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.     

Fungal diseases of birds of prey.

Aspergillosis and candidiasis are ranked among the most common infectious diseases in birds of prey. The prevention of these fungal diseases is often easier than treatment. Thus the clinician should strive to prevent infection by minimizing stress, maintaining a healthy environment, limiting long-term use of antibiotics and corticosteroids, and reducing exposure to fungal organisms. Although less commonly diagnosed among wild, free-ranging birds of prey, a high incidence in a free-ranging population should make the clinician think of an immunocompromising factor (i.e., toxins, human encroachment or low prey base) that may be contributing to infection. The diagnosis of aspergillosis and candidiasis often requires more than just the identification of the agent, as these ubiquitous organisms often are cultured from healthy birds of prey. In those birds of prey in which a fungal infection is highly suspected or proven, antifungal drugs remain the mainstay of treatment, although available drugs and modes of delivery have improved in recent years.     

Strains of avian paramyxovirus type 1 of low pathogenicity for chickens isolated from poultry and wild birds in Denmark

Twenty-one strains of avian paramyxovirus type 1 of low virulence for chickens were isolated in Denmark between 1996 and the beginning of 2003. The low virulence of the strains was demonstrated by sequencing the fusion (F) gene at the cleavage site motif and in some cases by determining the intracerebral pathogenicity index in day-old chicks. By using a panel of monoclonal antibodies it was shown that the isolates belonged to four different antigenic groups (five C2 isolates, six E isolates, six H isolates and four G/Q isolates). They were placed in three distinguishable genetic groups by phylogenetic analysis of a partial sequence of the F gene. The origin of the six E isolates was probably contaminated vaccines; the other viruses were isolated from wild birds and from poultry which probably came into contact with wild birds.     

Congo red medium to distinguish between invasive and non-invasive Escherichia coli pathogenic for poultry

In the course of our molecular studies of virulence factors associated with invasive avian Escherichia coli infections, it was first necessary to distinguish between common E. coli and those that cause septicemia in poultry. We found a direct correlation between the ability of clinical isolates of E. coli to bind Congo red dye (CR) and their ability to cause septicemic infection in chickens. This finding was supported by bacteriological studies of 30 broiler flocks (26 sick and 4 healthy) and by virulence studies in chickens and mice. All 144 isolates of E. coli from internal tissues of diseased birds were determined to be CR-positive (red colonies). Congo-red-positive E. coli colonies were isolated from air sacs, pericardium, liver, lung, joint fluid, and heart blood of chickens with lesions of colisepticemia. In contrast, of 170 E. coli isolates from the poultry house environment and from the trachea and cloaca of healthy birds, more than half were CR-negative (white colonies). No CR-negative (white) E. coli colonies were found in internal organs from birds with typical lesions of colisepticemia. We feel that these preliminary findings suggest that the CR dye binding could be used as a phenotypic marker to distinguish between invasive and noninvasive isolates.     

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.     

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.     

Articular aspergillosis of hip joints in turkeys

Aspergillosis is an important cause of morbidity and mortality in birds. Turkey poults are known to be particularly susceptible to fungal infection. Although the respiratory tract is the most commonly affected, dissemination can occur into virtually any organ. Here, we report an unusual outbreak of articular aspergillosis in a flock of meat turkeys with clinical signs of lameness. Between 7 and 11 weeks of age, turkeys had severe granulomatous osteoarthritis of the hip joints with necrosis of the femur head. Fungal morphology and PCR amplification and sequencing of the first ITS1-5.8S-ITS2 rDNA region identified Aspergillus fumigatus as the infectious agent. Concurrently, Staphylococcus spp. was isolated from the hip joints, which may have promoted the tropism of the fungus. Mild respiratory tract aspergillosis was observed in only one case. The findings suggest that fungal arthritis may present a specific disease entity in turkeys and should be considered as further cause of lameness in turkeys.     

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.     

