Detection of circovirus infection in pigeons by in situ hybridization using cloned DNA probes.

Degenerate primers were designed based on known sequence information for the circoviruses psittacine beak and feather disease virus and porcine circovirus and applied by polymerase chain reaction (PCR) to known virus-infected bursa of Fabricius (BF) from a pigeon. A 548-bp DNA fragment was amplified and shown to be specific to a novel circovirus, named pigeon circovirus (PiCV), and was used to produce sensitive and specific probes for detection of circovirus DNA by in situ hybridization (ISH). Using ISH on BF from 107 pigeons submitted for necropsy, infection was detected in 89%, compared with a histologic detection rate of 66%. Using the ISH technique, infected cells were also found in liver, kidney, trachea, lung, brain, crop, intestine, spleen, bone marrow, and heart of some birds. Large quantities of DNA were present in some of these tissues, and in the absence of BF, liver in particular is identified as a potentially useful organ to examine for presence of PiCV. This high prevalence of infection in diseased birds is noteworthy, emphasizing the need for studies to determine the precise role of this virus as a disease-producing agent.     

Prevalence of pigeon circovirus infections in feral pigeons in Ljubljana, Slovenia

Pigeon circovirus (PiCV) was detected by real-time PCR in cloacal swabs, pharyngeal swabs, and serum samples taken from 74 feral pigeons (Columba livia var. domestica) that were caught at various locations in the city of Ljubljana, Slovenia. PiCV infections were detected in the majority of the tested birds. The highest (74.3%) detection rate was observed in the cloacal swabs and the lowest (31.1%) in serum samples. PiCV DNA was more readily detected in the cloacal swabs, pharyngeal swabs, and serum samples of birds younger than 1 yr. Molecular analysis of partial open reading frame V1 sequences showed that PiCV strains detected in feral pigeons share high nucleotide and amino acid sequence identities with PiCV strains detected in ornamental, racing, meat, and feral pigeons.     

Development of a polymerase chain reaction-based in vivo method in the diagnosis of subclinical pigeon circovirus infection

This paper describes a polymerase chain reaction (PCR)-based method performed on blood samples and intestinal content to detect subclinical pigeon circovirus (PiCV) infection in live pigeons. In addition, two sets of primers (primer set 1 and 2), designed in two different regions of the viral genome, were used to provide evidence of possible differences in PCR responses. Blood and intestinal content samples were randomly collected from a total of 50 apparently healthy meat pigeons, aged 1 to 5 wk, which came from central Italy. Samples of primary lymphoid organs were also collected. Results showed a high level of PiCV infection, although clinical signs were not present. The results obtained with the two sets of primers showed that primer set 2 was able to detect a higher number of PCR-positive pigeons (45 of 50 pigeons) than primer set 1 (11 of 50 pigeons). In both cases an increase in positive results with pigeon age indicates that the major direction of transmission is likely horizontal. In these circumstances feces can play an important epidemiologic role, as supported by the consistent circovirus detection in intestinal content. The high sensitivity of this PCR test, which is able to detect very low amounts of viral DNA (5.5 x 10(-3) fg of plasmid containing the cloned PiCV genome), makes it suitable for possible application as an epidemiologic tool for identifying virus carriers for subsequent removal from lofts.     

Experimental infection of domestic pigeons with pigeon circovirus

Pigeon circovirus (PiCV) infection and young pigeon disease syndrome (YPDS), associated with high morbidity and mortality, have been recognized in young racing pigeons from large portions of Central Europe. There exist a number of data indicating that YPDS is a consequence of PiCV infection and subsequent immunosuppression. In order to prove PiCV to be one of the crucial factors of YPDS, an experimental infection with PiCV was performed under controlled conditions. Twenty-four domestic pigeons (Columba livia forma domestica) were divided into two groups with 12 pigeons each; an infection group and a control group. All birds were between their fourth to eighth week of life. Pigeons in the infection group were infected both intramuscularly and orally with PiCV purified from naturally infected birds, while pigeons in the control group received a placebo. To test a possible influence of the PiCV infection on the immune system, the animals in both groups were vaccinated simultaneously, on the same day, against PMV-1 (Lasovac plus, IDT, Dessau-Tornau, Germany). Weekly virologic testing showed a viraemic period, and excretion of the infection virus, in pigeons in the infection group. Replication of PiCV could be proved on the basis of histologic findings of multiglobular inclusion bodies, mainly observed in macrophages of the bursa of Fabricius. A PiCV, genetically distinct from the experimental virus, was detected in the control group by polymerase chain reaction (PCR) testing, but any histologic findings comparable to the infection group were absent. None of the pigeons revealed clinical signs of illness, or hints that immunosuppression had occurred, regardless of their group. The absence of stressful conditions, considered as a trigger for the development of YPDS, may be responsible for the failure of disease reproduction in our infection model.     

