Alternatives to zinc oxide in pig production.

Zinc oxide (ZnO) has been applied for many years in the production of pigs to reduce the number of diarrhoea in weaned piglets. In June 2022, the European Union banned the use of zinc oxide (ZnO) in pig feed. According to scientific reports, the may reason was the accumulation of this microelement in the environment of pig production. It has been shown that frequent application of ZnO can lead to increased antibiotic resistance in pathogenic swine microflora. The main alternatives to ZnO are probiotics, prebiotics, organic acids, essential oils, and liquid feeding systems. Alternatives to ZnO can be successfully used in pig production to reduce the number of diarrhoea among piglets during the postweaning period. Additional reports indicated that bacteriophage supplementation has a positive effect on the health of pigs. The article provides an overview of current ZnO substitutes that can be used in pig farming.

Vertical transmission of anti-ASFV antibodies as one of potential causes of seropositive results among young wild boar population in Poland.

The present study attempted to elucidate possible routes leading to the achievement of sero- positive results, among young (aged ≤1 year) wild boar population. In the years 2017-2018, the National Reference Laboratory (NRL) for African swine fever (ASF) in Poland examined nearly 27-thousand wild boar blood samples, collected during an active surveillance of ASF risk zones, for the presence of viral DNA and anti-ASFV antibodies. Out of all the examined samples, 420 were positive. However, in more than half of them (292 samples) antibodies against African swine fever virus (ASFV) were detected, while ASFV DNA was not detected in blood. Out of all 292 seropositive/PCR-negative samples, 126 belonged to young wild boars (aged ≤1 year). For this reason, the NRL in Poland has examined 10 selected seropositive wild boar carcasses to confirm or exclude post-mortem lesions for ASF as well as to investigate the presence of viral DNA in the internal organs. Neither pathological lesions for ASF nor the presence of genetic material of ASFV were found in the examined wild boars. To elucidate this outcomes, following hypotheses about possible reasons of the obtained results were drawn: the presence of convalescent animals, infection of low-virulent ASFV isolate and the vertical transmission of antibodies through the colostrum.

Effective inactivation of porcine epidemic diarrhea virus on contaminated surgery masks by low-concentrated sodium hypochlorite dispersion.

Coronaviruses present a considerable concern for humans and animals. The current world- wide pandemic of SARS-CoV-2 virus showed many gaps in understanding of coronaviruses spread and transmission. Because of lack of effective vaccine against SARS-CoV-2 the only preventive measures are represented by wearing protective masks and gloves thus limiting potential risk of contact with the airborne virus. Inversely, the limited time of protective function of the masks presents another drawback of their use. Therefore, the application of disinfection agent dispersed on the surface of protective masks may enhance their effectivity and safety of their application. The aim of the study was to examine the virucidal efficacy of low-concentra- ted sodium hypochlorite dispersed using ultrasonic humidifier on the surface of surgery masks. The study was conducted using SARS-CoV-2 surrogate virus, namely porcine epidemic diarrhea virus (PEDV) representing a model with similar biophysical properties and genomic structure to human coronaviruses. Five different concentrations of the disinfectant with different content of sodium hypochlorite were selected for the study. A final concentration of 0.228 g/L sodium hypochlorite effectively inactivated the PED virus and may support the biosafety of masks usage.

Virucidal effect of chosen disinfectants against African swine fever virus (ASFV) - preliminary studies.

Four commercial disinfectants were chosen for being generally accepted as effective against ASFV. Only two of them, based on sodium hypochlorite and potassium peroxymonosulfate, confirmed their effectiveness in selected concentrations. Taken together, our data supports the effectivenes of chemical disinfectants containing sodium hypochlorite (1%, 0.5% in low level soiling) and potassium peroxymonosulfate (1% in high level soiling). Furthermore, these results highlight the importance of pre-cleaning steps to remove soiling before proper disinfection which improves the effectiveness of tested disinfectants.

Comparison of loop-mediated isothermal amplification (LAMP) and cross-priming amplification (CPA) for detection of African swine fever virus.

