Dörte Lüschow

Comparison of the Specificity and Sensitivity of PCR, Nested PCR, and Real-Time PCR for the Diagnosis of Histomoniasis

Abstract Blackhead, also known as enterohepatitis, is caused by a protozoan parasite called Histomonas meleagridis. Clinical symptoms are nonspecific. Until now, diagnosis has been mainly based on postmortem lesions and microscopical and histopathological examination. In many cases, especially in layer flocks, these conventional methods are not sufficient, as the lesions are sometimes not clear. The technique for isolation of histomonads in vitro offers many advantages, but the confirmation of histomonads growing in culture may require a time-consuming procedure of rectal inoculation of culture material into chickens or turkeys. The aim of our investigation was to establish a conventional polymerase chain reaction (PCR), a nested PCR, and a real-time PCR, and to examine their specificity as well as sensitivity in the diagnosis of histomoniasis. The obtained results have shown that the conventional PCR is more sensitive than the real-time PCR. Furthermore, the sensitivity of the PCR can be increased by adding the nested PCR. However, the real-time PCR is more specific.

Prevalence of mycoplasmas in eggs from birds of prey using culture and a genus-specific mycoplasma polymerase chain reaction

Mycoplasmas are commensals and pathogens of different avian species, especially poultry and passeriforms. The role of mycoplasmas in raptors has not yet been completely determined, and especially not the possibility of vertical transmission. Therefore 424 raptor eggs were examined for the occurrence of mycoplasmas using culture, and 155 of these eggs with a Mycoplasma genus-specific polymerase chain reaction (PCR) assay. This PCR was tested for its sensitivity and specificity, especially for use in a bird population of unknown mycoplasma status (prevalence and species). The size of the amplified PCR product was large (1013 base pairs) to enable use of the product for species differentiation by sequencing. Culture and PCR yielded only one positive result, in an egg of a Northern Goshawk (Accipiter gentilis). The isolate was identified as Mycoplasma lipofaciens using an immunobinding assay, as well as by sequencing part of its 16S rRNA gene.

Protection and Virus Shedding of Falcons Vaccinated against Highly Pathogenic Avian Influenza A Virus (H5N1)

Virus shedding by vaccinated birds was markedly reduced.

Detection of Histomonas meleagridis DNA in Different Organs After Natural and Experimental Infections of Meat Turkeys

Abstract Histomonas meleagridis infection of turkeys is usually accompanied by a severe disease with unspecific clinical symptoms but with distinct pathological lesions in the ceca and liver. In the literature some macro- and microscopic evidence of the spread of histomonads to the other organs has been provided. The aim of the present investigations was to use real-time polymerase chain reaction (PCR) to demonstrate the dissemination of H. meleagridis DNA to different organs after natural and experimental infection of meat turkeys. Samples from several organs were collected from a meat-turkey flock, which proved to be naturally infected with histomoniasis, and examined for histomonad DNA by real-time PCR. Histomonad DNA was detected in all investigated ceca, livers, spleens, kidneys, and pooled brain swabs. Additionally it was found in 75% of investigated samples from bursae of Fabricius, in 50% of investigated duodenums, and in 40% of investigated jejunum samples. After experimental intracloacal infection of 3-wk-old turkey poults with 147,500 histomonads, similar samples were collected from all turkeys that died. After a 3-wk observation period the surviving birds, as well as the noninfected control group, were euthanatized and samples were taken. During the entire experimental period, 10 birds out the 20 infected birds died. Histomonad DNA was detected in all investigated ceca, livers, lungs, and hearts (100%) and almost all kidneys (90%) and bursae of Fabricius (80%). On the other hand, only 30% of examined spleens and 10% of brain samples revealed positive results. Surviving infected birds were euthanatized and necropsied; histomonad DNA was found in one out of 10 livers but not in any ceca. Also, histomonad DNA could not be detected in examined cecal and lung samples from the noninfected control group.

