Avian Bornavirus Associated with Fatal Disease in Psittacine Birds

Thanks to new technologies which enable rapid and unbiased screening for viral nucleic acids in clinical specimens, an impressive number of previously unknown viruses have recently been discovered. Two research groups independently identified a novel negative-strand RNA virus, now designated avian bornavirus (ABV), in parrots with proventricular dilatation disease (PDD), a severe lymphoplasmacytic ganglioneuritis of the gastrointestinal tract of psittacine birds that is frequently accompanied by encephalomyelitis. Since its discovery, ABV has been detected worldwide in many captive parrots and in one canary with PDD. ABV induced a PDD-like disease in experimentally infected cockatiels, strongly suggesting that ABV is highly pathogenic in psittacine birds. Until the discovery of ABV, the Bornaviridae family consisted of a single species, classical Borna disease virus (BDV), which is the causative agent of a progressive neurological disorder that affects primarily horses, sheep, and some other farm animals in central Europe. Although ABV and BDV share many biological features, there exist several interesting differences, which are discussed in this review.     

Venipuncture in psittacine birds

Techniques for jugular, basilic and medial metatarsal venipuncture in psittacine birds are discussed in detail. Although the number of animals used in the US for research annually is documented, birds and laboratory rats and mice are not included. It is presumed that the number of psittacine birds used for scientific purposes is relatively low. Therefore, only limited information is available about clinical techniques applicable to psittacine birds.     

Cyclopiazonic acid production by Aspergillus flavus and its effects on broiler chickens.

Cyclopiazonic acid (CPA) was purified from cultures of Aspergillus flavus, and ca. 14 g of the toxin was collected for use in feeding studies. Chicken rations were artificially contaminated with purified CPA at concentrations of 10, 50, and 100 ppm (microgram/g) and fed ad libitum to eight groups of chickens for 7 weeks. Chickens receiving feed with 100 ppm of CPA had high mortality, decreased weight gain, and poor feed conversion when compared with birds receiving other doses. Postmortem examination showed that chickens fed the two greatest doses of CPA had proventricular lesions characterized by mucosal erosion and hyperemia (100 ppm) and by thick mucosa and dilated proventricular lumens (50 ppm). Birds given 100 ppm of CPA in feed also had numerous yellow foci in their livers and spleens. Microscopic examination of tissues of birds that received 100 ppm of CPA revealed ulcerative proventriculitis, mucosal necrosis in the gizzard, and hepatic and splenic necrosis and inflammation. Birds given 50 ppm of CPA had hyperplasia of the proventricular mucosal epithelium. Birds given 10 ppm of CPA and control birds had no significant treatment-related lesions.     

Cyclopiazonic acid production by Aspergillus flavus and its effects on broiler chickens

Cyclopiazonic acid (CPA) was purified from cultures of Aspergillus flavus, and ca. 14 g of the toxin was collected for use in feeding studies. Chicken rations were artificially contaminated with purified CPA at concentrations of 10, 50, and 100 ppm (microgram/g) and fed ad libitum to eight groups of chickens for 7 weeks. Chickens receiving feed with 100 ppm of CPA had high mortality, decreased weight gain, and poor feed conversion when compared with birds receiving other doses. Postmortem examination showed that chickens fed the two greatest doses of CPA had proventricular lesions characterized by mucosal erosion and hyperemia (100 ppm) and by thick mucosa and dilated proventricular lumens (50 ppm). Birds given 100 ppm of CPA in feed also had numerous yellow foci in their livers and spleens. Microscopic examination of tissues of birds that received 100 ppm of CPA revealed ulcerative proventriculitis, mucosal necrosis in the gizzard, and hepatic and splenic necrosis and inflammation. Birds given 50 ppm of CPA had hyperplasia of the proventricular mucosal epithelium. Birds given 10 ppm of CPA and control birds had no significant treatment-related lesions.     

Cryptosporidiosis in zoo and pet birds.

