SARS-CoV-2 in Captive Nonhuman Primates, Spain, 2020-2023.

We conducted a serologic and molecular study to assess exposure of captive nonhuman primates (NHPs) to SARS-CoV-2 in Spain during the 2020-2023 COVID-19 pandemic. We found limited exposure of NHPs to SARS-CoV-2. Biosafety measures must be strictly maintained to avoid SARS-CoV-2 reverse-zoonotic transmission in the human-NHP interface.

Wild Animals in Captivity: An Analysis of Parasite Biodiversity and Transmission among Animals at Two Zoological Institutions with Different Typologies.

We have conducted a 10-year-long coprological study of the animals housed in two zoological institutions (ZooAquarium and Faunia, Madrid, Spain) to assess the parasite biodiversity, prevalence, and their relation with host class, diet, and enclosure type (soil type and level of isolation from wild fauna). A total of 4476 faecal samples from 132 mammal species and 951 samples from 86 avian species were examined. The results indicated that only 12.8% of avian species had parasites at least once during the study period, whereas 62.1% of mammal species tested positive. Predominantly, protists (Entamoeba, flagellates, and ciliates) and nematodes (mainly Trichuris) were identified in the findings. Carnivorous species were primarily infected by nematodes, while herbivorous and omnivorous species were mainly infected by protists. The number of infected herbivorous and omnivorous species was significantly greater than carnivorous species. Differences were observed based on soil type (artificial, natural, mixed) and isolation level (isolated/accessible), but these differences were not statistically significant. Several parasites (Entamoeba spp., Giardia spp., Balantidoides coli, Trichuris spp.) could potentially be transmitted between humans and some mammals and birds. Regular animal analyses and a personnel health program in the institutions would minimise transmission risks between zoo animals, wildlife, and humans.

Characterisation of cataracts and other ophthalmic findings in various species of captive penguins.

BACKGROUND: The identification of ophthalmic diseases that affect vision and/or cause discomfort should be a priority in captive penguins to improve their overall health and quality of life. METHODS: A routine ophthalmological examination was performed on 80 clinically normal penguins (160 eyes), and any lesions observed were recorded. RESULTS: Ocular lesions were frequent (56% of penguins), with cataracts being the most common (48.8% of penguins). Cortical cataracts (63% of eyes) and posterior subcapsular cataracts (27.4%) were the most commonly occurring. All maturation stages were found; incipient cataracts (52.1% of eyes with cataracts) were predominant, while Morgagnian cataracts (8.2%) were the least frequent. A correlation existed between lenticular changes and increasing age. Uveitis was present in 43.8% of eyes with cataracts, and ectropion uveae was the predominant clinical sign. Other ocular findings included blepharitis (3.8% of all eyes), corneal leukoma (5.6%) and posterior lens subluxation (7.5%). LIMITATIONS: The small number of birds of some species prevented the comparison of ophthalmic findings between species. CONCLUSION: This study corroborates the high prevalence of ocular lesions in captive penguins. Cataracts were frequent and age related. Most cataracts were cortical, and the predominant maturation stage was incipient. Lens-induced uveitis was a common finding. Lowered intraocular pressure was related to cataract formation.

Presence of Aspergillus fumigatus with the TR34/L98H Cyp51A mutation and other azole-resistant aspergilli in the air of a zoological park.

Antifungal-resistant fungi, including Aspergillus fumigatus and other Aspergillus species, pose an urgent threat to human and animal health. Furthermore, the environmental route of azole resistance selection due to the widespread use of azole fungicides in crop protection and other applications is a major public health issue. Although environmental surveillance of fungi is frequently performed in many zoological parks and wildlife rehabilitation centers, the antifungal susceptibility of recovered isolates is only rarely analyzed, which precludes a clear assessment of the threat posed by these fungi to captive animals. In this study, we assessed the presence of airborne azole-resistant Aspergillus spp., including the so-called 'cryptic species' (i.e., species which are phenotypically similar to more well-known aspergilli but clearly constitute different phylogenetic lineages) in a zoological park located in the city of Madrid, Spain. In general, our results revealed a low prevalence A. fumigatus and cryptic aspergilli with decreased susceptibility to azoles. However, we detected an A. fumigatus isolate with the TR34/L98H mutation in the gene encoding the lanosterol 14α-demethylase (Cyp51A), consisting of a tandem repeat of 34 base pairs in the promoter region and a lysine to histidine substitution at codon 98. Notably, this TR34/L98H mutation has been linked to the environmental route of azole resistance selection, thus highlighting the 'One Health' dimension of the emerging problem of antifungal resistance. In this context, continuous environmental surveillance of azole-resistant aspergilli in zoological parks and other similar animal facilities is recommended.

