Comparison of agar gel immunodiffusion test, enzyme-linked immunosorbent assay and PCR in diagnostics of enzootic bovine leukosis

Bovine leukaemia virus (BLV) is a retrovirus that induces a chronic infection in cattle. Once infected, cattle remain virus carriers for life and start to show an antibody response within a few weeks after infection. Eradication and control of the disease are based on early diagnostics and segregation of the carriers. The choice of a diagnostic method depends on the eradication programme, money resources and characteristics of the herd to be analysed. The agar gel immunodiffusion (AGID) test has been the serological test of choice for routine diagnosis of serum samples. Nevertheless, in more recent years, the enzyme-linked immunosorbent assay (ELISA) has replaced the AGID for large scale testing. For this purpose, commercially available BLV-ELISA kits were compared to the AGID and to the polymerase chain reaction (PCR) method performed with two sets of primers, amplifying env region. The ELISA kit based on the p24 core protein was found to be less specific and served as a screening test. The ELISA kit based on the envelope glycoprotein (gpSI) served as a verification test and gave a good correlation with the AGID test and PCR method. However, ELISA showed a higher sensitivity than AGID. The p24 based ELiSA was useful for screening a large number of samples, whereas gp51 based ELISA, AGID and PCR were more important for detecting the antibody response against the individual BLV-proteins and therefore for verification of the infection with BLV

Kontrola arteritisa konja na jednoj ergeli.

An epidemiology of infection with equine arteritis virus (EAV) on one stud farm with approximately 350 horses in the period from 1995 to 2008 was studied. Infection was detected by virological methods, using a virus neutralisation test (VNT) for EAV antibody detection in serum samples, and virus isolation and RT-PCR test for virus detection in semen. No clinical picture of the disease was observed. The highest seroprevalence (nearly 100%) was among stallions and old mares, while seroprevalence among young fi llies, before mating, was lower than 9%. A high incidence for seroconversion was detected among fi llies after mating. Virus was detected by RT-PCR and by a virus isolation test in the semen of 40.7% of 76 seropositive stallions. The 8 stallions, which were shedding EAV, were infected within the period of the first three years after birth, but the other 12 seropositive stallions, which were negative for EAV in semen samples, became firstly seropositive 5 years after birth. In this study we confirmed that the major transmission of EAV on the stud farm occurred from shedding stallions to fillies during the mating time, but an important role of virus transmission to other horses is also played by contact between different groups of animals. Virus positive stallions were castrated and a new breeding unit for young foals was established. EAV negative foals were vaccinated and were bred outside the farm up to 3 years of age.Prikazana je epizootiologija arteritisa konja na jednoj ergeli s 350 konja u razdoblju od 1995. do 2008. godine. Zaraza je bila dokazana na osnovi serološke pretrage virus neutralizacijskim testom (VNT), izdvajanja i identifikacije virusa te RT-PCR-om u sjemenu pastuha. Klinički znakovi bolesti nisu bili primijećeni. Najveća seroprevalencija (gotovo 100%) bila je dokazana u pastuha i starih kobila dok je seroprevalencija u ždrjebica prije pripusta bila manja od 9%. Visoka incidencija serokonverzije bila je dokazana u ždrjebica nakon pripusta. Virus je bio dokazan RT-PCR-om i izdvojen iz sjemena 40,7% od 76 serološki pozitivnih pastuha. Osam pastuha koji su izlučivali virus arteritisa bilo je zaraženo u prvim trima godinama života, a ostalih 12 serološki pozitivnih u kojih virus nije bio izdvojen iz sjemena postali su prvi put serološki pozitivni pet godina nakon ždrijebljenja. Potvrđeno je da se virus u najvećoj mjeri prenosio s pastuha koji su izlučivali virus na ždrjebice za vrijeme pripusta. Za prijenos virusa važan je bio i izravan dodir među različitim skupinama životinja. Pastusi pozitivni na virus bili su kastrirani te je osnovana nova uzgojna jedinica za ždrebad. Ždrebad negativna na virus arteritisa bila je cijepljena, a do treće godine držana izvan ergele

