Grzegorz Woźniakowski

Rapid Detection of Marek's Disease Virus in Feather Follicles by Loop-Mediated Amplification

SUMMARY. Mareks disease (MD) remains a serious problem in the production of poultry. The disease is caused by Mareks disease virus (MDV), and despite the ubiquitous use of vaccination to control losses, MD still affects poultry farming worldwide. The aim of this study was to develop a loop-mediated isothermal amplification (LAMP) method for the simple and inexpensive detection of MDV in feather tips of chickens. Two pairs of specific primers complementary to the meq oncogene of MDV were designed, targeting the sequence of the very virulent MDV strain, RB1B. Bst polymerase was used for the isothermal amplification of viral DNA at 65 C for 90 min in a water bath. The fluorescence signal was identified in MDV-positive samples after the addition of SYBR Green and ultraviolet (UV) illumination. The sensitivity of LAMP was 2 log 10 plaque-forming units (PFU)/ml of HPRS16 and 103 copies/ìl of plasmid containing the target gene (meq) and was equal in sensitivity to PCR amplification. Due to the use of three sets of primers, LAMP was highly specific for MDV-1 DNA. The developed LAMP technique is a rapid and simple tool for the specific detection of MDV in samples of feathers taken from live chickens. Since the use of thermocyclers is not necessary for LAMP assay, it can be conducted by small laboratories and even field veterinarians.

Loop-mediated isothermal amplification for the detection of goose circovirus

BackgroundGoose circovirus (GCV) presents an immunosuppressive problem in production of geese. The infection’s clinical symptoms include growth retardation or feathering disorders but the infection process may remain non-symptomatic what makes the infected birds more susceptible for secondary viral, bacterial and fungal infections. Diagnosis of GCV infection is made by histopathological examination, dot blot hybridization, polymerase chain reaction (PCR) and real-time PCR. However these techniques require application of thermocyclers and qualified staff which may be cost-consuming for some diagnostic units. The aim of this study was to develop loop-mediated isothermal amplification assay (LAMP) as a simple method of GCV detection.ResultsThe presented study has shown LAMP as a rapid tool of detecting DNA of goose circovirus (GCV) as soon in 30 min time. The method used three sets of primers: two outer primers (F3 and B3), two inner primers (FIP and BIP) and two loop primers (FL and BL) to accelerate the reaction. The optimum reaction temperature and the time were 61°C for 30 min, respectively. The results were analysed using SYBR Green dye and GelRedTM solutions. Thirty-eight isolates of GCV collected from geese flocks in Poland were examined. For comparison, real-time polymerase chain reaction with F3 and B3 primers and SYBR Green dye was conducted. The obtained results have shown GCV-LAMP as a sensitive, rapid and specific assay and alternative for PCR-based methods.ConclusionsThe developed technique due to its simplicity may be applied by any veterinary laboratory or even mobile diagnostics units for the routine detection of GCV.

Current status of African swine fever virus in a population of wild boar in eastern Poland (2014-2015).

African swine fever virus (ASFV) was detected in wild boar in eastern Poland in early 2014. So far, 65 cases of ASFV infection in wild boar have been recognised. The methods used for ASFV detection included highly specific real-time PCR with a universal probe library (UPL), enzyme-linked immunosorbent assay (ELISA), and an immunoperoxidase test (IPT) for identification of anti-ASFV antibodies. The positive ASF cases were located near the border with Belarus in Sokółka and Białystok counties. Some of the countermeasures for disease prevention include early ASF diagnosis by ASFV DNA identification as well as detection of specific antibodies by systematic screening. The aim of this study was to assess the current ASF status in a Polish population of wild boar during the last two years (2014-2015).

Occurrence of Chicken Parvovirus Infection in Poland

The aim of the foregoing study was the determination of the occurrence of parvovirus in chicken flocks from different regions of Poland during 2002-2011. The material used for this study originated from chickens showing clinical symptoms of stunting and emaciation. For the quick detection of genetic material of the viruses in field samples, real-time PCR was applied. The conducted study implied on the occurrence of parvoviral infections in Poland in approximately 18% of investigated chicken flocks. However, their exact role remains still unknown.

Molecular evolution of Marek's disease virus (MDV) field strains in a 40-year time period.

