Production of a rabbit anti-cockatiel immunoglobulin G and characterization of its cross-reactivities with immunoglobulin G of other psittacine species

The purpose of this work was to produce rabbit anti-cockatiel immunoglobulin G (IgG) and compare its cross-reactivity with sera from eight other psittacine birds: Quaker parakeet, budgerigar, green-wing macaw, blue-fronted Amazon parrot, eclectus parrot, African grey parrot, Patagonian conure, Moluccan cockatoo. Cockatiel IgG did not bind to protein A or G; therefore, these proteins could not be used in column chromatography to isolate the IgG. A combination of serum IgG precipitation by ammonium sulfate and yolk IgG extraction from egg was loaded in sodium dodecyl sulfate-polyacrylamide gel upon which the IgG was resolved by electrophoresis. The resolved IgG in sodium dodecyl sulfate-polyacrylamide gel was stained with Coomassie blue, cut, crushed in phosphate-buffered saline, and injected into rabbits. The rabbit anti-cockatiel IgG produced in this way reacted with a single protein in gel immunodiffusion assay with all nine psittacine bird sera but not with those of chicken and ostrich. Immunoelectrophoresis confirmed the cross-reactivity of different psittacine sera with the anti-cockatiel IgG serum but not with ostrich and chicken sera. This antiserum detected antibody responses in sera from cockatiels vaccinated against chlamydial major outer membrane protein in an immunoblot assay.     

Chlamydophila psittaci in free-living Blue-fronted Amazon parrots (Amazona aestiva) and Hyacinth macaws (Anodorhynchus hyacinthinus) in the Pantanal of Mato Grosso do Sul, Brazil

Chlamydophila psittaci (C. psittaci) infection was evaluated in 77 free-living nestlings of Blue-fronted Amazon parrots (Amazona aestiva) and Hyacinth macaws (Anodorhynchus hyacinthinus) in the Pantanal of Mato Grosso do Sul, Brazil. Tracheal and cloacal swab samples from 32 wild parrot and 45 macaw nestlings were submitted to semi-nested PCR, while serum samples were submitted to complement fixation test (CFT). Although all 32 Amazon parrot serum samples were negative by CFT, cloacal swabs from two birds were positive for Chlamydophila DNA by semi-nested PCR (6.3%); these positive birds were 32 and 45 days old. In macaws, tracheal and cloacal swabs were positive in 8.9% and 26.7% of the samples, respectively. Complement-fixing antibodies were detected in 4.8% of the macaw nestlings; macaw nestlings with positive findings were between 33 and 88 days old. These results indicate widespread dissemination of this pathogen in the two evaluated psittacine populations. No birds had clinical signs suggestive of chlamydiosis. To the best of our knowledge, this is the first report on C. psittaci in free-living Blue-fronted Amazon parrots and Hyacinth macaws in Brazil.     

Avian polyomavirus infection and disease in a green aracaris (Pteroglossus viridis).

Avian polyomavirus (APV) is one of the most significant pathogens of domestically raised psittacine birds (parrots). One or more APVs are suspected to infect nonpsittacine cage birds, but the relationship of these viruses to the APV infecting parrots remains unclear. In this report, for the first time, we fully document an APV infection in a nonpsittacine cage bird, a green aracaris (Pteroglossus viridis). Grossly, this bird evidenced generalized hemorrhage. Histologically, there was severe hepatic necrosis, splenic necrosis, and the presence of lightly basophilic to clear pannuclear inclusion bodies and karyomegaly in splenocytes and renal mesangeal cells, all characteristic lesions of APV infection in parrots. APV DNA was amplified directly from the liver by polymerase chain reaction and sequenced. The virus differed from the original APV sequence by only 24 base pairs (0.48% of the genome), demonstrating that it is a variant of the APV. A serologic survey of the remaining birds in the aviary demonstrated anti-APV antibody in two cockatoos, two cockatiels, a laughing kookaburra, a Lady Ross turaco, and five zebra finches. The remaining green aracaris was seronegative. The sequence and serologic data suggest that the APV that infected the green aracaris originated in a parrot and was capable of infecting birds from at least four orders.     

