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.

Skin mites in mice (Mus musculus): high prevalence of Myobia sp. (Acari, Arachnida) in Robertsonian mice.

Myobia sp. and Demodex sp. are two skin mites that infest mice, particularly immunodeficient or transgenic lab mice. In the present study, wild house mice from five localities from the Barcelona Roberstonian system were analysed in order to detect skin mites and compare their prevalence between standard (2n = 40) and Robertsonian mice (2n > 40). We found and identified skin mites through real-time qPCR by comparing sequences from the mitochondrial 16S rRNA and the nuclear 18S rRNA genes since no sequences are available so far using the mitochondrial gene. Fourteen positive samples were identified as Myobia musculi except for a deletion of 296 bp out to 465 bp sequenced, and one sample was identified as Demodex canis. Sampling one body site, the mite prevalence in standard and Robertsonian mice was 0 and 26%, respectively. The malfunction of the immune system elicits an overgrowth of skin mites and consequently leads to diseases such as canine demodicosis in dogs or rosacea in humans. In immunosuppressed mice, the probability of developing demodicosis is higher than in healthy mice. Since six murine toll-like receptors (TLRs) are located in four chromosomes affected by Robertsonian fusions, we cannot dismiss that differences in mite prevalence could be the consequence of the interruption of TLR function. Although ecological and/or morphological factors cannot be disregarded to explain differences in mite prevalence, the detection of translocation breakpoints in TLR genes or the analysis of TLR gene expression are needed to elucidate how Robertsonian fusions affect the immune system in mice.

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.

Demodicosis in a Free-Ranging Eurasian Brown Bear (Ursus arctos arctos) Cub in the Endangered Cantabrian Population, Spain.

A free-living female Cantabrian brown bear (Ursus arctos arctos) cub severely affected by mange in Asturias (northern Spain) represented the first report of demodicosis for this species. After antimicrobial, antiparasitic, anti-inflammatory, and analgesic therapy it recovered and was released back into the wild to the eastern Cantabrian brown bear subpopulation.

The first case of Demodex gatoi in Austria, detected with fecal flotation.

Feline demodicosis is a rare parasitic condition caused by three different species of mites (Demodex cati, Demodex gatoi, and an unnamed species). D. gatoi inhabits the superficial skin layer (stratum corneum) and is easily transmitted between individual cats. A 2-year-old female spayed Cornish Rex was presented with alopecia and pruritus. The dermatological examination revealed bilateral alopecia and excoriations on trunk, limbs, and belly. The second cat in the household, a 3-year-old female spayed Thai, showed no clinical signs. Superficial and deep skin scrapings were performed and cellophane tapes applied, and living D. gatoi mites could be detected in both cats. Oral ivermectin (0.25 mg/kg every other day) was subscribed. Feces were collected from both cats and fecal flotation with sugar and zinc solutions performed. When compared to skin scrapings and cellophane tapes, D. gatoi was detected more frequently and in higher numbers in fecal samples. Our findings suggest that D. gatoi can be efficiently diagnosed with coproscopy, particularly in asymptomatic carrier animals. DNA was extracted from the flotation liquid, and a PCR protocol for the species verification was designed. A fragment targeting a 325-bp DNA fragment of the D. gatoi mitochondrial 16S rDNA gene was amplified with a 100% similarity to the D. gatoi entry in GenBank® (GI 421920216). We report the first finding of D. gatoi in Austria and propose fecal flotation as a valuable tool for mite detection. Fecal flotation liquid is suitable for DNA extraction and PCR-based species verification of D. gatoi.

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.

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.

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 Darwin's 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.