Cryptosporidiosis is recognized as a primary disease in commercially raised chickens, turkeys, and bobwhite quail. Little is known about cryptosporidial infections in zoo and pet birds, although, infections of the small intestines, proventriculus, respiratory tract, and kidneys have been reported. In the present study, we reviewed cases of cryptosporidial infections in zoo and pet birds submitted to the State Veterinary Diagnostic Laboratory, Auburn, Alabama for necropsy or histopathologic examination. We identified infections in cockatiels, white-lored euphonias, bronze mannikin finches, and Australian diamond firetailed finches. Infections in the cockatiels occurred mostly in the small intestine, but parasites were also observed in the esophageal glands, air sacs, and proventriculus of some birds. Separate cases of small intestinal and proventricular infections were identified in the white-lored euphonias. Cryptosporidial parasites were found only in the proventriculus of the bronze mannikin finches and Australian diamond firetail finches. No cases of renal cryptosporidiosis were observed. Co-pathogens or other disease conditions were present in all birds. The Cryptosporidium species responsible for causing proventricular infection in zoo and pet birds may be different from C. meleagridis and C. baileyi.     

Characterization of Pasteurellaceae‐like bacteria isolated from clinically affected psittacine birds

Aims: The aim of the present investigation was to identify and characterize Pasteurella‐like isolates obtained from clinically affected psittacine birds. Methods and Results: A total of 37 isolates from psittacine birds tentatively classified with the family Pasteurellaceae were characterized phenotypically. The genetic relationship was investigated by sequencing of partial rpoB and 16S rRNA genes for selected isolates. The results obtained were compared with the data from 16 reference strains. Nine isolates were identified as Gallibacterium spp., 16 as Volucribacter spp. or Volucribacter‐like, while 11 isolates were classified as taxon 44 of Bisgaard. A single isolate was identified as Pasteurella multocida. Conclusions: Characterization of Pasteurellaceae by traditional methods is often inconclusive because of inconsistent reactions and phenotypic diversity. For the same reason, genotyping is essential to allow proper classification as demonstrated in the present study. Significance and Impact of the Study: Limited information exists on the isolation and significance of Pasteurellaceae associated with clinically affected psittacine birds showing signs of digestive and/or respiratory disorders. The present investigations demonstrated that these organisms are widely distributed among clinically affected birds, but isolation of these taxa cannot be unambiguously correlated with the symptoms observed.     

Duplex shuttle PCR for differential diagnosis of budgerigar fledgling disease and psittacine beak and feather disease

Two common viral diseases in psittacine birds including budgerigar fledgling disease (BFD), generally called avian polyomavirus (APV) infection, and psittacine beak and feather disease (PBFD) have similar clinical manifestations characterized by feather disorders. A duplex shuttle PCR was developed for detection of APV and PBFD virus (PBFDV). Two pairs of oligonucleotide primers were designed to amplify a 298-bp fragment of the t/T antigen region of APV genome and a 495-bp fragment of the capsid protein region encoded by open reading frame (ORF) C1 of PBFDV genome, respectively. In the present study, APV and PBFDV were detected simultaneously in one tube by duplex shuttle PCR using these two pairs of primers. The detection limits were 2 viral copies of APV and 3 viral copies of PBFDV. In the clinical application, we detected 16 APV-positive, 15 PBFDV-positive, and 3 mixed infected samples in 39 samples examined. Sequences of the amplified products were read. The t/T antigen region was conserved in the APV-positive samples as expected. ORF C1 of PBFDV genome showed diversity. Phylogenic analysis indicated that PBFDV ORF C1 consisted of 6 clusters which were related to subfamilies of psittacine birds. Our duplex shuttle PCR could be a useful method for differential diagnosis and molecular epidemiology of BFD and PBFD.     

THE ORIGIN OF STOMACH OIL IN THE PETRELS, WITH COMPARATIVE OBSERVATIONS ON THE AVIAN PROVENTRICULUS

The anatomy and histology of the proventriculus in several species of petrels were examined and compared with the conditions in other birds.
The proventriculus in petrels is comparatively very large and its mucosa is raised into longitudinal ridges; it thus has a much greater surface area than in other birds. The presence of lipoids other than triglycerides concentrated in large quantities, particularly in the outer parts of the epithelial cells of the proventricular glands of petrels, is demonstrated. Lipoids in the corresponding cells in other birds are minute in quantity.
The probability that these lipoids represent the stomach oil, or its precursor, immediately before secretion, is discussed and compared with other theories about the origin of the oil. The observations provide strong evidence, but not absolute proof, that the oil originates in the cells of the proventricular glands.
Suggestions about the possible function of petrel stomach oil are discussed. The oil may supplement the secretion of the preen gland; it may be an excreted by-product of the metabolism of excessively fat foods; or it may play an important part in water metabolism, especially in the nestling. Without further work, none of these theories can be upheld or refuted, and it is possible that all are partly correct, for they are not mutually exclusive.
Many petrels shoot stomach oil from the beak when they are disturbed. The possible origin of this habit from the escape reaction, found in many birds, of vomiting up the stomach contents when alarmed, is pointed out.     