Single or repeated immunization against GnRH fails to completely abolish spermatogenesis in dwarf bucks (Capra hircus).

In both captive wildlife and production animals is important to develop strategies for population control. Immunization against GnRH is an easy and inexpensive immunocastration method that reduces the concentration of testosterone and decreases sperm quality. However, its effectiveness depends on the species and repetition of the treatment. This study aimed to compare the effectiveness of a single treatment (initial immunization plus a booster with Improvac) vs repeated treatment (six doses of Improvac) to inhibit testicular function and maintain the contraceptive status during long periods in bucks. Three Dwarf bucks (Capra hircus) received two doses of Improvac, the first on Week 0, and the booster 4 weeks later (single immunization, group SI) while three Dwarf bucks received one dose of Improvac every 6 months during 3 consecutive years (repeated immunization, group RI). The other three Dwarf bucks remained untreated (control bucks, group CON). Bucks from RI had a greater decrease in scrotal circumference, testosterone concentration, male odor intensity, and sperm quality than SI bucks. However, there were no differences between SI and CON bucks in any of the variables studied. Overall, repeated treatment of Improvac decreased the testicular function of Dwarf bucks, although did not produce complete infertility. However, the repetition of the treatment produced more intensive negative effects, indicating that the strength of the effects of Improvac is rapidly lost in bucks.

Hepatitis E Virus Infections in Free-Ranging and Captive Cetaceans, Spain, 2011-2022.

Epidemiologic surveillance of hepatitis E virus in over 300 free-ranging and captive cetaceans in waters off Spain revealed extensive exposure to this pathogen. We suggest the persistent and widespread presence of hepatitis E in the marine environment off the coast of Spain may be driven by terrestrial sources of contamination.

Monitoring of hepatitis E virus in zoo animals from Spain, 2007-2021.

Hepatitis E virus (HEV, family Hepeviridae) is an important emerging and zoonotic pathogen. In recent decades, the number of human cases of zoonotic hepatitis E has increased considerably in industrialized countries and HEV has been detected in an expanding range of mammal species. Although domestic pigs and wild boar are considered the main reservoirs of zoonotic HEV genotypes, the role of other susceptible animals in the epidemiology of the virus is still poorly understood. A large-scale, long-term study was carried out (1) to assess HEV exposure in captive zoo animals in Spain and (2) to determine the dynamics of seropositivity in individuals that were sampled longitudinally during the study period. Between 2007 and 2021, serum samples from 425 zoo animals belonging to 109 animal species (including artiodactyls, carnivores, perissodactyls, proboscideans and rodents) were collected from 11 different zoological parks in Spain. Forty-six of these animals at seven of these zoos were also longitudinally sampled. Anti-HEV antibodies were detected in 36 (8.5%; 95% CI: 5.8-11.1) of 425 sampled zoo animals. Specific antibodies against HEV-3 and HEV-C1 antigens were confirmed in ELISA-positive animals using western blot assay. Two of 46 longitudinally surveyed animals seroconverted during the study period. Seropositivity was significantly higher in carnivores and perissodactyls than in artiodactyls, and also during the period 2012-2016 compared with 2007-2011. HEV RNA was not detected in any of the 262 animals that could be tested by RT-PCR. To the best of the author's knowledge, this is the first large-scale, long-term surveillance on HEV in different orders of zoo mammals. Our results indicate exposure to HEV-3 and HEV-C1 in zoo animals in Spain and confirm a widespread but not homogeneous spatiotemporal circulation of HEV in captive species in this country. Further studies are required to determine the role of zoo species, particularly carnivores and perissodactyls, in the epidemiology of HEV and to clarify the origins of infection in zoological parks.