PCR结合斑点杂交技术检测禽多瘤病毒方法的建立及应用

鹦鹉幼雏病是由禽类多瘤病毒(APV)引起的多种鹦鹉雏鸟死亡的急性病毒性传染病,严重危害鹦鹉养殖业的健康发展。为提高分子生物学方法检测APV的敏感性和特异性,对APV基因片段进行克隆和序列分析,设计合成1对特异性引物,以VP1基因为模板,经PCR扩增获得731 bp的核苷酸DNA,并用DIG标记,制备用于检测APV的特异性核酸探针;对制备的探针进行灵敏度检测,同时与普通PCR进行敏感性比较;使用制备的探针,对经分离鉴定和制备保存的其他7种禽病毒核酸进行特异性检测;用该核酸探针,对疑似感染APV的鹦鹉病料进行斑点杂交检测,并对鉴定为阳性的APV进行全基因组扩增和序列分析。结果显示:该探针可检测到2 pg量的APV特异性核酸片段;仅APV-VP1阳性核酸显色,呈现阳性反应,而阴性核酸和其他7种禽病毒核酸均不显色,呈阴性反应。结果表明:建立的核酸斑点杂交检测方法具有较高的灵敏度和特异性,可用于临床初步诊断。本方法的建立为我国开展APV分子流行病学调查及其感染的临床诊断提供了技术支撑。     

The pathogenicity of four avian influenza viruses for fowls, turkeys and ducks

Groups of 10 two-week-old chicks, turkey poults and ducklings were each infected by the intranasal route with one of four avian influenza viruses: a/fowl/Germany/34 (Hav 1N))--Rostock, A/FPV/Dutch/27 (Hav 1 Neq 1)--Dutch, A/fowl/Victoria/75 (Hav 1 Neq 1)--Australian, and A/parrot/Ulster/73 (Hav 1 N1)--Ulster. Eight hours after infection 10 birds of the same age and species were placed in contact with each group and allowed to mix. The clinical signs of disease and onset of sickness and death were recorded. Ulster virus was completely avirulent for all birds. Rostock, Dutch and Australian viruses were virulent for fowls and turkeys causing death in all birds with the exception of 3/10 in contact fowls from the Rostock virus group and 2/10 in contact fowls from the Australian virus group. Only Rostock virus caused sicked sickness or death in ducks, 9/10 intranasally infected and 6/7 in contact birds showed clinical signs and 2/10 intranasally infected and 3/7 in contact ducks died. Intranasal and in contact pathogenicity indices were calculated for each virus in each bird species and indicated quantitatively the differences in virulence of the four virus strains. Virus isolation and immune response studies indicated that surviving in contact fowls in the Rostock virus group had never been infected but that surviving Australian virus in contact fowls had recovered from infection. Infection was not established in Ulster virus in contact fowls and Australian virus intranasally infected and in contact ducks. The birds in all other groups showed positive virus isolations and a high incidence of positive immune response. The last virus isolation was made at 22 days after intranasal infection of ducks with Ulster virus.     

Comparative genomic analysis of eight Leptospira strains from Japan and the Philippines revealing the existence of four putative novel genomic islands/islets in L. interrogans serovar Lai strain 56601

Leptospirosis is one of the most widespread zoonotic diseases worldwide and can be considered an emerging health problem to both human and animal. Despite the importance of the disease, complete genome sequences are currently available for only three Leptospira interrogans strains: 56601, Fiocruz L1-130, and IPAV. Therefore, intra- and inter-species comparative genomic analyses of Leptospira are limited. Here, to advance current knowledge of the genomic differences within Leptospira species, next-generation sequencing technology was used to examine the genomes of eight L. interrogans strains belonging to six different serogroups isolated from humans and dogs in Japan and the Philippines. The genomic sequences were mapped to that of the reference strain, L. interrogans serovar Lai strain 56601. The results revealed the presence of four novel genomic islands/islets (GIs) in strain 56601. This study provides a deeper insight into the molecular basis and evolutionary perspective of the virulence of leptospires.     

