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.     

Rapid hematogenous dissemination of Aspergillus fumigatus and A. flavus spores in turkey poults following aerosol exposure

Cultures of blood samples taken from 3-week-old turkey poults exposed to aerosolized spores of either Aspergillus fumigatus or A. flavus for 15 min yielded organisms. Aspergillus fumigatus was also isolated from brain and liver tissue samples taken immediately after exposure. Exfoliated cells from the lungs of 10 poults exposed to aerosolized spores of A. fumigatus were allowed to attach to glass slides in tissue culture. Several types of the fixed and stained cells had attached or ingested spores of A. fumigatus. Macrophages were the predominant cell type with ingested spores, although other cell types may be involved in the transport of spores of A. fumigatus into the blood stream after aerosol exposure.     

Exposure of rabbits to spores of Aspergillus fumigatus or Penicillium sp: survival of fungi and microscopic changes in the respiratory and gastrointestinal tracts.

Rabbits were exposed to spores of Aspergillus fumigatus by 1 of 2 routes: exposure to aerosols of dry spores or introduction of liquid suspensions of spores directly into the stomach. Rabbits also were exposed to aerosols containing spores of a Penicillium sp. Cultural and microscopic examinations of tissues from the respiratory and gastrointestinal tracts indicated fungi were distributed throughout the gastrointestinal tract of the rabbits within 1 hour after exposure to aerosols of A fumigatus or Penicillum spores. Viable A fumigatus and Penicillium were detected in lung tissues of rabbits for 2 or 3 weeks after inhalation of spores. Aspergillus fumigatus was isolated from the gastrointestinal tract no more than 1 week after aerosol exposure, and Penicillium, not beyond 48 hours. However, when large numbers of A fumigatus spores were introduced directly into the stomach, fungi were isolated from tissues for as long as 16 days after exposure even though the intestinal contents were negative 4 to 7 days after introduction of spores. Tests for precipitating antibody were negative, with one exception, among 26 rabbits surviving for 2 weeks or more. Microscopic changes were more pronounced in rabbits exposed to spores of A fumigatus than in rabbits exposed to Penicillium spores.     

In vitro studies with dimethyldithiocarbamate, possible new antimicrobial for use in poultry

Tests were conducted to determine the in vitro efficacy of the dithiocarbamate analogue, dimethyldithiocarbamate (DmDTC), against selected poultry pathogens. Organisms studied were two bacteria, Pasteurella multocida and Escherichia coli, and a mold, Aspergillus fumigatus. Zone of inhibition and the minimum inhibitory concentration were determined for each organism. DmDTC was effective in vitro against all organisms tested, with A. fumigatus showing greatest overall sensitivity.     

Serum antibodies to the catalase antigen of Aspergillus fumigatus in cattle

Aspergillus fumigatus is an important agent of mycotic infection in cattle and a potent source of antigens. However, the efficacy of serological diagnosis of aspergillosis in cattle remains controversial. Corbel (1972) and Knudtson et al. (1974) considered a precipitin assay useful as a supplementary test in the diagnosis of mycotic abortion, whereas Wiseman et al. (1984) found the specificity too low to justify its routine use. We have studied 1) the antibody response to the catalase antigen of A. fumigatus in experimentally infected cattle and 2) the prevalence of catalase antibodies and A. fumigatus precipitins in healthy and diseased cattle. The aim was to ascertain how far detection of antibodies to a defined fungal antigen can contribute to the often difficult diagnosis of mycosis.     

Studies on the association of Aspergillus fumigatus with ocular infections in animals

The occurrence and aetiological significance of Aspergillus fumigatus, an opportunistic pathogen, have been studied in 93 animals with various ophthalmological problems. A total of 93 eye swabs collected from 35 mules, 26 dogs, 13 fowl, 11 cattle, five buffaloes and three camels were investigated mycologically for the presence of A fumigatus. The pathogen was isolated in pure and heavy growth from the swabs from two dogs, one bull, one mule and one fowl. The fungus was also demonstrated directly in clinical material by the potassium hydroxide technique. A fumigatus could not be cultured from the buffaloes and camels. All the five cases had been treated with broad spectrum antibiotics and cortisone and two had received traumatic injury to the eyes (one mule and a bull). The organism was not isolated in pure culture from the conjunctival swabs of 22 apparently healthy animals (11 dogs, six mules, three fowl and two cattle). Many other saprophytic fungi were recovered in mixed cultures but were considered to be contaminants. The clinical signs and diagnostic criteria of oculomycosis have been discussed.     