Occurrence and diagnostic relevance of virulence-associated factors in Streptococcus suis

Streptococcus suis (Sc. suis) can cause very different clinical entities. In contrast to Sc. suis-associated pneumonia, the induction of meningitis, septicemia, and polyarthritis by certain Sc. suis strains requires the expression of virulence factors that contribute to the invasiveness of the pathogen. In the presented study, we examined the occurrence of known virulence-associated factors in Sc. suis isolates from samples sent to the Institute of Microbiology, School of Veterinary Medicine Hannover, in order to evaluate their significance as potential virulence factors in different disease complexes in Northern Germany. The results show that (i) MRP + EF + serotype 2 and MRP* EF-serotype 9 strains are statistically significant associated with the disease complex meningitis/septicemia/arthritis and, thus, have to be considered invasive strains, (ii) serotyping alone is not sufficient for identification of virulent strains, (iii) there is a remarkable heterogeneity among pneumonia-associated Sc. suis strains and (iv) activity of haemolysin or suilysin appears to be not appropriate as virulence marker. Finally, it has to be noted that at present only half of the Sc. suis isolates from pigs with meningitis/septicemia/poyarthritis can be characterised by the detection of virulence-associated factors. Thus, the identification and characterisation of additional, serotype independent virulence factors of Sc. suis is a very important issue in future studies.     

Virulence of pigeon-origin Newcastle disease virus isolates for domestic chickens.

The virulence of six pigeon-origin isolates of Newcastle disease virus (NDV) was evaluated before and after passage in white leghorn chickens. Four isolates were defined as pigeon paramyxovirus-1 (PPMV-1) and two isolates were classified as avian paramyxovirus-1 (APMV-1) with NDV monoclonal antibodies. The four PPMV-1 isolates were passaged four times in chickens, and the APMV-1 isolates were passaged only once. Infected birds were monitored clinically and euthanatized. Tissues were collected for histopathology, in situ hybridization with a NDV matrix gene digoxigenin-labeled riboprobe, and immunohistochemistry with an anti-peptide antibody to the nucleoprotein. Mean death time, intracerebral pathogenicity index, and intravenous pathogenicity index tests performed before and after passage in chickens demonstrated increased virulence of the passaged PPMV-1 isolates and high virulence of the original isolates of APMV-1. Sequence analysis of the fusion protein cleavage site of all six isolates demonstrated a sequence typical of the virulent pathotype. Although the pathotyping results indicated a virulence increase of all passaged PPMV-1 isolates, clinical disease was limited to depression and some nervous signs in only some of the 4-wk-old specific-pathogen-free white leghorns inoculated intraconjunctivally. However, an increased frequency of clinical signs and some mortality occurred in 2 wk olds inoculated intraconjunctivally with passaged virus. Histologically, prominent lesions in heart and brain were observed in birds among all four groups inoculated with the PPMV-1 isolates. The behavior of the two pigeon-origin APMV-1 isolates when inoculated into chickens was characteristic of velogenic viscerotropic NDVs and included necro-hemorrhagic lesions in the gastrointestinal tract.     

Association between avian necrotic enteritis and Clostridium perfringens strains expressing NetB toxin

A novel toxin, NetB, has recently been identified in virulent avian Clostridium perfringens isolates and shown to be an essential virulence factor in a clinical necrotic enteritis isolate. To assess whether NetB is more generally associated with avian necrotic enteritis isolates we have screened a range of C. perfringens strains from geographically diverse locations for both the presence and expression of the netB gene. Forty-four isolates were derived from necrotic enteritis disease cases from Australia, Belgium, Denmark and Canada and 55 isolates from healthy chickens from Australia and Belgium. The majority of strains isolated from necrotic enteritis-affected birds were netB positive (70%) and there was an absolute correlation between the presence of netB and in vitro expression of the NetB protein. Only two of the C. perfringens isolates from healthy chickens carried netB. Sequencing of the netB gene from 23 positive isolates showed that NetB is highly conserved, with only one predicted amino acid (A168T) difference, in six isolates, compared to the published sequence. This change did not alter the in vitro activity of the NetB toxin. The gene encoding the recently discovered TpeL toxin was also screened using PCR and only found in a small proportion of NetB-positive isolates from diseased birds. A selection of NetB-negative isolates, originating from diseased birds, was unable to cause disease in a necrotic enteritis induction model. This study provides further evidence that NetB is important in pathogenesis and advances our current understanding of C. perfringens virulence factors in avian necrotic enteritis.     

