Christine A. Sundermann

EFFECTS OF COCCIDIAL AND MYCOPLASMAL INFECTIONS ON CAROTENOID-BASED PLUMAGE PIGMENTATION IN MALE HOUSE FINCHES

Abstract Carotenoid pigments produce the ornamental red, orange, and yellow integumentary coloration of many species of animals. Among individuals of a population, the hue and saturation of carotenoid-based ornaments can be extremely variable, and studies of fish and birds have shown that females generally prefer males that display the most saturated and reddest coloration. Consequently, there has been a great deal of interest in determining the proximate factors that affect individual expression of carotenoid-based pigmentation. Parasites might affect production of ornamental coloration, and the Hamilton-Zuk hypothesis proposes that parasitized males will show decreased expression of the secondary sexual traits preferred by females. We found that captive male House Finches (Carpodacus mexicanus) experimentally infected with Isospora spp. (coccidians) and/or Mycoplasma gallisepticum produced carotenoid-based plumage coloration that was significantly less red and less saturated than that of noninfected males. These observations validate a necessary condition of the Hamilton-Zuk hypothesis, but heritable resistance to the pathogens we examined remains to be demonstrated.

Inhibition of Cryptosporidium parvum in neonatal Hsd:(ICR)BR Swiss miceby polyether ionophores and aromatic amidines.

Cryptosporidicidal effects of two polyether ionophores (maduramicin and alborixin), a fluorinated 4-quinolone (enrofloxacin), and three analogs of pentamidine were evaluated in a suckling mouse bioassay. Treatment with all compounds except enrofloxacin and one of the pentamidine analogs [1,3-di(4-imidazolinophenoxy)propane] resulted in significant (P less than 0.05) reductions in oocyst excretion.

MOLECULAR PHYLOGENY OF THE OTHER TISSUE COCCIDIA: LANKESTERELLA AND CARYOSPORA

Nearly complete sequences were obtained from the 18S rDNA genes of Eimeria falciformis (the type species of the genus), Caryospora bigenetica, and Lankesterella minima. Two clones of the rDNA gene from C. bigenetica varied slightly in primary structure. Parsimony-based and maximum likelihood phylogenetic reconstructions with a number of other apicomplexan taxa support 2 major clades within the Eucoccidiorida, i.e., the isosporoid coccidia (consisting of Toxoplasma, Neospora, Isospora [in part], and Sarcocystis spp.) and a second clade containing Lankesterella and Caryospora spp., as well as the eimeriid coccidia (Cyclospora, Isospora [in part], and Eimeria spp.). Our observations suggest that Caryospora spp. may not belong in the family Eimeriidae but rather may be allied with the family Lankesterellidae with which they share molecular and life history similarities. This may be a third lineage of coccidian parasites that has independently evolved a unique heteroxenous transmission strategy.

Conventional and confocal microscopic studies of insulin receptor induction in Tetrahymena pyriformis

Tetrahymena pyriformis reportedly possesses binding structures for the vertebrate hormone insulin that are amplified in cells having prior exposure to the hormone. Conventional and confocal microscopic studies were conducted to verify the validity of the reports and to localize the binding sites. Logarithmic cultures were exposed to insulin concentrations of 0, 3, 6, and 12 micrograms/ml for 1 h (receptor induced, RI). After an additional culture period the cells were fixed, exposed to porcine insulin (antigen), immunocytochemically processed, and examined for staining intensity by video image analysis. Observations indicate that T. pyriformis does bind insulin whether or not the cells have prior exposure to insulin. Staining intensity increased at the two highest RI concentrations over 0 microgram/ml (P less than 0.01) but the staining intensity at 0 microgram/ml was not different from that at 3 micrograms/ml. The results confirm that T. pyriformis does bind insulin and that prior exposure to insulin increases the binding capacity for insulin in what may be a concentration-dependent manner. Confocal microscopy of RI cells that had been labeled with either fluorescein isothiocyanate-insulin or the immunocytochemical technique outlined above revealed labeling of the cytoplasm that appeared to be vesicular. Both techniques produced very similar labeling patterns when optical sections through the cells were viewed. Conventional fluorescence revealed ciliary labeling that could be decreased by incubation with excess unlabeled insulin. Further studies with the exo- mutant of T. thermophila, SB 255, showed that mucocyst discharge and capsule formation are not involved in insulin binding.

HOST SPECIFICITY OF CRYPTOSPORIDIUM SP. ISOLATED FROM CHICKENS

The host specificity of Cryptosporidium sp. infecting chickens was evaluated by oral inoculation of oocysts into 6 different species of neonatal rodents, adult nude mice (athymic), neonatal conventional and gnotobiotic pigs, turkeys, muscovy ducks and bobwhite quail. Examinations of tissue sections, ileal mucosal smears, fecal flotations and stained feces failed to reveal any infections in the mammalian species examined. Oocysts were observed in the feces, and developmental stages were observed in tissue sections, of turkeys and muscovy ducks but not bobwhite quail. This study indicates that Cryptosporidium sp. infections in avian species are probably not a zoonotic threat to humans.

