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.

Experimental diet-induced atherosclerosis in Quaker parrots (Myiopsitta monachus).

Spontaneous atherosclerosis is common in psittaciformes, and clinical signs associated with flow-limiting stenosis are encountered in pet birds. Nevertheless, a psittacine model of atherosclerosis has not been developed for research investigations. Sixteen captive-bred Quaker parrots (Myiopsitta monachus) were used in this study. While 4 control birds were fed a maintenance diet, 12 other birds were fed an atherogenic diet composed of 1% cholesterol controlling for a calorie-to-protein ratio for periods ranging from 2 to 8 months. The birds were euthanized at the end of their respective food trial period. Histopathology, transmission electron microscopy, and cholesterol measurement were performed on the ascending aorta and brachiocephalic and pulmonary arteries. Plasma lipoproteins, cholesterol, and triglycerides were also measured on a monthly basis. Significant atherosclerotic lesions were induced within 2 months and advanced atherosclerotic lesions within 4 to 6 months. The advanced lesions were histologically similar to naturally occurring lesions identified in the same parrot species with a lipid core and a fibrous cap. Ultrastructurally, there were extracellular lipid, foam cell, and endothelial changes. Arterial cholesterol content increased linearly over time. Plasma cholesterol and low-density lipoprotein (LDL) significantly increased over time by an average of 5- and 15-fold, respectively, with a shift from high-density lipoprotein to LDL as the main plasma lipoprotein. Quaker parrots also exhibited high plasma cholesteryl ester transfer protein activity that increased, although not significantly, over time. This experiment demonstrates that in Quaker parrots fed 1% cholesterol, advanced atherosclerosis can be induced relatively quickly, and lesions resemble those found in other avian models and humans.

Serum amyloid A, Serum Amyloid A1 and Haptoglobin in pregnant mares and their fetuses after experimental induction of placentitis.

Serum amyloid A (SAA) and Haptoglobin (Hp) are acute phase proteins, produced during inflammation, such as placentitis. In horses, SAA and SAA1 are protein coding genes. Objectives were to analyze SAA and Hp concentrations and relative abundance of SAA, SAA1 and Hp mRNA transcript in maternal and fetal tissues after experimental induction of placentitis or mares of a control group. Serum Amyloid A family proteins were in marked abundance in the stroma of the endometrium and chorioallantois associated with inflammatory cells. Maternal plasma SAA concentrations were greater (P = 0.01) in mares with experimentally induced placentitis compared to those of the control group. Maternal Hp from the groups were not different, but fetal Hp concentrations of mares with experimentally induced placentitis were greater (P = 0.02). Maternal plasma SAA and Hp concentrations were greater than fetal plasma concentrations in mares with experimentally induced placentitis (P < 0.05). Relative abundance of SAA mRNA transcript was greater in the maternal, fetal liver and chorioallantois of mares with experimentally induced placentitis (P < 0.05) compared to those in the control group. Interestingly, relative abundance of SAA1 mRNA transcript was greater in the chorioallantois of mares with experimentally induced placentitis (P < 0.05). The SAA and Hp concentrations, therefore, were greater in mares with induced placentitis. Furthermore, relative abundance of SAA1 mRNA transcript is specifically greater in the chorioallantois of mares with placentitis, which warrants further studies to elucidate the immunological response of SAA1 in the chorioallantois of mares with placentitis.

Serological survey for antibodies to Encephalitozoon cuniculi in cats within the United States.

Encephalitozoon cuniculi is an obligate intracellular microsporidian parasite that causes clinical and subclinical infection in many species. It is also considered a potential zoonotic disease. In the present study, a serological survey was conducted using samples from pet cats using an ELISA with a confirmed positive control sample from a cat with E. cuniculi related uveitis. Thirty-four of 127 serum samples or 26.8% were found to demonstrate reactivity with titers ranging from 1:32 to 1:1024. There were no significant differences by age or sex (p=0.99 and p=0.32, respectively). This is the first description of the use of an enzyme linked immunosorbent assay for the detection of E. cuniculi antibodies in cats in the United States.

The ability of murine cytomegalovirus and class I major histocompatibility complex-disparate parental cells to induce alterations characteristic of severe graft-versus-host reactions.