The reliable and rapid diagnosis of infectious animal diseases presents an exceptionally im- portant aspect when considering their control and prevention. The paper describes the compara- tive evaluation of two rapid isothermal amplification methods for diagnosis of African swine fever (ASF). The robustness of loop-mediated isothermal amplification (LAMP) and the cross-priming amplification (CPA) were compared using samples obtained from ASF confirmed animals. Both assays were evaluated in order to define their diagnostic capabilities in terms of ASF diagnosis and reproducibility of the results. Investigations showed no cross-reactivity for other pig patho- gens and no significant differences in the specificity of both assays. The sensitivity of LAMP reached 90%, while that of CPA was 70%. In conclusion, both methods are suitable for imple- mentation in preliminary ASF diagnosis but further improvements are required to enhance their diagnostic sensitivity.

Four years of African swine fever in Poland. New insights into epidemiology and prognosis of future disease spread.

Four and a half years of African Swine Fever (ASF) in population of free-ranging wild boars and domestic pigs revealed a number of novel insights into the disease epidemiology. Until No- vember 20th, 2018, in total 3048 cases in wild boars and 213 outbreaks in domestic pigs have been confirmed. In spite of low contagiosity as well as low rate of ASF spread in wild boars the disease has an enormous socio-economical impact on the production of pigs in Poland. One of the most important aspects which directly influences the dynamics of ASF spread is the unpredictable hu- man activity. Another important factor responsible for continuous ASF spread is fast recovery of wild boar population in spite of efforts taken by hunters. Assuming our scientific opinion ASF seems to be present in wildlife for the incoming few or several years. Therefore, extraordinary measures should be prepared and undertaken to limit the risk of the occurrence of future out- breaks in domestic pigs. One of the most crucial issues is implementation of strict biosecurity measures in all domestic pigs holdings.

Morphology of immune organs after very virulent plus strain of Marek's disease virus infection in vaccinated hens.

Marek's disease (MD) outbreaks in poultry flocks may be associated with overriding of vaccine immune protection by very virulent (vvMDV) or very virulent plus (vv+MDV) strains. This paper presents the study on lymphoid organ morphology in the latent phase of MD caused by vv+MDV which break post-vaccinal protection in hens. We also immunohistochemically examined B and T populations as well as B/T and CD4+/CD8+ ratio of lymphocytes in lymphatic organs and, as a background, in MD lymphomas from non-lymphatic organs. The number of antigen expressed cells was evaluated as a percentage of positive cells in the one power field. Organ samples were collected from 24 dead reproductive hens (Ross 308 line) in age between 35-56 weeks, infected with vv+MDV. The hens originated from farms with MD outbreaks, despite earlier routine vaccination with CVI988/Rispens + HVT. The control organ samples originated from 15 clinically healthy hens at the same age and line, subjected to the same vaccination schedule. The number of CD3+, CD8+ and TCRγδ+ cells was significantly lower in MDV infected thymus, spleen and cecal tonsils in comparison to that found in the control organs. The proportion of CD4+ was also distinctly reduced in the thymus and limited in the spleen of MDV infected hens. This study revealed that infection with field vv+MDV isolates might break post-vaccinal protection and influence the central and peripheral immune system. The decrease in CD8+ and TCRγδ+ cell number in the thymus, spleen and cecal tonsils suggests that primarily these cells are involved in cell-mediated cytotoxicity against MDV transformed cells during latency.

Development of cross-priming amplification for direct detection of the African Swine Fever Virus, in pig and wild boar blood and sera samples.

UNLABELLED: African swine fever (ASF) is considered a major threat to the production of pigs worldwide. The ASF aetiological agent, ASFV, is the sole member of the Asfivirus genus, belonging to the Asfarviridae family. An effective ASF vaccine is not currently available, thus the only measures of ASF spread control include, reliable and fast diagnosis. Officially approved, diagnostic methods include, virus isolation, serological assays, including enzyme-linked immunosorbent assay and immunoperoxidase assay (IPT) and different modifications of the polymerase chain reaction (PCR). This paper describes the first development and application of a cross-priming amplification method (CPA) for the direct detection of genetic ASFV material, in blood and sera from pigs and wild boars. This method is specific only to ASFV DNA. The study showed that CPA had equal sensitivity, in comparison to the official, universal probe library (UPL) real-time PCR and reached 7·2 copies of standard plasmid DNA, containing a p72 gene fragment. This method was capable of detecting ASFV DNA in all examined blood samples, originating from pigs; n = 10 and wild boars; n = 76. The obtained results were also confirmed by the officially approved, real-time PCR. The developed CPA might be further used by local and county veterinary officers, hunters or pig farmers, for preliminary ASF diagnosis. SIGNIFICANCE AND IMPACT OF THE STUDY: The spread of the African swine fever virus (ASFV) among infected pigs and wild boars, is currently one of the most important facets of virus transmission in eastern Europe. Cross-priming amplification (CPA) has been developed, for fast and direct development of genetic ASFV material in the blood and sera of infected pigs and wild boars. It has been shown that CPA is a rapid, sensitive and specific isothermal method for the detection of ASFV DNA, in directly collected blood or sera from pigs and wild boars.