Immunohistochemical diagnosis of Toxoplasma gondii: potential for cross-reactivity with Neospora caninum

A monoclonal antibody (MAB 3F5) was compared with a commercial polyclonal antibody for specificity for Toxoplasma gondii and cross-reactivity with Neospora caninum in paraffin-embedded tissue sections. Both antibody preparations reliably recognized T. gondii tachyzoites (RH isolate) in animal tissues but did not always label tissue cysts (ME-49 isolate). The commercial antibody strongly cross-reacted with N. caninum tachyzoites, but MAB 3F5 did not when the immunoperoxidase, immunofluorescence, or immunogold procedures were employed, nor did it cross-react with other apicomplexans, e.g., Isospora suis, Eimeria bovis. Sarcocystis cruzi, Hammondia hammondi, or Caryospora bigenetica. Immumoelectron microscopy revealed that MAB 3F5 bound to dense granules in extracellular T. gondii tachyzoites. In western blots of T. gondii tachyzoites, a major band at 38 kDa and a minor band at 32 kDa were labeled by MAB 3F5, but no labeling of the proteins of N. caninum tachyzoites or uninfected host cells occurred at the ultrastructural level or in immunoblots.

Genetic characterization of Toxoplasma gondii in wildlife from Alabama, USA

The genetic diversity of Toxoplasma gondii circulating in wildlife is of interest to understand the transmission of this parasite in the environment. In the present study, we genetically characterized five T. gondii isolates from different wild animals including two isolates from a bobcat (Lynx rufus), one from a red-shouldered hawk (Buteo lineatus), one from a white-tailed deer (Odocoileus virginianus), and one from a bald eagle (Haliaeetus leucocephalus). Genotyping of these samples using 11 PCR-restriction fragment length polymorphism markers (SAG1, 5′- and 3′-SAG2, alt.SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1, and Apico) revealed two types, including type I (ToxoDB#10) and type 12 (ToxoDB#5). This is the first report of genetic characterization of T. gondii strains in wildlife from Alabama and from a red-shouldered hawk.

CULTIVATION OF CRYPTOSPORIDIUM BAILEYI: STUDIES WITH CELL CULTURES, AVIAN EMBRYOS, AND PATHOGENICITY OF CHICKEN EMBRYO-PASSAGED OOCYSTS

Sporozoites of Cryptosporidium baileyi did not undergo development in primary cell cultures from either avian or mammalian hosts, or in mammalian cell lines. Oocysts of C. baileyi produced infections resulting in complete development to sporulated oocysts in chicken embryos and embryos of 8 other avian species examined. Inoculation of 4 X 10(5) oocysts was not pathogenic for avian embryos as evidenced by the lack of gross lesions or death. Oocysts obtained after C. baileyi had been passaged 10 times (first experiment) or 20 times (second experiment) in chicken embryos still caused clinical respiratory disease and gross airsacculitis when inoculated intratracheally into 2-day-old broiler chickens. Oocysts that had been passaged 10 times in chicken embryos were similarly pathogenic for 4-day-old turkeys after intratracheal inoculation.

Experimental infections in domestic ducks with Cryptosporidium baileyi isolated from chickens

Oral inoculation of 13 ducks (Anas platyrhynchos) with 1 x 10(6) Cryptosporidium baileyi oocysts produced patent infections but no clinical signs of disease. Intratracheal inoculation of 13 ducks with 1 x 10(6) C. baileyi oocysts produced only mild clinical signs of respiratory disease, no deaths, and gross lesions of airsacculitis in only three ducks. The distribution of developmental stages of C. baileyi in ducks was similar to that observed in experimentally infected chickens and turkeys. Results of this study indicate that ducks are more resistant to experimentally induced respiratory cryptosporidiosis caused by C. baileyi than are chickens and turkeys.

Post-translational glutamylation and tyrosination in tubulin of tritrichomonads and the diplomonad Giardia intestinalis.

Abstract. Glutamylated and tyrosinated tubulin were localized in Giardia intestinalis and selected trichomonads of the Tritrichomonadinae subfamily, using specific monoclonal antibodies directed at each of the post-translational modifications. Analysis was carried out using indirect immunofluorescence microscopy. Although trichomonad tubulins remained unlabeled by anti-tyrosine tubulin (TUB-1A2), the presence of the glutamylation motif (GT 335) was confirmed and found to differ in distribution among tritrichomonads. Tritrichomonas muris was most heavily labeled with GT 335, while T. foetus was the least so. Like trichomonads, Giardia was unreactive to anti-tyrosine tubulin; however, the GT 335 antibody produced marked fluorescence in Giardia trophozoites. This study is the first to report immunofluorescent localization of tubulin glutamylation in Giardia and confirms previously reported mass spectrometry data.