Astrid Vargas

Worldwide Occurrence of Feline Hemoplasma Infections in Wild Felid Species

ABSTRACT While hemoplasma infections in domestic cats are well studied, almost no information is available on their occurrence in wild felids. The aims of the present study were to investigate wild felid species as possible reservoirs of feline hemoplasmas and the molecular characterization of the hemoplasma isolates. Blood samples from the following 257 wild felids were analyzed: 35 Iberian lynxes from Spain, 36 Eurasian lynxes from Switzerland, 31 European wildcats from France, 45 lions from Tanzania, and 110 Brazilian wild felids, including 12 wild felid species kept in zoos and one free-ranging ocelot. Using real-time PCR, feline hemoplasmas were detected in samples of the following species: Iberian lynx, Eurasian lynx, European wildcat, lion, puma, oncilla, Geoffroys cat, margay, and ocelot. “Candidatus Mycoplasma haemominutum” was the most common feline hemoplasma in Iberian lynxes, Eurasian lynxes, Serengeti lions, and Brazilian wild felids, whereas “Candidatus Mycoplasma turicensis” was the most prevalent in European wildcats; hemoplasma coinfections were frequently observed. Hemoplasma infection was associated with species and free-ranging status of the felids in all animals and with feline leukemia virus provirus-positive status in European wildcats. Phylogenetic analyses of the 16S rRNA and the partial RNase P gene revealed that most hemoplasma isolates exhibit high sequence identities to domestic cat-derived isolates, although some isolates form different subclusters within the phylogenetic tree. In conclusion, 9 out of 15 wild felid species from three different continents were found to be infected with feline hemoplasmas. The effect of feline hemoplasma infections on wild felid populations needs to be further investigated.

Feline leukemia virus and other pathogens as important threats to the survival of the critically endangered Iberian lynx (Lynx pardinus)

Background The Iberian lynx (Lynx pardinus) is considered the most endangered felid species in the world. In order to save this species, the Spanish authorities implemented a captive breeding program recruiting lynxes from the wild. In this context, a retrospective survey on prevalence of selected feline pathogens in free-ranging lynxes was initiated. Methodology/ Principal Findings We systematically analyzed the prevalence and importance of seven viral, one protozoan (Cytauxzoon felis), and several bacterial (e.g., hemotropic mycoplasma) infections in 77 of approximately 200 remaining free-ranging Iberian lynxes of the Doñana and Sierra Morena areas, in Southern Spain, between 2003 and 2007. With the exception of feline immunodeficiency virus (FIV), evidence of infection by all tested feline pathogens was found in Iberian lynxes. Fourteen lynxes were feline leukemia virus (FeLV) provirus-positive; eleven of these were antigenemic (FeLV p27 positive). All 14 animals tested negative for other viral infections. During a six-month period in 2007, six of the provirus-positive antigenemic lynxes died. Infection with FeLV but not with other infectious agents was associated with mortality (p<0.001). Sequencing of the FeLV surface glycoprotein gene revealed a common origin for ten of the eleven samples. The ten sequences were closely related to FeLV-A/61E, originally isolated from cats in the USA. Endogenous FeLV sequences were not detected. Conclusions/Significance It was concluded that the FeLV infection most likely originated from domestic cats invading the lynxs habitats. Data available regarding the time frame, co-infections, and outcome of FeLV-infections suggest that, in contrast to the domestic cat, the FeLV strain affecting the lynxes in 2007 is highly virulent to this species. Our data argue strongly for vaccination of lynxes and domestic cats in and around lynxs habitats in order to prevent further spread of the virus as well as reduction the domestic cat population if the lynx population is to be maintained.

Heterozygosity-Fitness Correlations and Inbreeding Depression in Two Critically Endangered Mammals

The relation among inbreeding, heterozygosity, and fitness has been studied primarily among outbred populations, and little is known about these phenomena in endangered populations. Most researchers conclude that the relation between coefficient of inbreeding estimated from pedigrees and fitness traits (inbreeding-fitness correlations) better reflects inbreeding depression than the relation between marker heterozygosity and fitness traits (heterozygosity-fitness correlations). However, it has been suggested recently that heterozygosity-fitness correlations should only be expected when inbreeding generates extensive identity disequilibrium (correlations in heterozygosity and homozygosity across loci throughout the genome). We tested this hypothesis in Mohor gazelle (Gazella dama mhorr) and Iberian lynx (Lynx pardinus). For Mohor gazelle, we calculated the inbreeding coefficient and measured heterozygosity at 17 microsatellite loci. For Iberian lynx, we measured heterozygosity at 36 microsatellite loci. In both species we estimated semen quality, a phenotypic trait directly related to fitness that is controlled by many loci and is affected by inbreeding depression. Both species showed evidence of extensive identity disequilibrium, and in both species heterozygosity was associated with semen quality. In the Iberian lynx the low proportion of normal sperm associated with low levels of heterozygosity was so extreme that it is likely to limit the fertility of males. In Mohor gazelle, although heterozygosity was associated with semen quality, inbreeding coefficient was not. This result suggests that when coefficient of inbreeding is calculated on the basis of a genealogy that begins after a long history of inbreeding, the coefficient of inbreeding fails to capture previous demographic information because it is a poor estimator of accumulated individual inbreeding. We conclude that among highly endangered species with extensive identity disequilibrium, examination of heterozygosity-fitness correlations may be an effective way to detect inbreeding depression, whereas inbreeding-fitness correlations may be poor indicators of inbreeding depression if the pedigree does not accurately reflect the history of inbreeding.