Assessment of tuberculosis biomarkers in paratuberculosis-infected cattle

Introduction: Mycobacterium bovis and Mycobacterium avium subsp. paratuberculosis, respectively the causative agents of bovine tuberculosis (bTB) and bovine paratuberculosis (PTB), share a high number of antigenic proteins. This characteristics makes the differential diagnosis of the diseases difficult. The interferon gamma (IFN-γ), C-X-C motif chemokine ligand 10 (CXCL10), matrix metallopeptidase 9 (MMP9), interleukin 22 (IL-22) and thrombospondin 1 (THBS1) bovine genes have already been shown to be accurate transcriptional biomarkers of bTB. In order to improve the diagnosis of bTB and PTB, in the present study we evaluated the risk of false positivity of these bTB biomarkers in cattle with PTB. Material and methods: The transcription of these genes was studied in 13 PTB-infected cattle, using Mycobacterium avium subsp. paratuberculosis (MAP)-stimulated peripheral blood mononuclear cells (PBMC). Results: Overall, the levels of IFN-γ, CXCL10, MMP9 and IL-22 transcripts in MAP-stimulated PBMC failed to differentiate animals with PTB from healthy animals. However, as bTB-afflicted cattle do, the MAP-infected group also displayed a lower level of THBS1 transcription than the non-infected animals. Conclusion: The results of this study add new specificity attributes to the levels of transcription of IFN-γ, CXCL10, MMP9 and IL-22 as biomarkers for bTB.Instituto de BiotecnologíaFil: Klepp, Laura Ines. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Agrobiotecnología y Biología Molecular (IABIMO); ArgentinaFil: Klepp, Laura Ines. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Colombatti Olivieri, Maria Alejandra. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Agrobiotecnología y Biología Molecular; ArgentinaFil: Colombatti Olivieri, Maria Alejandra. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Moyano, Roberto Damian. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Agrobiotecnología y Biología Molecular; ArgentinaFil: Moyano, Roberto Damian. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Romano, Maria Isabel. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Agrobiotecnología y Biología Molecular; ArgentinaFil: Romano, Maria Isabel. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Malovrh, Tadej. University of Ljubljana. Veterinary Faculty. Institute for Microbiology and Parasitology; EsloveniaFil: Ocepek, Matjaž. University of Ljubljana. Veterinary Faculty. Institute for Microbiology and Parasitology; EsloveniaFil: Blanco, Federico Carlos. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Agrobiotecnología y Biología Molecular (IABIMO); ArgentinaFil: Blanco, Federico Carlos. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Bigi, Fabiana. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Agrobiotecnología y Biología Molecular (IABIMO); ArgentinaFil: Bigi, Fabiana. Consejo Nacional de investigaciones Científicas y Tecnológicas; Argentin

Japanese knotweed rhizome bark extract inhibits live SARS-CoV-2 in vitro

Coronavirus disease 2019 (COVID-19), a viral infectious respiratory disease, is caused by highly contagious severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and is responsible for the ongoing COVID-19 pandemic. Since very few drugs are known to be effective against SARS-CoV-2, there is a general need for new therapeutics, including plant-based drugs, for the prophylaxis and treatment of infections. In the current study, the activity of a 70% ethanolic(aq) extract of the rhizome bark of Japanese knotweed, an invasive alien plant species, was tested for the first time against the wild-type SARS-CoV-2 virus using a specific and robust virus neutralization test (VNT) on Vero-E6 cells, which best mimics the mechanism of real virus–host interaction. A statistically significant antiviral effect against SARS-CoV-2 (p-value < 0.05) was observed for the 50.8 µg mL−1 extract solution in cell medium. A suitable extract preparation was described to avoid loss of polyphenols throughout filtration of the extract, which was dissolved in cell medium containing fetal bovine serum (FBS). The significance of the differences between the sums of the test and control groups in the incidence of cytopathic effects (CPE) was determined using the one-way ANOVA test. A dose–response relationship was observed, with the cytotoxic effect occurring at higher concentrations of the extract (≥101.6 µg mL−1). The obtained results suggest possible use of this plant material for the production of various products (e.g., packaging, hygiene products, biodisinfectants, etc.) that would be useful against the spread of and for self-protection against COVID-19

