Natalia Sastre

Signatures of demographic bottlenecks in European wolf populations

Monitoring the loss of genetic diversity in wild populations after a bottleneck event is a priority in conservation and management plans. Here, we used diverse molecular markers to search for signatures of demographic bottlenecks in two wolf populations; an isolated population from the Iberian Peninsula and a non-isolated population from European Russia. Autosomal, mtDNA and Y-chromosomal diversity and the effective population size (Ne) were significantly lower in the Iberian population. Neutrality tests using mtDNA sequences, such as R2, Fu and Li’s F*, Tajima’s D and Fu’s Fs, were positively significant in the Iberian population, suggesting a population decline, but were not significant for the Russian population, likely due to its larger effective population size. However, three tests using autosomal data confirmed the occurrence of the genetic bottleneck in both populations. The M-ratio test was the only one providing significant results for both populations. Given the lack of consistency among the different tests, we recommend using multiple approaches to investigate possible past bottlenecks. The small effective population size (about 50) in the Iberian Peninsula compared to the presumed extant population size could indicate that the bottleneck was more powerful than initially suspected or an overestimation of the current population. The risks associated with small effective population sizes suggest that the genetic change in this population should be closely monitored in the future. On the other hand, the relatively small effective population size for Russian wolves (a few hundred individuals) could indicate some fragmentation, contrary to what is commonly assumed.

Phylogenetic relationships in three species of canine Demodex mite based on partial sequences of mitochondrial 16S rDNA

BACKGROUND The historical classification of Demodex mites has been based on their hosts and morphological features. Genome sequencing has proved to be a very effective taxonomic tool in phylogenetic studies and has been applied in the classification of Demodex. Mitochondrial 16S rDNA has been demonstrated to be an especially useful marker to establish phylogenetic relationships. HYPOTHESIS/OBJECTIVES To amplify and sequence a segment of the mitochondrial 16S rDNA from Demodex canis and Demodex injai, as well as from the short-bodied mite called, unofficially, D. cornei and to determine their genetic proximity. METHODS Demodex mites were examined microscopically and classified as Demodex folliculorum (one sample), D. canis (four samples), D. injai (two samples) or the short-bodied species D. cornei (three samples). DNA was extracted, and a 338 bp fragment of the 16S rDNA was amplified and sequenced. RESULTS The sequences of the four D. canis mites were identical and shared 99.6 and 97.3% identity with two D. canis sequences available at GenBank. The sequences of the D. cornei isolates were identical and showed 97.8, 98.2 and 99.6% identity with the D. canis isolates. The sequences of the two D. injai isolates were also identical and showed 76.6% identity with the D. canis sequence. CONCLUSION Demodex canis and D. injai are two different species, with a genetic distance of 23.3%. It would seem that the short-bodied Demodex mite D. cornei is a morphological variant of D. canis.

Detection of Leishmania infantum in captive wolves from Southwestern Europe

The aim of the present study was to determine the prevalence of Leishmania infantum infection in a wild reservoir host (Canis lupus) throughout an endemic area for the disease (Southern Europe). For that reason, the serum and peripheral blood samples of 33 captive wolves from the European Breeding of Endangered Species Programme (EEP) were analyzed using the enzyme-linked immunosorbent assay (ELISA) and real-time quantitative PCR (qPCR). L. infantum was detected in three samples from Central Portugal and Central and Northern Spain. Even though L. infantum infection in positive samples was low, surveillance of zoonotic leishmaniosis in this population is recommended as the parasite load could be higher in other tissues due to parasite tropism and most of the EEP institutions studied are located in endemic areas for canine leishmaniosis in Europe.

