Mark Krockenberger

Clonality and Recombination in Genetically Differentiated Subgroups of Cryptococcus gattii

ABSTRACT Cryptococcus gattii is a pathogenic yeast that together with Cryptococcus neoformans causes cryptococcosis in humans and animals. High numbers of viable C. gattii propagules can be obtained from certain species of Australian Eucalyptus camaldulensis trees, and an epidemiological link between Eucalyptus colonization and human exposure has been proposed. However, the highest prevalence of C. gattii cryptococcosis occurs in Papua New Guinea and in regions of Australia where the eucalypt species implicated to date are not endemic. This study investigated the population structure of three geographically distinct clinical and veterinary populations of C. gattii from Australia and Papua New Guinea. All populations that consisted of a genotype found frequently in Australia (VGI) were strongly clonal and were highly differentiated from one another. Two populations of the less common VGII genotype from Sydney and the Northern Territory had population structures inferring recombination. In addition, there was some evidence of reduced genetic differentiation between these geographically remote regions. In a companion study presented in this issue, VGII isolates were overwhelmingly more fertile than those of the VGI genotype, giving biological support to the indirect assessment of sexual exchange. It appears that the VGI genotype propagates clonally on eucalypts in Australia and on an unknown substrate in Papua New Guinea, with infection initiated by an unidentified infectious propagule. VGII isolates are completing their life cycles and may be dispersed via sexually produced basidiospores, which are also likely to initiate respiratory infection.

Retrospective study of feline and canine cryptococcosis in Australia from 1981 to 2001: 195 cases

A retrospective study of 155 cats and 40 dogs diagnosed with cryptococcosis between 1981 and 2001 was undertaken. Age, sex, breed, clinical findings, feline immunodeficiency virus and feline leukaemia virus status (in cats), species of Cryptococcus causing disease and region of domicile were recorded. Associations between variables were tested. Male and female cats were affected equally. Age ranged from 1 to 16 years, with a preponderance of cats aged between 2 and 3 years. Siamese, Himalayan and Ragdoll breeds were over-represented. Rural cats were more frequently infected with Cryptococcus gattii. Retroviral infection was not identified as a predisposing condition and was not correlated with either species of Cryptococcus or physical findings. Most cats had signs of nasal cavity infection, which was typically localised for a substantial period before invasion of adjacent structures or dissemination. Male and female dogs were affected equally. A marked preponderance of young, large breed dogs was noted. Border Collies, Boxers, Dalmatians, Dobermann Pinschers, Great Danes and German Shepherds were over-represented. Cryptococcus species involved was not affected by place of domicile. Although nasal cavity involvement was important, the canine cohort had a greater propensity to develop secondary central nervous system involvement and disseminated disease than feline cases. There were no clinical findings in either cats or dogs which could be reliably used to distinguish disease caused by Cryptococcus neoformans variety grubii from disease caused by Cryptococcus gattii. Both Cryptococcus species appear to be primary pathogens of cats and dogs, with the upper respiratory tract presumed to be the predominant primary site of inoculation in most but not all cases.

Evidence of Recombination in Mixed-Mating-Type and α-Only Populations of Cryptococcus gattii Sourced from Single Eucalyptus Tree Hollows

ABSTRACT Disease caused by the pathogenic yeast Cryptococcus gattii begins with the inhalation of an infectious propagule. As C. gattii is heavily encapsulated, this propagule is most likely to be a basidiospore. However, most C. gattii strains are infertile in laboratory crosses, and population studies indicate that recombination and dispersal are very restricted. In addition, strains of the α mating type predominate, which would not be expected in a mating population. C. gattii comprises four genetically distinct molecular genotypes, designated VGI to VGIV. C. gattii molecular type VGI has a strong association with Eucalyptus camaldulensis and can be found in high numbers in E. camaldulensis hollows. Previous work on isolates obtained from E. camaldulensis suggested that environmental populations of C. gattii are highly fragmented, have limited ability to disperse, and are confined to individual tree hollows. In the current study, we examined large numbers of isolates from three separate hollows for evidence of recombination. In two hollows, the α and a mating types were present in approximately equal numbers. The third hollow had α cells only and was from a region where a isolates have never been found. Statistical analysis of multilocus genotypes revealed recombining subpopulations in the three Eucalyptus hollows. Recombination was equally present in the α-a and α-only populations. This is consistent with recent studies that have found evidence suggestive of α-α mating in C. gattii and Cryptococcus neoformans and raises the possibility this may be a widespread phenomenon, allowing these fungi to recombine despite a paucity of a mating partners.

