Binding of curcumin to senile plaques and cerebral amyloid angiopathy in the aged brain of various animals and to neurofibrillary tangles in Alzheimer's brain

The binding of curcumin to senile plaques (SPs) and cerebral amyloid angiopathy (CAA) was examined in the aged brain of various animal species and a human patient with Alzheimer's disease (AD), together with its binding to neurofibrillary tangles (NFTs). Brain sections were immunostained with anti-amyloid β protein 1-42 (Aβ42) and anti-amyloid β protein 1-40 (Aβ40) antibodies. These sections were also stained with alkaline Congo red, periodic acid-methenamine silver (PAM), and curcumin (0.009% curcumin solution) with or without formic acid pretreatment. The sections from the AD brain were also immunostained for anti-paired helical filament-tau (PHF-tau), and were stained with Gallyas silver for NFTs. Some SPs in the AD, monkey, dog, bear, and amyloid precursor protein transgenic mouse (APP Tg-mouse) brains contained congophilic materials, and were intensely positive for curcumin. In addition, curcumin labeled some diffuse SPs negative for Congo red in the AD, monkey, bear, and APP Tg-mouse brains. In all animals, CAA was intensely positive for both Congo red and curcumin. The specific curcumin staining activity was lost by formic acid pretreatment. In the AD brain, NFTs positive for PHF-tau and Gallyas silver were moderately stained with curcumin. These findings indicate that curcumin specifically binds to the aggregated Aβ molecules in various animals, and further to phosphorylated tau protein, probably according to its conformational nature.     

Diseases of captive cheetahs (Acinonyx jubatus jubatus) in South Africa: a 20-year retrospective survey.

As part of an ongoing study to determine the basis for high prevalences of veno-occlusive disease, glomerulosclerosis, and chronic lymphoplasmacytic gastritis in cheetahs, a retrospective pathology survey of captive cheetahs in the Republic of South Africa (RSA) was conducted. The RSA population was selected because its genetic composition and captive management were similar to those of the cheetah population in U.S. zoos, in which these diseases are common. For this study, archived pathology materials at the University of Pretoria Faculty of Veterinary Sciences in Onderstepoort and the Faculty of Veterinary Science, MEDUNSA, from 69 cheetahs that died between 1975 and 1995 were reviewed, and prevalences of common lesions were compared with those in the U.S. population. Gastritis associated with Helicobacter-like organisms was the most prevalent disease, accounting for close to 40% of the mortalities, including several cheetahs < 3 yr old. Glomerulosclerosis and veno-occlusive disease also were major causes of mortality in RSA cheetahs. RSA cheetahs also had adrenal cortical hyperplasia, cardiac fibrosis, lymphocytic depletion of the spleen, systemic amyloidosis, and splenic myelolipomas. The presence in the captive RSA cheetah population of the same unusual diseases that are common in U.S. cheetahs suggests a species predilection to develop these diseases in captivity.     

Ageing changes in cat brains demonstrated by beta-amyloid and AT8-immunoreactive phosphorylated tau deposits

The life expectancy of domestic pet cats is increasing, along with the occurrence of geriatric-onset behavioural problems, such as cognitive dysfunction syndrome (CDS). While the cause of CDS is unclear, it has been suggested that it may result from age-related neurodegeneration. In aged and in particular senile human beings, histopathological changes may include the extracellular accumulation of plaque-like deposits of beta-amyloid (Abeta) protein and the intracellular accumulation of an abnormally hyperphosphorylated form of the microtubule-associated protein, tau. In severe cases, the latter may form into neurofibrillary tangles. Brain material was assessed from 19 cats, aged from 16 weeks to 14 years; 17 of which had clinical signs of neurological dysfunction. Immunohistochemical methods were used to detect Abeta and its intracellular precursor protein (amyloid precursor protein (APP)) and hyperphosphorylated-tau. APP was constitutively expressed, with diffuse staining of neurons and blood vessels being detected in all cats. More intense staining and diffuse extracellular Abeta staining deposits were found within the deep cortical areas of the anterior- and occasionally mid-cerebrum of seven cats, all of which were over 10 years of age. Neurons staining intensely positive for AT8-immunoreactivity were seen in two cats, aged 11 and 13 years. However, no mature neurofibrillary tangles were detected. This study demonstrated that extracellular Abeta accumulation and AT8-immunoreactivity within neurons are age-related phenomena in cats, and that they can occur concurrently. There are similarities between these changes and those observed in the brains of aged people and other old mammals.     