SUMMARY Mareks disease (MD) presents a serious threat in poultry production. The disease has been limited for over 40 yr by protective vaccination. The widely applied vaccination against MD is also one of the factors causing evolutionary pressure onto field Mareks disease virus (MDV) virulent strains. Molecular evolution of MDV genes involved in oncogenesis may increase the pathogenicity of MDV virulent strains. The goal of the presented study was to sum up the molecular evolution of MDV field strains isolated in the last 40 yr in Poland. In total, 85 field MDV strains collected between 1974 and 2012 were propagated in chicken embryo fibroblasts. After DNA extraction, three sets of primers were designed for PCR complementary to the MDV076 (RLORF7) region encoding the meq oncogene as well to the MDV077 (23 kDa protein binding alpha-enolase) and MDV077.5 (RLORF6) genes. The obtained 85 MDV076, 60 MDV077, and 58 MDV077.5 cloned fragments were sequenced and aligned with the sequences of reference MDV strains showing different pathogenicity levels. The retrieved nucleotide (nt) and deduced amino acid sequences of RLORF7, 23 kDa protein, and LORF6 of Polish field strains showed several mutations and substitutions homologous to those observed in reference strains with a determined pathogenicity. The observed changes indicated the continuous evolution of field MDV strains. The RLORF7 nt sequence of analyzed MDV isolates showed similarity to virulent and very virulent MDV reference strains. The obtained 23 kDa and LORF6 nt sequences provided more important data and were more similar to mildly pathogenic strains than to virulent and very virulent MDV. The specific nt motifs in all three genes may indicate an increase of MDV virulence and were found in strains starting from 2006. According to the obtained results, the strains isolated in 2012 are similar to the very virulent plus MDV group. The study showed that RLORF7, 23 kDa protein, and RLORF6 fragments harbor sequence motifs that may have some association with MDV pathogenicity level. However, the exact role of the investigated regions in pathogenicity should be further examined by knock-out MDV strains. Also, the true MDV pathotype may only be determined by traditional in vivo experiments. RESUMEN Evolución molecular de cepas de campo del virus de la enfermedad de Marek (MDV) en un período de 40 años. La enfermedad de Marek (MD) presenta una seria amenaza para la producción avícola. La enfermedad ha sido limitada durante más de 40 años por la protección inducida por la vacunación. La vacuna ampliamente aplicada contra la enfermedad de Marek es también uno de los factores causantes de la presión evolutiva sobre las cepas virulentas del virus de la enfermedad de Marek. La evolución molecular de los genes implicados en la oncogénesis del virus de Marek puede aumentar la patogenicidad de cepas virulentas. El objetivo del estudio presentado fue resumir la evolución molecular de cepas de campo del virus de Marek aisladas en los últimos 40 años en Polonia. En total, 85 cepas de campo recolectadas entre 1974 y 2012 se propagaron en fibroblastos de embrión de pollo. Después de la extracción de ADN, se diseñaron tres conjuntos de iniciadores para llevar a cabo un método de PCR complementario a las regiones MDV076 (RLORF7) que codifica para el oncogene meq y también para los genes MDV077 (proteína de unión alfa-enolasa de 23 kDa) y para el gene MDV077.5 (RLORF6). Los fragmentos clonados obtenidos, 85 para el gene MDV076, 60 para el gene MDV077 y 58 para el gene MDV077.5, fueron secuenciados y se alinearon con secuencias de referencia del virus de la enfermedad de Marek que muestran diferentes niveles de patogenicidad. Las secuencias de nucleótidos y de aminoácidos deducidos de RLORF7, de la proteína de 23 kDa y LORF6 de cepas de campo polacas mostraron varias mutaciones y sustituciones homólogas a los observados en las cepas de referencia con una patogenicidad determinada. Los cambios observados indican la continua evolución de las cepas de campo del virus de Marek. La secuencia de nucleótidos de RLORF7 de los aislamientos analizados mostró similitud con las cepas de referencia virulentas y muy virulentas. Las secuencias de nucleótidos de la proteína 23 kDa y de LORF6 proporcionaron datos más importantes y eran más similares a las cepas de baja patogenicidad en comparación con las cepas virulentas y muy virulentas. Los motivos de nucleótidos específicos en los tres genes pueden indicar un aumento en la virulencia del virus de Marek y se encontraron en las cepas a partir del año 2006. De acuerdo con los resultados obtenidos, las cepas aisladas en 2012 son similares al grupo muy virulento plus. El estudio mostró que los fragmentos RLORF7, de la proteína de 23 kDa y de RLORF6 albergan motivos de secuencias que pueden tener alguna relación con el nivel de patogenicidad del virus de Marek. Sin embargo, el papel exacto de las regiones investigadas sobre la patogenicidad debe ser examinada aún más mediante cepas con genes bloqueados. Además, el verdadero patotipo del virus de la enfermedad de Marek sólo puede ser determinado por experimentos in vivo tradicionales.

