Darja Barlič-Maganja

Human, porcine and bovine rotaviruses in Slovenia: evidence of interspecies transmission and genome reassortment

A surveillance of human, porcine and bovine rotaviruses was carried out in Slovenia in 2004 and 2005. Stool samples were collected from a total of 406 pigs (373 from asymptomatic animals), 132 cattle (126 from asymptomatic animals) and 241 humans (all with diarrhoea), tested for group A rotaviruses using RT-PCR and analysed by sequencing. The aims of the study were to determine the incidence of asymptomatic rotavirus infection in animals, to look for evidence of zoonotic transmission and to detect reassortment among rotaviruses. The rates of asymptomatic shedding of rotaviruses in pigs and cattle were 18.0 % (67/373) and 4.0 % (5/126), respectively. Evidence for zoonotic transmission was detected in one human rotavirus strain, SI-MB6, with the G3P[6] genotype combination, as the nucleotide and predicted amino acid sequences of the VP6, VP7, VP8* and NSP4 genes of strain SI-MB6 and of porcine strains showed high nucleotide and amino acid sequence identity. Two porcine rotavirus strains carried VP7 of probable human origin, suggesting an interspecies reassortment event in the past.

Detection and molecular characterisation of noroviruses and sapoviruses in asymptomatic swine and cattle in Slovenian farms

Acute infectious caliciviral gastroenteritis is a common illness in people all over the world. Two genera of the Caliciviridae family, Norovirus and Sapovirus, which usually cause disease in humans, can also be found in animals where they do not always cause clinical signs of gastroenteritis. To investigate the presence of norovirus (NoV) and sapovirus (SaV) strains in asymptomatic swine and cattle, a total of 525 faecal (406 pigs and 119 cattle) specimens were collected during 2004 and 2005 from 8 pig and 4 cattle farms geographically dispersed across Slovenia. RT-PCRs and sequencing were carried out using primers targeting RdRp and capsid regions of both NoVs and SaVs. NoV positivity was detected in both bovine (2/108 [1.9%]) and porcine (5/406 [1.2%]) faecal specimens while SaV positivity was present only in porcine (29/406 [7.1%]) specimens. All porcine NoV strains (n=5) detected were attributed to a single farm, while the porcine SaV strains (n=29) detected came from 5 different farms. Phylogenetic analysis of nucleotide sequences of partial RdRp fragments placed two of the bovine NoV strains in genogroup GIII. Of the 5 porcine NoV strains, 4 clustered with GII.11, while 1 strain showed the presence of GII.18. The majority [24/29, 82.7%] of the porcine SaV strains clustered in GIII within two separate lineages, while 5 strains clustered into recently identified genetic clusters GVII (3 strains), GVIII (1 strain) and unknown (1 strain), respectively. Although NoV and SaV strains in asymptomatic swine and cattle were detected at low levels, they were still phylogenetically placed in a common pattern within both genera showing great genetic variability. There were no detected human-like strains in this study.

Bovine viral diarrhoea (BVD) infections – control and eradication programme in breeding herds in Slovenia

A Slovenian BVD control and eradication programme was initiated in 1994, and the results from testing of bovine herds for antigen and antibodies in 1996 are presented. Samples originating from breeding herds, breeding herds for young bulls, and insemination stations were tested by antigen or antibody ELISA, or by PCR. Out of 7968 samples from 354 herds we found 18% of the animals antibody-positive. In one region situated in the north-east of Slovenia we found the herds to be almost nearly free of BVDV infections (5% prevalence). No positive antigen ELISA findings were done in 374 blood samples from recruitment herds for young bulls, whereas two out of 206 sera were investigated by PCR-reacted positive. The differences in seroprevalence found between regions is thought to be caused by differences in summer pasturing and husbandry practices.

Detection of infectious bursal disease virus in different lymphoid organs by single-step reverse transcription polymerase chain reaction and microplate hybridization assay

A rapid and sensitive method for the detection of infectious bursal disease virus (IBDV) RNA in different chicken lymphoid organs was developed. The method is based on a single-step reverse transcription polymerase chain reaction (RT-PCR) procedure and the enzyme-linked immunosorbent assay (ELISA) detection method of amplified products. Vaccinal IBDV strain and field isolates were used for the optimization of RT-PCR and for the determination of conditions for microplate hybridization and colorimetric detection of the amplicons. With this method, viral RNA could be detected in various stages of infection in samples of different lymphoid organs. Bursas and cecal tonsils were suitable organs for viral RNA detection at different times during IBDV infection. The RT-PCR/ELISA method can be applied for IBDV detection in routine diagnostic tests, which are not usually carried out because of the difficulties involved in isolating the virus.

Very Virulent Infectious Bursal Disease Virus in Southeastern Europe

SUMMARY. Clinical outbreaks of severe acute infectious bursal disease (IBD) were recorded since the mid- and late 1990s in several countries in the southeastern part of Europe. Epidemiologic data showed that both infectious bursal disease virus (IBDV)-vaccinated and IBDV-nonvaccinated chickens were affected with acute IBD and mortality up to 50% independent of the IBDV vaccination status of the appropriate parent flocks. For investigation of the causative agent of acute IBD, the variable region of VP2 was amplified, cloned, and sequenced. Nucleotide sequence analysis of polymerase chain reaction fragments showed several silent nucleotide exchanges in comparison with the sequence of the very virulent (vv) IBDV strain UK661. Also, restriction enzyme cleavage sites proposed specific for vvIBDV were present in all investigated strains. On the basis of clinical signs in affected flocks, recorded epidemiologic data, and sequence analysis, it is very likely the IBD-causing strains were of the vv phenotype.