Feline herpesvirus infection in a group of semi-captive cheetahs

Clinical disease caused by feline herpesvirus type-1 in wild felid species is similar to that in domestic cats. Herpesviruses are endemic in free-ranging lions in South Africa but actual clinical disease due to them has not been reported in free-ranging felids. The first reports of feline herpesvirus infection associated with clinical disease in wild felids came from Australia and the USA in 1970. Subsequent reports of clinical disease in cheetahs and other wild felid species were limited to captive animals. This report deals with clinical disease in a group of semi-captive cheetahs in which 18 animals were affected, and included 12 adult males, 4 adult females and 2 subadults. No mortalities occurred in this group, the most common clinical signs being sneezing, nasal discharge and loss of appetite.     

Renal lesions in MRL mice

Female MRL-Mp-lpr/lpr mice spontaneously develop autoimmune disease at three to five months of age and die most commonly from immune complex glomerulonephritis. Kidneys of two-month-old females appeared nearly normal by electron microscopy, and glomerular deposits of IgG an complement component 3 (C3) barely were detectable. In five-month-old females, immunofluorescence revealed numerous deposits of IgG and C3; glomerular mesangial cells were hypertrophic and hyperplastic and contained electron-dense material. There were subepithelial and subendothelial deposits of electron-dense material with swelling of epithelial cell cytoplasm. This disease has many features similar to the immune complex glomerulonephritis observed in New Zealand Black and White hybrid mice and in man.     

Radiographic kidney measurements in captive cheetahs (Acinonyx jubatus).

The prevalence of chronic renal disease is substantial among captive cheetahs (Acinonyx jubatus). The purpose of this study was to determine kidney measurements from radiographs of captive cheetahs (n = 15) with normal renal function. The ratio of kidney length to length of the body of the second lumbar vertebrae has been established for domestic cats with normal renal function. The mean ratio of renal length to length of the second lumbar vertebra was 1.81 +/- 0.14 in cheetahs. This baseline data may allow an objective evaluation of radiographic kidney size in cheetahs. However, evaluation of a small number of cheetahs with confirmed renal failure resulted in a similar ratio.     

In vitro responses of cheetah mononuclear cells to feline herpesvirus-1 and Cryptococcus neoformans.

In vitro T cell function by domestic cats and cheetahs to two common pathogens, feline herpesvirus-1 (FHV-1) and Cryptococcus neoformans, was assessed. Peripheral blood mononuclear cells (PBM) were stimulated with two strains of UV-inactivated FHV-1, whole heat-killed organisms or capsular antigen of Cryptococcus neoformans, and proliferative responses measured. As a group, cheetah PBM responded significantly poorer than domestic cat PBM when cultured with FHV-1. However, individual cheetah responses varied widely. Supplementation of cultures with exogenous interleukin 2 (IL-2) significantly increased the level of response of individual cheetahs to both strains of FHV-1. Cheetah sera contained slightly higher neutralizing antibody titers to FHV-1 than did domestic cat sera, suggesting that B cells function adequately in cheetahs. When stimulated with Cryptococcus neoformans, both species had similar incidences of positive proliferative responses. These data demonstrate that cheetahs exhibit heterogeneous responses to specific antigens, similar to domestic cats. However, a lower group response to FHV-1 in cheetahs suggests species differences occur. In addition, level of variability in major histocompatibility complex (MHC) class I-like genes, as determined by Southern blot hybridization, does not appear to correlate with a uniform response in in vitro functional assays. Therefore, additional mechanisms influence the final outcome of the immune response.     

Fecal cortisol metabolite analysis for noninvasive monitoring of adrenocortical function in the cheetah (Acinonyx jubatus).

