities of C. auribarbis while P. picta was the species most frequently observed, although both oestrids were located in the same host cavities. The earlier larviposition by C. auribarbis, and its faster larval development may reflect asynchronous life-cycles of both oestrids; this may decrease inter-specific competition between these sympatric species.

Genotypic identification of an undescribed spotted fever group rickettsia in Ixodes ricinus from southwestern Spain.

An undescribed rickettsia was directly analyzed with specific rickettsial molecular biology tools on Ixodes ricinus L. collected in different localities of the province of Cadiz (southwestern Spain). On the basis of the results of the citrate synthase (glta) gene, 190 kD-outer membrane protein (rOmpA) gene, and 16S ribosomal RNA (16S rRNA) gene partial sequence data, it was found that this rickettsia is sufficiently genetically distinct from other Rickettsia to be considered a distinct taxonomic entity. The isolation and culture of this organism, as well as comparative antigenic analysis, are required to ensure its conclusive taxonomic placement among spotted fever rickettsiae. The epidemiologic role of this new rickettsial agent and its possible pathogenicity to wild and domestic animals or humans is still unknown and needs to be investigated.

Prevalence and seasonality of Oestrus caucasicus Grunin, 1948 (Diptera: Oestridae) parasitizing the Spanish ibex, Capra pyrenaica (Mammalia: Artiodactyla).

Prevalence and intensity of parasitism by oestrid larvae in the Spanish ibex Capra pyrenaica were determined for a period of 2.5 yr in Sierra Nevada Natural Park, southern Spain. The most relevant result of this study was to find Oestrus caucasicus parasitizing this ungulate in a geographical location far from its previously known distribution, e.g., the Caucasus mountain range and central Asia. Larvae were found within 74% of the heads examined (n = 180). Prevalence was higher in females and in older animals. Lowest prevalence occurred during the summer months. The mean intensity (+/- SD) was 25.4 +/- 27.3 larvae and was similar in both host sexes. The number of larvae increased with host age and reached the highest intensities in winter, during December and January.

Pharyngeal bot flies (Oestridae) from sympatric wild cervids in southern Spain.

The prevalence of 2 species of pharyngeal bot flies parasitizing sympatric wild ungulates from southern Spain is given. Pharyngomyia picta was found in all red deer and fallow deer whereas Cephenemyia auribarbis was found in 59% of fallow deer and 81% of red deer examined. The mean number of P. picta larvae was 3-6 times more than that of C. auribarbis larvae. These data suggest that red deer are more heavily parasitized than fallow deer and that they are predominantly infested with P. picta larvae.

Estimation of gene diversity at the b locus of the constant region of the immunoglobulin light chain in natural populations of European rabbit (Oryctolagus cuniculus) in Portugal, Andalusia and on the Azorean Islands.

The minimal gene diversity at a locus of the antibody constant region, as estimated in natural populations of rabbit, revealed levels of heterozygosity similar to those reported for the major histocompatibility complex in human and murine populations. Sera of 416 wild rabbits were collected on the Iberian peninsula and on three islands of the Azorean archipelago and analyzed for the occurrence of the serological markers of the b locus of the immunoglobulin light chain. All four serotypes present in domestic rabbits were found in Portugal. They represented less than 50% of the gene pool. In Andalusia this was less than 15% and on the Azorean islands less than 10%. The pronounced and systematic hierarchy in allele frequencies, previously found in populations from the more recent distribution area of the species, was not observed. On the peninsula, the frequencies of the "domestic" alleles were similar, averaging 10%. The Portuguese sample revealed a total heterozygosity of at least 87%. This high value was supported by at least 11 serologically different alleles, none of them occurring at frequencies above 20%. These data are in agreement with an Iberian origin of the European rabbit and strongly suggest the coalescence of b locus allelic lines drawn from Iberian and western populations. The role of balancing selection in the evolution of the b locus polymorphism was further emphasized.