The present studies were undertaken to examine the ability of a viral pathogen to enhance immune alterations associated with parent----F1 graft-versus-host reactions (GvHR) across defined donor/recipient MHC genetic disparities. Murine cytomegalovirus (MCMV) was administered concurrently with a parental lymphoid inoculum into unirradiated F1 recipients in strain combinations limiting allogeneic differences to the entire MHC complex (class I/II), the H-2K region (class I), or H-2IA (class II) regions only. Alterations previously found to be associated with GvHR involving changes in the expression of Ly-6, Lyt-2, and L3T4 were examined to characterize the effects of MCMV. Mice receiving low numbers of class I/II-disparate parental cells or MCMV alone failed to exhibit significant GvHR-associated changes. In contrast, introduction of cells and virus resulted in marked alterations characteristic of F1 recipients injected with a large parental cell inoculum alone. Concurrent virus and parental cells could also induce marked changes when administered across differences involving only a class I-disparate--but not class II-disparate only--P----F1 combination. In addition to the phenotypic changes observed during the concurrent virus and class I GvHR, markedly reduced spleen cell proliferative activity and associated weight loss and mortality appeared to indicate that virus had enhanced this reaction. In total, these findings demonstrated that a donor/recipient class I MHC difference was necessary for virus and parental cells to induce the changes observed, and thus not all donor/recipient antigenic differences will result in a similar virus-induced effect. The results are discussed with respect to the potential mechanisms that may account for the apparent exacerbation of GvHR-associated alterations.

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.

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.

Regulation and selective expression of Ly-6A/E, a lymphocyte activation molecule, in the central nervous system.

The Ly-6 locus encodes a group of cell surface molecules found predominantly on lymphoid cells in the mouse. These proteins share several structural and functional characteristics with Thy-1, a molecule expressed in both lymphoid and neuronal tissue. Utilizing anti-Ly-6A/E monoclonal antibodies, the present results demonstrate in situ expression of these molecules in brain tissue. The findings also indicated that these molecules are not expressed during embryonic or neonatal stages of development. Moreover, although Ly-6b haplotype mice exhibited staining primary associated with vascular elements throughout the brain, Ly-6a mice exhibited staining predominantly associated with white matter limited to the hippocampal and midbrain regions. Although cultured glial and neuronal cells expressed marginally detectable levels of Ly-6A/E, the majority of GFAP+ cells in these cultures expressed high levels of Ly-6A/E following incubation with cytokines including rIFN-gamma. In addition, northern blot analysis of RNA from enriched astrocytic cultures corroborated the induction of Ly-6A/E expression. These findings have therefore established that Ly-6 is amongst those groups of genes expressed in both brain and lymphoid tissues.

Macrophage inhibitory factor-A3 derived from Mycobacterium avium serovar 2 inhibits candidacidal activity of murine peritoneal macrophages.

Macrophage inhibitory factor-A3 (MIF-A3), a fraction derived from Mycobacterium avium serovar 2 inhibited candidacidal activity in macrophages from C57BL/6, C57BL/10, C3H/HeJ and A/J strains of mice. Inhibition of candidacidal activity was demonstrated at MIF-A3 concentrations ranging from 100-400 micrograms/ml in macrophages without additional stimulators (exception C3H/HeJ macrophages) and in macrophages additionally stimulated with 200 U/ml interferon-gamma, 100 ng/ml phorbol myristate acetate and 0.4 ng/ml E. coli lipopolysaccharide from all mouse strains tested. The decreased candidacidal effect produced by MIF-A3 was dose-dependent and appeared greatest in macrophages treated with phorbol myristate acetate and lipopolysaccharide. This effect was neutralized by the addition of goat anti-MIF-A3 antiserum. Macrophages from the Bcgs mouse strains (C57BL/6 and C57Bl/100 were more sensitive to the effect(s) of MIF-A3 than macrophages from the Bcgr mouse strains (C3H/HeJ and A/J).

Mice immunosuppressed by murine retrovirus infection (MAIDS) are susceptible to cytomegalovirus retinitis.

Although various methods of immunosuppression have been used to enhance susceptibility of mice to murine cytomegalovirus (MCMV) retinitis, none reproduce the unique complexity of immune deficiency experienced by patients during the progression of AIDS. C57BL/6 mice are susceptible to a retrovirus-induced murine acquired immune deficiency syndrome (MAIDS), characterized by progressive immune dysfunction which shares many features with AIDS. We therefore evaluated the frequency and severity of MCMV retinitis in C57BL/6 mice with MAIDS. Following subretinal inoculation of MCMV, nearly 90% of mice with MAIDS developed a necrotizing retinitis 8 to 10 days postinfection, whereas retinitis was observed in only 8% of age-matched immunocompetent control mice. Histopathologic analysis of the retinitis that developed in mice with MAIDS revealed features similar to those found in AIDS-related CMV retinitis. Eyes from MAIDS animals also contained on average higher amounts of infectious virus when compared with eyes from control animals. We conclude that retrovirus-induced immunosuppression increases susceptibility of C57BL/6 mice to MCMV retinitis.

Hematologic, plasma protein, and biochemical profiles of brown pelicans (Pelecanus occidentalis).