A radioimmunoassay was validated for quantifying excreted cortisol metabolites in cheetah (Acinonyx jubatus) feces. High-performance liquid chromatography analysis indicated that immunoreactivity was associated with a water-soluble metabolite in fecal extracts from males and females. None of the immunoreactivity corresponded with free cortisol or corticosterone but rather was associated with a more polar, unidentified metabolite. To determine the biologic relevance of excreted immunoreactive cortisol metabolites, cheetahs were exposed to a variety of situations anticipated to increase cortisol secretion. First, to assess acute changes in adrenal activity, adrenocorticotropic hormone (ACTH; 400 IU i.m.) was administered to two adult males and two adult females. Pre-ACTH baseline serum cortisol and fecal cortisol metabolite concentrations varied among individuals. Serum cortisol concentrations were elevated above baseline within 10 min of ACTH injection, followed by corresponding increases in fecal cortisol metabolite concentrations (690-4,194% above baseline) 48 hr later in three of four cheetahs. In the fourth cheetah, a smaller increase (334% above baseline) in fecal cortisol metabolite excretion was observed 96 hr after ACTH injection. Seven cheetah females also were subjected to a variety of potentially stressful manipulations, including immobilization, translocation, and introduction to a male to assess the ability of this technique to detect physiologic changes in adrenal activity. Increased fecal corticoid metabolite excretion was observed 24-72 hr after exposure to these exogenous stressors. Results indicate that adrenocortical activity can be monitored noninvasively in the cheetah through analysis of these metabolites. This technique could be valuable for evaluating, and thus optimizing, environmental and management conditions and for investigating the role of stress in disease pathogenesis and the usually poor reproductive performance of this species in captivity.     

ULTRASONOGRAPHIC IDENTIFICATION AND CHARACTERIZATION OF SPLENIC NODULAR LIPOMATOSIS OR MYELOLIPOMAS IN CHEETAHS (ACINONYX JUBATUS)

A total of 44 live cheetahs were examined sonographically between 1989 and 1994. Seventeen of these animals originated from 4 European facilities, the other 27 cheetahs were examined in 3 facilities in Namibia. Evaluation of the cheetahs included a clinical examination, abdominal ultrasound, and complete blood work. Splenic myelolipomas were diagnosed sonographically in 11 cheetahs. In 7 cheetahs the diagnosis was confirmed at necropsy. Nine of the 14 cheetahs in Europe had myelolipomas in the spleen, while in the African cheetahs there were only 2 animals with myelolipomas. Histologically these lesions were characterized by fatty nodules. A more fitting term of Nodular Lipomatosis of the spleen is therefore suggested. All cheetahs with nodular lipomatosis of the spleen, had concurrent chronic diseases at necropsy. An indicator function of splenic nodular lipomatosis for chronic disease and/or stress is possible.     

Characterization of the gastric immune response in cheetahs (Acinonyx jubatus) with Helicobacter-associated gastritis

Captive cheetahs have an unusually severe progressive gastritis that is not present in wild cheetahs infected with the same strains of Helicobacter. This gastritis, when severe, has florid lymphocyte and plasma cell infiltrates in the epithelium and lamina propria with gland destruction, parietal cell loss, and, in some cases, lymphoid follicles. The local gastric immune response was characterized by immunohistochemistry in 21 cheetahs with varying degrees of gastritis. The character of the response was similar among types of gastritis except that cheetahs with severe gastritis had increased numbers (up to 70%) of lamina proprial CD79a+CD21- B cells. CD3+CD4+ T cells were present in the lamina propria, and CD3+CD8α+ T cells were within the glandular epithelium. Lymphoid aggregates had follicular differentiation with a central core of CD79a+/CD45R+ B cells and with an outer zone of CD3+ T cells that expressed both CD4 and CD8 antigens. MHC II antigens were diffusely expressed throughout the glandular and superficial epithelium. No cheetah had evidence of autoantibodies against the gastric mucosa when gastric samples from 30 cheetahs with different degrees of gastritis were incubated with autologous and heterologous serum. These findings indicate that T-cell distribution in cheetahs is qualitatively similar to that in other species infected with Helicobacter but that large numbers of lamina propria activated B cells and plasma cells did distinguish cheetahs with severe gastritis. Further research is needed to determine whether alterations in the Th1:Th2 balance are the cause of this more plasmacytic response in some cheetahs.     