雀形目野生鸟类REV感染状况的调查及分离株gp90基因序列分析

为了解雀形目野生鸟类感染禽内皮组织增生症病毒（Reticuloendotheliosis virus,REV）的情况,采集了352份样品,应用PCR方法进行了初筛。然后将阳性样品适当处理后接种CEF细胞,利用IFA等方法进一步鉴定。结果分离到2株病毒WB11042和WB11043,分别来自黄喉鹀和燕雀。对2株REV分离株的gp90基因进行扩增、测序和序列分析。结果显示,其gp90序列与REV亚型Ⅲ的代表毒株（CSV、APC-566）亲缘关系最近,高达99%以上;与亚型Ⅱ代表株（SNV）和亚型Ⅰ代表株（HA9901）的亲缘关系相对较远,在95.5%~97.1%之间。遗传进化树分析也表明这2株野生鸟类分离株属于REV亚型Ⅲ。结果表明,雀形目野生鸟类也可以感染REV。     

Chaunocephalus ferox in free-living white storks in central Spain

Ten out of 42 (23.8%) white storks (Ciconia ciconia) admitted to two rehabilitation centers in central Spain had lesions caused by the trematode Chaunocephalus ferox in the small intestinal wall. Fourteen of the examined birds were adults, five were subadults, and 23 were chicks of various ages. Parasitation was 32% (n = 8) in chicks and 13% (n = 2) in adult birds, whereas no juvenile bird was affected. Among dead birds, stork chicks affected by C. ferox lesions had a lower body weight (2196.1 g, SD = 814.2) than storks without lesions (2965.8 g, SD = 742.9, P < 0.05). Two chicks were additionally infected with Salmonella subspecies I serotype enteritidis 1,9,12: g, m:1, 7. Prevalence of the parasite in the examined birds was lower than in a population of Asian open-billed storks (Anastomus oscitans), in which it was pathogenic due to the destruction of the tunica muscularis and formation of large granulomatous lesions in the wall of the postduodenal portion of the small intestine. Pathogenic alterations caused by C. ferox are presumed to be related to numbers of adults present. Because storks admitted to rehabilitation centers suffer stress due to various reasons that may lower their immune response and exacerbate existing infections, the analysis of fecal sediments of white storks admitted for rehabilitation is recommended.     

Haemolytic Escherichia coli isolated from dogs with diarrhea have characteristics of both uropathogenic and necrotoxigenic strains

Twenty-four haemolytic Escherichia coli strains were isolated from dogs with diarrhea. The strains were serotyped and analysed by polymerase chain reaction (PCR) for genes encoding virulence factors associated with E. coli that cause diarrhea in animals. Adhesion antigen production was deduced from haemagglutination experiments. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of heat extracts was also used as an indication for the production of adhesive structures. The majority of the strains was shown to produce this type of virulence factor. Adhesion and invasion tests of the strains and Caco-2 cells showed that all strains adhered and that two were invasive. The two invasive strains were positive in the intimin PCR and one of them also contained genes encoding CS31A. The PCR for heat stable toxin (ST) was positive in only four strains, as was the presence of F17 fimbrial genes. Surprisingly, 19 strains had intact P fimbrial operons, coding for an adhesin involved in urinary tract infection (UTI). The cytotoxic necrotising factor 1 (CNF1) gene, also mainly found in UTI was likewise detected in these 19 strains. Cytolethal distending toxin (Cdt) genes were found in five strains. The high number of strains positive for CNF1 and P fimbriae prompted us to test the strains in a multiplex PCR used to test E. coli isolated from UTI in various species for 30 virulence associated genes. The data showed that the majority of the diarrhea isolates have virulence factor profiles highly similar to UTI E. coli isolates from dogs. This raises the question whether these isolates are real intestinal pathogens or "innocent bystanders". However, since CNF1 producing necrotoxic E. coli (NTEC) strains isolated from humans, pigs and calves with diarrhea appear to be highly related to our strains, it might be that in dogs this type of isolate is capable of causing not only UTI, but also diarrhea. If this is the case and this type of isolate is "bifunctional", domestic animals likely constitute a reservoir of NTEC strains which can be also pathogenic for humans.     