Cross-priming amplification for detection of bovine viral diarrhoea virus species 1 and 2.

AIMS: The aim of the study was the development of cross-priming amplification for ubiquitous detection of bovine viral diarrhoea virus (BVDV) species 1 and 2. METHODS AND RESULTS: Three and five specific primers, respectively, for the detection of BVDV-1 and BVDV-2, were designed on the basis of the sequences of the 5'UTR region. Incubation temperature and reaction time were determined. The optimal incubation conditions using water bath were 63°C for 75 min. Reverse transcription step (RT) was not required. The results were visualized under UV-light as a bright yellow fluorescence in positive samples. Additional method for results interpretation was agarose gel electrophoresis. Positive samples showed the presence of ladder-like banding patterns, formed by harpin-like cross-priming amplification (CPA) products. Sensitivity of CPA was compared with conventional RT-PCR and real-time RT-PCR. The CPA detection limit was 3500 copies for BVDV-1 and 80000 copies for BVDV-2 per reaction. For RT-PCR it was 350 and 80 copies for BVDV-1 and BVDV-2, respectively, and for real-time RT-PCR it was 35 copies for BVDV-1 and 80 copies for BVDV-2. The sensitivity of the developed method is sufficient to detect persistently infected (PI) animals. Positive results were found in 24 of 25 BVDV isolates belonging to species 1 and 2. Additionally, one false-negative result for BVDV-2 was detected. There were no false-positive results in negative samples and in the negative control. Both sets of primers used for the detection of BVDV-1 and BVDV-2 were not able to detect atypical pestiviruses. CPA positive results were confirmed by RT-PCR and real-time RT-PCR. CONCLUSIONS: CPA is a rapid method for the detection of BVDV-1 and BVDV-2 in field samples from PI animals. SIGNIFICANCE AND IMPACT OF STUDY: This is the first report on the application of the CPA method for the detection of BVDV.

Common occurrence of Gallid herpesvirus-2 with reticuloendotheliosis virus in chickens caused by possible contamination of vaccine stocks.

AIMS: The aim of this study was to investigate the common occurrence of reticuloendotheliosis virus (REV) among Gallid herpesvirus 2 (GaHV-2) infected chickens. The possible cause of this co-occurrence may be linked to contaminated vaccine stocks, which were also examined. METHODS AND RESULTS: The study was conducted on 25 field isolates of GaHV-2 collected between 2007 and 2013 from vaccinated chickens. Additionally, 10 commercial Marek's Disease vaccine stocks manufactured between 1993 and 2013, comprising of FC126 HVT, CVI988/Rispens and bivalent HVT + Rispens vaccines were examined. Chicken isolates were collected from the liver. Due to difficulties in differentiation between GaHV-2 and REV, by observation of clinical signs or lesions presented in liver or spleen, loop-mediated isothermal amplification (LAMP and RT-LAMP) as well as PCR-based methods were applied. CONCLUSIONS: The co-occurrence of GaHV-2 and REV genetic material was shown in 24 (96%) of 25 examined isolates. A marginal REV contamination was detected in three out 10 (30%) commercial vaccine stocks, mainly in bivalent HVT + Rispens vaccines produced between 2009 and 2012. SIGNIFICANCE AND IMPACT OF THE STUDY: Our results indicated the common occurrence of GaHV-2 and REV in Polish chicken flocks, which is probably linked to contaminated HVT + Rispens vaccine stocks. Reasons for the detection of a marginal REV contamination need to be further elucidated.

Expression of goose parvovirus whole VP3 protein and its epitopes in Escherichia coli cells.