北京地区1株鸽圆环病毒全基因组测定及遗传进化分析

[目的] 了解北京地区流行的鸽圆环病毒（Pigeon circovirus,PiCV）的基因组特征及变异规律。[方法] 以3只发病鸽的肝脏和脾脏组织为模板,采用PCR技术检测病原。以检测阳性的肝脏组织DNA为模板,应用PCR技术分段扩增PiCV的全基因序列。应用DNAStar和Mega 7.0软件对扩增得到的全序列进行拼接和核苷酸序列比对,并构建系统进化树,对病毒基因组的2个开放阅读框分别进行核苷酸和氨基酸序列比对,并构建系统进化树。[结果] 经PCR检测,3只病鸽中有1只病鸽的组织中检测到PiCV阳性,并未检出其他病毒。采用PCR分段扩增成功获得了1株PiCV的全基因组序列,命名为PiCV BJ,该病毒基因组大小为2 034 bp,包含有2个主要的开放阅读框（ORFs）,ORF-V1编码Rep蛋白,ORF-C1编码Cap蛋白。相似性比对结果显示,PiCV BJ株基因组序列与GenBank上登录的其他参考序列的核苷酸相似性在86.0%~97.0%之间,与2011年分离自波兰的PL53相似性为97.0%。遗传进化结果显示,PiCV BJ株与2014年分离自波兰的PL124在同一分支,亲缘关系较近;与其他禽源圆环病毒不在同一分支,亲缘关系较远。PiCV BJ株Cap基因的起始密码子为ATG,与2011年分离自比利时的11-08304株核苷酸、氨基酸序列相似性高达95.8%和85.2%;Rep基因与2011年分离自波兰的PL53相似性最高,核苷酸和氨基酸相似性分别高达94.6%和96.2%;Cap和Rep基因的进化树分析结果也显示,PiCV BJ株均与PL53和11-08304株在同一分支,亲缘关系较近,这与相似性分析的结果一致。[结论] PiCV BJ株来自国外,可能由赛鸽引种传入中国,提示在外部引种时要做好病毒监测。本研究丰富了PiCV的遗传学研究资料,为进一步探究PiCV的遗传变异及传播机制提供了参考依据,也为PiCV的防控提供了重要的理论基础。     

The epidemiology,molecular characterization and clinical pathology of circovirus infections in pigeons – current knowledge

The first cases of circovirus infections in pigeons were documented less than 25 years ago. Since then, circovirus infections have been reported on nearly all continents. The specificity of pigeon breeding defies biosecurity principles, which could be the reason for the high prevalence of PiCV infections. PiCV infections in pigeons lead to atrophy of immune system organs and lymphocyte apoptosis. Infected birds could be more susceptible to infections of the respiratory and digestive tract. PiCV has been associated with the young pigeon disease syndrome (YPDS). PiCVs are characterized by high levels of genetic diversity due to frequent point mutations, recombination processes in the PiCV genome and positive selection. Genetic recombinations and positive selection play the key role in the evolution of PiCV. A protocol for culturing PiCV under laboratory conditions has not yet been developed, and traditional vaccines against the infection are not available. Recombinant capsid proteins for detecting anti-PiCV antibodies have been obtained, and these antigens can be used in the production of diagnostic tests and subunit vaccines against PiCV infections. However, YPDS has complex etiology, and it remains unknown whether immunization against PiCV alone will contribute to effective control of YPDS.     

In situ hybridisation for the detection of Leishmania species in paraffin wax-embedded canine tissues using a digoxigenin-labelled oligonucleotide probe

The diagnosis of canine leishmaniosis (CanL) is currently predominantly achieved by cytological or histological identification of amastigotes in biopsy samples, demonstration of specific anti-Leishmania antibodies and PCR-based approaches. All these methods have the advantage of being sensitive and more or less specific; nevertheless, most of them also have disadvantages. A chromogenic in situ hybridisation (ISH) procedure with a digoxigenin-labelled probe, targeting a fragment of the 5.8S rRNA was developed for the detection of all species of Leishmania parasites in routinely paraffin wax-embedded canine tissues. This method was validated in comparison with traditional techniques (histology, PCR), on various tissues from three dogs with histological changes consistent with a florid leishmaniosis. Amastigote forms of Leishmania gave clear signals and were easily identified using ISH. Various tissues from 10 additional dogs with clinical suspicion or/and a positive serological test but without histological presence of amastigotes did not show any ISH signals. Potential cross-reactivity of the probe was ruled out by negative outcome of the ISH against selected protozoa (including the related Trypanosoma cruzi) and fungi. Thus, ISH proved to be a powerful tool for unambiguous detection of Leishmania parasites in paraffin wax-embedded tissues.     