Immunocontraception of male and female giraffes using the GnRH vaccine Improvac®.

The aim of this study was to develop protocols for contraception in both sexes of giraffes (Giraffa camelopardalis) by using the GnRH vaccine Improvac®. We evaluated the success of immunization by analyzing fecal reproductive hormone metabolites in female (n = 20) and male (n = 9) giraffes. Endocrine analysis provided the basis for the successful immunization protocol, as well as for assessing long-term effects. Reliable reduction of fecal steroid metabolites to baseline levels in female giraffes was achieved with three, and in males with four or five injections at 4-week intervals. Effective booster injections were administered at 2-month intervals in the first year of treatment and at three to 4-month intervals in the following years. In addition to endocrine analysis, we determined vaccination efficacy in bulls by assessing testicular atrophy. Long-term (>2 years) use in females was often accompanied by prolonged periods of persistent corpus luteum activity, although normal cycles were not observed. Problems might occur with reversibility, because in a few males and females, even after more than 2 years since treatment had been stopped, fecal hormone metabolites have not returned to pretreatment levels. The results are somewhat ambiguous, as reproduction can be suppressed by use of Improvac®, but the question of reversibility remains unsolved.

Monitoring of bluetongue virus in zoo animals in Spain, 2007-2019.

Bluetongue (BT) is an emerging and re-emerging communicable vector-borne disease of animal health concern. A serosurvey was performed to assess exposure to BT virus (BTV) in zoo animals in Spain and to determine the dynamics of seropositivity in longitudinally sampled individuals during the study period. Serum samples were collected from 241 zoo animals belonging to 71 different species in five urban zoos (A-E) in Spain between 2007 and 2019. Twenty-four of these animals were longitudinally surveyed at three of the sampled zoos (zoos B, C and E) during the study period. Anti-BTV antibodies were found in 46 (19.1%; 95% CI: 14.1-24.1) of the 241 captive animals analysed by commercial ELISA. A virus neutralization test confirmed specific antibodies against BTV-1 and BTV-4 in 25 (10.7%; 95% CI: 6.7-14.6) and five (3.0%; 95% CI: 0.3-4.0) animals, respectively. Two of the 24 longitudinally sampled individuals (one African elephant (Loxodanta africana) and one aoudad (Ammotragus lervia)) showed anti-BTV antibodies at all samplings, whereas seroconversions were detected in one mouflon (Ovis aries musimon) in 2016, and one Asian elephant (Elephas maximus) in 2019. To the best of the authors' knowledge, this is the first large-scale survey on BTV conducted in both artiodactyl and non-artiodactyl zoo species worldwide. The results confirm BTV exposure in urban zoo parks in Spain, which could be of animal health and conservation concern. Circulation of BTV was detected in yearling animals in years when there were no reports of BTV outbreaks in livestock. Surveillance in artiodactyl and non-artiodactyl zoo species could be a valuable tool for epidemiological monitoring of BTV.

DNA integrity and viability of testicular cells from diverse wild species after slow freezing or vitrification.