Virulence genes in bla(CTX-M) Escherichia coli isolates from chickens and humans

The aim of this study was to determine the presence of virulence genes in isolates of CTX-M Escherichia coli from diseased chickens, from healthy chickens and from urinary tract infections in people. Three CTX-M E. coli strains from three different instances of disease in poultry (two of which were E. coli related) were tested for bla(CTX-M) sequence type and replicon type. Additionally, they were tested for the presence of 56 virulence genes (encoding fimbriae, adhesins, toxins, microcins and iron acquisition genes) using a micro-array. Results were compared to the virulence genes present in isolates from 26 healthy chickens and from 10 people with urinary tract infections. All genes found in isolates from diseased birds, including the astA (heat stable toxin) and tsh (temperature sensitive haemagglutinin) genes which have previously been associated with colibacillosis in chickens, were also present in isolates from healthy birds. However, 6/10 of the virulence genes found were exclusive to isolates from humans. Genes exclusive to chicken isolates included ireA (sidephore receptor), lpfA (long polar fimbriae), mchF (microcin transporter protein) and tsh whilst genes exclusive to human isolates included ctdB (cytolethal distending toxin), nfaE (non-fimbrial adhesion), senB (plasmid encoded enterotoxin) and toxB (toxin B). The results support previous findings that CTX-M E. coli strains in chickens are generally different from those causing disease in humans, but genes such as astA and tsh in isolates from diseased birds with colisepticaemia were also present in isolates from healthy birds.     

Experimental infection of house sparrows (Passer domesticus) with West Nile virus isolates of Euro-Mediterranean and North American origins

West Nile virus (WNV) is a zoonotic arboviral pathogen transmitted by mosquitoes in a cycle involving wild birds as reservoir hosts. The virus has recently emerged in North America and re-emerged in Europe. North American WNV outbreaks are often accompanied by high mortality in wild birds, a feature that is uncommon in Europe. The reason for this difference is unknown, but the intrinsic virulence of the viruses circulating in each continent and/or the susceptibility to the disease of Palearctic as opposed to Nearctic wild bird species could play a role. To assess this question, experimental inoculations with four lineage 1 WNV strains, three from southern Europe (Italy/2008, Italy/2009 and Spain/2007) and one from North America (NY99) were performed on house sparrows (Passer domesticus), a wild passerine common in both continents. Non-significant differences which ranged from 0% to 25% were observed in mortality for the different WNV strains. Viremias lasted from 1 to 5–6 days post-inoculation (dpi) in all cases; individuals inoculated with NY99 had significantly higher titres than those inoculated with any of the Euro-Mediterranean strains. Remarkably, host competence was found to be higher for NY99 than for the other strains. Consequently, albeit being pathogenic for house sparrows, some Euro-Mediterranean strains had reduced capacity for replication in -and transmission from- this host, as compared to the NY99 strain. If applicable also to other wild bird host species, this relatively reduced transmission capacity of the Euro-Mediterranean strains could explain the lower incidence of this disease in wild birds in the Euro-Mediterranean area.     

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.     