Developing biological resource banks as a supporting tool for wildlife reproduction and conservation The Iberian lynx bank as a model for other endangered species

This work presents a Biological Resource Bank generated as a complementary supporting tool for the reproduction and the in situ and ex situ conservation of the Iberian lynx. In its design we prioritized the preservation of a maximum of the current genetic and biological diversity of the population, and the harmless collection of the samples. To provide future reproductive opportunities through any possible technique, we processed and cryopreserved germinal cells and tissues from dead animals, 7 males and 6 females, as well as somatic cells and tissues from 69 different individuals. This somatic cell reserve reflects a very important fraction of the population biodiversity which, furthermore, will allow the development of a wide variety of studies that can be easily extrapolated to the majority of the population. We have developed a new non-destructive method to isolate cells with stem-cell-like properties. If considered convenient in the future, and after proper research, such cells could permit therapeutic applications and perhaps be a good source to be used in somatic cell nuclear transfer. Samples of whole blood and its derivatives, hairs, urine and feces from many different individuals were also preserved. Proper storage of such samples is required to allow epidemiological studies to be performed for the testing of different etiological hypotheses or, in general, to develop any bio-sanitary study to improve conservation strategies within the natural habitat. This work describes the main aspects involved in the practical implementation of the Iberian lynx Biological Resource Bank, as a model that could be useful for the development of similar banks for other endangered species.

Importance of canine distemper virus (CDV) infection in free-ranging Iberian lynxes (Lynx pardinus).

Canine distemper virus (CDV) is a morbillivirus that is the etiological agent of one of the most important viral diseases affecting canids and an expanding range of other carnivores. Using real-time RT-PCR, CDV RNA was detected in organs of an Iberian lynx (Lynx pardinus) found dead in the Doñana National Park, Southwestern Andalusia, Spain. This finding may be of great importance for the conservation of the species; at present the Iberian lynx is the most critically endangered wild felid. The aim of the present study was to elucidate the significance of CDV for the Iberian lynx population. High viral loads were evident in the dead lynx, suggesting an etiological involvement of CDV in its death. When carnivores from the same region were analyzed by CDV RT-PCR, a stone marten (Martes foina) was positive. Phylogenetic analyses demonstrated high identity of the two detected CDVs and a close relationship to the European dog lineage of CDV. Antibodies to CDV were detected in 14.8% of 88 tested free-ranging Iberian lynxes. The sample seroprevalence was significantly higher in lynxes from the Doñana Natural Space (22.9%) than Sierra Morena (5%). The stone marten and a red fox (Vulpes vulpes) also tested seropositive. In conclusion, CDV is present in the Iberian lynx population, especially in the Doñana region, with sporadic cases of disease. To reduce the infectious pressure of CDV on this endangered population, a mass dog vaccination should be considered.

Assessment of semen quality, sperm cryopreservation and heterologous IVF in the critically endangered Iberian lynx (Lynx pardinus).

Semen traits and factors affecting sperm cryopreservation were assessed in the Iberian lynx (Lynx pardinus), a species regarded as the most endangered felid in the world. For cryopreservation, semen was washed, resuspended in a Tes-Tris-based diluent (TEST) or a Tris-based diluent (Biladyl), both with 20% egg yolk and 4% glycerol, loaded into straws, cooled to 5 degrees C using an automated programmable system and frozen on nitrogen vapour. Heterologous IVF of in vitro-matured domestic cat oocytes was used to test the fertilising ability of cryopreserved spermatozoa. Electroejaculates from five males were obtained. Characterisation of the electroejaculates revealed mean (+/- s.e.m.) values of 3.3 +/- 0.6 x 10(6) total spermatozoa, 73.6 +/- 4.6% motile spermatozoa, 23.7 +/- 4.0% morphologically normal spermatozoa and 40.7 +/- 2.3% spermatozoa with intact acrosomes. After thawing a higher percentage of motile spermatozoa was seen in TEST than in Biladyl (34.0 +/- 6.2% v. 7.5 +/- 4.8%, respectively; P < 0.05); however, there were no differences in the percentage of intact acrosomes between the two diluents. Iberian lynx spermatozoa fertilised domestic cat oocytes in vitro, with higher fertilisation rates observed for spermatozoa cryopreserved in TEST than in Biladyl, although the difference did not reach statistical significance (20.5 +/- 4.5% v. 11.5 +/- 6.8%, respectively). There were positive significant relations between the fertilisation rates and both the percentage of normal spermatozoa and the percentage of spermatozoa with an intact acrosome before cryopreservation (P = 0.04). This first report of the collection and cryopreservation of Iberian lynx semen and analysis of fertilising ability is an important step in the development of assisted reproductive techniques for this critically endangered felid species.