Protothecosis in 17 Australian dogs and a review of the canine literature

Systemic protothecosis was diagnosed in 17 Australian dogs between 1988 and 2005. There was a preponderance of young-adult (median 4 years), medium- to large-breed dogs. Females (12/17 cases) and Boxer dogs (7 cases, including 6 purebreds and one Boxer cross) were over-represented. Sixteen of 17 dogs died, with a median survival of four months. A disproportionate number of cases were from coastal Queensland. In most patients, first signs were referable to colitis (11/17 cases), which varied in severity, and was often present for many months before other symptoms developed. Subsequent to dissemination, signs were mostly ocular (12 cases) and/or neurologic (8 cases). Two dogs had signs due to bony lesions. Once dissemination was evident, death or euthanasia transpired quickly. Prototheca organisms had a tropism for the eye, central nervous system (CNS), bone, kidneys and myocardium, tissues with a good blood supply. Microscopic examination and culture of urine (5 cases), cerebrospinal fluid (CSF;1 case), rectal scrapings (4 cases), aspirates or biopsies of eyes (5 cases) and histology of colonic biopsies (6 cases) as well as skin and lymph nodes (2 cases) helped secure a diagnosis. Of the cases where culture was successful, P wickerhamii was isolated from two patients, while P zopfii was isolated from five. P zopfii infections had a more aggressive course. Treatment was not attempted in most cases. Combination therapy with amphotericin B and itraconazole proved effective in two cases, although in one of these treatment should have been for a longer duration. One surviving dog is currently still receiving itraconazole. Protothecosis should be considered in all dogs with refractory colitis, especially in female Boxers.

The Trehalose Synthesis Pathway Is an Integral Part of the Virulence Composite for Cryptococcus gattii

ABSTRACT The trehalose pathway is essential for stress tolerance and virulence in fungi. We investigated the importance of this pathway for virulence of the pathogenic yeast Cryptococcus gattii using the highly virulent Vancouver Island, Canada, outbreak strain R265. Three genes putatively involved in trehalose biosynthesis, TPS1 (trehalose-6-phosphate [T6P] synthase) and TPS2 (T6P phosphatase), and degradation, NTH1 (neutral trehalose), were deleted in this strain, creating the R265tps1Δ, R265tps2Δ, and R265nth1Δ mutants. As in Cryptococcus neoformans, cellular trehalose was reduced in the R265tps1Δ and R265tps2Δ mutants, which could not grow and died, respectively, at 37°C on yeast extract-peptone-dextrose agar, suggesting that T6P accumulation in R265tps2Δ is directly toxic. Characterizations of the cryptococcal hexokinases and trehalose mutants support their linkage to the control of glycolysis in this species. However, unlike C. neoformans, the C. gattii R265tps1Δ mutant demonstrated, in addition, defects in melanin and capsule production, supporting an influence of T6P on these virulence pathways. Attenuated virulence of the R265tps1Δ mutant was not due solely to its 37°C growth defect, as shown in worm studies and confirmed by suppressor mutants. Furthermore, an intact trehalose pathway controls protein secretion, mating, and cell wall integrity in C. gattii. Thus, the trehalose synthesis pathway plays a central role in the virulence composites of C. gattii through multiple mechanisms. Deletion of NTH1 had no effect on virulence, but inactivation of the synthesis genes, TPS1 and TPS2, has profound effects on survival of C. gattii in the invertebrate and mammalian hosts. These results highlight the central importance of this pathway in the virulence composites of both pathogenic cryptococcal species.

Cryptococcus neoformans var. gattii in the koala (Phascolarctos cinereus): a review of 43 cases of cryptococcosis.

Details of 11 previously reported cases and 32 new cases of cryptococcosis in captive and wild koalas were analysed. Cryptococcus neoformans var. gattii accounted for all 29 cases in which varietal status was determined. No age or sex predisposition was observed. The respiratory tract was the primary focus of disease in 77% of cases. Although the lower respiratory tract was affected most commonly (60% of cases), 30% of cases had upper respiratory tract lesions and 14% had both. Dissemination was common, especially to the central nervous system (37% cases). Local extension to surrounding tissues was a feature of upper respiratory tract disease. Other tissues showing cryptococcal invasion included lymph nodes (19%), gastrointestinal tract (12%), kidneys (12%), spleen (9%) and skin (7%). Only three cases (7%) had no respiratory tract or central nervous system involvement, two cases of primary skin inoculation and one case of primary lymphadenopathy. Late presentation was a likely factor in the high proportion of cases with disseminated disease (40%). The proportion of koala cases with involvement of the central nervous system, lower respiratory tract and skin, parallels what has been reported for immunocompetent people. Cryptococcosis in the koala appears to be an excellent naturally occurring model for examination of the cryptococcal host-parasite relationship in all species.

A review of neosporosis and pathologic findings of Neospora caninum infection in wildlife.

Highlights • The importance of Neospora caninum-associated disease in wildlife is reviewed.• There are only 12 reports of clinical neosporosis in wildlife species to date.• The best practice guidelines to follow for reporting wildlife cases of neosporosis are presented.