Pathologic, hematologic, and serologic changes in rabbits given T-2 mycotoxin orally and exposed to aerosols of Aspergillus fumigatus conidia

The influence of immunosuppression by T-2 mycotoxin on the fungal disease aspergillosis was investigated in rabbits. Four groups of rabbits (groups 1A, 1B, 3A, and 3B) were given 0.5 mg of T-2 toxin/kg of body weight/day, PO; in addition, rabbits of groups 3A and 3B were exposed to aerosols of Aspergillus fumigatus conidia from days 7 through 16. Rabbits of groups 2A and 2B were exposed to A fumigatus aerosols, but were not given T-2 toxin, and rabbits of group 0 served as controls. Two rabbits of group 1A, 1 rabbit of group 1B, and 1 rabbit of group 3A died before scheduled necropsy. Rabbits of groups 1A, 2A, and 3A were killed and necropsied on day 17, and the remaining rabbits (groups 0, 1B, 2B, and 3B) were killed and necropsied on day 28. Changes caused by T-2 toxin included leukopenia, marginal anemia, and increased number of and morphologic changes in nucleated erythrocytes by day 21, followed by a regenerative hematologic response. Serum alkaline phosphatase and sorbitol dehydrogenase activities and antibody response to A fumigatus (as measured by an indirect hemagglutination test) were decreased by T-2 toxin ingestion. Rabbits with aspergillosis had leukocytosis, increased PCV, and increased antibody response to A fumigatus. Histologic lesions consisting of centrilobular hepatocellular swelling, portal and periportal fibrosis, and lymphocyte necrosis and/or depletion within secondary lymphoid tissue were observed in most rabbits treated with T-2 toxin. Normal defense mechanisms against A fumigatus infection were compromised by T-2 treatment, as evidenced by the severity and extent of lung lesions, greater number of hyphal elements observed, and greater number of colonies of A fumigatus isolated from rabbits of groups 3A and 3B. There were no significant changes in group-0 rabbits.     

Chronic obstructive pulmonary disease (COPD) in horses: aetiological studies: responses to intradermal and inhalation antigenic challenge.

Micropolyspora faeni and Aspergillus fumigatus were identified as common causes of respiratory hypersensitivity in horses affected with chronic obstructive pulmonary disease (COPD). Rye grass pollen and an Actinomycete evoked respiratory allergy in a few horses. Not infrequently, individual horses were found to have respiratory hypersensitivity to two or more antigens. The methods used to examine for allergy were intradermal testing and inhalation challenge with environmental antigens. An intradermal test using an M faeni extract was demonstrated to be suitable for diagnostic use in horses previously accurately diagnosed as suffering from COPD. In contrast, the A fumigatus antigen used proved unsatisfactory for such a purpose. Skin reaction to M faeni and A fumigatus extracts by horses affected with COPD indicated that the hypersensitivity was a dual one--a weak response shortly after injection followed by an Arthus-like response 4 to 8 hours later. As a parameter for monitoring responses to inhalation challenge, maximum intrathoracic pressure change (max delta Ppl) proved satisfactory, whereas changes in partial pressure of arterial oxygen (PaO2) did not.     

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.     

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.     

Pathogenesis of Aspergillus fumigatus infection in pigeons in the Sudan.

The pathogenesis of a pigeon isolate of Aspergillus fumigatus in a local breed of pigeons in the Sudan was tested. The spores inoculated intravenously resulted in an acute disease with 100% of mortality within six days. At necropsy, pinpoint and miliary lesions were prominent in the liver, spleen, lungs and kidneys. Histopathological examination detected lesions in the liver, heart, lungs, kidneys, spleen and brain. Hyphae and/or spores were encountered in all these organs. The presence of A. fumigatus was confirmed by reisolation from the liver, lungs and kidneys.     

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.     

Cavitary pulmonary lesion associated with Aspergillus fumigatus infection in a German shepherd dog

A two-year-old female German shepherd dog was presented with chronic cough and haemoptysis. Thoracic radiographs revealed a thin-walled cavitary lesion within a consolidated left cranial lung lobe. Bronchoalveolar lavage confirmed a concurrent bacterial infection; however, despite antibiotic and anthelmintic therapy the clinical signs failed to resolve. A left cranial lung lobectomy was performed. Histopathology and fungal culture confirmed the presence of Aspergillus fumigatus. The necrotic cavity had features compatible with a bronchial origin, possibly a form of cystic bronchiectasis, arising either as a congenital anomaly or acquired secondary to infection. Surgery provided resolution of clinical signs for just over a year before the dog deteriorated again and was subsequently euthanised. Necropsy was declined by the owners. This case report presents a unique presentation in which the predominant clinical sign was coughing due to pulmonary involvement. Aspergillus fumigatus was isolated from the left cranial lung lobe.     