Sex identification of wolf (Canis lupus) using non-invasive samples

We have developed new specific primers for sex determination from forensic samples of wolves (Canis lupus), such as hair, saliva, faecal, tooth and urine samples. In order to improve molecular sexing, we performed a multiplex semi-nested polymerase chain reaction (PCR) and several replicated amplifications per sample to avoid errors in low quantity DNA samples, such as allelic dropout and false alleles. The sex of individuals is automatically determined by capillary electrophoresis with a fluorescently labelled internal sex-specific primer from each pair. Our method yielded sex identification on 100% of invasive samples and 93% of forensic samples, being one of the highest success rates obtained from wild animals.

Survey of infectious agents in the endangered Darwin's fox (Lycalopex fulvipes): High prevalence and diversity of hemotrophic mycoplasmas

Very little is known about the diseases affecting the Darwins fox (Lycalopex fulvipes), which is considered to be one of the most endangered carnivores worldwide. Blood samples of 30 foxes captured on Chiloé Island (Chile) were tested with a battery of PCR assays targeting the following pathogens: Ehrlichia/Anaplasma sp., Rickettsia sp., Bartonella sp., Coxiella burnetti, Borrelia sp., Mycoplasma sp., Babesia sp., Hepatozoon canis, Hepatozoon felis, Leishmania donovani complex, and Filariae. Analysis of the 16S rRNA gene revealed the presence of Mycoplasma spp. in 17 samples (56.7%, 95% Confidence Intervals= 38.2-73.7). Of these, 15 infections were caused by a Mycoplasma belonging to the M. haemofelis/haemocanis (Mhf/Mhc) group, whereas two were caused by a Mycoplasma showing between 89% and 94% identity with different Candidatus Mycoplasma turicensis from felids and rodents hemoplasmas. The analysis of the sequence of the RNA subunit of the RNase P gene of 10 of the foxes positive for Mhf/Mhc showed that eight were infected with M. haemocanis (Mhc), one with a Mycoplasma showing 94% identity with Mhc, and one by M. haemofelis (Mhf). One of the foxes positive for Mhc was infected with a Ricketssia closely related to R. felis. All foxes were negative for the other studied pathogens. Our results are of interest because of the unexpectedly high prevalence of Mycoplasma spp. detected, the variability of species identified, the presence of a potentially new species of hemoplasma, and the first time a hemoplasma considered to be a feline pathogen (Mhf) has been identified in a canid. Though external symptoms were not observed in any of the infected foxes, further clinical and epidemiological studies are necessary to determine the importance of hemoplasma infection in this unique species.

Development of a PCR technique specific for Demodex injai in biological specimens

The identification of Demodex injai as a second Demodex species of dog opened new questions and challenges in the understanding on the Demodex–host relationships. In this paper, we describe the development of a conventional PCR technique based on published genome sequences of D. injai from GenBank that specifically detects DNA from D. injai. This technique amplifies a 238-bp fragment corresponding to a region of the mitochondrial 16S rDNA of D. injai. The PCR was positive in DNA samples obtained from mites identified morphologically as D. injai, which served as positive controls, as well as in samples from three cases of demodicosis associated with proliferation of mites identified as D. injai. Furthermore, the PCR was positive in 2 out of 19 healthy dogs. Samples of Demodex canis and Demodex folliculorum were consistently negative. Skin samples from seven dogs with generalized demodicosis caused by D. canis were all negative in the D. injai-specific PCR, demonstrating that in generalized canine demodicosis, mite proliferation is species-specific. This technique can be a useful tool in the diagnosis and in epidemiologic and pathogenic studies.

Identification of a third feline Demodex species through partial sequencing of the 16S rDNA and frequency of Demodex species in 74 cats using a PCR assay

