Multi-Residue Method for Pesticides Determination in Dried Hops by Liquid Chromatography Tandem Mass Spectrometry.

In this study a multi-residue determination method for 36 pesticides in dried hops was reported. The sample preparation procedure was based on the acetate buffered QuEChERS method. A few mixtures of dispersive solid phase extraction (dSPE) sorbents consisting PSA, C18, GCB, Z-Sep and Z-Sep+ were investigated to clean-up the supernatant and minimize matrix co-extractives. The degree of clean-up was assessed by gravimetric measurements, which showed the best results for mixtures containing the Z-Sep+ sorbent. This is the first study to apply Z-Sep+ sorbent for hops material and the first to improve the method for pesticide residues determination in hops. Samples were analysed using liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) and the procedure was validated according to the SANTE/11813/2017 document at four concentration levels: 0.02, 0.05, 0.1 and 1 mg/kg. The limits of quantification (LOQ) were in the range of 0.02-0.1 mg/kg. For all active substances, the trueness (recovery) ranged from 70 to 120% and the precision (RSDr) value was <20%. Specificity, linearity and matrix effect were also evaluated. The validated method was applied to the analysis of 15 real dried hop samples and the relevant data on detected residues were included.

A Diagnostic Device for In-Situ Detection of Swine Viral Diseases: The SWINOSTICS Project.

In this paper, we present the concept of a novel diagnostic device for on-site analyses, based on the use of advanced bio-sensing and photonics technologies to tackle emerging and endemic viruses causing swine epidemics and significant economic damage in farms. The device is currently under development in the framework of the EU Commission co-funded project. The overall concept behind the project is to develop a method for an early and fast on field detection of selected swine viruses by non-specialized personnel. The technology is able to detect pathogens in different types of biological samples, such as oral fluids, faeces, blood or nasal swabs. The device will allow for an immediate on-site threat assessment. In this work, we present the overall concept of the device, its architecture with the technical requirements, and all the used innovative technologies that contribute to the advancements of the current state of the art.

African Swine Fever Epidemic, Poland, 2014-2015.

In Poland, African swine fever (ASF) emerged in February 2014; by August 2015, the virus had been detected in >130 wild boar and in pigs in 3 backyard holdings. We evaluated ASF spread in Poland during these 18 months. Phylogenetic analysis indicated repeated incursions of genetically distinct ASF viruses of genotype II; the number of cases positively correlated wild boar density; and disease spread was very slow. More cases were reported during summer than autumn. The 18-month prevalence of ASF in areas under various animal movement restrictions was 18.6% among wild boar found dead or killed by vehicles and only 0.2% in hunted wild boar. Repeated introductions of the virus into the country, the primary role of wild boar in virus maintenance, and the slow spread of the disease indicate a need for enhanced biosecurity at pig holdings and continuous and intensive surveillance for fast detection of ASF.

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 Sokólka and Bialystok 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).

Rapid detection of Mycoplasma synoviae by loop-mediated isothermal amplification.

Mycoplasma synoviae (MS) remains a serious concern in production of poultry and affects world production of chickens and turkeys. Loop-mediated isothermal amplification (LAMP) of DNA has been recently used for the identification of different economically important avian pathogens. The aim of this study was to develop LAMP for simple and inexpensive detection of MS strains in poultry using specifically designed primers targeting hemagglutin A (vlh) gene. The assay was conducted in a water bath for 1 h at 63 °C. The results were visualized after addition of SYBR Green(®) fluorescent dye. LAMP was specific exclusively for MS without cross-reactivity with other Mycoplasma species. The sensitivity of LAMP was determined as 10(-1) CFU/ml and was 1,000 times higher than MS-specific polymerase chain reaction. LAMP assay was conducted on 18 MS field strains to ensure its reliability and usefulness. This is the first report on LAMP development and application for the rapid detection of MS isolated from chickens. This simple method may be applied by diagnostic laboratories without access to expensive equipment.

Application of cross-priming amplification (CPA) for detection of fowl adenovirus (FAdV) strains.