猪圆环病毒2型原位杂交检测技术的建立与应用

参照GenBank发表的猪圆环病毒2型（PCV2）ORF2基因序列设计引物，利用PCR扩增得到PCV2BF株341bp的核酸片段，用随机引物法制备出地高辛标记的核酸探针。制备的探针与PCV1、PRRSV、PPV、PRV等不发生反应，可检测的最低PCV2DNA含量为1．78Pg。对30份临床组织样本进行了检测，并与PCR比较，结果表明，阴性符合率为100％，阳性符合率为88．9％。应用原位杂交技术分析了PCV2在人工感染仔猪主要组织中的分布，结果表明，感染后3d，从仔猪的淋巴结、胸腺、肺脏、脾脏、鼻黏膜可检测到阳性信号，感染后21d，肝脏、肾脏、胰腺和回肠可检出阳性信号，至感染后42d，可从心脏、胃、脑检出阳性信号。在整个试验过程中会厌软骨、膀胱、皮肤、肌肉等组织均为阴性。本研究结果表明，建立的PCV2原位杂交技术具有良好的敏感性和特异性，可用于PCV2的实验室诊断和感染靶细胞的定位分析。     

Detection of Minchinia occulta in samples of pearl oysters Pinctada maxima infected by Haplosporidium hinei

Objective   To determine if juvenile pearl oysters ( Pinctada maxima ) infected with Haplosporidium hinei are also infected with another haplosporidian parasite, Minchinia occulta .
Design   Archived samples of pearl oysters infected with H. hinei were examined using polymerase chain reaction (PCR) assays and in situ hybridisation (ISH) to analyse and identify haplosporidians. A 144-bp and 220-bp region of Minchinia DNA were targeted by PCR and amplified DNA from formalin-fixed H. hinei -infected pearl oyster samples was sequenced. A 25-bp oligonucleotide probe targeting a variable section of the parasite's small subunit rRNA gene was used in ISH.
Results   The results of DNA-based diagnostic assays supported each other. The sequences obtained by PCR were found to be almost identical to M. occulta from rock oysters and the ISH assay demonstrated infection with M. occulta in affected pearl oysters. ISH indicated a prevalence of infection of 26.7% in one of the previous outbreaks.
Conclusion   Pearl oyster spat are susceptible to infection by a Minchinia parasite, most likely M. occulta , which was recently identified in rock oysters within the pearl-producing zones of Western Australia and is associated with mortalities of up to 80% in this species. The occurrence of haplosporidian co-infections in pearl oysters suggests the immunocompetence of juvenile oysters may be an important factor in preventing infection and therefore preventing mortalities such as those occurring in the recent outbreaks of pearl oyster oedema disease.     

Prevalence and characteristics of intimin- and Shiga toxin-producing Escherichia coli from gulls,pigeons and broilers in Finland

The role of birds as sources of Shiga toxin-and intimin-producing Escherichia coli was studied. Fecal samples from live gulls (n=86), pigeons (n=33) and broiler chickens (n=199) from 23 flocks were analyzed for stx and eae by PCR. No stx positive samples were detected. In contrast, eae E. coli were highly prevalent among gulls (40%), and was also found in pigeons (7%) and chickens (57% of the flocks contaminated). The eae positive isolates were analyzed genetically and O-serogrouped. One isolate from a pigeon was found to have stx (2f). The isolates of gulls differed from those of pigeons and chickens, and all eae E. coli isolates from birds differed from human pathogenic strains by the lack of EHEC-hlyA and bfp/EAF as well as distribution of O-serogroups. Thus, birds cannot be regarded as important carriers of zoonotic stx or eae E. coli in Finland.     

Chlamydophila psittaci DNA detection in the faeces of cage birds

In this study, we investigated the shedding of Chlamydophila psittaci in faecal samples from cage birds using PCR testing. A total of 47 faeces samples were collected from four different aviaries. Main symptoms determined after clinical investigation and owner histories of the birds showed that the birds had respiratory system problems changing from mild to severe. They also showed conjunctivitis, diarrhoea or no symptoms at all. DNA extractions from faeces were performed with the QIAamp DNA Stool Mini Kit. Following PCR with Cp. psittaci specific primers, 43 (91.5%) samples were determined to harbour-specific DNA. Only one bird from each aviary was found to be negative by PCR. As all the samples from birds showing clinical signs were PCR positive, these signs could be correlated to psittacosis in these birds. Cp. psittaci shedding in faeces was detected in all the aviaries. After restriction analysis of PCR amplicons with AluI enzyme, all the isolates showed the same RFLP (Restriction Fragment Length Polymorphism) patterns with the control Cp. psittaci DNA. PCR following QIAamp DNA stool mini kit extraction of faecal samples was found to be a rapid, specific, sensitive, reproducible test, which did not need additional nested PCR of samples.     