A new eimerian species (Apicomplexa: Eimeriidae) from the blue-fronted Amazon parrot Amazona aestiva L. (Aves: Psittacidae) in Brazil

The Neotropical psittacine species Amazona aestiva, commonly known as the blue-fronted Amazon, is one of the most common and best-known psittacine birds kept as a pet worldwide. However, very little is known about the diseases or parasites of these birds. In this study, we describe a new species, Eimeria aestivae, associated with these parrots. The new species is characterized by: ovoid smooth oocysts (n = 60), 36.8 (33.2-41.5) × 23.7 (21.7-25.7) μm, length/width ratio = 1.55; polar granule present; ellipsoidal sporocysts (n = 25), 19.8 (17.5-21.6) × 9.3 (8.3-9.9) μm; Stieda, sub-Stieda body, and sporocyst residuum present. Sporozoites (n = 20), 2 per sporocyst, elongate and curved, 17.6 (15.8-19.2) × 3.8 (3.2-4.8) μm; each with 2 refractile bodies. The oocysts of the other 2 eimerian species described for Amazona are larger than those of the presented species, but they all seem to be closely related because of some similarities among them.     

Guidelines and ethical considerations for housing and management of psittacine birds used in research

The Psittaciformes are a large order of landbirds comprising over 350 species in about 83 genera. In 2009, 141 published studies implicated parrots as research subjects; in 31 of these studies, 483 individuals from 45 different species could be considered laboratory animals. Amazons and budgerigars were by far the most represented psittacine species. The laboratory research topics were categorized as either veterinary medicine and diagnostics (bacteriology, hematology, morphology, and reproduction; 45%) or behavioral and sensory studies (behavior, acoustics, and vision; 17%). Confinement of psittacine species for research purposes is a matter of concern as scientifically based species-specific housing guidelines are scarce. The aim of this article is to provide scientific information relevant to the laboratory confinement of Psittaciformes to promote the refinement of acquisition, housing, and maintenance practices of these birds as laboratory animals. We briefly discuss systematics, geographical distribution, legislation, and conservation status as background information on laboratory parrot confinement. The following section presents welfare concerns related to captive containment (including domestication status) and psittacine cognition. We then discuss considerations in the acquisition of laboratory parrots and review important management issues such as nutrition, zoonoses, housing, and environmental enrichment. The final section reviews indications of distress and compromised welfare.     

Pepsinogen Induction in Chick Stomach Epithelia by Reaggregated Proventricular Mesenchymal Cells In Vitro

In vitro organ culture system which permits embryonic chick proventriculus (glandular stomach) to synthesize pepsinogen de novo was developed. Explants of the proventricular rudiment were cultured on Millipore filters in Medium 199 with Earle's salts supplemented with 50% 12-day embryo extract at 38°C in 95% air and 5% CO₂.
In these culture conditions, pepsinogen, a functional marker protein of proventriculus, was first detected after 3 days of cultivation of 6-day chick proventricular rudiment. When recombined and cultured with 6-day proventricular mesenchyme, 6-day oesophageal, proventricular or gizzard (muscular stomach) epithelium expressed pepsinogen while small intestinal epithelium did not. These results were consistent with the previous results obtained by chorioallantoic membrane (CAM) grafting, and showed that the culture conditions are permissive for pepsinogen expression.
When recombined and cultured with reaggregated mesenchymal cells isolated from 6-day proventricular mesenchymal fragments, both 6-day proventricular and gizzard epithelia formed glandular structure and expressed pepsinogen. This indicates that the proventricular mesenchymal cells retain the ability to induce morphogenesis and cytodifferentiation of the proventricular epithelium even if the normal organization of proventricular mesenchyme is once destroyed.     

Evidence for the existence of two new members of the family Chlamydiaceae and proposal of Chlamydia avium sp. nov. and Chlamydia gallinacea sp. nov.