Fowl adenoviruses (FAdVs) are widely distributed among chickens. Detection of FAdVs is mainly accomplished by virus isolation, serological assays, various polymerase chain reaction (PCR) assays, and loop-mediated isothermal amplification (LAMP). To increase the diagnostic capacity of currently applied techniques, cross-priming amplification (CPA) for the detection of the FAdV hexon gene was developed. The single CPA assay was optimised to detect all serotypes 1-8a-8b-11 representing the species Fowl aviadenovirus A-E. The optimal temperature and incubation time were determined to be 68 °C for 2 h. Using different incubation temperatures, it was possible to differentiate some FAdV serotypes. The results were recorded after addition of SYBR Green I(®) dye, which produced a greenish fluorescence under UV light. The CPA products separated by gel electrophoresis showed different "ladder-like" patterns for the different serotypes. The assay was specific for all serotypes of FAdV, and no cross-reactivity was observed with members of the genus Atadenovirus, duck atadenovirus A (egg drop syndrome virus EDS-76 [EDSV]) or control samples containing Marek's disease virus (MDV), infectious laryngotracheitis virus (ILTV) or chicken anaemia virus (CAV). The results of the newly developed FAdV-CPA were compared with those of real-time PCR. The sensitivity of CPA was equal to that of real-time PCR and reached 10(-2.0) TCID50, but the CPA method was more rapid and cheaper than the PCR systems. CPA is a highly specific, sensitive, efficient, and rapid tool for detection of all FAdV serotypes. This is the first report on the application of CPA for detection of FAdV strains.

Visual detection of goose haemorrhagic polyomavirus in geese and ducks by loop-mediated isothermal amplification.

Goose haemorrhagic polyomavirus (GHPV) is an aetiological agent of haemorrhagic nephritis and enteritis of geese occurring in geese (Anser anser). GHPV may also infect Muscovy ducks (Carina mochata) and mule ducks. Early detection of GHPV is important to isolate the infected birds from the rest of the flock thus limiting infection transmission. The current diagnosis of haemorrhagic nephritis and enteritis of geese is based on virus isolation, histopathological examination, haemagglutination inhibition assay, ELISA and polymerase chain reaction (PCR). Recently, real-time PCR assay was developed which considerably improved detection of GHPV. In spite of many advantages, these methods are still time-consuming and inaccessible for laboratories with limited access to ELISA plate readers or PCR thermocyclers. The aim of our study was to develop loop-mediated isothermal amplification (LAMP) that may be conducted in a water bath. Two pairs of specific primers complementary to VP1 gene of GHPV were designed. The results of GHPV LAMP were recorded under ultraviolet light. Our study showed LAMP was able to specifically amplify VP1 fragment of a GHPV without cross-reactivity with other pathogens of geese and ducks. LAMP detected as little as 1.5 pg of DNA extracted from a GHPV standard strain (150 pg/µl). The optimized LAMP was used to examine 18 field specimens collected from dead and clinically diseased geese and ducks aged from 1 to 12 weeks. The positive signal for GHPV was detected in three out of 18 (16.6%) specimens. These results were reproducible and consistent with those of four real-time PCR. To the best of our knowledge this is the first report on LAMP application for the GHPV detection.

First survey of the occurrence of duck enteritis virus (DEV) in free-ranging Polish water birds.

Duck plague (DP) caused by anatid herpesvirus 1, also called duck enteritis virus (DEV), presents one of the most important concerns in mass waterfowl production. Apart from geese and ducks, free-ranging water birds are the most notorious infection carriers. The epidemiology of DEV in Western Europe remains unknown. Therefore, it was reasonable to conduct a study on its occurrence using modern but simple real-time loop-mediated isothermal amplification (LAMP). Analysis of 132 field isolates showed the presence of DEV in 96 birds (72.7 %), and it was found predominantly in wild ducks (Anas platyrhynchos) and mute swans (Cygnus olor). This virus was also found in graylag geese (Anser anser), tundra bean geese (Anser fabalis), and grey herons (Ardea cinerea). The results were recorded as green colour of positive samples, fluorescence under ultraviolet light, and florescent curves in a real-time PCR system. This study indicates the high prevalence of DEV among free-ranging water birds in Poland and the possible transmission to other birds settling in the water environment. This is the first report of DEV detection among free-ranging water birds in Poland.

Direct detection of Marek's disease virus in poultry dust by loop-mediated isothermal amplification.