Isolation,in vitro propagation,genetic analysis,and immunogenic characterization of an Ehrlichia canis strain from southeastern Brazil

Amplification of the 16S rRNA gene from a blood sample obtained from a dog in southeastern Brazil was used to confirm a naturally acquired Ehrlichia (E.) canis infection. Following isolation and culturing of the new bacterial strain called Uberlândia, partial sequences of the dsb and p28 genes were obtained. The dsb partial sequence of the novel strain was 100% similar to dsb gene sequences of E. canis obtained from different geographic areas around the world. Conversely, the p28 partial sequence for the E. canis Uberlândia strain differed at several nucleotides from other sequences available in GenBank. To confirm the antigenic profile of the Uberlândia strain, an indirect immunofluorescence assay against E. canis antigens was performed using dog sera collected from two different areas in Brazil (Uberlândia and São Paulo). The results suggest that both antigens were able to identify animals seropositive for E. canis in Brazil since these Brazilian strains appear to be highly conserved.     

Multi-locus sequence typing of Salmonella enterica subsp. enterica serovar Enteritidis strains in Japan between 1973 and 2004

Salmonella enterica subsp. enterica serovar Enteritidis (S. Enteritidis) was responsible for a worldwide pandemic during the 1980s and 1990s; however, changes in the dominant lineage before and after this event remain unknown. This study determined S. Enteritidis lineages before and after this pandemic event in Japan using multilocus sequence typing (MLST). Thirty S. Enteritidis strains were collected in Japan between 1973 and 2004, consisting of 27 human strains from individual episodes, a bovine strain, a liquid egg strain and an eggshell strain. Strains showed nine phage types and 17 pulsed-field profiles with pulsed-field gel electrophoresis. All strains had homologous type 11 sequences without any nucleotide differences in seven housekeeping genes. These MLST results suggest that S. Enteritidis with the diversities revealed by phage typing and pulsed-field profiling has a highly clonal population. Although type 11 S. Enteritidis may exhibit both pleiotropic surface structure and pulsed-field type variation, it is likely to be a stable lineage derived from an ancestor before the 1980s and/or 1990s pandemic in Japan.     

Phylogenetic analysis of small ruminant lentiviruses detected in Slovenia

Small ruminant lentiviruses (SRLV), which belong to the Retroviridae family, infect goats and sheep worldwide. The aim of this study was to characterize the SRLV strains circulating in Slovenia, by phylogenetic analysis of two genomic regions, 1.8 kb gag-pol fragment and 1.2 kb pol fragment. The results of our study revealed that Slovenian SRLV strains are highly heterogeneous, with ovine strains belonging to genotype A and caprine strains to genotypes A and B. The closest relatives of sheep virus sequences from two flocks that clustered together (SLO 35, 36) were found to be in subtype A5. A cluster composed of four sheep virus sequences (SLO 31) was clearly divergent from all other subtypes in group A and could not be assigned to any of them. The virus sequences from one goat flock belonged solely to subtype B1, whereas virus sequences from more than one genotype were found to circulate within the other two goat flocks, belonging to subtype B1 (SLO 1 and SLO 37) and to genotype A (SLO 2 and 78–88 g). Two goat virus sequences (SLO 2) were found to belong to genotype A and could not be assigned to existing subtypes. One goat virus sequence (37–88 g) from flock 37 was clearly different from other sequences of this flock and was more closely related to genotype A sequences. We propose two new subtypes within genotype A, subtype A14 (SLO 2) and A15 (SLO 31).     

