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Cryptococcosis in ferrets from Spain and Portugal: the veterinarians' role
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Cryptococcosis is a fungal disease affecting more than one million people per year worldwide. The main etiological agents of cryptococcosis are the two sibling species Cryptococcus neoformans and Cryptococcus gattii that present numerous differences in geographical distribution, ecological niches, epidemiology, pathobiology, clinical presentation and molecular characters. Genotyping of the two Cryptococcus species at subspecies level supplies relevant information to understand how this fungus has spread worldwide, the nature of its population structure, and how it evolved to be a deadly pathogen. At present, nine major molecular types have been recognized: VNI, VNII, VNB, VNIII, and VNIV among C. neoformans isolates, and VGI, VGII, VGIII, and VGIV among C. gattii isolates. In this paper all the information available in the literature concerning the isolation of the two Cryptococcus species has been collected and analyzed on the basis of their geographical origin, source of isolation, level of identification, species, and molecular type. A detailed analysis of the geographical distribution of the major molecular types in each continent has been described and represented on thematic maps. This study represents a useful tool to start new epidemiological surveys on the basis of the present knowledge.
This study provides a comprehensive picture of the C. neoformans/C. gattii molecular types most often associated with human cryptococcosis in Portugal and assesses the impact of C. gattii in these infections. One hundred and twenty-two clinical isolates, from distinct patients, were identified as C. neoformans and genotyped by URA5-RFLP, with the molecular types VNI (45.5 %) and VNIII (30.9 %) being the most commonly found ones. The molecular types VNII (11.4 %) and VNIV (11.4 %) were less abundant. One patient was found to be infected with a VGII isolate. This patient exhibited unusual clinical symptoms of cryptococcosis, reinforcing the suspicion for the presence of a different genotypic pattern, as determined afterwards. This case was detected in 2007 and is the first report of a potential autochthonous C. gattii infection case in Portugal, as the patient revealed no historical record of travelling outside the country.
Over the past two decades, several fungal outbreaks have occurred, including the high-profile 'Vancouver Island' and 'Pacific Northwest' outbreaks, caused by Cryptococcus gattii, which has affected hundreds of otherwise healthy humans and animals. Over the same time period, C. gattii was the cause of several additional case clusters at localities outside of the tropical and subtropical climate zones where the species normally occurs. In every case, the causative agent belongs to a previously rare genotype of C. gattii called AFLP6/VGII, but the origin of the outbreak clades remains enigmatic. Here we used phylogenetic and recombination analyses, based on AFLP and multiple MLST datasets, and coalescence gene genealogy to demonstrate that these outbreaks have arisen from a highly-recombining C. gattii population in the native rainforest of Northern Brazil. Thus the modern virulent C. gattii AFLP6/VGII outbreak lineages derived from mating events in South America and then dispersed to temperate regions where they cause serious infections in humans and animals.
This communication describes the consensus multi-locus typing scheme established by the Cryptococcal Working Group I (Genotyping of Cryptococcus neoformans and C. gattii) of the International Society for Human and Animal Mycology (ISHAM) using seven unlinked genetic loci for global strain genotyping. These genetic loci include the housekeeping genes CAP59,GPD1, LAC1, PLB1, SOD1, URA5 and the IGS1 region. Allele and sequence type information are accessible at http://www.mlst.net/.
Until recently, Cryptococcus gattii infections occurred mainly in tropical and subtropical climate zones. However, during the past decade, C. gattii infections in humans and animals in Europe have increased. To determine whether the infections in Europe were acquired from an autochthonous source or associated with travel, we used multilocus sequence typing to compare 100 isolates from Europe (57 from 40 human patients, 22 from the environment, and 21 from animals) with 191 isolates from around the world. Of the 57 human patient isolates, 47 (83%) were obtained since 1995. Among the 40 patients, 24 (60%) probably acquired the C. gattii infection outside Europe; the remaining 16 (40%) probably acquired the infection within Europe. Human patient isolates from Mediterranean Europe clustered into a distinct genotype with animal and environmental isolates. These results indicate that reactivation of dormant C. gattii infections can occur many years after the infectious agent was acquired elsewhere.