Glomerulonephropathy in aged captive Key Largo woodrats (Neotoma floridana smalli)

A retrospective review of mortality records of Key Largo woodrats (Neotoma floridana smalli) in a captive breeding program revealed chronic renal disease in 5 of 6 woodrats older than 4 years of age. Two of the 5 woodrats with chronic renal disease also had clinical evidence of diabetes mellitus. Kidneys from all 5 woodrats were examined via light microscopy, histochemical staining, immunohistochemical staining, and transmission electron microscopy. The dietary histories of the affected animals were examined as well. The most striking histopathologic abnormality in the affected kidneys was the presence of large protein casts within cortical and medullary tubules in combination with lesions of membranous glomerulopathy and glomerulosclerosis. Transmission electron microscopy revealed thickening and undulation of the tubular and glomerular mesangial basement membranes with the variable presence of electron-dense deposits within the capillary endothelial basement membrane. Patchy glomerular immunoreactivity for IgG was noted in 2 cases, but IgA and IgM immunoreactivity were not present. The pathologic changes in the kidneys of the Key Largo woodrats mirrored many of the features of chronic progressive nephropathy commonly diagnosed in laboratory rats. Woodrats in the captive population were fed an ad libitum high-protein diet similar to diets that have been shown in laboratory rats to exacerbate the development and progression of chronic progressive nephropathy. It is concluded that Key Largo woodrats develop glomerulonephropathy with features similar to chronic progressive nephropathy described in laboratory rats. Age, concomitant disease, and dietary factors may contribute to the development and severity of this potentially age-limiting disease in Key Largo woodrats.     

Efficacy and long-term outcome of gastritis therapy in cheetahs (Acinonyx jubatus).

A prospective clinical trial evaluating efficacy and long-term outcome of treatments for lymphoplasmacytic gastritis in cheetahs (Acinonyx jubatus) was conducted. The study evaluated efficacy of 11 different antibiotic and antiinflammatory treatment protocols in 32 cheetahs (19 male, 13 female) for reducing gastric inflammation and Helicobacter colonization and monitored the course of disease through histologic grading of gastric biopsies. All cheetahs were biopsied up to I wk before treatment and then rebiopsied within 1 mo after treatment. Most animals were reassigned to a second treatment regimen within 6 mo. Each animal received from one to three treatments during the study period. After the trial, gastric biopsies were obtained from each cheetah annually until death or transfer from the facility to assess disease progression. The trial and follow-up period spanned 10 yr. At onset of the trial, all 32 cheetahs had some degree of gastritis, and 26 cheetahs (81%) were colonized with Helicobacter. Inflammatory lesions worsened regardless of treatment or the presence of Helicobacter. No treatment had a significant effect on inflammatory changes except the lansoprazole/clarithromycin/amoxicillin treatment group, which produced a short-term decrease in inflammation when compared to controls. Prednisone had no effect on gastric inflammation. Overall, 65% of colonized cheetahs were initially cleared of histologic evidence of Helicobacter by treatment, with short-term eradication occurring in 100% of the animals treated with omeprazole/clarithromycin/amoxicillin or tetracycline/metronidazole/Pepto-Bismol for 28 days. Long-term follow-up of treated animals in this study clearly demonstrated that these treatments had little effect on life-long progression of gastritis or on Helicobacter burden in individual cheetahs, although some treatments provided short-term reduction in gastritis and Helicobacter. These results provide evidence that Helicobacter alone is not the cause of gastritis in cheetahs and do not support the use of antibacterial treatments in cheetahs unless significant clinical signs (e.g., frequent vomiting/regurgitation, weight loss) are apparent.     

In vitro mitogen responses and lymphocyte subpopulations in cheetahs.