Pathogenicity and transmission studies of H5N2 parrot avian influenza virus of Mexican lineage in different poultry species

In 2004, a low pathogenic H5N2 influenza virus (A/parrot/CA/6032/04) was identified in a psittacine bird for the first time in the United States. Sequence and phylogenetic analysis of the hemagglutinin gene grouped the parrot isolate under the Mexican lineage H5N2 viruses (subgroup B) with highest similarity to recent chicken-origin isolates from Guatemala. Antigenic analysis further confirmed the close relatedness of the parrot isolate to Mexican lineage viruses, the highest cross-reactivity being demonstrated to Guatemala isolates. In vivo studies of the parrot isolate in chickens, ducks and turkeys showed that the virus, though did not cause any clinical signs, could replicate to high titers in these birds and efficiently transmit to contact control cage mates. The possibility that the parrot harboring the virus was introduced into the United States as a result of illegal trade across the border provides additional concern for the movement of foreign animal diseases from neighboring countries. Considering the potential threat of the virus to domestic poultry, efforts should be continued to prevent the entry and spread of influenza viruses by imposing effective surveillance and monitoring measures.     

Immune-mediated hemolytic anemia in an eclectus parrot

CASE DESCRIPTION: A 2-year-old female Solomon Island eclectus parrot (Eclectus roratus) was evaluated by a veterinarian because of a 4-day history of progressive lethargy, weakness, poor appetite, and inactivity. The bird was referred to a veterinary teaching hospital for further examination. CLINICAL FINDINGS: Clinicopathologic analyses revealed that the parrot had marked regenerative anemia, autoagglutination, and biliverdinuria. Small, rounded RBCs (thought to be spherocytes) were detected in blood smears. The abnormal findings met the diagnostic criteria for dogs with primary immune-mediated hemolytic anemia. However, analyses of blood samples for lead and zinc concentrations and plasma bile acids concentrations; the use of PCR assays for Chlamydophila psittaci, psittacine circovirus 1 (causative agent of beak and feather disease), and polyomavirus; and microbial culture and Gram staining of feces did not reveal a cause for the hemolytic anemia. TREATMENT AND OUTCOME: Although administration of immunosuppressive doses of cyclosporine was initiated, there was a rapid progression of disease, which lead to death of the parrot before this treatment could be continued long-term. Lack of an identifiable underlying disease (confirmed by complete histologic examinations at necropsy) supported the diagnosis of primary immune-mediated hemolytic anemia. CLINICAL RELEVANCE: Primary immune-mediated hemolytic anemia has not been widely reported in psittacine birds. A comprehensive evaluation and complete histologic examination of tissues to rule out underlying disease processes are required to definitively establish a diagnosis of primary immune-mediated hemolytic anemia in parrots. Primary immune-me-diated hemolytic anemia should be considered as a differential diagnosis for regenerative anemia in a parrot.     

Genomic characterization and distribution of bovine foamy virus in Japan

Bovine foamy virus (BFV) is distributed through worldwide cattle herds. Although the biological features of BFV are not well understood, appearance of clinical manifestation by superinfection with other microorganisms is inferred. In Japan, reports of genomic characterizations and epidemiology of this virus are limited. In this study, we performed whole genomic sequencing of BFV strains Ibaraki and No.43, which were isolated in this country. Additionally, we investigated BFV in geographically distant four daily farms in Japan, to estimate the distribution of BFV and its correlation to bovine leukemia virus (BLV). BFV was distributed throughout Japan; the average positive rate was 12.7%. The nucleotide sequence identities of the isolates were 99.6% when compared with BFV strain isolated in the USA. The phylogenetic tree using env gene sequence showed strains Ibaraki, No.43 and Kagoshima were sorted in the same cluster including the USA and Chinese strains, while Hokkaido strain was in the other cluster including European strains. Although no clear correlation between BFV and BLV could be found, BFV and BLV infections were likely to increase with ages. Our data on epidemiology and characteristics of BFV will provide important information to reveal biological features of BFV.     