A domestic ferret (Mustela putorius furo) was presented with lymphadenopathy and acute bilateral blindness. Cytologic evaluation and biopsy of an affected lymph node
revealed pyogranulomatous lymphadenitis with intralesional yeast consistent with Cryptococcus sp. Subsequent studies demonstrated Cryptococcus gattii serotype B VGI/AFLP4 as the causative agent. The ferret was treated with fluconazole and prednisone. After one month of therapy,
an improvement of the clinical symptoms was detected although blindness persisted. Seven months after presentation, the disease
progressed to a severe neurologic condition, and it was euthanized. Postmortem exam revealed disseminated cryptococcosis with
prominent neurologic involvement. Nasal swabs of other ferrets and humans from the same household revealed that two ferrets
and two humans to be asymptomatic carriers of the same strain of cryptococcus as the necropsied ferret. These findings stress
the importance of veterinary diagnostic work with pets and epidemiological investigations for disease prevention in them and
in their owners.
The in vitro susceptibilities of a worldwide collection of 350 Cryptococcus gattii isolates to seven antifungal drugs, including the new triazole isavuconazole, were tested. With amplified fragment length polymorphism (AFLP) fingerprinting, human, veterinary, and environmental C. gattii isolates were subdivided into seven AFLP genotypes, including the interspecies hybrids AFLP8 and AFLP9. The majority of clinical isolates (n = 215) comprised genotypes AFLP4 (n = 76) and AFLP6 (n = 103). The clinical AFLP6 isolates had significantly higher geometric mean MICs for flucytosine and fluconazole than the clinical AFLP4 isolates. Of the seven antifungal compounds examined in this study, isavuconazole had the lowest MIC(90) (0.125 μg/ml) for all C. gattii isolates, followed by a 1 log(2) dilution step increase (MIC(90), 0.25 μg/ml) for itraconazole, voriconazole, and posaconazole. Amphotericin B had an acceptable MIC(90) of 0.5 μg/ml, but fluconazole and flucytosine had relatively high MIC(90)s of 8 μg/ml.
Cryptococcus gattii and C. neoformans cause pulmonary and systemic cryptococcosis. Recently, C. gattii was recognized as a distinct pathogen of humans and animals. We analyzed information from 400 publications (1948-2008) to examine whether the fungus occurs globally. Known distribution of C. gattii is possibly limited because specialized reagents for differentiation from C. neoformans are not readily available and not always used, and environmental surveys are patchy. However, autochthonous reports of C. gattii cryptococcosis have now been recognized from tropical and temperate regions. An ongoing outbreak in western Canada strengthens the case that the range of the pathogen has expanded. A few studies have highlighted differences in cryptococcosis between C. gattii and C. neoformans. More than 50 tree species have yielded C. gattii especially from decayed hollows suggesting a possible ecologic niche. This pathogen merits more attention so its environmental occurrence and role in cryptococcosis can be accurately determined.