OBJECTIVE: To establish hematologic and biochemical reference values for the brown pelican (Pelecanus occidentalis). ANIMALS: 31 captive, healthy, but permanently disabled pelicans and 35 wild-caught, healthy pelicans from a rehabilitation facility on the east coast of Florida. PROCEDURES: Samples of venous blood were collected from each pelican, and hematologic, plasma biochemical, and electrophoretic protein analyses were performed. Student t-tests were used to compare blood values between captive versus wild-caught, adult male versus adult female, and adult versus juvenile pelicans. RESULTS: Hematologic and electrophoretic values were similar between male and female, adult and juvenile, and captive and wild-caught pelicans. Significant sex differences existed for plasma calcium and triglyceride concentrations. Plasma concentrations of calcium, cholesterol, and CO2 content differed between captive and wild-caught adults. No significant differences were found between wild-caught adult and juvenile pelicans. CONCLUSIONS AND CLINICAL RELEVANCE: Our plasma biochemical results are similar to those of other brown pelicans and confamilial species. Additional studies on seabirds are encouraged, as age, sex, reproductive status, feeding habits, and captivity are important variables for health assessment in this and other aquatic species.

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.

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.

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.

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.

Protein electrophoresis as a diagnostic and prognostic tool in raptor medicine.

Plasma proteins of 139 healthy adult birds of prey from 10 species were separated by electrophoresis to characterize and document normal reference ranges and species-specific electrophoretic patternsand to evaluate the value of this technique for health screening, disease diagnosis, and prognostic indication. Species studied included bald eagle (Haliaeetus leucocephalus), red-tailed hawk (Buteo jamaicensis), barn owl (Tyto alba), great horned owl (Bubo virginianus), turkey vulture (Cathartes aura), Harris' hawk (Parabuteo unicinctus), Stellar's sea eagle (Haliaeetus pelagicus), barred owl (Strix varia), screech owl (Otus asio), and black vulture (Coragyps atratus). Several clinical cases show the diagnostic/therapeutic value of protein electrophoresis in raptors. This study establishes species-specific reference ranges for several birds of prey and discusses the benefit of electrophoresis as a diagnostic technique in health screens, as a diagnostic aid in conjunction with other tests, and as a prognostic indicator in clinical evaluation of raptors.

Seroprevalence of Encephalitozoon cuniculi in dogs in the United States.

Encephalitozoon cuniculi is an obligate intracellular microsporidian parasite that can result in clinical and subclinical infection in many species. It is also considered the cause of a potential zoonotic disease. In the present study, a serological survey was conducted using samples from pet dogs in the state of Florida. Twenty-seven of 125 serum samples, or 21.6%, were found to demonstrate reactivity by ELISA, with titers ranging from 1:32 to 1:512. There were no significant differences by age or sex. This is the first report on the detection of E. cuniculi antibodies in dogs in the United States.

Evaluation of methods to improve the diagnosis of systemic inflammation in alpacas.

BACKGROUND: The stoic nature of alpacas and limitations of current diagnostic tests make early recognition of inflammatory diseases in this species challenging. OBJECTIVES: In a model of mild systemic inflammation, this study evaluated the utility of different clinical and clinicopathologic variables as accurate predictors of inflammation in alpacas. ANIMALS: Twelve clinically healthy alpacas were randomly assigned to equal-sized treatment (TG) and control (CG) groups. After collection of initial blood samples (0 hour), lipopolysaccharide (LPS; 20 µg/kg/24 h) or saline was administered by SC osmotic mini-pumps (OMP) for 96 hours. Additional blood samples were collected at 12, 18, 24, 36, 48, 72, 96, 120, 144, and 240 hours and differential leukocyte counts and concentrations of globulin, albumin, iron, haptoglobin, and serum amyloid A were measured. RESULTS: Mild swelling was observed at OMP implantation sites in both groups. Other clinical signs of systemic inflammation were not observed. Total leukocytes, neutrophils, albumin, and globulin concentrations were not significantly different between groups. Compared with CG-alpacas, TG-alpacas had fewer lymphocytes (P = .0322), more band neutrophils (P = .0087), and higher neutrophil/lymphocyte ratios (P = .0295) during the first 96 hours of the study. During LPS administration, serum iron concentrations were significantly decreased in TG-alpacas (P < .0001). Haptoglobin concentrations of TG-animals exceeded those of CG-animals after removal of OMP (P = .0056). Serum amyloid A was not detectable in alpacas in this study. CONCLUSION AND CLINICAL IMPORTANCE: These results indicated that neutrophil/lymphocyte ratios and serum iron concentrations are early indicators of inflammation in alpacas. Additional research is needed to evaluate the acute phase protein responses of alpacas.