Comparison of diagnostic tools for the detection of aspergillosis in blood samples of experimentally infected falcons.

Antemortem diagnosis of avian aspergillosis is very challenging. Diagnostic assays using blood samples would aid in an early and more definitive diagnosis. In the current study, detection of anti-Aspergillus antibodies, Aspergillus antigen, and Aspergillus toxin (fumigaclavine A), protein electrophoresis and measurement of acute-phase protein concentrations were performed on serum of 18 adult and plasma of 21 juvenile gyr-saker hybrid falcons (Falco rusticolus x Falco cherrug). Adult (n = 15) and juvenile (n = 18) falcons were experimentally inoculated with different dosages of the same strain of Aspergillus fumigatus and an additional three falcons from each age group were used as uninfected control animals. Blood samples were collected prior to inoculation and at 28 days postinoculation. Of the 33 inoculated falcons, 16 demonstrated clinical signs (vomiting, greenish urates, dyspnea, ruffled feathers) commonly associated with aspergillosis and in 14 falcons necropsy revealed aspergillosis granulomas confirmed by mycology and histopathology. Positive galactomannan results were rare, with only 3/15 positive samples from adult falcons and none in the juvenile birds. Most of the inoculated falcons showed an increase of serum amyloid A (66.7%) and haptoglobin (70.4%), but fumigaclavine A was not detected in the blood from any of the experimental animals. Elevated antibody indices were detected in 96.7% of the inoculated birds, but also in 66.7% of the controls. Significant decreases in albumin:globulin ratio were obvious in 81.5% of the inoculated birds, including 100% of the birds with granulomas. Blood from falcons with granulomas demonstrated significantly increased concentration values of alpha 2 and ß globulins, decreased percentages of prealbumin and albumin, and increased percentages of alpha 2 and ß globulins compared to inoculated falcons without granulomas. In conclusion, acute-phase proteins and the electrophoretic profile of birds challenged with A. fulmigatus show significant alterations, which in combination with other diagnostic procedures, assist in the early diagnosis of avian aspergillosis.

Hematological effects and metal residue concentrations following chronic dosing with tungsten-iron and tungsten-polymer shot in adult game-farm mallards.

The U.S. Fish and Wildlife Service required a chronic dosing study that assessed the health and reproductive effects of tungsten-iron and tungsten-polymer shot in adult game-farm mallards (Anas platyrhynchos) prior to granting permanent approval of the shot for waterfowl hunting. Herein, we present the effects of tungsten-iron and tungsten-polymer shot on various hematologic parameters and metal residue concentrations in the femur, liver, kidneys, and gonads. Thirty-two-bird groups (sexes equal) of adult mallards were dosed orally with eight #4 steel shot (control), eight #4 tungsten-iron shot, or eight #4 tungsten-polymer shot on days 0, 30, 60, 90, and 120 of a 150 day trial (26 January 1998 to 25 June 1998). An additional 12 mallards (sexes equal) received eight #4 lead shot (positive control) on day 0 of the study. Lead-dosed mallards had significantly decreased hematocrit, hemoglobin concentration, and whole-blood delta aminolevulinic acid dehydratase activity on day 7, as well as significant changes in a number of plasma chemistry parameters compared to ducks in the control, tungsten-iron, or tungsten-polymer groups. Mallards dosed with tungsten-iron or tungsten-polymer shot had occasional significant differences in hematocrit and plasma chemistry values when compared to control mallards over the 150 day period, but these changes were not considered to be indicative of deleterious effects. Low concentrations of tungsten were detected in gonad and kidney samples from males and females and in liver samples from females dosed with tungsten-polymer shot. Tungsten was also detected in femur samples from tungsten-polymer-dosed mallards. Higher concentrations of tungsten were detected in femur, liver, kidney, and gonad samples from tungsten-iron-dosed ducks. Tungsten-iron or tungsten-polymer shot repeatedly administered to adult mallards did not cause adverse hematological effects during the 150 day trial. Concentrations of tungsten in the femur, liver, kidneys, and gonads were generally higher in tungsten-iron-dosed ducks when compared to tungsten-polymer-dosed ducks.

Immune status of free-ranging green turtles with fibropapillomatosis from Hawaii.