Whole blood and tissue fungal DNA quantification in the diagnosis of canine sino-nasal aspergillosis

Various combinations of tests are used to confirm the diagnosis of canine sino-nasal aspergillosis (SNA) because false-positive and false-negative results can occur with each test. Therefore, the aim of this study was to evaluate whether detection of fungal DNA in blood and nasal tissue samples was of value in the clinical diagnosis of this disease. Four groups were included in the study (dogs with SNA, lymphoplasmacytic rhinitis or nasal neoplasia, and control animals). Real-time PCR assays detecting DNA from all Penicillium and Aspergillus species (PenAsp assay) or species-specific DNA from A. fumigatus, A. terreus, A. flavus and A. niger were applied to whole blood and nasal tissue samples. Results obtained by PCR were compared between the groups. Sensitivity, specificity, positive and negative predictive values (PPV and NPV) for fungal DNA detection were compared with those for alternative diagnostic procedures including histopathology, serology and fungal culture. Significantly more fungal DNA was detected by the PenAsp assay in tissue biopsies from dogs with SNA than in the three other groups. Sensitivity, specificity, PPV and NPV for this method were 1.00, 0.06, 0.32 and 1.00. A. fumigatus DNA was detected in seven tissue biopsies from dogs with SNA and in one biopsy from a dog with a nasal tumour. Sensitivity, specificity, PPV and NPV for this diagnostic test were 0.50, 0.97, 0.87 and 0.82. No significant difference was found between the groups with respect to the amount of DNA detected in blood by the PenAsp assay. Sensitivity, specificity, PPV and NPV for this method were 0.71, 0.24, 0.31 and 0.64. A. fumigatus DNA was detected in the blood of three dogs with SNA and sixteen dogs without SNA. Sensitivity, specificity, PPV and NPV for this diagnostic tool were 0.21, 0.45, 0.15 and 0.54. Detection of A. fumigatus DNA in nasal tissue had the highest specificity, PPV and NPV but sensitivity of this method was low. Detection of fungal DNA in whole blood was of no value in the diagnosis of SNA.     

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.     

肠道出血综合征研究进展

肠道出血综合征(HBS)是近年来发现的反刍动物的肠道疾病,其原因不甚明了。而从俄勒冈大学的实验中意外地发现,在HBS患病奶牛和对照奶牛的饲料、胃肠内容物、胃肠壁、淋巴结和血液等病料中都检测到烟曲霉菌和梭菌,怀疑这两种微生物可能是HBS的致病病原。经进一步研究表明,在HBS病牛和对照奶牛中都检测到梭菌毒素基因,而烟曲霉菌只在患病奶牛中检测到,对照牛则呈阴性。由此可见,烟曲霉菌与HBS之间具有密切的联系,烟曲霉菌很可能就是HBS的一个重要的病原学因素。目前,已有一种称为OmniGen-AF的产品可有效预防HBS的发生。     

Aspergillus fumigatus infection in an ostrich (Struthio camelus)

An 11-month-old female ostrich (Struthio camelus) had become gradually emaciated over a 2-week period and subsequently died. Necropsy revealed white to green mold growth on the walls of caseous thickened air sac membranes and multiple white necrotic foci in the lungs and liver. Histologically, the multiple exudative, necrotic and granulomatous lesions were compatible with mycotic infection in the air sacs and lungs, and hyphae positively reacted with a monoclonal antibody (Mab-WF-AF-1) to Aspergillus fumigatus wall fractions. Multifocal hepatic necrosis was also found, and several spores were observed in the blood vessels. Fungal culture of these lesions yielded pure growth of A. fumigatus. This is an established case of fatal A. fumigatus infection in an ostrich reared in Japan.     

Bovine mycotic abortion in Denmark

A survey is given of the occurrence of mycotic infections associated with abortion in Danish cattle. During a period of six years a total of 748 samples of placenta material were examined. Mycotic abortion was demonstrated in 101 cases (14%). The case rate was significantly higher (21%) during the winter months from December through February than during the rest of the year (10%). Abortions occurred as from the 135th day of pregnancy, with maximum in the eighth month, where 45% of all cases were observed. A. fumigatus was demonstrated in 77 cases, Mucor spp. in 11 cases, Absidia spp. in 5 cases, and a mixed infection with A. fumigatus and Mucor spp. in 3 cases. In 5 cases the fungi were demonstrated by microscopic and histological examination, but not by culture. The annual occurrence of mycotic abortion varied from 10% to 24% of cases of abortion examined. The latter figure was recorded in a year following a particularly heavy rainfall in the month of June.     

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.     

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.