Pathological, clinical and mycological findings in experimental aspergillosis infections of starlings

A group of 24 wild starlings (Sturnus vulgaris), in undefined age categories but at least post-pubertal, constituted the material of this study. Six starlings were kept as a control group and 18 starlings served as the infection group. The starlings in the infection group were infected with inoculums of 1.35 x 10(6)/0.2 ml Aspergillus fumigatus via intratracheal route whereas the control group was administered only placebo in the same way. Six, four, two, four and two birds died on 2, 3, 4, 5 and 6 days post inoculation respectively. At the necropsy of the dead birds, caseous foci were determined in the lungs, on the air sacks, myocardium, thoracic wall and abdominal serosa. In the histopathological examination of the white-yellowish caseous foci ranging from pinhead to chick pean in size, necrotic granulomatous foci consisting of macrophages, heterophil leukocytes and gigant cells encapsulated with a fibrous tissue were observed. Hyphae and spores of A. fumigatus were determined in these foci using the Gridley staining method.     

Susceptibility of fungi isolated from the respiratory tract of falcons to amphotericin B, itraconazole and voriconazole

The minimum inhibitory concentrations (mics) of fungi isolated from the air sacs of falcons before (group 1), and during antifungal treatment with amphotericin B nebulisation and oral itraconazole or voriconazole (group 2), or with itraconazole alone (group 3) or voriconazole alone (group 4) were determined. Before treatment, 95 per cent of the isolates, including Aspergillus fumigatus, Aspergillus flavus, Aspergillus niger and Aspergillus terreus, were susceptible to voriconazole at mics up to 0.38 microg/ml, and all the isolates were susceptible at mics up to 1 microg/ml. Before treatment, 21 per cent of the isolates, including A fumigatus (27.6 per cent), A flavus (16.6 per cent), A niger (100 per cent) and A terreus (23 per cent), were resistant (mic > or =1 microg/ml) to itraconazole; 51 per cent of the isolates, including A fumigatus (31 per cent), A flavus (78 per cent), A niger (14 per cent) and A terreus (77 per cent), had mics of over 1 microg/ml to amphotericin B, and after treatment their mics increased significantly. In contrast, there were no significant differences between the mics of voriconazole and itraconazole for the different Aspergillus species before and during treatment with these antifungal agents.     

Development of a method for the identification, using the polymerase chain reaction, of Aspergillus fumigatus isolated from ostriches

Objective To develop a method for identifying DNA of Aspergillus fumigatus from ostriches, using the polymerase chain reaction (PCR). A fumigatus is the principal causative agent of avian aspergillosis.
Design A biochemical trial.
Sample population Twelve Aspergillus fumigatus isolates and three other Aspergillus species.
Procedure PCR primers that were based on the sequence of the alkaline protease gene from human isolates of A fumigatus were used.
Results We successfully tested the method on ostrich isolates from five states and showed that the test is specific for A fumigatus.
Conclusions In most cases the DNA sequence of A fumigatus isolates from ostriches is similar to that of human isolates. DNA sequences vary significantly among A fumigatus isolates, including those from affected ostriches in the same flock. The genetic variation may be used to trace aspergillus infections in ostrich flocks and determine if the disease is transmitted by contact with infected birds.     

Aspergillus fumigatus infection in two wild Eurasian black vultures (Aegypius monachus Linnaeus) with carbofuran insecticide poisoning: a case report

Aspergillus spp. are opportunistic pathogens which cause pulmonary aspergillosis in animals and humans with compromised immune systems. Two Eurasian black vultures (Aegypius monachus Linnaeus) were found dead or clinically ill from carbofuran insecticide during the winter of 2004. Carbofuran was detected in the stomach contents by gas chromatograph-mass spectrometry. Gross lesions showed severe granulomatous pneumonia and serofibrinous pleuropneumonia in both birds, with most lesions restricted to the pulmonary system. Histological lesions included pyogranulomatous pneumonia and suppurative parabronchiolitis/pleuritis/air sacculitis with a number of septated fungal hyphae, suggesting severe pulmonary aspergillosis. Fungal isolates from each vulture were identified as Aspergillus fumigatus by both lactophenol cotton blue staining and genetic analysis. This is the first report of pulmonary aspergillosis caused by A. fumigatus in wild Eurasian black vultures and suggests that Aspergillus infection could be an important cause of death in these birds which migrate from Mongolia to Korea during the winter. The incidence of the disease may be related to impaired immunity caused directly or indirectly by carbofuran poisoning.     