Marek's disease virus (MDV) is a serious concern for poultry production and represents a unique herpesvirus model. MDV can be shed by doubly infected chickens despite vaccination. The fully infectious MDV particles are produced in the feather follicle epithelium (FFE), and MDV remains infectious for many months in fine skin particles and feather debris. Molecular biology methods including PCR and real-time PCR have been shown to be valuable for the detection of MDV DNA in farm dust. Recently, loop-mediated isothermal amplification (LAMP) was found to be useful in the detection of MDV in feathers and internal organs of infected chickens. LAMP is also less affected by the inhibitors present in DNA samples. Taking into account the advantages of LAMP, direct detection of MDV DNA in poultry dust has been conducted in this research. The detection of MDV DNA was possible in 11 out of the 12 examined dust samples without DNA extraction. The DNA was retrieved from dust samples by dilution and incubation at 95 °C for 5 min. The direct detection of MDV DNA in the dust was possible within 30 min using a water bath and UV light. The results were confirmed by electrophoresis and melting curve analysis of the LAMP products. Our results show that LAMP may be used to test for the presence of virulent MDV in poultry farm dust without DNA extraction.

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

Marek's 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 Marek's 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.

Hemorrhagic nephritis and enteritis in a goose flock in Poland--disease course analysis and characterization of etiologic agent.

Hemorrhagic nephritis enteritis of geese (HNEG) is an epizootic viral disease caused by infection with goose hemorrhagic polyomavirus (GHPV) that affects domestic geese. This study describes the epizootic analysis, laboratory diagnosis, and molecular characterization of GHPV isolates associated with HNEG cases in Poland. HNEG symptoms persisted in infected flocks for 2 wk with a 32% mortality rate. Primary gross lesions included hemorrhaging of the kidneys, intestines, and lungs. Histopathologic examination confirmed HNEG and identified that the causative agent was similar to other GHPV isolates and identical to the Toulouse 2008 isolate.

Effect of ß-hydroxybutyrate acid on gene expression levels of antioxidant biomarkers and growth hormone-related genes in liver cell culture.

Introduction: In dairy cattle, oxidative stress is a predominant problem associated with diseases and reproductive health issues. This study aimed to detect the variation in the antioxidant biomarkers by adding different concentrations of ß-hydroxybutyric acid (BHBA) and sought to elucidate its effects on the gene expression levels of growth hormone (GH) and antioxidant biomarkers in bovine hepatocytes. Material and Methods: Four antioxidant biomarkers, namely malondialdehyde (MDA), superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSH Px) were evaluated using commercially available bovine ELISA kits. The expression levels of the bovine GH, its receptor (GHR), insulin-like growth factor (IGF), IGF-1, IGF-1 receptor, CAT, SOD, GSH-Px and ß-actin (as a reference) genes in liver cell culture were determined by reverse transcriptase-PCR assay. Results: With the increase of BHBA concentration and culture time, the activities of SOD, CAT, and GSH Px biomarkers in hepatocytes decreased. However, the content of MDA in hepatocytes increased gradually with the increase of hepatocyte culture time and BHBA concentration. The qPCR results revealed that after adding BHBA, gene expression levels of GSH-Px, SOD and IGF biomarkers in hepatocytes began to differ in the culture groups at 12 h, whereas the gene expression level of the CAT and GHR biomarkers in hepatocytes began to differ at 6 h. Conclusion: Quantitative PCR results showed that the BHBA significantly downregulated the expression levels of the GHR gene and CAT, GSH Px and SOD antioxidant biomarker genes.

Label-Free Detection of African Swine Fever and Classical Swine Fever in the Point-of-Care Setting Using Photonic Integrated Circuits Integrated in a Microfluidic Device.

Swine viral diseases have the capacity to cause significant losses and affect the sector's sustainability, a situation further exacerbated by the lack of antiviral drugs and the limited availability of effective vaccines. In this context, a novel point-of-care (POC) diagnostic device incorporating photonic integrated circuits (PICs), microfluidics and information, and communication technology into a single platform was developed for the field diagnosis of African swine fever (ASF) and classical swine fever (CSF). The device targets viral particles and has been validated using oral fluid and serum samples. Sensitivity, specificity, accuracy, precision, positive likelihood ratio (PLR), negative likelihood ratio (NLR), and diagnostic odds ratio (DOR) were calculated to assess the performance of the device, and PCR was the reference method employed. Its sensitivities were 80.97% and 79%, specificities were 88.46% and 79.07%, and DOR values were 32.25 and 14.21 for ASF and CSF, respectively. The proposed POC device and PIC sensors can be employed for the pen-side detection of ASF and CSF, thus introducing novel technological advancements in the field of animal diagnostics. The need for proper validation studies of POC devices is highlighted to optimize animal biosecurity.