Animal herpesviruses and their zoonotic potential for cross-species infection

Herpesviruses of humans and animals cause severe diseases that influence not only the health and epidemiological status but are also economically important in the context of food production. The members of Herpesviridae are host specific agents that also share many properties that potentially make them capable of crossing the species barriers. The objective of presented review paper was to summarize the relationship between herpesviruses of animals and humans and their zoonotic potential. In humans, the most epidemiologically important herpesviruses are represented by Human herepesvirus-1 and Human herpesvirus-2, which are commonly known as herpes simplex virus type 1 and 2, varicella-zooster virus (VZV), Epstein-Barr virus (EBV), Kaposis Sarcoma-associated herpesvirus (KSHV), cytomegalovirus (CMV), as well as Human herpesviruses: HHV-6A, HHV-6B, and HHV-7. However, in terms of the potential to cross the species barrier, there are a few herpesviruses, including B virus disease (CeHV-1), Mareks disease virus (MDV), Equid herpesvirus-1 (EHV-1) or pseudorabies virus (PRV), which are potentially able to infect different hosts. To summarize, in advantageous conditions the host specific herpesviruses may pose a threat for public health but also may exert a negative impact on the economical aspects of animal production. The most probable of these are zoonotic infections caused by B virus disease; however, close contact between infected animal hosts and humans may lead to transmission and replication of other Herpesviridae members.

Phylogenetic analysis of Columbid herpesvirus-1 in rock pigeons, birds of prey and non-raptorial birds in Poland.

BackgroundThe identity of herpesviruses isolated in Europe from domestic pigeons (Columbid herpesvirus-1 - CoHV-1) as well as falcons and owls remains unknown. All these herpesviruses are antigenically and genetically related. The falcons and owls are thought to have become infected during the ingestion of pigeon meat thus suggesting the virus’s capacity to infect a wide range of hosts. The aim of the conducted study was to detect the occurrence of CoHV-1 and estimating the similarities and differences in the DNA-dependent DNA polymerase gene of herpesviruses isolated from domestic pigeons, birds of prey and non-raptorial free-ranging birds in Poland.ResultsThe study has shown the presence of CoHV-1 in 20.4% (18/88) in the examined birds. In case of one CoHV-1, infected Peregrine Falcon (Falco peregrinus), neurological signs were observed. Nucleotide sequencing of the DNA-dependent DNA polymerase gene, showed a high similarity among Polish strains (100%), independently from the species of the affected birds. Only one compared CoHV-1 strain - KP 21/23 originating from Germany showed a slightly lower similarity at a level of 99.1%. Further analysis has shown the identity of DNA-dependent DNA polymerase of CoHV-1 strains and other herpesviruses present in poultry as well as other birds ranged from 35.4 to 44.9%. Interestingly CoHV-1 infection was also confirmed for the first time in four non-raptorial birds.ConclusionsThe current study has shown a high similarity of CoHV-1 strains and the possible transmission of herpesviruses between domestic rock pigeons and free-ranging birds including raptors and non-raptorial birds. Further studies focused on cloning and the analysis of the whole CoHV-1 genome which is needed to explain the role of the observed similarities and differences between field strains of columbid herpesviruses.

Genetic variance of Derzsy's disease strains isolated in Poland

The aim of this study was the assessment of the genetic variance of Derzys disease (GPV) strains isolated from cases occurring in Poland. The nucleotide and predicted aminoacid sequences of VP2 and VP3 surface proteins of the Polish GPV strains were compared with other strains previously isolated in Hungary, France, Germany, China and Taiwan. The observed genetic variance of the aminoacid sequence within the group of Polish strains was low and reached 5% of the overall analysed sequence. Considerable differences in aminoacid sequence were found in the case of Polish field GPV strains and Muscovy duck parvovirus strain MDPV FM which was also analysed in this study. The conducted investigations confirmed the presupposition that Polish GPV strains and strains previously isolated in Hungary and France share a common origin.

Comparison of Loop-Mediated Isothermal Amplification and PCR for the Detection and Differentiation of Marek's Disease Virus Serotypes 1, 2, and 3