Adenovirus of psittacine birds: investigations on isolation and development of a real-time polymerase chain reaction for specific detection

Liver samples of psittacine birds with a histological suspicion of an adenovirus infection, confirmed by electron microscopy examination, were subjected to virus isolation attempts using a heterologous cell culture system and a homologous cell culture system in the form of chicken embryo liver cells and psittacine embryo fibroblasts, respectively. Whereas isolation in chicken embryo liver cells failed, virus was isolated successfully in the psittacine embryo fibroblasts cell culture system. Molecular investigations identified the virus as a specific psittacine adenovirus (PsAdV). Additionally, on the basis of the hexon gene sequence data obtained, a real-time polymerase chain reaction (PCR) for specific detection of PsAdV was developed. To ensure an exclusive hybridization with PsAdV, selected primers were located within the variable L1 region of the hexon gene. Furthermore, the specificity of the real-time PCR was confirmed by investigation of a panel of different avian adenoviruses and unrelated DNA viruses. Using this PCR, the threshold cycle values obtained support the propagation of PsAdV in the homologous cell culture system in comparison with the chicken cell culture system. Moreover, the developed PCR represents a reliable method for specific and sensitive detection of PsAdV in clinical samples.

Detection of Avian coronavirus infectious bronchitis virus type QX infection in Switzerland

Infectious bronchitis, a disease of chickens caused by Avian coronavirus infectious bronchitis virus (IBV), leads to severe economic losses for the poultry industry worldwide. Various attempts to control the virus based on vaccination strategies are performed. However, due to the emergence of novel genotypes, an effective control of the virus is hindered. In 1996, a novel viral genotype named IBV-QX was reported for the first time in Qingdao, Shandong province, China. The first appearance of an IBV-QX isolate in Europe was reported between 2003 and 2004 in The Netherlands. Subsequently, infections with this genotype were found in several other European countries such as France, Italy, Germany, United Kingdom, Slovenia, and Sweden. The present report describes the use of a new set of degenerate primers that amplify a 636-bp fragment within the S1 gene by reverse transcription polymerase chain reaction to detect the occurrence of IBV-QX infection in Switzerland.

Avian influenza virus risk assessment in falconry

BackgroundThere is a continuing threat of human infections with avian influenza viruses (AIV). In this regard falconers might be a potential risk group because they have close contact to their hunting birds (raptors such as falcons and hawks) as well as their avian prey such as gulls and ducks. Both (hunting birds and prey birds) seem to be highly susceptible to some AIV strains, especially H5N1. We therefore conducted a field study to investigate AIV infections in falconers, their falconry birds as well as prey birds.FindingsDuring 2 hunting seasons (2006/2007 and 2007/2008) falconers took tracheal and cloacal swabs from 1080 prey birds that were captured by their falconry birds (n = 54) in Germany. AIV-RNA of subtypes H6, H9, or H13 was detected in swabs of 4.1% of gulls (n = 74) and 3.8% of ducks (n = 53) using RT-PCR. The remaining 953 sampled prey birds and all falconry birds were negative. Blood samples of the falconry birds tested negative for AIV specific antibodies. Serum samples from all 43 falconers reacted positive in influenza A virus-specific ELISA, but remained negative using microneutralisation test against subtypes H5 and H7 and haemagglutination inhibition test against subtypes H6, H9 and H13.ConclusionAlthough we were able to detect AIV-RNA in samples from prey birds, the corresponding falconry birds and falconers did not become infected. Currently falconers do not seem to carry a high risk for getting infected with AIV through handling their falconry birds and their prey.