The family Chlamydiaceae with the recombined single genus Chlamydia currently comprises nine species, all of which are obligate intracellular organisms distinguished by a unique biphasic developmental cycle. Anecdotal evidence from epidemiological surveys in flocks of poultry, pigeons and psittacine birds have indicated the presence of non-classified chlamydial strains, some of which may act as pathogens. In the present study, phylogenetic analysis of ribosomal RNA and ompA genes, as well as multi-locus sequence analysis of 11 field isolates were conducted. All independent analyses assigned the strains into two different clades of monophyletic origin corresponding to pigeon and psittacine strains or poultry isolates, respectively. Comparative genome analysis involving the type strains of currently accepted Chlamydiaceae species and the designated type strains representing the two new clades confirmed that the latter could be classified into two different species as their average nucleotide identity (ANI) values were always below 94%, both with the closest relative species and between themselves.     

Detection of Avian bornavirus 5 RNA in Eclectus roratus with feather picking disorder

Avian bornavirus (ABV) was discovered recently in parrots with proventricular dilatation disease (PDD), a fatal neurological disease. Although ABV has been shown to be a causative agent of PDD, its virological characteristics are largely unknown. Here we report the detection of ABV genotype 5 RNA in an Eclectus roratus with feather picking disorder (FPD). Interestingly, although the bird was persistently infected with ABV5 for at least 8 months, it had no clinical signs of PDD. Although it remains unclear whether ABV5 is associated with FPD, these findings raise the importance of epidemiological studies of birds with diseases other than PDD.     

Localization of DNA-synthesizing cells and cell proliferation pattern in developing proventricular (glandular stomach) epithelium of embryonic and hatched chickens

Localization of DNA-synthesizing cells in the developing proventricular (glandular stomach) epithelium of embryonic and hatched chickens was investigated. DNA-synthesizing cells were scattered throughout the proventricular epithelium during all developmental stages studied. The results indicate that there is no clear proliferative zone in the proventricular epithelium of the chicken. The labeling indices (LI) of proventricular epithelial cells were measured. On the 6.5th day of incubation, the LI of glandular epithelium reached 29.5 ± 1.5%. the highest value of all the stages studied. This extremely rapid cell proliferation can be considered to be a driving force for the elongation of the proventricular glands during the following stages. Just after hatching, the LI of both the glandular and luminal (non-glandular) epithelia significantly increased from those on the 18th day of incubation. It is suggested that the rise in LI possibly reflected proventricular growth to fit in the change in the method of nourishment after hatching. In 2 week old chickens, the LI of both the glandular and luminal epithelia were reduced to approximately 1%. The active production of embryonic chicken pepsinogen in all glandular epithelial cells of the embryonic chicken revealed that proliferation and differentiation are not necessarily exclusive during the embryonic stages of proventricular development.     

Antibody selection for immunocytochemical characterization of the male reproductive system in Psittaciformes

The success of breeding programs is limited by the sparse knowledge about endocrine regulation and biochemical reactions in the psittacine male tract. The immunocytochemical analysis of parrots' testicular tissues provides an insight into their reproductive system but is often hampered by the lack of reliable antibodies. In the present study, we tested a large panel of antibodies raised against steroid receptors, steroidogenic enzymes, relaxin peptides including their receptors, and proliferation markers on paraffin sections of testicular tissue from eight psittacine genera representing three continents. All investigated species displayed the tested markers in somatic and germ cells of testis and epididymis, even though cell distribution was partly heterogenous and in species-specific patterns. The 17β-hydroxysteroid-dehydrogenase-2, 3β-hydroxysteroid-dehydrogenase, and smooth muscle actin allowed the cross-species differentiation between active and nonactive gonads. The remaining steroidogenic enzymes, steroid receptors, relaxin peptides, and Ki67 proved to be suitable to define reproductive activity depending on the parrot species. Adapting immunocytochemical methods to different psittacines was successful, though various cellular expression patterns do not allow the transfer of results among different parrot species. However, the availability of a reliable repertory of sexual markers is important to examine reproductive biology of psittacine birds.     