The aim of this study was the expression of goose parvovirus capsid protein (VP3) and its epitopes in Escherichia coli cells. Expression of the whole VP3 protein provided an insufficient amount of protein. In contrast, the expression of two VP3 epitopes (VP3ep4, VP3ep6) in E. coli, resulted in very high expression levels. This may suggest that smaller parts of the GPV antigenic determinants are more efficiently expressed than the complete VP3 gene.

The development and evaluation of cross-priming amplification for the detection of avian reovirus.

AIMS: The aim of this study was to develop and evaluate cross-priming amplification (CPA) for the detection of avian reovirus (ARV). METHODS AND RESULTS: Five specific primers were designed, on the basis of the σNS sequence of the S1133 ARV strain. Incubation temperature and primer concentrations were determined. The optimal incubation conditions in a water bath were 61.3°C for 45 min. No reverse transcription stage was required. The results were recorded under UV light illumination as a bright, greenish fluorescence in positive samples, and through the lack of this in negative controls and samples. Additionally, the gel electrophoresis performed during analysis showed the presence of ladder-like patterns, formed by hairpin-like CPA products. The developed CPA method was compared to reverse-transcription polymerase chain reaction (RT-PCR) and real-time RT-PCR. Sensitivity of CPA was estimated using seven dilutions of standard S1133 strain and reached 0.05 log10 TCID50 ml(-1). RT-PCR sensitivity reached 2.5 log10 TCID50 ml(-1) and was 1000 times lower than for CPA, whereas real-time RT-PCR sensitivity reached 1.5 log10 TCID50 ml(-1). Analysis of 32 RNAs extracted from field specimens showed the presence of an ARVσNS fragment in 4 (12.5%) samples. Interestingly, the positive samples originated from flocks affected by Marek's disease (MD) or fowl adenovirus (FadV). RT-PCR was unable to detect ARV, due to its lower sensitivity. However, the real-time RT-PCR that was conducted confirmed the CPA study. CONCLUSIONS: CPA is a very sensitive and rapid method, which allows ARV detection using simple laboratory equipment. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report on the application of the CPA method for detection of ARV, using simple laboratory equipment.

Detection of CoHV-1 by loop-mediated amplification (LAMP). Application of LAMP for CoHV-1 incidence monitoring in domestic pigeons.

UNLABELLED: The occurrence of Columbid herpesvirus-1 (CoHV-1) in domestic pigeons presents a potential threat for their husbandry and trade. CoHV-1 causes Smadel disease among pigeons but may not be manifested by any clinical signs and complicates secondary infections. The material for our study originated from 42 domestic pigeons sent to private veterinary practice in Lublin, Poland, between 2011 and 2013. Some of birds showed clinical signs similar to Smadel disease. The cytological examination also indicated on CoHV-1 infection. The incidence of CoHV-1 was tested in DNA extracted from liver of birds by loop-mediated isothermal amplification (LAMP). LAMP was used for the monitoring of CoHV-1 presence among pigeons in Poland. Our study showed that LAMP was capable of detecting CoHV-1 presence in 8 (19%) of 42 examined birds without the use of any advanced laboratory equipment. The results were confirmed by real-time PCR and virus isolation in chicken embryo fibroblasts. This is the first report on LAMP application for successful detection of CoHV-1 in domestic pigeons. SIGNIFICANCE AND IMPACT OF THE STUDY: The incidence of Columbid herpesvirus-1 (CoHV-1) in pigeons was examined for the first time by loop-mediated isothermal amplification (LAMP). The study showed the presence of CoHV-1 in 8 of 42 examined domestic pigeons. LAMP technique developed within this study may be used by not well-equipped veterinary laboratories.

Occurrence of reovirus infection in Muscovy ducks (Cairina moschata) in south western Poland.

During the summer 2012 an incidence of high mortality, above 44 percent, in two flocks of Muscovy ducklings in Poland was noted. The clinical signs included considerable weight loss and inability to walk. During the post-mortem evaluations dehydration and enteritis, gouty kidneys as well as hemorrhagic liver and spleen lesions were found. The laboratory diagnosis included agar gel precipitation assay (AGP) as well as polymerase chain reaction (PCR) or reverse transcription PCR for the presence of goose parvovirus (GPV), duck circovirus (DuCV), duck reovirus (DRV) and avian reovirus (ARV). Interestingly, the examinations performed by AGP showed partial reactivity of liver homogenates from Muscovy ducklings with chicken S1133 antiserum. The presence of duck reovirus RNA was also detected by real-time RT-PCR targeting the chicken reovirus sigma NS fragment, while the sequencing showed major similarity to chicken S1133, 1733, GX/2010/1 and TARV-MN2 reovirus strains. The virus sequence was also related to a previously isolated TH11 strain from Muscovy ducks in China. Further study is needed in order to explain the particular epidemiology of the reovirus infection of Muscovy ducklings.