Influenza A Virus Monitoring in Urban and Free-Ranging Pigeon Populations in Germany, 2006–2008

SUMMARY. Tracheal and cloacal swabs as well as blood samples from 408 feral urban (Columba livia forma domestica) and 170 free-ranging wood pigeons (Columba palumbus) in Germany were tested for infection with avian influenza viruses (AIVs). Neither influenza A virus was isolated in the swab samples nor influenza A virus RNA detected using real-time reverse transcriptase–PCR (RT-qPCR). In three urban feral pigeons, avian paramyxovirus (APMV) serotype 1 was isolated. Two of 123 serum samples from hunted free-ranging wood pigeons contained specific antibodies against influenza A virus but not against the subtypes H5 and H7. In conclusion, our study indicates that even after the occurrence of zoonotic highly pathogenic avian influenza virus (HPAIV) subtype H5N1 in the area of investigation in Germany, pigeons do not play a major part in the transmission of influenza viruses. The risk of AIV infection for humans from urban and free-ranging wood pigeons is, if at all, of minimal importance.

Quantitative PCR as a Tool to Determine the Reticuloendotheliosis Virus–Proviral Load of Fowl Poxvirus

Abstract Sequences of the reticuloendotheliosis virus (REV), an avian retrovirus, are integrated into the genome of fowl poxvirus (FPV). We developed and evaluated a quantitative multiplex real-time PCR (multiplex qPCR) assay to determine the REV-proviral load of FPV strains. Amplification efficiencies were 98.7% for the amplification of the FPV DNA and 88.7% for the amplification of REV-proviral DNA. The ratio between FPV DNA and REV-proviral DNA was calculated from the PCR efficiencies and the threshold cycle deviation of the unknown samples vs. a standard. The intraassay variation was determined by investigating triplets of different dilutions of the standard. The coefficient of variation between the threshold cycles was below 0.05 in all tested dilutions. The ratios of the triplet had a coefficient of variation of 0.201. Generally, the method overestimated the relative amount of REV-proviral DNA. Skin lesions from fowlpox outbreaks were investigated with the multiplex qPCR. The FPV:REV ratio was between 1:0.803 and 1:1.411 in samples with sufficient DNA to allow a conclusion. In addition, the investigation of cell culture material of several passages of a FPV field isolate showed a complete loss of the REV provirus after 36 passages. The loss rate of the REV provirus was approximately 50% per passage. In conclusion, we established the multiplex qPCR assay as a convenient and reliable method to determine the REV-proviral load of FPV. The first results we obtained with it show that it is of value for further investigations about the significance of the integration of the REV provirus into the genome of FPV.

Survey for zoonotic pathogens in Norway rat populations from Europe

BACKGROUND The Norway rat Rattus norvegicus is an important reservoir of various zoonotic pathogens, such as cowpox virus and Leptospira, but also for agents of no or unknown zoonotic potential. We describe a survey of 426 Norway rats originating from five European countries and different habitats for Leptospira spp., rickettsiae, orthopoxvirus (OPV), avian metapneumovirus subtypes A and B (aMPV) and rat polyomavirus (rat PyV). RESULTS Leptospira DNA was detected in 60 out of 420 (14.3%) rats, and Rickettsia DNA was found in three out of 369 (0.8%) rats investigated. PCR-based typing resulted in the identification of L. interrogans sequence type 17, which corresponds to the serogroup Icterohaemorrhagiae, and Rickettsia helvetica respectively. Rat PyV DNA was detected in 103 out of 421 (24.5%) rats. OPV DNA and aMPV RNA were detected in none of the rats, but OPV-specific antibodies were detected in three out of 388 (0.8%) rats. The frequency of single Leptospira and rat PyV infections and coinfections was, independent of sex, greater for adults compared with juveniles/subadults and greater at rural sites compared with urban areas. CONCLUSIONS Study results indicate a broad geographical distribution of Leptospira DNA in rats within Europe, underlining the need to investigate further the potential mechanisms leading to increased prevalence in rural habitats and to assess the relevance to public health. In contrast, rickettsia and OPV infections rarely occurred in wild rat populations. The potential influence of rat PyV on the susceptibility to infections with other pathogens should be investigated in future studies.