Application of loop-mediated isothermal amplification (LAMP) assays for the detection of bovine herpesvirus 1.

Bovine herpesvirus-1 (BoHV-1), a causative agent of Infectious Bovine Rhinotracheitis (IBR), is responsible for high economic losses in cattle farming industry. The use of testing methods that allow early detection of BoHV-1-infected animals is a key element of each program of IBR eradication. The aim of the study was to design and evaluate two variants of LAMP isothermal tests with SYBR Green fluorescence probes, specific to the genes encoding gD and gE glycoproteins of BoHV-1. LAMP gE BoHV-1 assay was able to distinguish between gE- and gE+ strains of the virus. Both LAMP gD and gE assays were specific to BoHV-1 and did not react with other related to BoHV-1 alphaherpesviruses. Sensitivity of LAMP gD was 2x104 copies of the viral genome whereas for LAMP gE it was 2x105. Diagnostic sensitivity calculated for LAMP gD was 64.7% whereas for LAMP gE it was 80%. Diagnostic specificity for LAMP gD and LAMP gE was 78.9% and 89.3%, respectively. LAMP assay can be a rapid and simple method of diagnosis of acute BoHV-1 infections and discrimination of gE- strains. However, relatively low diagnostic sensitivity of the method can limit its use in routine diagnostics.

Variability of non-structural proteins of equine arteritis virus during persistent infection of the stallion.

The genetic stability of ORF1a encoding non-structural proteins nsp1, nsp2, nsp3 and nsp4 of equine arteritis virus (EAV) has been analysed for nearly seven years in a persistently infected stallion of the Malopolska breed. Between November 2004 and June 2011, 11 semen samples were collected. Viral RNA extracted from semen of this carrier stallion was amplified, sequenced and compared with the sequences of the other known strains of EAV. Sequence analysis of ORF1a showed 84 synonymous and 16 non-synonymous mutations. The most variable part of ORF1a was the region encoding nsp2 protein with 13 non-synonymous substitutions. The degree of amino acid identity between isolates ranged from 98.91 to 100%. Only single non-synonymous mutations were detected in nsp1 (one substitution) and nsp4 (two substitutions). The most stable was nsp3 in which no amino acid substitutions were observed during the whole period of observation.

Application of real-time PCR for evaluation of distribution of equine herpesvirus type 1 in tissues of aborted fetuses.

A highly sensitive and specific real-time PCR assay was used for detection and quantitation of equine herpesvirus type 1 (EHV-1) in the different internal organs of aborted fetuses. Tissue samples from 23 aborted fetuses submitted to the Department of Virology of the National Veterinary Research Institute in Pulawy between 2012 and 2013 were used for testing. Total DNA was extracted using a phenol-chloroform-isoamyl alcohol standard protocol. A real-time PCR with forward and reverse primers encompassing a highly conserved region encoding viral glycoprotein B was adapted for diagnosis of EHV-1 infection. The detection limit of the assay was shown to be 6.0 × 10° of viral DNA copies and the obtained standard curve exhibited a linear range from 10° to 107 molecules. Sixteen out of twenty three aborted fetuses (69.5%) were positive for EHV-1 in real-time PCR. The highest EHV-1 DNA load was obtained for liver (mean Ct value: 15.7) and lung (18.2) samples, while the lowest was in the thymus (29.6) and placenta (28.4).

First report on equine herpesvirus type 4 isolation in Poland--evaluation of diagnostic tools.

Upper respiratory tract infections are still a serious problem in breeding and racing horses. The most common virological factors are EHV1 and EHV4, which are both a major cause of secondary infections. High EHV4 seroprevalence in Polish horses indicates a high transmission rate of this pathogen among horses and increases the need for proper diagnostics. The aim of this study was to develop a reliable laboratory diagnostic scheme for upper respiratory tract infections and to describe the first isolation of EHV4 in Poland. Twenty one nasal swabs collected from young horses under the age of 2 years showing clinical signs of equine rhinopneumonitis were tested with duplex PCR for simultaneous detection and differentiation between EHV1/EHV4. Positive samples were then subjected to virus isolation in Vero cells. Additionally, real-time PCR was developed which allowed viral copy numbers to be quantified and enabled defining that a DNA load below 10(3) copies per 1 ml of the sample reflected latent infection or decline of the disease. However, the sensitivity of traditional PCR proved to be sufficient in the diagnostic of the lytic infections and allowed identification of 10 EHV4 infected horses from which 3 strains were successfully isolated in cell culture. Another four EHV4 positive results were obtained by real-time PCR; but, a high Ct (threshold cycle) and a low virus DNA copy number suggested a latent infection. This report describes the first successful isolation of EHV4 from Polish horses.

Predominance of bovine viral diarrhea virus 1b and 1d subtypes during eight years of survey in Poland.