Lack of genetic variability and apparent susceptibility of cheetahs (Acinonyx jubatus jubatus) to coronavirus infection has lead to speculation that this species may have immune system deficits. To establish a foundation for evaluation of the immune function, cheetah peripheral blood mononuclear cells (PBM) were stimulated by a panel of six mitogens, and responses compared with those of domestic cat PBM. Individual responses in both species were variable, but evenly distributed throughout the range of stimulation for each mitogen. Proliferation by PBM from domestic cats occurred within the same range as that of the cheetahs. However, a significantly lower response to peanut agglutinin (PNA) was observed with domestic cat PBM. Although responses varied between animals, certain individual cheetahs were consistent low responders. The decreased values could not be explained by lack of IL-2 responsiveness since exogenous IL-2 significantly enhanced mitogen-stimulated proliferation in 11 of 12 cheetahs tested. The phenotypic distribution of domestic cat and cheetah lymphocyte subpopulations was similar as assessed by immunofluorescence staining for surface immunoglobulin (sIg) and cytotoxic T (Tc) cells (using a specific monoclonal antibody, FT2). Values for B cells (31.2% sIg+) and Tc (28.7% FT2+) were slightly higher in domestic cats as compared with cheetah PBM (13.3% sIg+; 19.0% FT2+). Even though no species-specific deficits were detected, a significant negative correlation between PHA-stimulated proliferation and percent FT2+ (Tc) cheetah cells was observed. This indicates that proliferation can be used indirectly to assess relative numbers of functional T helper cells in cheetahs. Our studies suggest that these aspects of the cheetah's immune system are comparable with the domestic cat, and establish a basis for in vitro assays evaluating antigen-specific responses.     

The Pathogenesis of Reactive Systemic Amyloidosis

The diagnosis of amyloidosis is based on the presence of extracellular tissue deposits of proteinaceous material that demonstrate a characteristic green color when stained with Congo red and viewed under polarized light. Several different proteins are amyloidogenic but, in domestic animals, spontaneously occurring systemic amyloidosis is reactive in nature and characterized by the presence of amyloid protein AA. This type of systemic amyloidosis may occur secondary to chronic inflammatory or neoplastic disease, but in many instances no predisposing disease is found. A sustained increase in the serum concentration of serum amyloid A protein (SAA) is necessary but not sufficient for the development of reactive amyloidosis. Other inherited and acquired host-related factors are likely to be important in the development of reactive amyloidosis because this condition develops in few patients with chronic inflammatory disease. The tissue tropism of amyloid deposits varies with the amyloid protein itself and species affected. The consequences of amyloidosis for the host depend upon the tissues involved and the response of these tissues to the presence of the amyloid deposits. In domestic animals, reactive systemic amyloidosis is nephropathic, leading to end-stage renal disease, and the clinical presentation is that of uremia.     

Measurement of glomerular filtration rate, renal plasma flow, and endogenous creatinine clearance in cheetahs (Acinonyx jubatus jubatus).

Glomerular filtration rate (GFR), renal plasma flow (RPF), and the endogenous creatinine clearance (CCr) rate were determined in 13 captive cheetahs, Acinonyx jubatus jubatus (seven females and six males, 1.5-7.5 yr of age, x = 5.02 yr), during general anesthesia with Telazol and isoflurane by measuring the urinary clearances of inulin, para-aminohipppuric acid, and endogenous creatinine, respectively. Methods to determine GFR, RPF, and endogenous CCr in captive cheetahs were evaluated, and the relationship between GFR and CCr for this species was determined. The GFR and the RPF were stable during the procedure, with mean values of 1.59+/-0.17 ml/min/kg body weight and 5.12+/-1.15 ml/min/kg body weight, respectively. Although the mean value for CCr (1.47+/-0.20 ml/min/kg body weight) was significantly less than the corresponding value for GFR, the mean difference (0.11+/-0.02 ml/min/kg weight) between the two measurements was slight, and the values were highly correlated (R2 = 0.928; P < 0.0001). The measurement of CCr in cheetahs should provide a reliable estimate of GFR, facilitating the early detection of renal disease in this species.     