Cryptococcus neoformans and Cryptococcus gattii are closely related pathogenic fungi that cause pneumonia and meningitis in both immunocompromised and immunocompetent hosts and are a significant global infectious disease risk. Both species are found in the environment and are acquired via inhalation, leading to an initial pulmonary infection. The infectious propagule is unknown but is hypothesized to be small desiccated yeast cells or spores produced by sexual reproduction (opposite- or same-sex mating). Here we characterize the morphology, germination properties, and virulence of spores. A comparative morphological analysis of hyphae and spores produced by opposite-sex mating, same-sex mating, and self-fertile diploid strains was conducted by scanning electron microscopy, yielding insight into hyphal/basidial morphology and spore size, structure, and surface properties. Spores isolated by microdissection were found to readily germinate even on water agarose medium. Thus, nutritional signals do not appear to be required to stimulate spore germination, and as-yet-unknown environmental factors may normally constrain germination in nature. As few as 500 CFU of a spore-enriched infectious inoculum (approximately 95% spores) of serotype A C. neoformans var. grubii were fully virulent (100% lethal infection) in both a murine inhalation virulence model and the invertebrate model host Galleria mellonella. In contrast to a previous report on C. neoformans var. neoformans, spores of C. neoformans var. grubii were not more infectious than yeast cells. Molecular analysis of isolates recovered from tissues of infected mice (lung, spleen, and brain) provides evidence for infection and dissemination by recombinant spore products. These studies provide a detailed morphological and physiological analysis of the spore and document that spores can serve as infectious propagules.
A domestic ferret was presented for episodic regurgitation. Cytologic examination and culture of an enlarged submandibular lymph node revealed Cryptococcus neoformans variety grubii (serotype A). The ferret was successfully treated with itraconazole. This is the first documented case of Cryptococcus neoformans variety grubii in a ferret in the United States.
From 2002 to 2007, 23 ferrets from Europe and the United States were diagnosed with systemic pyogranulomatous inflammation resembling feline infectious peritonitis (FIP). The average age at the time of diagnosis was 11 months. The disease was progressive in all cases, and average duration of clinical illness was 67 days. Common clinical findings were anorexia, weight loss, diarrhea, and large, palpable intra-abdominal masses; less frequent findings included hind limb paresis, central nervous system signs, vomiting, and dyspnea. Frequent hematologic findings were mild anemia, thrombocytopenia, and hypergammaglobulinemia. Grossly, whitish nodules were found in numerous tissues, most frequently the mesenteric adipose tissue and lymph nodes, visceral peritoneum, liver, kidneys, spleen, and lungs. One ferret had a serous abdominal effusion. Microscopically, pyogranulomatous inflammation involved especially the visceral peritoneum, mesenteric adipose tissue, liver, lungs, kidneys, lymph nodes, spleen, pancreas, adrenal glands, and/or blood vessels. Immunohistochemically, all cases were positive for coronavirus antigen using monoclonal antibody FIPV3-70. Electron microscopic examination of inflammatory lesions identified particles with coronavirus morphology in the cytoplasm of macrophages. Partial sequencing of the coronavirus spike gene obtained from frozen tissue indicates that the virus is related to ferret enteric coronavirus.
Resumo: Com o objectivo de caracterizar o potencial nosoló-gico das contaminações dos espaços públicos de Lisboa por Cryptococcus spp, procedeu-se à pesquisa destas leveduras em 148 amostras colhidas de plantas ornamentais (folhas, flores e pólen) e de fezes de pombos, em parques, jardins e praças públicas. De cada amostra prepararam-se e inocula-ram-se alíquotas numa gelose específica (Staib) e incubaram-se a 30 ºC, durante cinco dias. Detectaram-se quatro espécies de Cryptococcus nos diferentes tipos de amostra. Os teores de Cryptococcus laurentii variaram entre 10 2 a 10 6 células/g em: flores de eucaliptos (50% positivos). As amostras de acácias (flores), tília (flores), jacarandá (flores) e pinheiro (pólen) fo-ram 100% positivas; Nas fezes de pombo, aquela levedura foi encontrada em 40 % das amostras. C. neoformans foi encon-trado em 10 das 22 amostras de eucaliptos e em 24 das amos-tras fecais de pombos (24,0%); os respectivos teores oscilaram entre 10 2 e 10 6 células/g. C. albidus e