Species-specificity of equine and porcine Lawsonia intracellularis isolates in laboratory animals

Lawsonia intracellularis infection causes proliferative enteropathy (PE) in many mammalian species, with porcine and equine proliferative enteropathy (PPE and EPE) known worldwide. Hamsters are a well-published animal model for PPE infection studies in pigs. There is no laboratory animal model for EPE infection studies and it is not known whether there is species-specificity for equine or porcine isolates of L. intracellularis in animal models. The objective of this study was to determine whether it is possible to generate typical EPE lesions in hamsters after inoculation with an equine strain of L. intracellularis (EPE strain) and whether it is comparatively possible to generate PPE lesions in rabbits after inoculation with a porcine strain of L. intracellularis (PPE strain). In 2 separate trials, 4-week-old and 3-week-old weanling golden Syrian hamsters were challenged with EPE strains and compared to uninfected (both trials) and PPE-infected controls (Trial 2 only). Concurrently, 6 female New Zealand white juvenile rabbits were infected with PPE strain and observed concomitantly to 8 similar rabbits infected with EPE strain for a different experiment. Hamsters and rabbits were observed for 21 to 24 days post-infection (DPI), depending on the experiment. Neither infected species developed clinical signs. The presence of disease was assessed with diagnostic techniques classically used for pigs and horses: immune-peroxidase monolayer assay on sera; quantitative polymerase chain reaction (qPCR) detection of molecular DNA in feces; and hematoxylin and eosin (H&E) stain and immunohistochemistry (IHC) on intestinal tissues. Our results showed that EPE-challenged hamsters do not develop infection when compared with PPE controls (IHC, P = 0.009; qPCR, P = 0.0003). Conversely, PPE-challenged rabbits do not develop typical intestinal lesions in comparison to EPE-challenged rabbits, with serological response at 14 DPI being significantly lower (P = 0.0023). In conclusion, PPE and EPE strains appear to have different host-specificities for hamsters and rabbits, respectively.     

Genetic diversity in twenty variants of the avian polyomavirus.

To determine if different pathotypes of the avian polyomavirus (APV) exist and to compare the genomes of APVs originating from different geographic areas, dates, and species of birds, the partial sequences of 18 APVs were determined. New viral sequences were compared with three published APV sequences. Two of the new viruses had identical sequences. Forty point mutations were found at 31 loci. A 27-bp deletion was found in the VP2 and VP3 open reading frames of one virus. A duplication of the putative origin of replication and adjacent enhancer region was previously reported in one APV. Smaller duplications involving the origin in one APV and a second enhancer region in another were discovered. All duplications were in tissue culture-adapted viruses, suggesting they occurred during the isolation process. Excluding duplications and the deletion, maximum variation between viruses was small (11 bp). A maximum parsimony tree was constructed that contained three major branches. The three earliest isolates were on separate branches. The European viruses were confined to branch I, but APVs from the United States were on all three branches. Lovebird, budgerigar, and macaw APVs were also on each of the three branches, suggesting that species-specific pathotypes have not developed. Most nonsynonymous mutations occurred in a small portion of the VP2 and VP3 open reading frames, demonstrating a selection for these mutations. That a glycine at VP2 221 will inhibit virus replication in chicken embryo fibroblasts (CEFs) has been previously reported. In contrast, six of seven of the new APVs isolated in CEFs had a glycine at VP2 221.     

福建省部分地区鹦鹉喙羽病的分子流行病学调查及其病原Cap蛋白抗原表位预测

为了解2017—2018年鹦鹉喙羽病在福建省某些地区鹦鹉中的流行情况,收集了福建省部分地区298份鹦鹉粪便样品,采用PCR方法进行粪便样品检测,对其中鹦鹉喙羽病病毒检测呈阳性的5个地区的样品进行衣壳蛋白(Cap)基因测序后比较其同源性,绘制系统进化树,分析氨基酸序列,并通过生物信息学及序列分析软件预测Cap蛋白二级结构及B细胞抗原表位。结果显示:鹦鹉喙羽病病毒的平均阳性率为41.28%,福州市某动物救助站和福州动物园的阳性率较高,分别为65.17%和64.29%,其次为三明动物园、福州市花鸟市场和福州市某鹦鹉繁殖基地,南平动物园的阳性率最低;所测5个毒株Cap基因与GenBank中新西兰株(AY518913)亲缘关系较近;Cap蛋白有丰富的二级结构和多处抗原指数较高的区域,具有潜在的B细胞抗原表位,位于5~27、110~127和141~153位氨基酸残基或其附近。研究结果对防控鹦鹉喙羽病,保障鹦鹉健康养殖有重要参考意义。     