First molecular identification and subtype distribution of Blastocystis sp. isolated from hooded crows (Corvus cornix) and pigeons (Columba livia) in Tehran Province,Iran

Blastocystis is a common intestinal parasite among humans and animals such as non-human primates, pigs, cattle, birds, amphibians, and less frequently, rats, reptiles and insects. Since Blastocystis is a widely transmissible parasite between humans and mammals or birds, it is prominent to determine whether newly secluded non-human isolates are zoonotic. There are no comprehensive studies in Iran assessing the prevalence and molecular identification of Blastocystis infection in birds, especially in pigeons and crows. So, the aim of this study was to identify Blastocystis subtypes (STs) in crows and pigeons in Tehran province, Iran, using Nested PCR-RFLP and sequencing. Overall, 300 Blastocystis isolates from birds (156 pigeons and 144 crows) were subtyped by PCR, and the homology among isolates was then confirmed by RFLP analysis of the 18S rRNA gene. The prevalence of Blastocystis infection was detected 42.9% in pigeons and 44.4% in crows. All positive pigeons were owned by ST13 (100%). Among crows, 46 samples (71.8%) like pigeons were ST13, and 13 samples (20.3%) were ST14. Five samples (7.9%) remained unknown. This study was the first report of ST13 and ST14 of Blastocystis from birds. In the present study, our data revealed a high prevalence of Blastocystis sp. in pigeon’s and crow’s samples and the isolates from these birds were classified into two genetically distinct STs. Therefore, birds appear to be infected with various STs. It is important to determine the phylogenetic relationships between unknown STs from these birds and the multiple STs of Blastocystis.     

Detection of EHV-1 and EHV-4 in placental sections of naturally occurring EHV-1- and EHV-4-related abortions in the UK: use of the placenta in diagnosis

REASONS FOR PERFORMING STUDY: EHV-1 and EHV-4 abortion diagnosis is based upon detailed examination of the aborted fetus. However, in some cases, only the placenta is available for examination. Furthermore, the contribution of lesions in the placenta to pathogenesis and diagnosis of EHV-1 and EHV-4 abortion has been neglected. OBJECTIVES: To assess the utility of placental examination in equine herpesvirus-1 (EHV-1) and EHV-4 abortion diagnosis. METHODS: Sections of allantochorion from 49 herpesvirus abortions were analysed by PCR, in situ hybridisation and immunostaining. RESULTS: Virus-specific nested PCR confirmed the presence of viral DNA in 46 cases; 41 cases were EHV-1-positive and 5 EHV-4-positive. Microscopic changes were nonspecific. Examination of the PCR-positive sections of allantochorion revealed EHV-1 DNA by in situ hybridisation (ISH) in 21 cases and EHV-4 in 4 cases. In 2 samples, DNA of both viruses was present on PCR and ISH. Viral antigen was found by immunohistology in 15 cases. Regarding the localisation of virus in the placentae, both viral DNA and antigen of EHV-1 and EHV-4 were found in endothelial cells of chorionic villi and, occasionally, in trophoblast epithelium. In the stromal endothelium, only EHV-1 was found. CONCLUSIONS: The data indicate that examination of placentae is a useful diagnostic aid in EHV-1 and EHV-4 abortion diagnosis. POTENTIAL RELEVANCE: Virological examination of the placenta should become standard practice in equine abortion investigations, particularly in those cases where the fetus is not available for examination.     

Evaluation of different diagnostic methods for diagnosis of Lumpy skin disease in cows

Viral isolation, polymerase chain reaction (PCR), dot blot hybridization (DBH), and indirect enzyme-linked immunosorbent assay (iELISA) were used for the diagnosis of lumpy skin disease in clinically infected, fevered, and apparently normal dairy cows. Lumpy skin disease virus (LSDV) was isolated from skin biopsies and blood samples collected from clinically infected cows in percentages of 72% and 20%, respectively. The virus recovered from blood samples collected from fevered cows in percentage of 33.3%. Both PCR and DBH detected viral DNA in 100% of skin biopsies collected from clinically infected cows whereas the detection rates in blood samples collected from clinically infected animals were 100% and 84% using PCR and DBH, respectively. Viral DNA was detected in blood samples collected from fevered cows using PCR and DBH in percentages of 77.8% and 66.6%, respectively. Only 19.1% of blood samples collected from in-contact cows was positive for both of PCR and DBH. Detection rates of antibodies against LSDV using iELISA in serum samples collected from clinically infected and fevered cows were 56% and 11.1%, respectively, whereas all in-contact cows had no antibodies against the virus.     