Diagnostics procedures in rabies

Rabies is a major zoonosis for which diagnostic techniques can only be performed in the laboratory. Laboratory techniques are preferably oriented on tissue removed from the cranium: hippocampus (Ammon's horn), cerebellum and the medulla oblongata or tissue liquids. Clinical observation may only lead to a suspicion of rabies. The only way to perform a reliable diagnosis of the disease is to identify the virus or some of its specific components using laboratory tests such as histological identification of characteristic cell lesions, immunochemical identification of rabies virus antigen and virus isolation. Serological tests are rarely used in epidemiological surveys but much more frequently in control of the vaccination programs (e.g. oral vaccination). Most commonly used serological tests are the virus neutralization test on cell culture (FAVN), virus neutralization in mice and ELISA

The effects of feed naturally contaminated with Fusarium mycotoxins on the thymus in suckling piglets

In this study, feed naturally containing Fusarium mycotoxins was fed to gilts during the perinatal period, and the effects on the thymus were investigated in one-week-old piglets. Twenty gilts were divided into equal control (0.26 mg deoxynivalenol, DON) and experimental (5.08 mg DON, 0.09 mg zearalenone and 21.61 mg fusaric acid per kg of feed) groups. One suckling piglet from each litter (n = 20) was sacrificed at one week of age to obtain thymus samples for further analysis. The cortex to medulla ratio of the thymus was morphometrically analysed using NIS Elements BR (Nikon) software. Paraffin-embedded thymus sections were stained to quantify apoptosis (with terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling – TUNEL method), cellular proliferation (Ki-67) and macrophages (MAC 387). The results showed that the thymus cortex (P = 0.023) to medulla (P = 0.023) ratio was significantly lower in the experimental group. The number of apoptotic cells (cortex, P = 0.010, medulla, P = 0.001) and the number of proliferating cells in the thymus cortex (P = 0.001) and medulla (P < 0.001) were significantly higher in the experimental group. Our results indicate that feeding Fusarium mycotoxins to a parent animal during the perinatal period induces significant alterations in the thymus of one-week-old piglets, which indicates an immunosuppressive effect in piglets

Presence of antibodies against rabies in wild boars

Serum samples of 746 shot wild boars collected throughout Slovenia during the hunting season of 2005/2006 were examined for the presence of antibodies against rabies virus: 541 samples were collected in areas subjected to yearly antirabies vaccination, and 205 samples were collected in areas where preventive antirabies vaccination was not practised. Using a modified enzyme-linked immunosorbent assay (ELISA), in 209 out of 746 sera (28%) the levels of antibodies against rabies virus were higher than 0.5 IU/ml and deemed positive. A total of 173/541 (32%) and 36/205 (18%) samples were positive in the vaccinated and nonvaccinated areas, respectively. Further analysis of 191 out of the 746 samples using the fluorescent antibody virus neutralisation (FAVN) test revealed the presence of antibodies against rabies virus in 122/191 (64%) samples. This is the first extended research reporting that antibodies against rabies virus that originate from preventive oral vaccination targeting the fox population are present in wild boar

Experiences with rabies eradication programs

Oral vaccination as a method of rabies eradication in the field was first started in Switzerland in 1978 and after 1984 several other EU countries followed this practice. Due to oral vaccination some European countries are now rabies-free in terrestrial animals. In Slovenia, after the first experimental oral vaccination and study of vaccination models from 1988 -1992, the spring-autumn campaigns have been carried out since 1995. The model of oral vaccination of wildlife requires 16-20 baits per km2 in the vaccination area. The baits were distributed by plane. They were dispersed from a height of 300-500 m. The aeroplanes' paths were 1000 metres apart. In the vaccination campaigns two vaccines were used. Lysvulpen®, produced by the Bioveta company at the Czech Republic, was laid down in the southwestern part of the country, and Fuchsoral®, produced by the German company Impfstoffwerk Dessau-Tornau, was placed in the eastern part of Slovenia. A rapid decline of rabies was evidenced from 1995 to 1999, when the oral vaccination program in the whole territory using the aircraft baits distributing system was practiced. In 1999, only 6 rabies cases were laboratory-confirmed, whereas in 1995, 1089 rabies cases were documented. Of the 14 rabies cases detected in 1998, 12 were found as an island in a circle with a radius of 30 km in the centre of the vaccinated area. In 2000 and 2001, rabies incidence increased again, so it was decided to change the baits distribution system in the year 2001. The vaccination by crossing flights in certain areas was introduced. In the next year (2002), after changing the vaccination strategy, positive cases rapidly dropped and only 15 cases in 2002, and 8 cases were found in 2003, near the non-vaccinated border with Croatia