Prevalence of Bartonella infection in wild African lions (Panthera leo) and cheetahs (Acinonyx jubatus)

Bartonella species are emerging pathogens that have been isolated worldwide from humans and other mammals. Our objective was to estimate the prevalence of Bartonella infection in free-ranging African lions (Panthera leo) and cheetahs (Acinonyx jubatus). Blood and/or serum samples were collected from a convenience sample of 113 lions and 74 cheetahs captured in Africa between 1982 and 2002. Whole blood samples available from 58 of the lions and 17 of the cheetahs were cultured for evidence of Bartonella spp., and whole blood from 54 of the 58 lions and 73 of the 74 cheetahs tested for the presence of Bartonella DNA by TaqMan PCR. Serum samples from the 113 lions and 74 cheetahs were tested for the presence of antibodies against Bartonella henselae using an immunofluorescence assay. Three (5.2%) of the 58 lions and one (5.9%) of the 17 cheetahs were bacteremic. Two lions were infected with B. henselae, based on PCR/RFLP of the citrate synthase gene. The third lion and the cheetah were infected with previously unidentified Bartonella strains. Twenty-three percent of the 73 cheetahs and 3.7% of the 54 lions tested by TaqMan PCR were positive for Bartonella spp. B. henselae antibody prevalence was 17% (19/113) for the lions and 31% (23/74) for the cheetahs. The prevalence of seropositivity, bacteremia, and positive TaqMan PCR was not significantly different between sexes and age categories (juvenile versus adult) for both lions and cheetahs. Domestic cats are thus no longer the only known carriers of Bartonella spp. in Africa. Translocation of B. henselae seronegative and TaqMan PCR negative wild felids might be effective in limiting the spread of Bartonella infection.     

Spontaneous differentiation of adult rat marrow stromal cells in a long-term culture

It is well recognized that bone marrow stromal cells (MSCs) can differentiate into neuron-like cells when supplemented with growth factors and/or chemical treatments. We demonstrated that primary MSCs obtained from adult rats could spontaneously differentiate into neural precursor cells after long-term culture. During the outset of in vitro culture, less than 0.1% of adult rat primary MSCs expressed nestin, the common protein of neural precursors. These MSCs didn't show neuronal morphology nor express neuronal antigens. In contrast, after continuous maintenance for 6 weeks, a significant subpopulation of MSCs formed cellular clumps and expressed nestin (32.3 +/- 6.3%). Less than 0.1% of cells expressing immature neuron marker betaIII-tubulin could be detected in these prolonged cultured MSCs. After serum deprivation and growth factor supplement, these nestin-positive cells could express neuron-like morphology and neuron-specific markers NF-H, betaIII-tubulin, tau, and neurotransmitter GABA. In contrast, the MSCs without prolonged culture didn't show neuronal morphology nor neuronal markers even after serum withdrawal and growth factors stimulation. These results demonstrated that neural precursors could be obtained from long-term cultured MSCs, and suggested that MSCs should be useful as a potential source for treatment of neurological disease.     

Diet composition and blood values of captive cheetahs (Acinonyx jubatus) fed either supplemented meat or commercial food preparations.

Nutrition most certainly affects health and may play a role in the etiology of growth and reproductive problems in captive cheetah (Acinonyxjubatus) populations. The objective of our research was to examine nutritional differences between two dietary regimens and quantify their physiologic effects on cheetahs held in captivity. Twelve cheetahs were randomly assigned to either a commercial diet (COM) or a supplemented meat diet (SMD) group. These cats were physically examined and had blood samples taken three times over the course of a year. Representative samples of COM and four separate components of the SMD treatment were analyzed over the same time frame for proximate nutrient composition, digestibility, and concentrations of taurine, fat-soluble vitamins, and selected minerals. Concentrations of fat, vitamins A and E, Se, Fe, Cu, Na, and Mn were significantly higher in COM compared with those in SMD samples, with the exception of fat content in turkey. Mg content was lower in COM than in SMD; other nutrients did not differ. Mean concentrations of vitamins A and E in COM were markedly higher than in SMD samples (408,140 vs. 29,696 IU/kg dry matter [DM] and 431 vs. 48 IU/kg DM, respectively) and varied dramatically between sampling periods. Percent crude protein and protein-to-fat ratios were high for SMD compared with either whole prey-based or commercial food preparations. Blood urea nitrogen and serum creatinine levels were above normal reference means for domestic cats. Plasma concentrations of vitamins A, D, and E were significantly higher in COM-fed than in SMD-fed cheetahs. Both plasma retinol and tocopherol levels were almost three times higher in COM-fed cats (1.26 +/- 0.06 vs. 0.53 +/- 0.03 microg/ml and 17.5 +/- 0.7 vs. 6.4 +/- 0.02 microg/ml, respectively) and exceeded the normal ranges expected for domestic felids. Significant differences between male and female cheetahs were found for plasma concentrations of vitamin E, Se, and Fe after allowing for effects of diet and time of collection. Excess fat-soluble dietary vitamins can result in direct toxicities as well as nutrient antagonisms and may be linked to reproductive and health issues in captive cheetahs. The high protein levels found in SMD may be linked to chronic renal disease, which was detected in some of these cheetahs.     