A diagnostic method based on MGB probes for rapid detection and simultaneous differentiation between virulent and vaccine strains of avian paramyxovirus type 1.

Newcastle disease virus (NDV), also designated avian paramyxovirus type 1 (APMV-1), is a serious pathogen of poultry, causing highly contagious Newcastle disease (ND), with high morbidity or mortality, depending on the strain. Accordingly, rapid and reliable detection of APMV-1 and differentiation between vaccine and virulent strains is of crucial importance for ND diagnosis and plays an important role in effective control of the disease. In this study, two real-time reverse transcription polymerase chain reaction (real-time RT-PCR) assays using minor groove-binding (MGB) probes were developed for broad range detection and simultaneous pathotyping of APMV-1. The two assays were evaluated for their ability to detect in allantoic fluids viral RNA of all known APMV-1 lineages. Additionally, the applicability of the developed assays was assessed by the detection and pathotype prediction of APMV-1 in swabs and organs. The assays demonstrated high analytical specificity, sensitivity and good reproducibility, with coefficients of variation ranging from 0.2% to 3.9% and from 0.6% to 7.2% for intra-assay and inter-assay variability, respectively. The results indicated the suitability of both assays as a complementary method for rapid screening and typing of APMV-1.

Control of Rabies in Slovenia

Red foxes (Vulpes vulpes) are the main reservoir of rabies in Slovenia, whereas cases of rabies in other wildlife species occur sporadically. In 1995, a program of oral vaccination of wildlife in Slovenia was initiated; baits with oral vaccine were distributed by air at a density of 20 baits/km2. During 1995, when the oral vaccination program was started, 1,089 cases of rabies (including both wild and domestic animals) were reported. Five years later (1999), only six positive animals were detected among 1,195 tested (0.5%). Despite an increase in bait density (25 baits/km2) during the years 2000 and 2001, reported rabies cases increased to 115 and 135, respectively. In 2003, following initiation of a new bait-dropping strategy, which incorporated perpendicular rather than parallel flight lines, the number of rabies cases decreased to eight.

Evidence of avian influenza virus and paramyxovirus subtype 2 in wild-living passerine birds in Slovenia

A total of 670 cloacal swabs were taken from 37 species of wild-living passerine birds in years 2004, 2005, and 2006. The cloacal swabs were pooled into pools of three swabs for analysis. Isolation of avian influenza virus (AIV) and avian paramyxoviruses (APMV) was done on chicken embryos. One-step reverse-transcription polymerase chain reaction (RT-PCR) was used to detect AIV RNA. AIV nucleic acid was detected by RT-PCR in a sample of one common starling (Sturnus vulgaris). All attempts of AIV isolation from wild-living passerine birds’ samples were negative. APMV 2 was isolated in one robin (Erithacus rubecula) sample. In light of the presented results and literature data, passerines appear to play a minor role as potential disseminators of AIV and APMV; however, it seems that some wild-living passerine species could act as occasional carriers.

Detection of Foot and Mouth Disease Virus by RT-PCR and Microplate Hydridization Assay Using Inactivated Viral Antigens

A single step RT-PCR was tested for detection of foot and mouth disease virus (FMDV) and immunoenzymatic determination of amplified products in a microplate hybridization assay. Inactivated reference strains (ELISA antigen) of all seven serotypes were used to optimize the test. Oligonucleotide primers were selected from two different genomic regions coding for RNA polymerase and VP1 protein, respectively. The RT-PCR used to amplify the polymerase gene specific RNA detected FMDV strains A, C, O, Asia1 and SAT1, and the identity of the fragments obtained was confirmed with a specific internal biotin-labelled capture probe. For the amplification of the VP1 genome region, two sets of oligonucleotide primers were used. One primer pair was successfully applied for the detection of serotypes A, C, O and Asia1 and a second one for serotypes SAT1, SAT2, SAT3. The specific probe enabled the detection of all the amplified products in a PCR ELISA test. By comparison with antigen ELISA, the PCR ELISA method allowed the detection of smaller amounts of FMDV in the inactivated material examined. The application of molecular diagnostic methods to inactivated antigens offers a good alternative procedure for developing and optimizing a sensitive method for detection of FMDV in laboratories that are not allowed to work with viable FMDV.

Fusion and Matrix Protein Gene Sequence Analysis of Paramyxoviruses of Type 1(PMV-1) Isolated from Pigeons in Slovenia

Paramyxoviruses of type 1 (PMV-l) isolated from pigeons were genetically analyzed. A part of the fusion and the matrix protein genes were amplified and sequenced, Typical amino acid sequences associated with virulence were determined at the fusion protein cleavage site in all PMV-1 isolates. All Slovene pigeon PMV-1 strains share high amino acid sequence similarity with other pigeon strains. In the phylogenetic tree, they are clustered together with pigeon PMV-1 isolates with moderate pathogenicity. Phylogenetic analysis obtained from the fusion and the matrix protein gene alignments showed the same branching order. Viruses circulating among pigeons were found to form quite unique lineage of virulent NDV strains.