Introduction and objective: Cryopreservation of testicular tissues offers new possibilities to protect endangered species, genetically valuable individuals or even the fertility potential of prepubertal individuals who have died unexpectedly. However, the use of this technique still remains a challenge. In this study, slow freezing and vitrification of testicular tissue was investigated to find out which cryopreservation method could better preserve the viability and DNA integrity of testicular germ cells in diverse wild species. Methods: Testes were obtained post-mortem from 18 artiodactyls (wild boar, roe deer, dwarf goat, mhor gazelle, European mouflon, African forest buffalo, Malayan tapir, dorcas gazelle, Iberian ibex, gnu, red river hog), 5 primates (colobus monkey, capuchin monkey, mandrill), 8 carnivores (gray wolf, Persian leopard, binturong, European mink, American black bear, suricata), and 2 rodents (Patagonian mara). The testicles belonged to adult individuals and were cut into small pieces and cryopreserved by needle immersed vitrification or uncontrolled slow freezing using a passive cooling device. After warming or thawing, testicular tissues were enzymatically digested and two germ cell types were differentiated based on their morphology: rounded cells (spermatogonia, spermatocytes, and early spermatids) and elongated cells (elongated spermatids and spermatozoa). Cell viability was assessed by SYBR-14/propidium iodide while DNA fragmentation by TUNEL assay with fluorescence microscope. Results and discussion: Our preliminary results revealed that our uncontrolled slow freezing method better preserved the viability and DNA integrity of elongated cells than vitrification. Such trend was observed in all species, being significant in artiodactyls, carnivores, and primates. Similarly, the viability and DNA integrity of rounded cells was also better maintained in primates by uncontrolled slow freezing, while in carnivores, vitrification by needle immersion showed better results in this type of cells. In artiodactyls and rodents both techniques preserved the viability of rounded cells in a similar manner, although the DNA integrity of these cells was greater after needle immersed vitrification in artiodactyls. Conclusions: In conclusion, the effectiveness of each cryopreservation method is affected by the phylogenetic diversity between species and cell type.

Caretaker Score Reliability for Personality Assessment of Bottlenose Dolphin (Tursiops truncatus).

The evaluation of zoo animals' personalities can likely lead to a range of benefits, including improving breeding success, creating stable social groups, and designing and developing environmental enrichment programmes. The goal of this study was to use caretakers scores to evaluate personality in bottlenose dolphins and to assess the reliability of scores within each rater and among raters from each centre. To this end, 24 caretakers from 3 countries (Spain, France, and Argentina), including a total of 5 dolphinariums and 6 groups of dolphins, used a questionnaire based on the Five-Factor Model of Personality to score bottlenose dolphins on a number of personality traits in three different contexts. Each caretaker evaluated the animals under their care twice, ensuring that raters did not share thoughts nor impressions with other raters. Our findings showed a good degree of agreement between each rater's scores and a fair degree of agreement among scores of raters from the same centre. We also identified which raters and centres had significant mean score differences and detected that 4 out of 24 raters from two different centres showed such differences systematically. The evaluation of raters' reliability and the identification of particular inconsistent raters and centres is critical to make more appropriate and realistic management decisions that, in turn, directly impact animals' welfare.

Sperm Cryopreservation in American Flamingo (Phoenicopterus Ruber): Influence of Cryoprotectants and Seminal Plasma Removal.

The American flamingo is a useful model for the development of successful semen cryopreservation procedures to be applied to threatened related species from the family Phoenicopteridae, and to permit genetic material banking. Current study sought to develop effective sperm cryopreservation protocols through examining the influences of two permeating cryoprotectants and the seminal plasma removal. During two consecutive years (April), semen samples were collected and frozen from American flamingos. In the first year, the effect of two permeating cryoprotectants, DMA (dimethylacetamide) (6%) or Me2SO (dimethylsulphoxide) (8%), on frozen-thawed sperm variables were compared in 21 males. No differences were seen between DMA and Me2SO for sperm motility, sperm viability, and DNA fragmentation after thawing. In the second year, the role of seminal plasma on sperm cryoresistance was investigated in 31 flamingos. Sperm samples were cryopreserved with and without seminal plasma, using Me2SO (8%) as a cryoprotectant. The results showed that samples with seminal plasma had higher values than samples without seminal plasma for the following sperm variables: Straight line velocity (22.40 µm/s vs. 16.64 µm/s), wobble (75.83% vs. 69.40%), (p < 0.05), linearity (62.73% vs. 52.01%) and straightness (82.38% vs. 73.79%) (p < 0.01); but acrosome integrity was lower (55.56% vs. 66.88%) (p < 0.05). The cryoresistance ratio (CR) was greater in samples frozen with seminal plasma than without seminal plasma for CR-progressive motility (138.72 vs. 54.59), CR-curvilinear velocity (105.98 vs. 89.32), CR-straight line velocity (152.77 vs. 112.58), CR-average path velocity (122.48 vs. 98.12), CR-wobble (111.75 vs. 102.04) (p < 0.05), CR-linearity (139.41 vs. 113.18), and CR-straightness (124.02 vs. 109.97) (p < 0.01). This research demonstrated that there were not differences between Me2SO and DMA to successful freezing sperm of flamingos; seminal plasma removal did not provide a benefit for sperm cryopreservation.