Cell-mediated and humoral immune status of free-ranging green turtles (Chelonia mydas) in Hawaii (USA) with and without fibropapillornatosis (FP) were assessed. Tumored and non-tumored turtles from Kaneohe Bay (KB) on the island of Oahu and from FP-free areas on the west (Kona/Kohala) coast of the island of Hawaii were sampled from April 1998 through February 1999. Turtles on Oahu were grouped (0-3) for severity of tumors with 0 for absence of tumors, 1 for light, 2 for moderate, and 3 for most severe. Turtles were weighed, straight carapace length measured and the regression slope of weight to straight carapace length compared between groups (KB0, KB1, KB2, KB3, Kona). Blood was assayed for differential white blood cell count, hematocrit, in vitro peripheral blood mononuclear cell (PBMC) proliferation in the presence of concanavalin A (ConA) and phytohaemagglutinin (PHA), and protein electrophoresis. On Oahu, heterophil/lymphocyte ratio increased while eosinophil/monocyte ratio decreased with increasing tumors score. Peripheral blood mononuclear cell proliferation indices for ConA and PHA were significantly lower for turtles with tumor scores 2 and 3. Tumor score 3 turtles (KB3) had significantly lower hematocrit, total protein, alpha 1, alpha 2, and gamma globulins than the other four groups. No significant differences in immune status were seen between non-tumored (or KB1) turtles from Oahu and Hawaii. There was no significant difference between groups in regression slopes of body condition to carapace length. We conclude that turtles with severe FP are imunosuppressed. Furthermore, the lack of significant difference in immune status between non-tumored (and KB1) turtles from Oahu and Kona/Kohala indicates that immunosuppression may not be a prerequisite for development of FP.

Altered in vitro immune responses in green turtles (Chelonia mydas) with fibropapillomatosis.

The immune competence of green sea turtles (Chelonia mydas) with fibropapillomatosis was assessed using in vitro techniques to measure lymphocyte proliferation in response to mitogens. In comparison with captive, healthy green sea turtles, those afflicted with fibropapillomas demonstrated diminished proliferation with Concanavalin A, phytohemagglutinin (T-cell mitogens), and lipopolysaccharide (B-cell mitogen). Also, markedly decreased proliferative responses to the lymphocyte polyclonal stimulator combination of ionomycin and phorbol myristate acetate were observed. Total circulating white blood cell counts were not statistically different between the two groups, although an overall decrease in lymphocyte number was observed in the papilloma group. The albumin/globulin ratio was decreased in the papilloma group because of decreased albumin and increased gamma globulins.

Acute effects of lead, steel, tungsten-iron, and tungsten-polymer shot administered to game-farm mallards.

Sixteen-bird groups (sexes equal) of adult mallards (Anas platyrhynchos) were orally dosed with eight #4 steel short, eight #4 lead shot, eight BB-size tungsten-iron shot, eight BB-size tungsten-polymer shot, or were sham-dosed and maintained for 30 days (16 January 1996 to 15 February 1996). Half of the lead-dosed ducks (five males, three females) died during the study, whereas no ducks died in the other dosage groups. For lead-dosed ducks, hematocrit and hemoglobin concentration were decreased on day 15 of the trial, but not on day 30. Delta aminolevulinic acid dehydratase activity in lead-dosed ducks was lower when compared to steel-dosed ducks only. Plasma activities of selected enzymes were elevated in lead-dosed ducks when compared to enzyme activities of ducks in the other groups. For lead-dosed ducks, relative heart, liver, and kidney weights increased in comparison to relative weights of those organs of ducks in other groups. Histology of tissues indicated that renal nephrosis accompanied by biliary stasis was present in the eight lead-dosed ducks that died. For the eight lead-dosed ducks that survived, six had mild to severe biliary stasis. Mild biliary stasis was noted in five tungsten-iron dosed ducks and three tungsten-polymer dosed ducks. Amounts of lead in the femur, liver, and kidneys were higher in lead-dosed ducks than in ducks of the other four groups. Small amounts of tungsten were detected in the femur and kidneys of two tungsten-polymer dosed ducks. Higher concentrations of tungsten were detected in the femur, liver, and kidneys of all tungsten-iron dosed ducks. The rate of shot erosion was highest (80%) for the tungsten-polymer shot, followed by tungsten-iron (55%), lead (50%), and steel shot (33%). Results indicated that tungsten-iron or tungsten-polymer shot (8 shot/duck) orally administered to mallards did not adversely affect them during a 30-day trial.

Concurrent MCMV infection augments donor antihost-specific activity and alters clinical outcome following experimental allogenic bone marrow transplantation.

