Patricia S. Chavey

BIOMEDICAL EVALUATION OF FREE-RANGING RING-TAILED LEMURS (LEMUR CATTA) IN THREE HABITATS AT THE BEZA MAHAFALY SPECIAL RESERVE, MADAGASCAR

Abstract Complete physical examinations and biomedical sample collection were performed on 70 free-ranging ring-tailed lemurs (Lemur catta) from three different habitats in the Beza Mahfaly Special Reserve (BMSR), in southern Madagascar, to assess the impact of humans and habitat on lemur health. Lemurs were chemically immobilized with ketamine and diazepam administered via blow darts for concurrent biomedical, morphometric, and behavioral studies. Subsets of the animals had blood analyzed for hematology, serum chemistry, micronutrients, fat-soluble vitamins (vitamins A, D, and E), measures of iron metabolism, and polymerase chain reaction assays (PCR) for Toxoplasma gondii, Hemoplasma spp., Bartonella spp., Ehrlichia spp., Anaplasma phagocytophilum, and Neorickettsia risticii. Results were compared on the basis of gender and the habitats at the study site: reserve (intact gallery forest), degraded (human inhabited and altered), and marginal (dry didieracea forest with heavy grazing and tree cutting). Levels of vitamin D, triglycerides, and cholesterol, and measures of iron metabolism for BMSR lemurs were greater than those previously reported for a free-ranging lemur population (Tsimanampetsotsa Strict Nature Reserve, Madagascar) with less access to foods of anthropogenic origin. BMSR ring-tailed lemurs from a habitat with less water (marginal) had higher sodium (P = 0.051), chloride (P = 0.045), osmolality (P = 0.010), and amylase (P = 0.05) levels than lemurs from other BMSR habitats, suggesting that these lemurs were less hydrated. Vitamin D levels of male lemurs were higher (P = 0.011) than those of females at BMSR, possibly because of differences in sunning behavior or differential selection of food items. The biological significance is uncertain for other parameters with statistically significant differences. All samples tested (n = 20) were negative for the pathogens tested using PCR assays. Continued concurrent biomedical and ecological research is needed at BMSR to confirm these results and determine their association with population mortality and fecundity rates.

Thin layer chromatography of erythrocyte membrane glycolipids from type A and type B cats

Erythrocyte membrane glycolipids from blood type A and type B cats were examined by thin layer chromatography. The results indicate that the major erythrocyte membrane glycolipid of type A cats is NeuGc-NeuGc-Galactose-Glucose-Ceramide ([NeuGc]2GD3), where NeuAc represents N-glycolyl-neuraminic acid. In contrast, the major erythrocyte membrane glycolipid of type B cats is NeuAc-NeuAc-Galactose-Glucose-Ceramide ([NeuAc]2GD3), where NeuAc represents N-acetylneuraminic acid. These major erythrocyte membrane glycolipids may be the blood group antigens for type A and type B cats, respectively. All type A cats may have enzymes to synthesise erythrocyte membrane glycolipids with terminal NeuAc, whereas type B cats may lack the gene for N-acetylneuraminic acid hydroxylase, the enzyme that converts NeuAc into NeuGc.

SURVEY AND CLINICAL APPLICATION OF SERUM IRON, TOTAL IRON BINDING CAPACITY, TRANSFERRIN SATURATION, AND SERUM FERRITIN IN CAPTIVE BLACK AND WHITE RUFFED LEMURS (VARECIA VARIEGATA VARIEGATA)

Abstract Serum samples from 63 clinically normal captive black and white ruffed lemurs (Varecia variegata variegata) were analyzed to survey serum iron, total iron binding capacity, transferrin saturation, and serum ferritin levels. Data analysis showed no differences in these analytes attributable to sex, but significantly higher levels of serum iron, transferrin saturation, and serum ferritin in older animals. The survey data were examined in light of two black and white ruffed lemurs that were treated for iron overload with serial phlebotomies. Prior to therapy, both phlebotomized lemurs had excess hepatic iron deposition, but had serum iron, transferrin saturation, and serum ferritin below the upper limits observed in the survey animals, suggesting that some clinically normal animals included in the survey may have accumulated excess systemic iron. Serial phlebotomy therapy reduced serum iron, transferrin saturation, and serum ferritin in both animals. Three years after the conclusion of therapy in the one remaining case, serum iron and transferrin saturation have risen substantially, whereas serum ferritin has risen slightly. Serum iron, transferrin saturation, and serum ferritin may be useful predictors of systemic iron stores in this species, though the correlation between these parameters and systemic iron stores needs to be determined.

Reactivity of lichen lectins with blood typed canine erythrocytes.

Extracts from 69 species of lichens were tested for their ability to agglutinate untreated and enzyme-modified erythrocytes from a panel of blood typed dogs. Forty-three lichen species reacted positively with either untreated or enzyme-modified cells. Many extracts exhibited differential agglutination among red cells tested. The patterns of differential agglutination observed with the lichen extracts did not correspond to known canine blood groups present on the test red cell panel.