Intestinal Protists in Captive Non-human Primates and Their Handlers in Six European Zoological Gardens. Molecular Evidence of Zoonotic Transmission.

We assessed the occurrence, genetic diversity, and zoonotic potential of four protozoan (Cryptosporidium spp., Entamoeba histolytica, Entamoeba dispar, Giardia duodenalis), one stramenopile (Blastocystis sp.), one microsporidia (Enterocytozoon bieneusi), and two ciliate (Balantioides coli, Troglodytella abrassarti) intestinal parasite or commensal protist species in captive non-human primates (NHP) and their zookeepers from six European zoological gardens in France (n = 1), Germany (n = 1), and Spain (n = 4). Faecal samples from NHP (n = 454) belonging to 63 species within 35 genera and humans (n = 70) were collected at two sampling periods in each participating institution between October 2018-August 2021. Detection and species identification was accomplished by PCR and Sanger sequencing of the ssu rRNA and/or ITS genes. Sub-genotyping analyses using specific markers were conducted on isolates positive for G. duodenalis (gdh, bg, tpi) and Cryptosporidium spp. (gp60). Overall, 41.0% (186/454) and 30.0% (21/70) of the faecal samples of NHP and human origin tested positive for at least one intestinal protist species, respectively. In NHP, Blastocystis sp. was the most prevalent protist species found (20.3%), followed by G. duodenalis (18.1%), E. dispar (7.9%), B. coli and T. abrassarti (1.5% each), and Cryptosporidium spp. and E. bieneusi (0.9% each). Occurrence rates varied largely among NHP host species, sampling periods, and zoological institutions. The predominant protist species found in humans was Blastocystis sp. (25.7%), followed by Cryptosporidium spp. (2.9%), E. dispar (1.4%), and G. duodenalis (1.4%). Sequencing of PCR-positive amplicons in human and/or NHP confirmed the presence of Cryptosporidium in six isolates (C. hominis: 66.7%, C. parvum: 33.3%), G. duodenalis in 18 isolates (assemblage A: 16.7%, assemblage B: 83.3%), Blastocystis in 110 isolates (ST1:38.2%, ST2:11.8%, ST3: 18.2%, ST4: 9.1%, ST5: 17.3%, ST8: 2.7%, ST13: 0.9%), and E. bieneusi in four isolates (CM18: 75.0%, Type IV: 25.0%). Zoonotic transmission events involving Blastocystis ST1-ST4 were identified in four zoological institutions. Zoonotic transmission of C. hominis was highly suspected, but not fully demonstrated, in one of them. Monitoring of intestinal protist species might be useful for assessing health status of captive NHP and their zookeepers, and to identify transmission pathways of faecal-orally transmitted pathogens.

Cryopreservation of ferret (Mustela putorius furo) sperm collected by rectal massage and electroejaculation: Comparison of a decelerating and an accelerating freezing rate protocol.

The domestic ferret (Mustela putorius furo) provides a good model for developing new reproductive technologies for use with threatened related species. Such technologies could also be used in the reproductive management of this pet species. The present work reports an improved freezing protocol for ferret sperm. Semen was collected by electroejaculation plus rectal massage (in an attempt to reduce the electrical stimulation necessary) from five adult male ferrets, and then subjected to one of two freezing protocols: (a) from 5 to -35°C at 40°C/min, then from -35 to -65°C at 17°C/min, and finally from -65 to -85°C at 3°C/min-a decelerating freezing rate; and (b) from 5 to - 10°C at 5°C/min, and then from -10 to -130°C at 60°C/min-an accelerating freezing rate. After thawing, the viability and acrosomal integrity of the sperm frozen via the two-step accelerating method were better than those frozen via the three-step decelerating method (43.3 ± 3.5% and 71.2 ± 3.4% compared with 29.7 ± 3.7% and 58.8 ± 3.4% respectively; p < .05). No differences were seen between the methods with respect to sperm motility variables; most sperm (>90%) remained static with both freezing methods. In conclusion, although the method with accelerating freezing rate was associated with better post-thaw sperm viability and acrosome integrity values, neither of the two freezing methods tested provided adequate motility results after thawing. Combining rectal massage with electrical stimuli seemed to reduce the number of the latter required for successful sperm collection.