Interactions between Marek's disease virus Rispens/CVI988 vaccine strain and adenovirus field strain in chicken embryo fibroblast (CEF) cultures.

The aim of the study was to determine the influence of adenovirus infection on the replication of Marek's disease virus vaccine strain Rispens/CVI988 during in vitro co-infection studies. Adenovirus field strain JN-5/10j was isolated from sick chickens. The study was conducted in chicken embryo fibroblast cultures (CEF). Monolayers of CEFs were infected with Rispens strain and field adenovirus strain JN-5/10j with different doses (10(1.0)-10(3.0) TCID50) in the following manner: a) simultaneously, b) first, infection with Rispens strain and after 24 h infection with adenovirus strain JN-5/10j and c) infection with adenovirus strain JN-5/10j 24 h before infection with Rispens strain. After 18, 24, 48, 72, and 96 h of incubation, the copy number of the pp38 gene of Rispens strain was determined using Real-time PCR. The results indicated that the Adenovirus infection before the infection with Rispens strain reduced the replication of the pp38 gene after 48 h by 2 log10.

Incidence of parvoviruses in chickens infected with Marek's disease virus.

The aim of the study was to determine co-occurrence of Marek's disease virus (MDV) and chicken parvovirus (ChPV) co-occurrence in field chicken flocks. The materials for the study derived from 115 broiler chickens or layer hens originated from 23 farms with suspicion of Marek's disease (MD). Dual infection with MDV and ChPV was found in 23 (20%) examined chickens. The results obtained suggest a possibility of influence of ChPV infection on efficacy of the MD vaccines.

Viral infections in goose flocks in Poland.

The aim of this study was to determine the infectious agents isolated from infection - suspected geese sent for the diagnostic examination to National Veterinary Research Institute. The birds were sent from goose flocks localized in different parts of Poland. Totally, 1,013 birds from 122 flocks were examined. The presence of goose parvovirus (GPV), goose haemorrhagic polyomavirus (GHPV), and goose circovirus (GoCV) was detected by triplex PCR. The presence of GPV DNA was shown in 36 flocks. The disease was most frequently diagnosed in goslings aging 3.5 weeks (ten flocks), and 2.5 weeks (six flocks). The analysis of the nucleotide sequence of VP1 encoding region has shown close similarity of Polish GPV strains within the group which ranged from 92% to 100%. Moreover, the similarity level of these strains with GPV isolated in Europe was from 91.3% to 100%. The occurrence of GoCV DNA was shown in 25 goose flocks. The presence of GoCV DNA was found among geese aged from 2 to 6 weeks, but predominantly in those aging 3.5 (three flocks) and 5 weeks (five flocks). The sequence analysis of PCR products from the sequenced region of ORFC1 capsid protein of GoCV has shown that Polish isolates share from 85% to 91% similarity with the sequences of GoCV strains isolated in other countries. The presence of DNA of GHPV was found in 3-week-old geese. During the last 2 years the presence of GHPV was confirmed in three flocks of goslings at the age from 3 to 3.5 weeks. During the last 12 years the occurrence of co-infection with GPV and GoCV was detected in six flocks aging from 5 to 6 weeks.

Sequence analysis of meq oncogene among Polish strains of Marek's disease.

The main goal of the present study was to analyse the nucleotide and amino acid sequence of Meq--the main oncoprotein of Marek's disease virus. The conducted analysis of the nucleotide sequences of MDV has shown a significant similarity between Polish and reference strains of MDV assigned to the vvMDV and vv+MDV pathotypes. Some of the detected nucleotide point mutations were specific only for Polish strains. The sequence analysis has shown a number of single substitutions and two poly-amino acid insertions among strains defined as mild-pathogenic (mMDV) and one attenuated vaccine strain CVI988--Rispens. These results indicate that there is a close relation between changes in nucleotide or amino acid sequence of meq and the virulence of Polish MDV strains.