SUMMARY.  The previously conducted study on loop-mediated isothermal amplification (LAMP) has shown its usefulness for the detection of Mareks disease virus (MDV) virulent field strains. The current study improves the previously designed LAMP method with an additional pair of loop primers, which accelerates the reaction, and describes two other LAMP procedures for the specific detection of FC126 strain of turkey herpesvirus and nonpathogenic SB-1 strain. The developed LAMP procedures were also confirmed and compared with PCR. Each LAMP reaction used three pairs of specific primers designed to target the nucleotide sequence of the very virulent MDV strain, the SB-1 strain of MDV-2, and turkey herpesvirus, respectively. All LAMP reactions were flexible and provided reliable results at a wide range of incubation temperatures from 54.0 to 62.3 C in 15 to 90 min. LAMP does not need any thermocyclers, because all assays were conducted in a water bath. The green fluorescence signal was recorded under ultraviolet illumination in LAMP samples containing virulent MDV and turkey herpesvirus where SYBR Green was added to the reaction mixture, whereas the SB-1–positive samples presented orange illumination after GelRed™ staining solution. The sensitivity of the three LAMP reactions ranged from 2 log10 plaque-forming units (PFU)/ml of the virulent MDV HPRS-16 strain and turkey herpesvirus (HVT) to 3 log10 PFU/ml of the SB-1 nonpathogenic strain. The sensitivity of the compared PCR was lower by 1–2 log10 PFU/ml. The conducted studies have shown that developed LAMP methods may be used instead of PCR for the detection and differentiation of virulent and nonpathogenic MDV strains used in prophylaxis against MD. LAMP may be conducted without access to thermocyclers. RESUMEN.  Nota de Investigación—Comparación de la amplificación isotérmica mediada por gazas y la reacción en cadena de la polimerasa para la detección y diferenciación de los serotipos 1, 2 y 3 del virus de la enfermedad de Marek. Un estudio llevado a cabo previamente sobre la amplificación isotérmica mediada por gazas (LAMP), demostró su utilidad para la detección de las cepas virulentas de campo del virus de la enfermedad de Marek (MDV). El estudio actual mejoró el método LAMP previamente diseñado mediante un par adicional de iniciadores de gaza, lo que acelera la reacción. También este estudio describe otros dos procedimientos LAMP para la detección específica de la cepa FC126 del virus herpes del pavo y de la cepa no patógena SB-1. Los procedimientos LAMP desarrollados también fueron confirmados y comparados con la PCR. Cada reacción LAMP utilizó tres pares de iniciadores específicos diseñados para detectar las secuencias de nucleótidos de la cepa de Marek muy virulenta, de la cepa SB-1 perteneciente al serotipo 2, y del herpesvirus de pavo, respectivamente. Todas las reacciones LAMP fueron flexibles y proporcionaron resultados confiables en un amplio rango de temperaturas de incubación desde 54.0 a 62.3 C en 15 a 90 min. Los procedimientos LAMP no necesitan de termocicladores, porque todos los ensayos se llevaron a cabo en un baño de agua. La señal de fluorescencia de color verde se registró bajo iluminación ultravioleta en muestras procesadas mediante el procedimiento LAMP que contenían al virus muy virulento de Marek y herpesvirus de pavo y donde se añadió SYBR Green a la mezcla de reacción, mientras que las muestras positivas para el virus SB-1 presentaron iluminación anaranjada después de la tinción con GelRed™. La sensibilidad de las tres reacciones LAMP tuvo un rango desde 2 log10 unidades formadoras de placas (UFP)/ ml para la cepa virulenta HPRS-16 y para el herpesvirus de pavo (HVT) y de hasta 3 log10 UFP /ml para la cepa no patógena SB-1. Por su parte, la sensibilidad de la PCR fue inferior en 1 a 2 log10 UFP/ ml. Los estudios realizados han demostrado que los métodos LAMP desarrollados pueden ser usados en lugar de la PCR para la detección y diferenciación de cepas virulentas o no patógenas del virus de Marek que son utilizadas en la profilaxis contra esta enfermedad. Los procedimientos LAMP pueden llevarse a cabo sin la necesidad de termocicladores.

Polymerase cross-linking spiral reaction (PCLSR) for detection of African swine fever virus (ASFV) in pigs and wild boars

The study reports the development of a polymerase cross-linking spiral reaction (PCLSR) for the detection of African swine fever virus (ASFV) DNA in blood collected from infected pigs and wild boars. The method uses 3 specifically designed primers. Two outer-spiral primers comprising of 3′ sequences complementary to ASFV p72 gene sequence and 5′end sequences complementary to exogenous gene of black widow alpha-latrotoxin as well as additional ASFV specific cross-linking primer. The method is specific exclusively to ASFV DNA without cross-reactions with cDNA of classical swine fever virus (CSFV), porcine reproductive respiratory syndrome (PRRSV) or porcine epidemic diarrhea virus (PEDV). The sensitivity of this technique reached 7.2 × 102 copies per μl−1 of plasmid containing p72 gene. The PCLSR was conducted at 65 °C creating cross-linked complex structures. The results of PCLSR were visualized using SYBR Green I dye, gel electrophoresis while the reaction progress was traced using real-time PCR system that resulted in registration of fluorescent curves and melting peaks at 85.3 °C. The developed PCLSR was examined using blood or tissue samples collected from selected 17 ASF cases from infected wild boars and 3 outbreaks in pigs. Further tests have been also conducted using 55 tissue samples from 23 outbreaks and 22 cases. These results showed that PCLSR might be further used for preliminary and cost-effective detection and surveillance of ASFV.