Complement resistance,as determined by viable count and flow cytometric methods,and its association with the presence of iss and the virulence of avian Escherichia coli

Previous work in our labs has shown that avian Escherichia coli virulence is correlated with resistance to complement. Also, our studies have revealed that the presence of the increased serum survival gene (iss), known to contribute to the complement resistance and virulence of mammalian E. coli, may predict the virulent nature of an avian E. coli isolate. This relationship warrants further research, but further clarification of the relationship among virulence, complement resistance, and iss sequences requires use of complement susceptibility assays. Such assays, unfortunately, are labor-intensive, expensive, and difficult to perform. In the present study, the results of two complement susceptibility assays for 20 E. coli isolates, 10 incriminated in avian colibacillosis and 10 from the intestinal tracts of apparently healthy birds, were compared in an attempt to determine if flow cytometric analysis was a reasonable alternative to a viable count assay. In addition, the virulence of these isolates for chick embryos was determined, and each isolate was examined for the presence of iss using amplification techniques. The flow cytometric method was found to be repeatable for most isolates, and its results showed moderate agreement with those obtained through viable counts. All intestinal isolates of healthy birds proved avirulent using the embryo lethality assay; however, not all isolates from sick birds were demonstrated to be virulent. Possible explanations of these results include that the methods originally used to isolate these organisms failed to detect the illness-inciting strains or that the virulence of these strains had declined following initial isolation. Additionally, we must consider the possibility that the embryo lethality assay of virulence used here might not be sensitive enough to detect differences between these two groups of isolates. Also, it should be noted that virulence assays, such as the one used here, fail to account for predisposing host or environmental conditions, enabling a less virulent isolate to cause disease under natural conditions. Interestingly, the complement resistance of a strain was significantly associated with its lethality in embryos, and iss-containing isolates were significantly more likely than those lacking iss to be classified as complement-resistant and virulent. Such results, at least for this group of avian E. coli, suggest that there is a compelling but imperfect relationship among complement resistance, virulence, and the presence of iss. These results also suggest that the flow cytometric assay may be a reasonable alternative to the viable count method of determining complement resistance.     

Toxoplasma gondii antibodies in the white stork Ciconia ciconia

The prevalence of Toxoplasma gondii in chicks of wild birds and captive individuals was studied in the Poznań environs and in the Poznań Zoological Garden in the years 2002-2003. Bird blood was tested for T. gondii antibodies by an indirect fluorescent antibody test. T. gondii antibodies were detected from 5.8% of 205 analysed white stork chicks and 13.6% of 44 analysed adult storks in the zoo. Because toxoplasmosis is one of the more common parasitic zoonoses worldwide, we briefly discuss the potential epidemiological importance of stork toxoplasmosis to humans.     

Experimental Aspergillus fumigatus infection in quails and results of treatment with itraconazole

This study was performed to investigate (i). the clinical, histopathological and biochemical changes in quails (Coturnix coturnix japonica) with experimentally induced aspergillosis; and (ii). the efficiency of itraconazole treatment on these infected birds. A total of 18021-day-old male quails was randomly divided into three groups (control, infected untreated and infected treated), each containing 60. The experimental infection was set by intratracheal inoculation of 0.2 ml inoculum of Aspergillus fumigatus (CBS 113.26 strain) consisting of approximately 2.7 x 106 spores/ml. Two days after the inoculation, general clinical signs of aspergillosis in the respiratory tract were observed. In the histopathological examination, caseous foci were found in lungs, trachea and on airsacs. All quails died in the infected untreated group. Aspergillus fumigatus was isolated from the various organs of all dead quails. There was no significant change in serum aspartate aminotransferase (AST) and gamma-glutamyltransferase (GGT) activities in infected untreated birds compared with controls. However, alanine aminotransferase (ALT) activity, albumin and calcium levels, and albumin/globulin (A/G) ratio were lower while phosphorus and globulin levels were higher in the infected untreated group than in controls. Each quail in the infected treated group was given 10 mg/kg/day itraconazole via drinking water for 7 days immediately after the first clinical findings. Although all quails died in the infected untreated group, 41 quails survived in the itraconazole treatment group. Biochemical values also returned approximately to the control levels after the treatment. The conclusion was drawn that aspergillosis in the quails might cause economical losses because of high mortality. Oral itraconazole treatment of aspergillosis might lower the mortality rate in quails.