Evaluation of plasma (1-->3) beta-D-glucan concentrations in birds naturally and experimentally infected with aspergillus fumigatus

Avian aspergillosis, most often caused by Aspergillus fumigatus, is a common and devastating disease affecting a range of bird species. Early diagnosis is difficult and often unreliable. The current study evaluated the utility of measuring (1-->3)-beta-D-glucan (BG) concentrations in avian plasma samples to aid in the diagnosis of aspergillosis. We evaluated a commercially available BG assay (Fungitell, Beacon Diagnostics) using 178 plasma samples from naturally infected, experimentally infected, and aspergillosis-free birds. Although there was variation in BG concentration, as reflected by high standard deviations, seabirds with confirmed aspergillosis had the highest mean BG concentrations (M = 3098.7 pg/dl, SD = 5022.6, n = 22) followed by companion avian species and raptors with confirmed aspergillosis (M = 1033.8 pg/dl, SD = 1531.6, n = 19) and experimentally infected Japanese quail (Coturnix japonica; M = 1066.5 pg/dl, SD = 1348.2, n = 17). Variation in severity of disease, differences among species of birds with and without disease, and also different levels in environmental exposure likely contribute to the differences among avian groups. The overall sensitivity and specificity of the BG test for diagnosis of aspergillosis in birds was 60.0 and 92.7%, respectively, with an overall optimized avian cut-offvalue of > or = 461 pg/dl for positive disease. Our findings suggest that, although BG concentrations are highly variable between and within different avian groups, it could serve as a useful adjunctive diagnostic test for aspergillosis that is applicable to multiple avian species in some settings, particularly as a negative predictor of infection.     

Absence of protection against challenge with aspergillus fumigatus by adoptive transfer of splenocytes from convalescent turkeys

Only limited protective immunity against aspergillosis after experimental immunization of turkeys has been previously demonstrated. No studies evaluating the efficacy of transfer of immunity in preventing aspergillosis in birds have been reported. This study consisted of two trials assessing the level of protection against Aspergillus fumigatus challenge afforded by transfer of splenocytes from convalescent turkeys. Three treatment groups of 12-to-14-wk-old Beltsville small white (BSW) turkeys comprising the splenocyte donors were prepared by one of the following: 1) intra-air sac (IA) challenge with A. fumigatus conidia 5 wk prior to transfer; 2) IA challenge and then intravenous (i.v.) injection of killed conidia 1 wk prior to transfer; or 3) sham inoculations. Splenocytes from each group were pooled, enriched for mononuclear leukocytes by density gradient centrifugation, and diluted in cell culture medium (CM). Cell viability was assessed by dye exclusion. Each splenocyte preparation was administered intravenously to one of three recipient groups consisting of 10 BSW turkeys each. A control group (n = 10) was given cell-free CM. Recipients were challenged with viable A. fumigatus conidia 16 hr after splenocyte transfer by unilateral IA (trial 1) or i.v. (trial 2) inoculation. Lesion scores postchallenge revealed no differences between turkeys given splenocytes from convalescent vs. naive (control) turkeys. IA exposure produced ipsilateral lesions in air sacs and lung, whereas i.v. exposure produced severe miliary hepatitis. Donor cell function was confirmed by mitogen blastogenesis; however, cells were nonresponsive to A. fumigatus antigens, regardless of previous exposure status.     