Low-Grade Alimentary Lymphoma: Clinicopathological Findings and Response to Treatment in 17 Cases:

Low-grade alimentary lymphoma (LGAL) was diagnosed by histological and immunohistochemical evaluation of full-thickness biopsies from multiple regions of the gastrointestinal tract collected during exploratory laparotomy in 17 cats. The most common clinical signs were weight loss (n=17) and vomiting and/or diarrhoea (n=15). Clinical signs were chronic in 11 cases. Abdominal palpation was abnormal in 12 cats, including diffuse intestinal thickening (n=8), an abdominal mass due to mesenteric lymph node enlargement (n=5) and a focal mural intestinal mass (n=1). The most common ultrasonographic finding was normal or increased intestinal wall thickness with preservation of layering. Ultrasound-guided fine-needle aspirates of mesenteric lymph nodes (n=9) were incorrectly identified as benign lymphoid hyperplasia in eight cats, in which the histological diagnosis from biopsies was lymphoma. There was neoplastic infiltration of more than one anatomic region of the gastrointestinal tract in 16/17 cats. The jejunum (15/15 cats) and ileum (13/14 cats), followed by the duodenum (10/12 cats), were the most frequently affected sites. Twelve cats were treated with oral prednisolone and high-dose pulse chlorambucil, two with a modified Madison–Wisconsin multiagent protocol and three with a combination of both protocols. Thirteen of the 17 cats (76%) had complete clinical remission with a median remission time of 18.9 months. Cats that achieved complete remission had significantly longer median survival times (19.3 months) than cats that did not achieve complete remission (n=4) (4.1 months; P=0.019). The prognosis for cats with LGAL treated with oral prednisolone in combination with high-dose pulse chlorambucil is good to excellent.

Metabolites released by Cryptococcus neoformans var. neoformans and var. gattii differentially affect human neutrophil function

Differences in the ability of Cryptococcus neoformans var. neoformans (CNVN) and var. gattii (CNVG) to establish localized lesions in the lungs of healthy humans remain unexplained. In this study, CNVG infection in a rat model was characterized by early neutrophil invasion into lung tissue, but phagocytosis of cryptococci was not observed. The chemical composition of non-enzymic components secreted by one strain of each variety (heat-inactivated supernatants from CNVN and CNVG, termed vns and vgs, respectively) were compared, using magnetic resonance spectroscopy. Effects on human neutrophil viability and functions at both pH 5.5 and 7.0 were investigated, as the pH of cryptococcomas was found to be 5.4-5.6 in vivo. The supernatants were similar in composition, although metabolites in vns were generally present in higher concentrations. In addition, vgs contained two novel metabolites-acetoin and dihydroxyacetone. Polyphosphate was observed in cells from both varieties and may be a source of extracellular inorganic phosphate. Superoxide production in the presence of phorbol ester was enhanced by treatment with vns and decreased by vgs. At pH 5.5, vns caused high levels of necrosis in neutrophils, as well as increased adhesion/migration through A549 lung epithelial cell monolayers. Individual supernatant components such as polyols, acetoin, dihydroxyacetone, and gamma-aminobutyric acid exhibited both pro- and anti-inflammatory properties. Overall, we found that vgs was potentially less pro-inflammatory than vns. Inhibition of neutrophil function by products of CNVG may promote survival of extracellular organisms, and local multiplication to form cryptococcomas.

Disseminated Mycobacterium avium infection in young cats: overrepresentation of Abyssinian cats

Disseminated Mycobacterium avium-intracellulare complex (MAC) infection was diagnosed in 10 young cats (1–5 years of age) from Australia or North America between 1995 and 2004. A further two cats with disseminated mycobacteriosis (precise agent not identified) were recognised during this period. Of the 12, 10 were Abyssinian cats, one was a Somali cat and one was a domestic shorthair cat. None of the cats tested positive for either FeLV antigen or FIV antibody. The clinical course of these infections was indolent, with cats typically presenting for weight loss, initially in the face of polyphagia, with a chronicity of up to several months. Additional clinical features included lower respiratory tract signs and peripheral lymphadenomegaly. A marked diffuse interstitial pattern was evident in thoracic radiographs, even in cats without overt respiratory involvement. Hair clipped to perform diagnostic procedures tended to regrow slowly, if at all. Diagnosis was generally made by obtaining representative tissue specimens from mesenteric lymph nodes, liver or kidney at laparotomy, or from a popliteal lymph node. The primary antecedent event was most likely colonisation of either the alimentary or respiratory tract, followed by local invasion and eventual lymphatic and haematogenous dissemination. Nine cases were treated using combination therapy with agents effective for MAC infection in human patients. Two cats are still undergoing initial therapy and have responded. Of the remaining seven, all responded during long courses (5–14 months) of clarithromycin combined with either clofazimine or rifampicin, and a fluoroquinolone or doxycycline. Of these, three cats remain well (with durations between 2 months and 2 years following therapy); two developed recurrent disease (at 3 months and 2 years, respectively, following therapy) and have restarted therapy. The remaining two cats improved 1 year and 5 months, respectively, after diagnosis but ultimately succumbed. The two cats in which therapy was restarted have improved dramatically. Certain lines of Abyssinian and Somali cats likely suffer from a familial immunodeficiency that predisposes them to infection with slow-growing mycobacteria such as MAC.