BACKGROUND Demodex cati and Demodex gatoi are considered the two Demodex species of cats. However, several reports have identified Demodex mites morphologically different from these two species. The differentiation of Demodex mites is usually based on morphology, but within the same species different morphologies can occur. DNA amplification/sequencing has been used effectively to identify and differentiate Demodex mites in humans, dogs and cats. HYPOTHESIS/OBJECTIVES The aim was to develop a PCR technique to identify feline Demodex mites and use this technique to investigate the frequency of Demodex in cats. METHODS Demodex cati, D. gatoi and Demodex mites classified morphologically as the third unnamed feline species were obtained. Hair samples were taken from 74 cats. DNA was extracted; a 330 bp fragment of the 16S rDNA was amplified and sequenced. RESULTS The sequences of D. cati and D. gatoi shared >98% identity with those published on GenBank. The sequence of the third unnamed species showed 98% identity with a recently published feline Demodex sequence and only 75.2 and 70.9% identity with D. gatoi and D. cati sequences, respectively. Demodex DNA was detected in 19 of 74 cats tested; 11 DNA sequences corresponded to Demodex canis, five to Demodex folliculorum, three to D. cati and two to Demodex brevis. CONCLUSIONS AND CLINICAL IMPORTANCE Three Demodex species can be found in cats, because the third unnamed Demodex species is likely to be a distinct species. Apart from D. cati and D. gatoi, DNA from D. canis, D. folliculorum and D. brevis was found on feline skin.

Detection, Prevalence and Phylogenetic Relationships of Demodex spp and further Skin Prostigmata Mites (Acari, Arachnida) in Wild and Domestic Mammals

This study was conceived to detect skin mites in social mammals through real-time qPCR, and to estimate taxonomic Demodex and further Prostigmata mite relationships in different host species by comparing sequences from two genes: mitochondrial 16S rRNA and nuclear 18S rRNA. We determined the mite prevalence in the hair follicles of marmots (13%) and bats (17%). The high prevalence found in marmots and bats by sampling only one site on the body may indicate that mites are common inhabitants of their skin. Since we found three different mites (Neuchelacheles sp, Myobia sp and Penthaleus sp) in three bat species (Miotis yumanensis, Miotis californicus and Corynorhinus townsendii) and two different mites (both inferred to be members of the Prostigmata order) in one marmot species (Marmota flaviventris), we tentatively concluded that these skin mites 1) cannot be assigned to the same genus based only on a common host, and 2) seem to evolve according to the specific habitat and/or specific hair and sebaceous gland of the mammalian host. Moreover, two M. yumanensis bats harbored identical Neuchelacheles mites, indicating the possibility of interspecific cross-infection within a colony. However, some skin mites species are less restricted by host species than previously thought. Specifically, Demodex canis seems to be more transmissible across species than other skin mites. D. canis have been found mostly in dogs but also in cats and captive bats. In addition, we report the first case of D. canis infestation in a domestic ferret (Mustela putorius). All these mammalian hosts are related to human activities, and D. canis evolution may be a consequence of this relationship. The monophyletic Demodex clade showing closely related dog and human Demodex sequences also supports this likely hypothesis.

On the path to extinction: Inbreeding and admixture in a declining grey wolf population

Allee effects reduce the viability of small populations in many different ways, which act synergistically to lead populations towards extinction vortexes. The Sierra Morena wolf population, isolated in the south of the Iberian Peninsula and composed of just one or few packs for decades, represents a good example of how diverse threats act additively in very small populations. We sequenced the genome of one of the last wolves identified (and road‐killed) in Sierra Morena and that of another wolf in the Iberian Wolf Captive Breeding Program and compared them with other wolf and dog genomes from around the world (including two previously published genome sequences from northern Iberian wolves). The results showed relatively low overall genetic diversity in Iberian wolves, but diverse population histories including past introgression of dog genes. The Sierra Morena wolf had an extraordinarily high level of inbreeding and long runs of homozygosity, resulting from the long isolation. In addition, about one‐third of the genome was of dog origin. Despite the introgression of dog genes, heterozygosity remained low because of continued inbreeding after several hybridization events. The results thus illustrate the case of a small and isolated wolf population where the low population density may have favoured hybridization and introgression of dog alleles, but continued inbreeding may have resulted in large chromosomal fragments of wolf origin completely disappearing from the population, and being replaced by chromosomal fragments of dog origin. The latest population surveys suggest that this population may have gone extinct.