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

BACKGROUND: The 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. RESULTS: The 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. CONCLUSIONS: The 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.

Comparison of loop-mediated isothermal amplification and PCR for the detection and differentiation of Marek's disease virus serotypes 1, 2, and 3.

The previously conducted study on loop-mediated isothermal amplification (LAMP) has shown its usefulness for the detection of Marek's 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.

African Swine Fever: Transmission, Spread, and Control through Biosecurity and Disinfection, Including Polish Trends.

African swine fever is a contagious disease, affecting pigs and wild boars, which poses a major threat to the pig industry worldwide and, therefore, to the agricultural economies of many countries. Despite intensive studies, an effective vaccine against the disease has not yet been developed. Since 2007, ASFV has been circulating in Eastern and Central Europe, covering an increasingly large area. As of 2018, the disease is additionally spreading at an unprecedented scale in Southeast Asia, nearly ruining China's pig-producing sector and generating economic losses of approximately USD 111.2 billion in 2019. ASFV's high resistance to environmental conditions, together with the lack of an approved vaccine, plays a key role in the spread of the disease. Therefore, the biosecurity and disinfection of pig farms are the only effective tools through which to prevent ASFV from entering the farms. The selection of a disinfectant, with research-proven efficacy and proper use, taking into account environmental conditions, exposure time, pH range, and temperature, plays a crucial role in the disinfection process. Despite the significant importance of ASF epizootics, little information is available on the effectiveness of different disinfectants against ASFV. In this review, we have compiled the current knowledge on the transmission, spread, and control of ASF using the principles of biosecurity, with particular attention to disinfection, including a perspective based on Polish experience with ASF control.

Nine Years of African Swine Fever in Poland.

(1) Background: African swine fever (ASF) is a highly contagious and fatal haemorrhagic disease in domestic pigs and wild boars, causing significant economic loss to the swine industry in the European Union. The genotype II of African swine fever has spread in many European countries since the virus was detected in 2007 in Georgia. In Poland, the genotype II of the ASF virus was confirmed on 17 February 2014 in the eastern part of the country and appeared to have been transmitted to Poland from Belarus. Poland has been particularly affected by ASF epidemics in the last decade, resulting in a significant decline in the Polish pig population. Wild boars are the main reservoir of the African swine fever virus (ASFV), but human activities such as transportation and illegal animal trade are the primary reasons for the long-distance transmission of the disease. (2) Conclusions: During the nine years of ASF in Poland, multiple measures have been taken to prevent the spread of the virus among the wild boar population via the passive and active surveillance of these animals. With regard to pig farms, the only effective measure for preventing the spread of ASF is the efficient enforcement by state authorities of the biosecurity standards and the farmers' compliance with them.

Some aspects of the diagnosis and treatment of eosinophilic granuloma in cats.

Introduction: Eosinophils represent the most active cells in mammals that show protective and assistive activity in the host immune defence against helminth parasites. These cells are also responsible for the reduction of allergic and inflammatory reactions. The eosinophils play a key role in allergic reactions by secretion of different chemical molecules leading to swelling, lesions and granuloma onset. Material and Methods: The study was carried out on 30 cats with inflammatory skin lesions. The cats ranged in age from seven months to 13 years, and had an average age of three years. The research methodology included information on the disease, dermatological conclusions, concomitant disorders, medical and laboratory data and the treatment strategy. Results: In total, 30 cats were diagnosed with eosinophilic granuloma complex. The distribution of lesions was 87.1% in the skin and 12.9% at the skin-mucosal junction. The lesions increased and decreased with the seasons of spring and summer, and the onset of the disease usually coincided with exposure to fleas. Conclusion: Eosinophilic granuloma complex in cats is a serious pathology and frequently requires lifelong treatment, so it is important to diagnose it quickly and accurately to ensure optimal treatment of affected animals.

Loop-mediated isothermal amplification for the detection of goose circovirus.

BACKGROUND: Goose 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. RESULTS: The 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 GelRed(TM) 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. CONCLUSIONS: The developed technique due to its simplicity may be applied by any veterinary laboratory or even mobile diagnostics units for the routine detection of GCV.