鸽圆环病毒SYBR GreenⅠ荧光定量PCR方法的建立及应用

为建立一种针对鸽圆环病毒(PiCV)的快速诊断方法,试验根据PiCV的Rep保守基因序列,设计合成一对特异性引物,建立了PiCV荧光定量PCR检测方法,对其敏感性、特异性、重复性进行评价并利用该方法对临床样本进行检测。结果表明:在最佳反应条件下,所建立的PiCV荧光定量PCR方法在1.43×10²~1.43×10⁸copies/μL标准品范围内具有良好的线性相关性,线性相关系数为0.998;敏感性试验结果显示,该方法最低可检测到1.43×10²copies/μL标准品,是常规PCR检测方法的1000倍;特异性试验结果显示,该方法与其他常见的鸽病病原不发生交叉反应;重复性评价结果显示,批内与批间的变异系数均小于0.8%;所建立PiCV荧光定量PCR方法对临床鸽血清样品检测结果显示,PiCV阳性率达38.7%,高于常规PCR方法的检测阳性率(28.7%),说明该方法具有良好的适用性。研究结果PiCV的流行病学调查、病原学监测及定量研究奠定了基础。     

Detection and characterization of Shiga toxin-producing Escherichia coli in feral pigeons

Escherichia coli strains producing a variant of Shiga toxin 2 (Stx2), designated Stx2f, have been recently described in the stools of feral pigeons. During 1997-1998, 649 pigeons were trapped and examined in three different squares of Rome. Stool samples were collected from each bird and enrichment cultures were examined for the presence of Stx by the vero cell assay. Stx-producing E. coli (STEC) were isolated from the positive cultures and characterized by serotyping and PCR analysis of stx and other virulence-related genes. Stx was detected in 10.8% of the stool enrichment cultures. The percentage of positive birds did not differ significantly for the three flocks considered and the season of sample collection. Conversely, STEC carriage was significantly more frequent in young than in adult birds (17.9 versus 8.2%). None of the birds examined showed signs of disease. STEC strains were isolated from 30 of 42 Stx-positive cultures examined. All the strains produced Stx2f, and most of them possessed genes encoding for intimin and the cytolethal distending toxin (CLDT). Six serogroups were identified, but most of the isolates belonged to O45, O18ab, and O75. Molecular typing indicated that most of the isolates within a flock were clonally-related. This work confirms that pigeons represent a natural reservoir of STEC strains characterized by the production of the toxin variant Stx2f, and by the frequent presence of eae and cldt genes. Further work is needed to clarify whether these STEC may represent a cause of avian disease or even a potential health hazard for humans.     

The detection of Salmonella typhimurium varatio copenhagen DT 2 in purebred pigeons

On the occasion of a large exhibition of pure-breed fancy pigeons 398 animals from 49 different dovecotes were examined for Salmonella shedding. Faecal samples were taken after caging of the birds for the exhibition and after 3 days, before the end of the exhibition. Salmonella were detected in faeces of 28 out of 398 pigeons (7.04%). 10 birds were Salmonella positive only after caging for the exhibition, 10 other animals only before the end of the exhibition, and 8 pigeons at both occasions. The Salmonella positive birds originated from 15 different dovecotes, i.e. in ca. 30% of the dovecotes at the exhibition at least 1 Salmonella positive pigeon was identified. The share of positive birds in these dovecotes varied between 5% and 83%. All Salmonella isolates belonged to the serovar Typhimurium variant copenhagen and were of phage type DT 2. The results of this study do not provide complete evidence on the spreading of Salmonella organisms from birds infected at time of caging to other pigeons during the exhibition, however, such transmission cannot be excluded. In only 18 dovecotes pigeons were immunised against Salmonella Typhimurium. However, in these dovecotes all breeder birds but only 13% of the young pigeons had been immunised. Among the vaccinated breeder pigeons the number of Salmonella positive birds was considerably lower (not significant) than among the non-vaccinated breeders. There is epidemiological evidence that vaccination of pigeons has a considerable protective effect against Salmonella exposure. However, in order to effectively reduce Salmonella findings in pure-breed fancy pigeons it is recommended to provide vaccination to pigeons in a greater number of dovecotes and to include the progeny, too.     

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.