Encephalitozoon hellem in Two Eclectus Parrots (Eclectus roratus): Identification from Archival Tissues

ABSTRACT Members of the phylum Microspora are obligate, intracellular, single-celled parasites identified in a wide range of vertebrate and invertebrate hosts. Only a few cases of microsporidial infections have been documented in psittacine birds including peach-faced, masked, and Fischer's lovebirds ( Agapornis roseicollis, A. personata , and A. fischeri. respectively), budgerigars ( Melopsittacus undulatus ), and a double yellow-headed Amazon parrot ( Amazona ochrocephala ). Parasite identification has typically been limited to phylum or genus, and no avian species of microsporidia has clearly been described. In this report, microsporidia were identified in the kidney and intestine of a new host, the eclectus parrot ( Eclectus roratus ). Parasites were identified as Encephalitozoon hellem using morphologic, ultrastructural, and small subunit ribosomal RNA gene sequence data obtained from archived tissues. This parasite species was first identified in immunocompromised humans and may be a potential zoonotic pathogen. The epidemiology and prevalence of this parasite in humans and birds should be further explored.     

Developmental changes in the ability to express embryonic pepsinogen in the stomach epithelia of chick embryos

Summary The avian stomach is subdivided into two parts, the proventriculus and the gizzard. It has been shown that the gizzard epithelium can express embryonic chick pepsinogen (ECPg) antigen, a marker protein of the proventricular epithelium, as well as normal proventricular epithelium, under the appropriate experimental conditions. To study the possible mechanisms involved in the suppression of ECPg synthesis in the gizzard epithelium during normal development, we carried out heterotypic and heterochronic recombination experiments of the epithelium and mesenchyme of these two organ rudiments. When recombined and cultured with 6-day proventricular mesenchyme, gizzard epithelium of 3.5- to 12-day embryos expressed pepsinogen at all stages tested. However, the ratio of ECPg-positive cells to total epithelial cells in the gizzard epithelium decreased rapidly when epithelium older than 7 days was cultured with proventricular mesenchyme. In contrast to proventricular mesenchyme, 6-day gizzard mesenchyme did not allow ECPg expression in associated proventricular epithelium of 3.5- to 7-day embryos. These results indicate that gizzard epithelium does not express pepsinogen in normal development because of both a decrease in ability to express the enzyme in itself in the course of development and a repressive influence of gizzard mesenchyme.     

Immunotyping of Chlamydia psittaci by indirect immunofluorescence antibody test with monoclonal antibodies

Monoclonal antibodies against pigeon and budgerigar strains of Chlamydia psittaci were used to classify the immunotypes of C. psittaci strains by an indirect immunofluorescence antibody (IFA) test. Thirty-three C. psittaci strains from pigeons and 24 from budgerigars were divided into three immunotypes (P-I, P-II, and P-III) and (B-I, B-II, and B-III), respectively. Two strains from human psittacosis patients were identified as P-III and B-I, coinciding with the epidemiological evidence of each human infection. Two strains from psittacine birds, a parrot and a parakeet, were identical to the B-II immunotype. Other mammalian strains were quite distinct from avian strains in their IFA reaction with the monoclonal antibodies.     

Gizzard epithelium of chick embryos can express embryonic pepsinogen antigen,a marker protein of proventriculus

Summary The avian stomach is composed of two distinct organs, the proventriculus and the gizzard. Pepsinogen expression in the proventricular and gizzard epithelia of chick embryos was investigated immunohistochemically with anti-embryonic chick pepsinogen (anti-ECPg) antiserum. In normal development, the ECPg antigen was expressed only in the glandular epithelial cells of the embryonic proventriculus from the 8th day of incubation onwards. However, both proventricular and gizzard epithelia of 6-day embryos expressed the ECPg antigen when recombined and cultured with the proventricular mesenchyme. Chronological studies revealed that the ECPg antigen was first detected in a few epithelial cells at 3 days of cultivation. The percentage of ECPg-positive cells among the total epithelial cells in each recombinant increased with the length of the culture period and all the glandular epithelial cells were positive at 9 days. During this process, the percentage of ECPg-positive cells in each cultured recombinant was similar in proventricular and gizzard epithelia. Moreover, both epithelia could express the ECPg antigen when recombined and cultured with the oesophageal or small-intestine mesenchyme for 9 days, though the percentage of ECPg-positive cells in each cultured recombinant was much lower than that in the cultured recombinant with the proventricular mesenchyme. These results indicate that the gizzard epithelium of 6-day chick embryos possesses a similar potential for pepsinogen expression as the proventricular epithelium of the same age.