The genetic diversity of bovine viral diarrhea virus (BVDV) was determined from 65 animals persistently infected with BVDV and diagnosed between 2004 and 2011 in Poland. The samples originated from 28 herds in 12 provinces, where over 90% of the whole cattle population of Poland is reared. Phylogenetic analysis based on the fragments of two genomic regions of BVDV namely, 5'-untranslated region (5'-UTR) and N(pro) was performed. All the BVDV isolates belonged to BVDV-1 species and were further divided into four subtypes. There were 31 viruses of BVDV-1b subtype (47.6%) present in 12 herds, 24 of BVDV-1d subtype (36.9%) in 9 herds, 8 of BVDV-1f subtype (12.3%) in 5 herds and 2 BVDV-1g subtype (3.0%) in 2 herds. Neither BVDV-1a subtype, nor BVDV-2 species or any atypical bovine pestivirus were found among isolates tested. Despite increasing import of live cattle in the recent years, genetic diversity of Polish BVDV isolates was rather low.

First report of Schmallenberg virus infection in cattle and midges in Poland.

Two outbreaks of Schmallenberg virus (SBV) infection that coincided with the introduction of two bulls imported from France into two herds located in West Pomerania and Silesia provinces in Poland are described in detail. The first SBV real-time RT-PCR-positive result was obtained during routine testing of one of the imported bulls. The second bull and the affected farms were tracked by further investigation. Transmission of SBV into Polish cattle herds where the bulls were imported was confirmed by viral RNA detection in real-time RT-PCR, virus isolation followed by immunoperoxidase (IPX) staining and seroconversion. SBV RNA was detected also in Culicoides obsoletus pools caught in a trap located 5 km from one of the outbreaks. Testing nearly 900 samples collected prior to the two outbreaks from the same areas or provinces neighbouring with Germany where SBV cases had previously been detected gave negative results for the presence of SBV or specific antibodies. These cases are the first ones detected in cattle in Poland and provide evidence of recent transmission of the pathogen into the country and involvement of midge vectors.

Protein expression changes in cells inoculated with equine influenza virus: antibody microarray analysis.

Changes in the level of cellular proteins in cells inoculated with equine influenza virus H7N7 and H3N8 were studied with microarray technique. H3N8 induced pro-apoptotic proteins while H7N7 induced both pro- as well as anti-apoptotic factors. The higher level of some cytoskeleton components and proteins involved in the protein quality control was recorded. Relatively high number of proteins involved in the regulation of transcription was down-regulated. The pattern of changes observed for H7N7 and H3N8 may reflect differences in the biological properties of both serotypes.

Seroprevalence of BHV-1 (bovine herpesvirus type 1) among non-vaccinated dairy cattle herds with respiratory disorders.

The objective of this study was to estimate a herd-level seroprevalence of bovine herpesvirus type 1 (BHV-1) in herds with clinical symptoms of the respiratory tract. Eighty-three herds with suspected BHV-1 infection were selected and divided into two categories with respect to their size: small (n = 27) and large herds (n = 56). Samples were collected from calves, heifers and cows older than 24 months. Seroprevalence was determined using the gB ELISA test. The herd level seroprevalence was estimated as 53% (44/83) in the tested herds, 11.1% (3/27) in the small herds and 73.2% (41/56) in the large herds. Our study suggests that the current biosecurity measures still warrant improvement.

Atypical status of bovine spongiform encephalopathy in Poland: a molecular typing study.

The aim of this study was to analyze molecular features of protease-resistant prion protein (PrP(res)) in Western blots of BSE cases diagnosed in Poland with respect to a possible atypical status. Confirmed cases were analyzed by Western blotting with several monoclonal antibodies directed at N-terminal and core epitopes of prion protein (PrP). Most cases showed the classical glycoprofile characterized by the dominance of the di- over the monoglycosylated PrP(res) band, yielding di-/mono- ratios well above 2 and by reactivity with antibodies having their epitopes in bovine PrP region 110-242 (C-type cases). Surprisingly, seven cases of BSE were atypical. Six were classified as L-type based on a slightly lower molecular mass (M(r)) of the non- glycosylated band with respect to C-types and a conspicuously low di-/mono- ratio of glycosylated PrP(res) bands approaching unity. One case was classified as H-type because of a higher M(r) of PrP(res) bands on the blot when compared with C-type cases. A characteristic epitope of H-type PrP(res) occurred in the 101-110 region of PrP for which only antibody 12B2 had a sufficient affinity. The occurrence of atypical cases only in animals 9 years of age and older raises questions about the mechanisms of prion diseases and the origin of BSE.

Rabies surveillance, trends in animal rabies and human post-exposure treatment in Poland, 1990 -2004.

This paper describes recent changes in the epizootical and epidemiological situation of rabies in Poland. Analysis of routine surveillance data on animal cases and human post-exposure treatment was performed in order to examine the impact of introduction of cell culture vaccine for human use and the implementation of the fox immunisation programme. The success of the immunisation programme for wild animals has become evident during the past 3 years, as a 9-fold decrease in animal rabies cases has been observed. To date, however, the downward trend in animal rabies cases has had no effect on the frequency of administration of the post-exposure treatment for humans. Moreover, two cases of locally acquired human rabies have occurred in patients who did not receive post-exposure vaccination. These cases prove that rabies should be still considered a public health concern in Poland.