The present studies were undertaken to examine whether concurrent MCMV infection during allogeneic bone marrow transplantation (BMT) could alter the developing donor-host immune interactions and affect the overall outcome of the transplant. In order to determine the effect of MCMV on antihost activity arising following an allogeneic BMT, specific donor antihost cytotoxicity was examined. The results demonstrated that concurrent virus infection in mice receiving a BMT from donors either H2-matched and non-MHC-mismatched or mismatched at both MHC and non-MHC transplantation loci, augmented antihost cytotoxic activity mediated by CD8+ T cells assayed directly from the recipient's spleen 10-14 days posttransplant. Notably, allogenic BMT recipients receiving either lethal or nonlethal numbers of donor T cells and inoculated with MCMV exhibited more rapid and profound weight loss compared with uninfected allogeneic and syngeneic BMT recipients. Concurrent virus presence also resulted in a markedly increased incidence of mortality in allogeneic BMT recipients of nonlethal numbers of T cells. We conclude from these findings that when virus is present early after allogeneic BMT, the resulting interactions can potentiate T cell-mediated donor-antirecipient--i.e., graft vs. host-reactivity. In total, the results support the notion that pathogens could complicate allogeneic BMT by contributing to the development of graft vs. host disease.

Prostate, adrenocortical, and brown adipose tumors in fetal globin/T antigen transgenic mice.

Targeted oncogenesis in transgenic mice has unexpectedly produced predictable tissue-specific tumors. We previously showed that hybrid gene constructs of the human fetal G gamma- or mouse embryonic beta h1-globin promoter linked to the viral simian virus 40 T antigen (G gamma/T and beta h1/T) expressed appropriately in embryonic erythroid tissue, with some unexpected expression elsewhere. Tumors arising in the G gamma/T and beta h1/T transgenic mice were identified by histology, electron microscopy, cell culture, and RNase protection analyses. In one G gamma/T transgenic line, males developed prostate tumors that showed mixed neuroendocrine and epithelial cell features, whereas females developed adrenocortical tumors. In several other G gamma/T lines, brown adipose tumors, or hibernomas, developed in the subcutaneous interscapular neck and shoulder area, as well as internally in the periadrenal and pericardial areas. Little or no expression of T antigen was detected in adult animals before visible tumor formation. In contrast, beta h1/T transgenic mice developed only choroid plexus tumors. Transient transfection assays in prostate and adrenocortical tumor-derived cell lines showed that the G gamma-globin promoter is 7-to 10-fold more active than the beta h1-globin promoter. Activity of 5' G gamma-globin promoter-deletion DNA plasmids was analyzed by transient transfection in a variety of human prostate cancer cell lines. The G gamma-globin promoter region between -140 and -201 also showed high activity in the androgen-independent human prostate cancer cell lines DU-145 and PPC-1, but low activity in the androgen-responsive human prostate cell line LNCaP. We conclude that tumor formation in the G gamma/T transgenic lines apparently results from cryptic positive DNA cis elements active in prostate and adrenocortical cells. Because G gamma-globin promoter activity is highest in embryonic tissue, tumors in adult transgenic mice may result from expression of T antigen in embryonic prostate, adrenal glands, and brown adipose tissue.

Virus-associated immune responses in mice undergoing GVHR exacerbated by concurrent MCMV infection.

It has been proposed that virus-induced immune responses early postinjection of donor cells can result in the exacerbation of GVH reactions. Previously, we and others have shown that introduction of MCMV together with class I-disparate donor cells results in the development of severe GVH reactions. The present studies were performed to examine the nature of the immune responses induced by concurrent MCMV infection early during GVHR. Within 3 days and continuing through day 10 postinjection, spleens from recipients of virus + GVHR inocula exhibited enhanced cytotoxic activity against YAC-1 target cells effected by a Thy1-Lyt-2-ASGM1+NK1.1+ population. Notably, within one week after virus and GVHR injection, sera from those animals were found to contain specific anti-MCMV IgM antibody at levels comparable to those induced in mice injected only with virus. However, in contrast to recipients of MCMV alone, sera from virus + GVHR animals never contained anti-MCMV IgG antibody. To determine the effect of virus on a discrete donor antihost response, antihost cytotoxic activity was examined. By 7 days postinjection, the spleens of virus + GVHR but not GVHR-only recipients contained marked levels of specific antihost cytotoxic activity, mediated by a Thy1+Lyt-2+ASGM1+NK1.1- population. In total, these findings support the hypothesis that early virus-induced immune responses may promote the development of severe graft-versus-host responses including the enhancement of donor antihost specific cytotoxic T cells.