Severe outbreak of disease in the southern chamois (Rupicapra pyrenaica) associated with border disease virus infection

An outbreak of a previously unreported disease affecting southern chamois (Rupicapra pyrenaica) in the central Pyrenees (NE Spain) was recorded in 2001 and 2002. There was a marked temporal distribution, most animals being found between February and June. After the outbreak, the population was found to have decreased by about 42%, most probably due to the disease. We examined 20 affected chamois. Clinical manifestations included depression, weakness and movement difficulties in all cases. Three chamois presented abnormal behaviour, with absence of flight reaction, and 16 showed different degrees of alopecia with skin hyperpigmentation. At necropsy cachexia was observed in all animals, four chamois had abscesses in different parts of the body, four had pneumonia, one had an extensive subcutaneous infection on the head and neck and one had severe orchitis. Microscopic lesions were found in the brain, mainly edema, gliosis, espongiosis, cariorrexis and neuronal multifocal necrosis. A perivascular mononuclear inflammatory infiltrate was present in three of them. Skin lesions included marked follicular atrophy, mild to moderate epidermal hyperplasia with orthokeratotic hyperkeratosis and follicular hyperkeratosis, and hypermelanosis. In 13 chamois there were haemosiderin deposits in the spleen, and in three individuals kidney "cloissone" was observed. Intraeritrocitic parasites were detected either by direct observation or PCR in 8 of 17 chamois. A pestivirus was isolated and detected by RT-PCR from 12 of 13 affected chamois and antigenic characterized as border disease virus by monoclonal antibodies. This is the first time a border disease virus has been associated with an outbreak of a high-mortality disease in a wild species.     

Dietary isoflavone absorption, excretion, and metabolism in captive cheetahs (Acinonyx jubatus)

Dietary isoflavones, capable of influencing reproductive parameters in domestic cats (Felis catus), have been detected in commercial diets fed to captive cheetahs (Acinonyx jubatus). However, the absorptive and metabolic capacity of cheetahs towards isoflavones has not yet been studied. Experiments were designed to describe the plasma concentration-time curve, metabolite profile, and urinary and fecal excretion of genistein and daidzein in cheetahs following consumption of isoflavones. Four adult cheetahs were administered a single oral bolus of genistein and daidzein, and five juvenile cheetahs consuming a milk replacer formula found to contain isoflavones were also included. Urine was collected from all animals, and blood and feces were also collected from adult cheetahs following isoflavone exposure. Samples were analyzed for isoflavone metabolite concentration by liquid chromatography-electrospray ionization-multiple reaction ion monitoring mass spectrometry and high-performance liquid chromatography. Sulfate conjugates were the primary metabolites detected of both genistein and daidzein (60-80% of total isoflavones present) in the plasma and urine of cheetahs. A smaller proportion of daidzein was detected as conjugates in the urine of juvenile cheetahs, compared to adult cheetahs. Other metabolites included unconjugated genistein and daidzein, O-desmethylangolensin, and dihydrodaidzein, but not equol. Only 33% of the ingested genistein dose, and 9% of daidzein, was detected in plasma from adult cheetahs. However, of the ingested dose, 67% of genistein and 45% of daidzein were detected in the feces of adults. This study revealed that cheetahs appear efficient in their conjugation of absorbed dietary isoflavones and only a small fraction of ingested dose is absorbed. However, the capacity of the cheetah to conjugate genistein and daidzein with sulfate moieties appears lower than reported in the domestic cat. This may confer greater opportunity for biologic activity of isoflavones in the cheetah than would be predicted from findings in the domestic cat. However, further investigation is required.