Molecular epidemiology and virulence assessment of Aspergillus fumigatus isolates from white stork chicks and their environment

Aspergillus fumigatus is a common pathogen in poultry and captive wild birds and an emerging opportunistic fungal pathogen in immunocompromised humans. Although invasive aspergillosis is frequently reported in free-ranging wild birds, the incidence and epidemiology of the disease in a natural setting is unknown. We recently reported endemic outbreaks of invasive aspergillosis at white stork nesting sites close to human habitation in Germany with significant subsequent breeding losses. Therefore, we hypothesized that A. fumigatus strains with higher virulence in birds may have evolved in this environment and performed the first epidemiological analysis of invasive aspergillosis in free-ranging wild birds. Sixty-one clinical and environmental A. fumigatus isolates from six affected nesting sites were genotyped by microsatellite analysis using the STRAf-assay. The isolates showed a remarkable high genomic diversity and, contrary to the initial hypothesis, clinical and environmental isolates did not cluster significantly. Interestingly, storks were infected with two to four different genotypes and in most cases both mating types MAT-1.1 and MAT-1.2 were present within the same specimen. The majority of selected clinical and environmental strains exhibited similar virulence in an in vivo infection model using embryonated chicken eggs. Noteworthy, virulence was not associated with one distinct fungal mating type. These results further support the assumption that the majority of A. fumigatus strains have the potential to cause disease in susceptible hosts. In white storks, immaturity of the immune system during the first three weeks of age may enhance susceptibility to invasive aspergillosis.     

Use of tetracycline sorbate for the treatment of Aspergillus fumigatus infection in broiler chicks.

The efficacy of tetracycline sorbate (Pimafungil; contains 20% tetracycline sorbate w/v) was evaluated for treatment of Aspergillus fumigatus in naturally infected broiler chicks. The drug was administered to a group of 160 infected broiler chicks at 200 mg/l of drinking water, daily and consecutively for 5 days. Another group of 100 infected broiler chicks was kept as untreated controls. The chicks of the treated group showed appreciable improvement in clinical symptoms, mortality, body weight and recovery as shown by absence of gross and histopathological lesions and of the fungus.     

Pathogenicity differences of multiple isolates of Aspergillus fumigatus in turkeys.

Sixteen Aspergillus fumigatus isolates of environmental, mammalian, and avian origin were used to assess: 1) intra-air-sac inoculation as a viable challenge alternative to aerosol exposure, and 2) isolate variability in pathogenicity. Development of lesions, antibody response in survivors, mortality, and weight gains were assessed. Turkey poults were challenged with equal numbers of viable conidia. Total number of conidia given per experimental group varied markedly and did not influence mortality. Antibody response as measured by the enzyme-linked immunosorbent assay and agar gel immunodiffusion test was erratic, although most poults with high antibody scores had marked lesions and low weight. Lesions were characterized by necrogranulomatous pneumonia and airsacculitis with marked visceral involvement. The source of the isolate was not a factor in mortality, although this was biased by the high numbers of isolates from birds with aspergillosis. The single environmental isolate produced no mortality.     

Use of posaconazole in the management of invasive orbital aspergillosis in a cat

Orbital infection with Aspergillus fumigatus was diagnosed in a Persian cat that was presented with chronic third eyelid protrusion and exophthalmos. Evidence of nasal, sinus, or disseminated aspergillosis was not detected in this cat. Complete surgical excision of diseased tissues was not possible during orbital exenteration, and infection subsequently extended into the tissues of the oral cavity. Oral therapy with itraconazole and parenteral therapy with amphotericin B were ineffective in resolving the infection. Oral therapy with a novel triazole, posaconazole, was curative.     

Experimental Salmonella enteritidis phage type 4 infection and egg transmission in Japanese quails

Japanese quails were inoculated orally with 1 x 10(10) colony-forming units of Salmonella enteritidis phage type 4 isolated from chicks to obtain information concerning S. enteritidis infection and egg transmission. The inoculation resulted in a bacteraemic infection with seeding of the liver, spleen, intestine, peritoneum, ovule, ovary and oviduct. Some infected birds showed diarrhoea, ruffled feathers, depression, loss of appetite and death. However, most infected birds remained clinically normal with normal egg production. Salmonella enteritidis was isolated from the albumen of 7, yolk of 15, shell of 13, and shell membrane of 15 of 164 eggs. The experiment suggests that S. enteritidis phage type 4 is invasive for Japanese quails and the infected eggs laid by S. enteritidis infected quails are probably the result of transovarian infection.     