C. hungaricus também foram encontrados com elevada frequência em todas as amostras, mas os respectivos teores foram sempre inferiores. Este conjunto de resultados confirma a relevância de alguns elementos ambientais como fontes de contágio por Crypto-cocci, agentes cuja patogenicidade assume especial importân-cia nos animais e nos humanos imunocomprometidos. Summary: The aim of this work was to evaluate the potential hazard for Human and Animal health of of Cryptococcus spp. as contaminant of ornamental trees and pigeons faecal mate-rials on the public areas of Lisbon. These yeasts were research in 148 samples of different materials coming from leaves, flowers, pollen of decorative trees and from pigeons faeces. From each sample an aliquot was prepared and cultured on a specific media (Staib Agar), for 5 days, at 30 ºC. Four diffe-rent Cryptococcus species were detected on all kind of samples. The levels of contamination with C. laurentii, ranged from 10 2 to 10 6 cells/g. Eucalyptus-trees flowers, revealing 50% positive; All samples of Acacia-trees flowers, Jacaranda flowers (8), Lindens flowers(3) and in pinus pollen (2) were contaminated with this yeast (100%), at levels that ranged from 10 2 to 10 5 cells/g. In pigeons faeces this Cryptococcus was found in 40% of the samples. C. neoformans was found in 24% of the pigeons faeces samples (100), with levels ranging from 10 2 to 10 6 cells/g. This agent has been also found on 10 samples of eucalyptus flowers. C. albidus and C. hungaricus were also regularly found but at inferior levels. These results confirm the importance of many urban environ-mental elements, like flowers of ornamental trees, its pollens and pigeons faeces, as Cryptococci sources.
Since their introduction as pets several decades ago, ferrets have become an increasingly popular household pet. Great strides have been made in improving their diet and understanding common diseases (eg, insulinoma, hyperadrenocorticism, lymphoma) that affect them. With the frequency with which these conditions are seen, it sometimes is easy to forget that ferrets can be affected by other diseases. Some of these diseases, such as cryptococcosis, are known, but may be increasing in incidence and range, whereas others, such as hypothyroidism and pure red cell aplasia, may be underrecognized or underreported. This review highlights new and emerging diseases not already well reviewed in the literature.
Recent Cryptococcus gattii infections in humans and animals, including several outbreaks in goats, were the basis of this environmental survey in six provinces of Spain. A total of 479 samples from 20 tree species were studied. Cryptococcus gattii was found for the first time in autochthonous Mediterranean trees in Spain. Fourteen isolates of this pathogen were obtained from seven trees of three different species: 12 from carob (Ceratonia siliqua), one from Mediterranean stone pine (Pinus halepensis) and another from eucalyptus (Eucalyptus camaldulensis). All C. gattii isolates were genotype AFLP4/VGI and mating type alpha, and were found to be genetically identical with C. gattii strains isolated from humans and animals in Spain. This supports the hypothesis that these trees may be a natural source for infection of humans and mammals in the Mediterranean area.
A 4-year-old castrated male domestic ferret from central Massachusetts was evaluated for weight loss over a 1.5-month period and for 2 days of retching, diarrhea, and signs of lethargy. It had been housed indoors, with 2 other ferrets, 2 cats, and humans that lacked signs or symptoms of disease.
Physical examination revealed a thin body condition, tachypnea, an increase in respiratory effort, and retching. Splenomegaly was detected during abdominal palpation. Clinicopathologic analysis revealed lymphopenia, lactic acidosis, hypoglycemia, hypocalcemia, hypoalbuminemia, and hyperglobulinemia. A pulmonary bronchointerstitial pattern was evident on radiographs, and abdominal ultrasonography revealed a suspected pancreatic mass and mesenteric lymphadenopathy.
After 2 weeks of medical treatment and once clinical signs resolved, an exploratory laparotomy was performed and a lymph node biopsy specimen was collected. Histologic evaluation of the specimen revealed Cryptococcus-like organisms. Antifungal treatment was initiated with itraconazole (PO) and amphotericin B (IV). The ferret died after 2 days of treatment. A full necropsy was performed, revealing multicentric cryptococcosis affecting the lungs, brain, spleen, and multiple lymph nodes. Paraffin-embedded, formalin-fixed lung tissue was submitted for DNA extraction, and the organism was identified as Cryptococcus neoformans var grubii.