Specific immunoprophylaxis of some viral diseases in cattle.

Specific immunoprophylaxis has played a key role in prevention and control of animal infectious diseases for many years. Vaccines are commonly used to diminish economic losses in animal production caused by infectious agents. Currently, respiratory diseases are the main health problem in cattle. BHV1, BRSV and BVD-MD viruses are among the most important pathogens worldwide. They can cause a disease themselves or they can be one of many agents causing a respiratory syndrome. The following article describes the specific immunoprophylaxis of cattle viral diseases caused by such viruses.

Experimental inoculation of calves with laboratory strains of bovine viral diarrhea virus.

Diarrhea, erosions and ulcers of the oral mucosa, with conjunctival and nasal discharges, were observed in six calves inoculated with a mixture of two laboratory cytopathic reference strains of bovine viral diarrhea virus (BVDV)-Oregon C24 V and NADL. The clinical picture was accompanied by biphasic body temperature elevation, transient leukopenia and a decrease in the number of lymphocytes. High dose of viruses and multiple routes of inoculation promoted the development of clinical and hematological changes typical for BVDV infection although laboratory strains were used.

Prevalence of bovine viral diarrhea virus infection in bulls in artificial insemination centers in Poland.

This study was directed at the evaluation of the prevalence of bovine viral diarrhea virus (BVDV) infection in bulls in artificial insemination centers. Both serological and virological examinations were performed. Blood samples were tested in micro-seroneutralization test for BVDV antibodies. Virus isolation was performed in cell culture and BVDV antigen was detected in an indirect immunofluorescence assay with monoclonal antibodies. One hundred and seventy-five serum samples and 219 whole blood samples for virus isolation were tested. Neutralizing antibodies were found in 86% of the bulls. Persistent infection (PI) was detected in 0.9% of the analyzed blood samples.

[Personal experience in clinical use of low power laser therapy]. / Wlasne doswiadczenia w klinicznym wykorzystaniu lasera niskoenergetycznego.

The results of our own clinical trials regarding therapeutical effects of low power lasers in the treatment of some diseases of motional system, skin and lesions of peripheral circulation were presented in the paper. The laser therapy was used in over 320 patients in period of last 3 years. The infra-red laser (wave-length 904 nm, mean power 4 mW) was used. The procedures were performed daily, for 5 days a week in 2 stages: the scan of sore region (3-min.) and the irradiation of trigger points (2-3 min. each). As a result of 10-25 procedures a significant clinical improvement in 65-90% patients was obtained. On the base of obtained results one can conclude that low-power laser therapy could make a useful supplementary method of treatment of overloading syndrome of motional system, postthrombotic syndrome, lymphatic edema and trophic lesions of skin.

[Use of magnetic field in treatment of trophic leg ulcers]. / Wykorzystanie pola magnetycznego w leczeniu owrzodzenia podudzi.

Theoretical basis and favourable therapeutical application of magnetic field in case of trophic ulcerations of the lower limbs are discussed. As a clinical example of such action a case of a 39-year old female patient with trophic ulceration of the leg is presented. Practical advantages of magnetotherapy in similar to the described case patients are suggested.

[Usefulness of determining creatine kinase BB isoenzyme activity in early diagnosis of myocardial infarction]. / Ocena przydatnosci oznaczania aktywnosci izoenzymu BB kinazy kreatynowej we wczesnej diagnostyce zawalu serca.

The value of radioimmunoassay for creatine kinase BB isoenzyme (CK-BB) determination in early diagnosis of acute myocardial infarction was estimated. The clinical material consisted of 35 randomly selected patients admitted to a coronary care unit during the 4 h after chest pain suspected for myocardial infarction. In all patients standard 12 lead electrocardiograms were obtained on admission and at 24, 48 and 72 hours after admission. Blood samples for CK-BB analysis were collected on admission and at 4 hourly intervals for 48 h after admission. Aspartate aminotransferase (GOT) activity was determined in all patients on admission and in samples obtained at 24 and 48 hours. The patients were classified to 3 groups according to electrocardiographic and clinical findings. The first group consisted of 10 patients whose electrocardiograms fulfilled the criteria of transmural infarction. The electrocardiograms of 10 patients of the second group fulfilled the criteria of subendocardial infarction. The remaining 15 patients made up a third group of coronary insufficiency. The activity of CK-BB at various time intervals after chest pain in all groups was compared in order to estimate the value of this method for differentiation of the 3 causes of coronary pain. The frequency of positive results (the values exceeding upper limits of normal range for healthy people) at various time intervals after pain obtained with both enzymatic methods was compared in order to estimate the sensitivity of analysed method.(ABSTRACT TRUNCATED AT 250 WORDS)