Slower rates of accumulation of DNA damage in leukocytes correlate with longer lifespans across several species of birds and mammals.

Although there is previous evidence showing an increase in various types of DNA damage with aging in mice and humans, a comparative study determining accumulation rates of DNA double strand breaks, as determined by presence of phosphorylated histone H2AX (γH2AX), in leukocytes of individuals of different ages from phylogenetically distinct species from birds to mammals was lacking. Here, we demonstrate that the rate of accumulation of DNA damage as measured by the DNA damage marker γH2AX correlates with species longevity in dolphins, goats, reindeer, American flamingos, and griffon vultures. In particular, we find that species that show slower rates of accumulation of the DNA damage marker γH2AX also live longer.

Telomere shortening rate predicts species life span.

Telomere shortening to a critical length can trigger aging and shorter life spans in mice and humans by a mechanism that involves induction of a persistent DNA damage response at chromosome ends and loss of cellular viability. However, whether telomere length is a universal determinant of species longevity is not known. To determine whether telomere shortening can be a single parameter to predict species longevities, here we measured in parallel the telomere length of a wide variety of species (birds and mammals) with very different life spans and body sizes, including mouse (Mus musculus), goat (Capra hircus), Audouin's gull (Larus audouinii), reindeer (Rangifer tarandus), griffon vulture (Gyps fulvus), bottlenose dolphin (Tursiops truncatus), American flamingo (Phoenicopterus ruber), and Sumatran elephant (Elephas maximus sumatranus). We found that the telomere shortening rate, but not the initial telomere length alone, is a powerful predictor of species life span. These results support the notion that critical telomere shortening and the consequent onset of telomeric DNA damage and cellular senescence are a general determinant of species life span.

Potentially human-virulent Vibrio vulnificus isolates from diseased great pompano (Trachinotus goodei).

Vibrio vulnificus is an opportunistic human pathogen responsible for the majority of seafood-associated deaths worldwide and is also a relevant fish pathogen for the aquaculture industry. In addition to infections in aquatic livestock, V. vulnificus also represents a risk to aquarium animals. For the first time, this work describes an important mortality outbreak in Trachinotus goodei in a zoo aquarium, with the isolation of Vibrio vulnificus (Vv) from the internal organs of the diseased fish. The isolates were identified by MALDI-TOF MS, serotyped and characterized by pulsed-field gel electrophoresis (PFGE). Although the isolates from great pompanos did not belong to pathovar piscis (formerly biotype 2) or to any of the fish-related serovars, they all had identical phenotypes, antimicrobial susceptibility profiles and PFGE patterns, which together with their isolation in pure culture from internal organs is strongly indicative of their clinical significance. Moreover, Vv isolates harboured important genetic markers of human virulence potential: they had the clinical variant of the vcg gene, gave the 338 bp DNA amplification product of the pilF gene and resisted the bactericidal activity of human serum. All these results strongly suggest that these Vv isolates should be considered potentially virulent for humans. These results extend the range of fish species affected by V. vulnificus, confirm the threat that this pathogen represents to aquatic animals and highlight the risk that this bacterial pathogen poses to human health.

Using Farm Animal Welfare Protocols as a Base to Assess the Welfare of Wild Animals in Captivity-Case Study: Dorcas Gazelles (Gazella dorcas).