To the authors' knowledge, this is the first report of disseminated cryptococcosis in a North American ferret. This case is unique in that the ferret lived indoors, in a geographic region in which reports of cryptococcosis are rare. The genotyping technique used to identify the Cryptococcus strain can aid in better understanding the epidemiology of cryptococcosis.
En este trabajo realizamos una revisión de la criptococosis en los animales de compañía. La criptococosis es una micosis esporádica y poco frecuente en los animales domésticos. El agente etiológico más frecuentemente aislado es Cryptococcus neoformans, aunque también Cryptococcus gattii ha sido aislado de distintas especies animales. A diferencia de otras micosis sistémicas, es más frecuente en el gato que en el perro y otras especies animales. En este trabajo nos centramos en la criptococosis felina y canina, describiendo la sintomatología clínica más habitual y las diferentes formas clínicas que presenta en cada una de estas especies. Abordamos también el diagnóstico y el tratamiento de la criptococosis. El diagnóstico suele incluir una serie de pruebas complementarias como el examen citológico de las muestras, la determinación del antígeno capsular y el aislamiento e identificación de las especies de Cryptococcus involucradas. Respecto al tratamiento de la criptococosis, revisamos los antifúngicos más habituales y la situación en que cada uno de ellos se emplea. Finalmente, se analiza la situación de la criptococosis en animales domésticos en España y se discute el papel que juega esta enfermedad como zoonosis y sus implicaciones en la salud pública.
Cryptococcosis was diagnosed in seven ferrets (five from Australia; two from western Canada) displaying a wide range of clinical signs. Two of the ferrets lived together. One (5-years-old) had cryptococcal rhinitis and presented when the infection spread to the nasal bridge. Its sibling developed cryptococcal abscessation of the right retropharyngeal lymph node 12 months later, soon after developing a severe skin condition. DNA fingerprinting and microsatellite analysis demonstrated that the two strains isolated from these siblings were indistinguishable. Two ferrets (2- to 3-years-old) developed generalised cryptococcosis: one had primary lower respiratory tract disease with pneumonia, pleurisy and mediastinal lymph node involvement, while in the other a segment of intestine was the primary focus of infection with subsequent spread to mesenteric lymph nodes, liver and lung. The remaining three ferrets (1.75 to 4-years-old) had localised disease of a distal limb, in one case with spread to the regional lymph node. Cryptococcus bacillisporus (formerly C. neoformans var gattii) accounted for three of the four infections in Australian ferrets where the biotype could be determined. The Australian ferret with intestinal involvement and the two ferrets from Vancouver had C. neoformans var grubii infections.
The pathogenesis of Cryptococcus spp. infection following nasal colonization is unclear. This article reports follow-up data on a cohort of seven cats and five dogs identified in a previous study as sub-clinically infected with Cryptococcus spp. or colonized by C. gattii. Two cats progressed to clinical disease within four to six months of initial detection of antigenemia and nasal cavity colonization. The ten other animals remained asymptomatic but many were repeatedly positive on cryptococcal antigen testing or nasal fungal culture suggesting protracted infection or colonization. The results indicate that asymptomatically infected animals may clear the organism, remain sub-clinically infected or progress to clinical disease. Factors influencing the transition from exposure to disease require further investigation.
More than 110 years of study of the Cryptococcus neoformans and Cryptococcus gattii species complex has resulted in an enormous accumulation of fundamental and applied biological and clinical knowledge. Recent developments in our understanding of the diversity within the species complex are presented, emphasizing the intraspecific complexity, which includes species, microspecies, hybrids, serotypes and genotypes. Each of these may have different roles in disease. An overview of obsolete and current names is presented.
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