In vivo studies with dimethyldithiocarbamate, a possible new antimicrobial for use against Aspergillus fumigatus in poultry

Dimethyldithiocarbamate (DmDTC), the carbamate analogue, was tested for therapeutic efficacy in a series of in vivo challenge trials using 5- and 10-week-old white leghorn chickens. Challenge organisms were Pasteurella multocida X-73, Escherichia coli O1:K1, and Aspergillus fumigatus. Birds were evaluated for survival rates, lesion scores, and the rate at which the bacteria or mold could be reisolated following challenge. Results showed DmDTC to be ineffective against P. multocida and E. coli at the treatment levels and in the form used in these trials, but DmDTC significantly reduced lesion scores and inhibited the rate of isolation of A. fumigatus compared with untreated infected birds.     

Germination of Aspergillus fumigatus inside avian respiratory macrophages is associated with cytotoxicity

ABSTRACT: Although aspergillosis is one of the most common diseases in captive birds, the pathogenesis of avian aspergillosis is poorly known. We studied the role of avian respiratory macrophages as a first line of defense against avian aspergillosis. The phagocytic and killing capacities of avian respiratory macrophages were evaluated using pigeon respiratory macrophages that were inoculated with Aspergillus fumigatus conidia. On average, 25% of macrophage-associated conidia were phagocytosed after one hour. Sixteen percents of these cell-associated conidia were killed after 4 h and conidial germination was inhibited in more than 95% of the conidia. A. fumigatus conidia were shown to be cytotoxic to the macrophages. Intracellularly germinating conidia were located free in the cytoplasm of necrotic cells, as shown using transmission electron microscopy. These results suggest that avian respiratory macrophages may prevent early establishment of infection, unless the number of A. fumigatus conidia exceeds the macrophage killing capacity, leading to intracellular germination and colonization of the respiratory tract.     

A case of aspergillosis in a broiler breeder flock

A case of aspergillosis in a broiler breeder flock having respiratory and nervous system problems caused by Aspergillus fumigatus and Aspergillus niger is documented. Dyspnea, hyperpnea, blindness, torticollis, lack of equilibrium, and stunting were observed clinically. On postmortem examination of the affected birds, white to yellow caseous nodules were observed on lungs, thoracic air sacs, eyes, and cerebellum. Histopathologic examination of lungs and cerebellum revealed classic granulomatous inflammation and cerebellar lesions, necrotic meningoencephalitis, respectively. No lesions were noted in the cerebrum histopathologically. Aspergillus hyphae were observed in stained sections prepared from lesioned organs. Fungal spores and branched septate hyphae were observed in direct microscopy. Aspergillus fumigatus and A. niger were isolated from the inoculations prepared from the suspensions of organs showing lesions.     

Sinonasal and sino-orbital aspergillosis in 23 cats: aetiology, clinicopathological features and treatment outcomes

Aetiology, clinicopathological findings and treatment outcomes were documented in 23 cats (1.5-13 years of age) with sinonasal (SNA, n=6) or sino-orbital (SOA, n=17) aspergillosis. Cases recruited retrospectively and prospectively were included if fungal hyphae were identified on cytological or histological examination and the fungal pathogen was identified by PCR and DNA sequencing (ITS1 or ITS1-5.8S-ITS2 regions, rDNA gene cluster). Fungal culture was positive in 22/23 cases. In cases of SNA, the fungal pathogen was Aspergillus fumigatus (n=4), Neosartorya fischeri or A. lentulus (n=1) or a non-speciated Neosartorya spp. (n=1). In all cases of SOA (n=17), the fungal pathogen was identified as Neosartorya spp. Nine cats had brachycephalic conformation. Cats with SNA were more likely to be infected with A. fumigatus and had a better prognosis than cats with SOA.     