Membranous glomerulonephritis in the Iberian lynx (Lynx pardinus).

Abstract The Iberian lynx is the most endangered felid species in the world, confined nowadays to two isolated metapopulations in the southwest of Spain, where less than 200 individuals survive. Little is known about the diseases that affect these animals in the wild or in captivity. Kidney samples from necropsies of 27 Iberian lynxes, wild and captive, were examined by histopathology, immunohistochemistry (IgG, IgM, IgA, laminin, type IV collagen, and fibronectin), electron microscopy (n =8) and immunogold labelling for IgM, IgG and IgA in one case, in order to characterize the glomerulopathy prevalent in this species. Urinalyses from records were available for 9 of the necropsied animals and blood and urine samples from 23 free ranging and captive Iberian lynxes were prospectively obtained in order to evaluate the renal function of the living population. A focal, diffuse membranous glomerulonephritis (MGN) that progressed with age was diagnosed in all but one of the animals in different stages not associated to concurrently known infectious diseases. Positive immunoexpression of IgM and IgG was observed in the glomerular capillary basement membranes and intramembranous electron-dense deposits, compatible with immune complexes (ICs) were seen with electron microscopy. The immunogold labelling was also positive for IgM and IgG in the electron-dense areas. The serum biochemistry and urinalyses also revealed signs of mild chronic kidney disease in 16 of the 23 animals evaluated. In conclusion, the membranous glomerulopathy affecting the Iberian lynx is a progressive disease of immune origin. We postulate a possible genetic predisposition towards the disease, enhanced by inbreeding and a possible connection to an immune-mediated systemic disease.

Feline leukemia virus infection: a threat for the survival of the critically endangered Iberian lynx (Lynx pardinus).

Abstract The Iberian lynx (Lynx pardinus) is considered the most endangered felid species in the world. To date, less than 200 animals remain in the wild. Low numbers and genetic uniformity may contribute to render this species particularly susceptible to infectious diseases. Different pathogens have been identified in Iberian lynxes; including several feline bacterial and viral agents. Within a 6-month period starting in December 2006, 12 lynxes living in the northern part of the Doñana area were found to be infected with feline leukemia virus (FeLV). Eleven of these animals were antigenemic, and four of them died in the wild in less than 6 months since the first infected animal had been discovered. The remaining viremic lynxes were captured and allocated to a quarantine center to stop the spread of the infection. An additional three animals died shortly in the quarantine center due to acute anemic disease. Sequencing of the envelope surface unit gene revealed a common origin for the FeLV found in all lynxes. The sequences were closely related to FeLV-A/61E, originally isolated from cats in the USA. Our data demonstrate that, similarly to FeLV, the introduction of a new or particularly pathogenic infection brought into the small population of Iberian lynxes by other wild carnivores or feral cats and dogs roaming in the same habitats have severe consequences. It could result in epidemics that have the potential to eradicate the entire lynx population.

Feline leukemia virus outbreak in the critically endangered Iberian lynx (Lynx pardinus): high-throughput sequencing of envelope variable region A and experimental transmission.

The Iberian lynx is the most endangered felid species. During winter/spring 2006/7, a feline leukemia virus (FeLV) outbreak of unexpected virulence killed about 2/3 of the infected Iberian lynxes. All FeLV-positive animals were co-infected with feline hemoplasmas. To further characterize the Iberian lynx FeLV strain and evaluate its potential virulence, the FeLV envelope gene variable region A (VRA) mutant spectrum was analyzed using the Roche 454 sequencing technology, and an in vivo transmission study of lynx blood to specified-pathogen-free cats was performed. VRA mutations indicated weak apolipoprotein B mRNA editing enzyme and catalytic polypeptide-like cytidine deaminase (APOBEC) restriction of FeLV replication, and variants characteristic of aggressive FeLV strains, such as FeLV-C or FeLV-A/61C, were not detected. Cats exposed to FeLV/Candidatus Mycoplasma haemominutum-positive lynx blood did not show a particularly severe outcome of infection. The results underscore the special susceptibility of Iberian lynxes to infectious diseases.