Production and Characterisation of Murine Monoclonal Antibodies to Feline Erythrocyte A and B Antigens

Abstract: Anti-A antiserum from blood type B cats, the current reagent used to detect blood type A cats, is expensive, labour intensive to produce, and can vary in sensitivity between preparations. In contrast, monoclonal antibodies are produced easily in large quantities and pure form. We produced six IgM class murine monoclonal antibodies, four specific for feline blood type A and two that detect feline blood type B, by injection of mice with liposomes incorporating type A or B erythrocyte membrane antigens. Specificities of each monoclonal antibody were characterised by high performance thin layer chromatography of feline erythrocyte membrane glycolipids and by immunoblotting of feline erythrocyte membrane proteins separated by SDS-PAGE. The anti-A monoclonal antibodies specifically detected feline blood type A by direct agglutination of blood-typed samples from many cats. Each anti-A monoclonal antibody agglutinated some, but not all, feline blood type AB samples. Two anti-A monoclonal antibodies appeared identical and recognised [NeuGc]2GD3, the major glycolipid antigen of type A blood. The other two also appeared identical to each other and recognised a slower migrating glycolipid band, which may be [NeuGc]GT3. The two anti-B monoclonal antibodies detected feline blood type B by direct agglutination and both recognised [NeuAc]2GD3, the major glycolipid antigen of type B blood. None of the monoclonal antibodies recognised erythrocyte membrane glycoproteins specific for either feline type A or type B blood. The ability of the anti-A monoclonal antibodies produced in this study to specifically detect feline blood type A makes them useful replacements for anti-A antiserum for blood typing of cats. The inability of each anti-A antibody to agglutinate blood from every type AB cat suggests a difference between the A antigen of some type A and some type AB cats.

ENZYME-LINKED IMMUNOSORBENT ASSAY TO QUANTITATE SERUM FERRITIN IN BLACK AND WHITE RUFFED LEMURS (VARECIA VARIEGATA VARIEGATA)

Abstract Lemurs in captivity progressively accumulate iron deposits in a variety of organs (hemosiderosis) including duodenum, liver, and spleen throughout their lives. When excessive, the toxic effects of intracellular iron on parenchymal cells, particularly the liver, can result in clinical disease and death. The pathogenesis of excessive iron storage in these species has been attributed to dietary factors related to diets commonly fed in captivity. Tissue iron stores can be directly estimated by tissue biopsy and histologic examination, or quantitated by chemical analysis of biopsy tissue. However, expense and risk associated with anesthesia and surgery prevent routine use of tissue biopsy to assess iron status. A noninvasive means of assessing total body iron stores is needed to monitor iron stores in lemurs to determine whether dietary modification is preventing excessive iron deposition, and to monitor potential therapies such as phlebotomy or chelation. Serum ferritin concentration correlates with tissue iron stores in humans, horses, calves, dogs, cats, and pigs. Serum ferritin is considered the best serum analyte to predict total body iron stores in these species and is more reliable than serum iron or total iron binding capacity, both of which may be affected by disorders unrelated to iron adequacy or excess including hypoproteinemia, chronic infection, hemolytic anemia, hypothyroidism, renal disease, and drug administration. We have developed an enzyme-linked immunosorbent assay to measure serum ferritin in lemurs. The assay uses polyclonal rabbit anti-human ferritin antibodies in a sandwich arrangement. Ferritin isolated from liver and spleen of a black and white ruffed lemur (Varecia variegata variegata) was used as a standard. Ferritin standards were linear from 0 to 50 μg/L. Recovery of purified ferritin from lemur serum varied from 95% to 110%. The within-assay variability was 4.5%, and the assay-to-assay variability for three different samples ranged from 10% to 17%. The assay also measures serum ferritin in several other lemur species.

EVALUATION OF SERUM FERRITIN AND SERUM IRON IN FREE-RANGING BLACK RHINOCEROS (DICEROS BICORNIS) AS A TOOL TO UNDERSTAND FACTORS AFFECTING IRON-OVERLOAD DISORDER

Abstract Iron overload disorder (IOD) is a significant health issue for captive black rhinoceros (Diceros bicornis). Measurement of serum ferritin with a validated rhinoceros ferritin ELISA has been used extensively to detect animals in U.S. zoos that are at risk of developing IOD. However, there is limited information on serum ferritin levels in free-ranging black rhinoceros using this same assay. Serum ferritin, iron, and gamma-glutamyl transpeptidase (GGT) were determined in 194 black rhinoceros from southern Africa. Mean ferritin in free-ranging black rhinoceros (290.54 ±247.4 ng/ml) was significantly higher than in free-ranging white rhinoceros (64.0 ± 102.4 ng/ml) sampled in this study from Kruger National Park, South Africa. However, there were no significant differences between genders or age groups. Ferritin values varied with geographical location of the black rhinoceros, although this was not clinically significant. Serum iron values were also higher in black rhinoceros (40.4 ± 19.1 μmol/L) compared to white rhinoceros (29.7 ± 10.7 μmol/L). There was no association between ferritin and GGT. This study provides serum ferritin, iron, and GGT values from free-ranging black rhinoceros that can be used for as comparative target values for captive animals.