There is a lack of protocols specifically developed for the assessment of welfare of wild animals in captivity, even when it is known that providing good standards of welfare is important. The aim of this study was the development and the application of a protocol for the assessment of welfare in captive dorcas gazelles. The protocol was mainly developed taking into account the protocol for the assessment of welfare in cattle from the Welfare Quality® project, the available literature of the biology of this species and the Husbandry Guidelines developed for captive breeding and management of this species. The protocol was specifically developed for dorcas gazelles and included four principles, 10 criteria and 23 animal and environmental-based indicators. To test its utility, this protocol was applied to five different groups of gazelles from three different zoos. Its application made possible to detect areas for improvement in all groups assessed.

First report of Leishmania infantum infection in the endangered orangutan (Pongo pygmaeus pygmaeus) in Madrid, Spain.

BACKGROUND: Some wild animals have been recognized as potential reservoirs of Leishmania infantum infection (e.g. carnivores, lagomorphs, rodents, etc.). Leishmania infantum was also identified infecting humans and lagomorphs (i.e. hares and rabbits) over the period of 2009-2016, with the latter acting as the main reservoirs involved in the human leishmaniosis outbreak in Madrid. RESULTS: Two cases of clinical leishmaniosis are reported in orangutans (Pongo pygmaeus pygmaeus) housed at two different centres in Madrid. The first is the case of a 36-year-old male orangutan with severe weight loss and apathy. A complete blood count and biochemical profile revealed anaemia, neutropenia, hypoalbuminaemia and elevated transaminases. Hepato-splenomegaly was also observed. Four months later, due to worsening of clinical signs (mainly bilateral epistaxis), blood and bone marrow samples were collected. Amastigotes of L. infantum were detected in macrophages from a bone marrow aspirate and by specific polymerase chain reaction. The second case was a 34-year-old female orangutan with severe weight loss and apathy and no other apparent clinical signs. A complete blood count and biochemical profile revealed anaemia, pancytopenia and hypoalbuminaemia. Splenomegaly and pericardial effusion were also observed. As leishmaniosis was included in the differential diagnosis, both blood and bone marrow samples were collected. Leishmania infantum infection was confirmed by microscopy, molecular diagnosis and serology (immunofluorescence antibody test). Both animals were treated daily with oral miltefosine for 28 days; allopurinol was also given uninterruptedly in Case 2 for at least 6 months. During follow-up, though good clinical recovery was clear, a lack of parasitological cure was confirmed molecularly in both blood and bone marrow samples from the two orangutans. In both habitats, the presence of the sand fly vector identified as Phlebotomus perniciosus was confirmed. CONCLUSIONS: To our knowledge, this is the first report of L. infantum infection in great apes and in the endangered species P. p. pygmaeus. We are presently looking for L. infantum in other non-human primates living in the same peri-urban areas. If detected, we will examine the impacts of this serious disease on these critically endangered species.

Composition of marsupial zona pellucida: a molecular and phylogenetic approach.

The zona pellucida (ZP) is an extracellular matrix that surrounds mammalian oocytes. In eutherians it is formed from three or four proteins (ZP1, ZP2, ZP3, ZP4). In the few marsupials that have been studied, however, only three of these have been characterised (ZP2, ZP3, ZP4). Nevertheless, the composition in marsupials may be more complex, since a duplication of the ZP3 gene was recently described in one species. The aim of this work was to elucidate the ZP composition in marsupials and relate it to the evolution of the ZP gene family. For that, an in silico and molecular analysis was undertaken, focusing on two South American species (gray short-tailed opossum and common opossum) and five Australian species (brushtail possum, koala, Bennett's wallaby, Tammar wallaby and Tasmanian devil). This analysis identified the presence of ZP1 mRNA and mRNA from two or three paralogues of ZP3 in marsupials. Furthermore, evidence for ZP1 and ZP4 pseudogenes in the South American subfamily Didelphinae and for ZP3 pseudogenes in two marsupials is provided. In conclusion, two different composition models are proposed for marsupials: a model with four proteins (ZP1, ZP2 and ZP3 (two copies)) for the South American species and a model with six proteins (ZP1, ZP2, ZP3 (three copies) and ZP4) for the Australasian species.