In vitro and in vivo characterization of avian reoviruses. II. Clinical evaluation of chickens infected with two avian reovirus pathotypes

The effect of two avian reovirus isolates (2408 and 1733) on digestion and nutrient metabolism in infected chickens was assessed by an in vitro absorption assay and clinical blood chemistry analysis. Birds of various ages were inoculated orally and intratracheally with reovirus and sampled periodically for the respective assays. Transitory malabsorption was observed in the duodenum of birds infected with reovirus 2408. Conversely, increased absorption was detected in the ileum of these same birds. Clinical blood chemistry analyses of birds infected with both isolates revealed that severely affected birds had abnormally elevated plasma total protein, plasma albumin, and calcium levels. Decreases were found in percent bone ash and, due to abnormally high globulin levels, in albumin:globulin (A:G) ratios. A significant (P less than 0.05) correlation between body weights and total protein, albumin, A:G ratio, and bone ash was found in infected birds. The most pronounced metabolic and physiologic changes occurred in the severely affected birds, and, in general, pathogenicity of the isolates was reflected by the degree of metabolic change.     

Effect of ochratoxin A on Escherichia coli-challenged broiler chicks

A study was conducted to evaluate the effects of ochratoxin A (OA) on Escherichia coli-challenged broiler chickens. Day-old broiler chicks were separated into two groups of 92 chicks each, with one group fed a control mash diet, and the other fed a mash diet containing 2 ppm OA. On day 14, each group was further separated into two groups, with one group inoculated with E. coli O78 (1 x 10(7) colony-forming units/0.5 ml), whereas the other group was not inoculated with E. coli. After E. coli inoculation on day 14, four birds from each group were euthanatized at 1, 2, 3, 5, 7, 10, 14, and 21 days postinoculation. Escherichia coli infection caused dullness, depression, huddling, and diarrhea. Mortality was 14.3% in chicks infected with E. coli but fed no OA. Mortality increased to 35.7% in chicks fed OA and infected with E. coli. Decreased body weight and reduced feed intake were observed in chicks fed OA, and the effects were more pronounced in chicks fed OA and infected with E. coli. Increased serum levels of aspartate aminotransferase, alanine aminotransferase, uric acid, and creatinine and decreased levels of total proteins, albumin, globulins, calcium, and phosphorus were observed in OA-fed birds. Escherichia coli infection did not cause significant alteration in any of the serum biochemical parameters. The presence of OA in poultry rations increased mortality and the severity of an E. coli infection.     

Treatment of Mycotic Rhinitis With Itraconazole in Three Horses

Itraconazole, a third-generation azole, was evaluated for treatment of resistant nasal mycotic infections in horses. Two horses with Aspergillus spp nasal granulomas and 1 horse with Conidiobolus coronatus nasal infection were treated with itraconazole (3 mg/kg PO bid). One of the horses with nasal aspergillosis was also treated by surgical resection of the nasal septum. The treatment time for the horses ranged from 3 to 4.5 months. No adverse effects were noted in any of the horses during the treatment period. Peak and trough serum itraconazole concentrations were < 0.5 μg/mL in all 3 horses. Itraconazole (3 mg/kg PO bid) appears to be effective in the treatment of nasal Aspergillus spp infections in horses because the fungal infection was eliminated in both horses. One horse still had excessive nasal sounds during exercise and was retired from training, whereas the other horse returned to normal. The nasal C. coronatus infection appeared resistant to itraconazole treatment in the affected horse because the granulomas were still present after 4.5 months of treatment.     

Bronchoscopic and serologic diagnosis of Aspergillus fumigatus pulmonary infection in a bottlenose dolphin (Tursiops truncatus).

A 4-yr-old male bottlenose dolphin (Tursiops truncatus) developed an Aspergillus fumigatus pneumonia. Fungal elements were identified by cytology and microbiology from endoscopic bronchoalveolar lavage and brushings of a raised yellow endobronchial lesion. The results of qualitative immunodiffusion serology, a technique that identifies specific circulating antibodies to Aspergillus fumigatus, were suggestive of an active infection. The dolphin was treated with itraconazole for over 2 yr, which resulted in remission of clinical signs. Pneumonia caused by Aspergillus sp. accounts for the large majority of pulmonary mycoses in marine mammals. Bronchoscopy facilitated an early definitive diagnosis, accurate treatment, and remission.