Butcherbird polyomavirus isolated from a grey butcherbird (Cracticus torquatus) in Queensland,Australia

A novel avian polyomavirus was detected in peri-ocular skin lesions collected from a grey butcherbird (Cracticus torquatus), using a combination of multiply primed rolling circle amplification, nested PCR and long range PCR. The sequence of Butcherbird polyomavirus was determined by combining next generation sequencing and primer walking techniques. The circular double-stranded DNA genome of Butcherbird polyomavirus consisted of 5084 bp, and encoded six open reading frames (ORF-X, VP2, VP3, VP1, small T-antigen and large T-antigen). Phylogenetic analysis placed it amongst other members of the genus Avipolyomavirus, most closely related to Crow polyomavirus. Next generation sequencing enabled the detection of DNA fragments similar to, but distinct from, Canarypox virus within the same lesion from which Butcherbird polyomavirus was amplified, thus confirming an avipolyomavirus-avipoxvirus co-infection in the peri-ocular skin lesions of this grey butcherbird.     

Avian pathogenic Escherichia coli MT78 invades chicken fibroblasts

Avian pathogenic Escherichia coli (APEC) are responsible for extraintestinal diseases, called colibacillosis, in avian species. The most severe manifestation of the disease is colisepticemia that usually starts at the respiratory tract and may result in bird death. However, it is not yet clear how APEC cross the respiratory epithelium and get into the bloodstream. In this work, we studied the interaction between 8 APEC strains (UEL31, UEL17, UEL13, UEL29, MT78, IMT5155, IMT2470, A2363) and a chicken non-phagocytic cell, the fibroblast CEC-32 cell line. We investigated the association profile, the invasion capability, the cytotoxicity effect and the induction of caspase-3/7 activation in an attempt to understand the way the pathogen gains access to the host bloodstream. Association to cells was determined after 1 h of infection, while cell invasion was determined after 4 and 24 h of infection. The cytotoxic effect of bacterial infection was measured by lactate dehydrogenase (LDH) release and the activation of the apoptotic program was verified by caspase-3/7 activation. Also, the presence of genes for adhesins, invasins and other related virulence-associated factors was verified by PCR. All bacterial strains showed similarity in relation to adhesion, LDH release and caspase-3/7 activation. However, one APEC strain, MT78, showed high invasion capability, comparable to the invasive Salmonella typhimurium strain SL1344. Since an APEC strain was capable of invading non-phagocytic cells in vitro, the same may be happening with the epithelial cells of the avian respiratory tract in vivo. CEC-32 monolayers can also provide a useful experimental model to study the molecular mechanisms used by APEC to invade non-phagocytic cells.     

Ocular teratoid medulloepithelioma in a northern red-shouldered macaw: case report and literature review

A 4-mo-old northern red-shouldered macaw (Diopsittaca nobilis) was admitted to the veterinary hospital of the Arruda Câmara Zoo, in the State of Paraiba, Brazil, for investigation of an orbital mass. Given rapid progression and lack of response to treatment, the bird was euthanized, and an autopsy was performed. Histologically, the mass consisted of a retrobulbar invasive tumor characterized by tubular and rosette-like structures, with interspersed heteroplastic tissues, such as aggregates of neuroglial cells and islands of hyaline cartilage. The tumor was immunopositive for pancytokeratin, GFAP, NSE, and S100. These findings were compatible with an ocular teratoid medulloepithelioma, a neoplasm best described in humans but also reported rarely in young cockatiels and African Grey parrots.