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.

Rotaviral RNA found on various surfaces in a hospital laundry.

The aim of this investigative study was to determine the presence of rotaviral RNA at various control points (CP) of a hospital laundry. One of the possible sources of hospital infections is inappropriately laundered and disinfected hospital textiles. RT-PCR and nested PCR for gene amplification using specific primers following RNA isolation were used to determine the presence of rotaviral RNA on swabs. In addition, rotavirus suspensions were inoculated on marked surfaces as positive controls for different surfaces (cotton textiles, folding table and industrial dryer). Rotaviral RNA was found on various laundry surfaces: technical equipment, storage shelves, transport vehicles, personnel's hands, damp textiles, and folded laundry. Rotaviral RNA was also detected at all positive controls on tested surfaces after 24h. Based on the results, it is very important to take into consideration the proper handling of textiles after washing as one of the precautions against hospital-acquired infections. This paper reports the presence of rotaviral RNA for the first time on surfaces in laundries and equipment, as well as textiles.

Interactions of macrophages with probiotic bacteria lead to increased antiviral response against vesicular stomatitis virus.

Macrophages are an important cellular component of the innate immune system and are normally rapidly recruited and/or activated at the site of virus infection. They can participate in the antiviral response by killing infected cells, by producing antiviral cytokines such as nitric oxide and by producing chemokines and immunoregulatory cytokines that enable the adaptive immune response to recognize infected cells and perform antiviral effector functions. Probiotics, as a part of the normal gut intestinal flora, are important in supporting a functional yet balanced immune system. Improving our understanding of their role in the activation of macrophages and their stimulation of proinflammatory cytokine production in early viral infection was the main goal of this study. Our in vitro model study showed that probiotic bacteria, either from the species Lactobacillus or Bifidobacteria have the ability to decrease viral infection by establishing the antiviral state in macrophages, by production of NO and inflammatory cytokines such as interleukin 6 and interferon-gamma. These effects correlated with the mitochondrial activity of infected macrophages, therefore, the measurements of mitochondrial dehydrogenases activity could be implied as the first indicator of potential inhibitory effects of the probiotics on virus replication. The interactions between probiotic bacteria, macrophages and vesicular stomatitis virus (VSV), markedly depended on the bacterial strain studied.

Rotaviral RNA found in wastewaters from hospital laundry.

The aim of this prospective study was to determine the presence of rotaviral RNA in water from a hospital laundry. Since rotaviruses are known as major causal agents of diarrhoea in humans, it is necessary that laundering hospital textiles results in efficient chemo-thermal disinfection, thus minimizing the possibility of transmission of rotaviruses to immune-compromised patients in hospitals. RT-PCR and second round PCR for gene amplification using specific primers, succeeding ultra-filtration and RNA isolation, was used to determine the presence of rotaviral RNA in water samples. The results show that rotaviral RNA was found in wastewater after the washing process, thus confirming an inadequate disinfecting effect of the examined laundering procedures.

The porcine trophoblastic interferon-gamma, secreted by a polarized epithelium, has specific structural and biochemical properties.

At the time of implantation in the maternal uterus, the trophectoderm of the pig blastocyst is the source of a massive secretion of interferon-gamma (IFN-gamma), together with lesser amounts of IFN-delta, a unique species of type I IFN. This trophoblastic IFN-gamma (TrIFN-gamma) is an unprecedented example of IFN-gamma being produced spontaneously by an epithelium. We therefore studied some of its structural and biochemical properties, by comparison with pig IFN-gamma from other sources, either natural LeIFN-gamma (from adult leucocytes), or recombinant. Biologically active TrIFN-gamma is a dimeric molecule, of which monomers are mainly composed of a truncated polypeptide chain with two glycotypes, unlike LeIFN-gamma which is formed of at least two polypeptide chains and four glycotypes. TrIFN-gamma collected in the uterus lumen was enzymatically deglycosylated and analysed by mass spectrometry (MALDI-TOF). The data revealed that the more abundant polypeptide has a mass of 14.74 kDa, corresponding to a C-terminal cleavage of 17 residues from the expected 143-residue long mature sequence. A minor polypeptide, with a mass of 12.63 kDa, corresponds to a C-terminal truncation of 36 amino acids. MALDI-TOF analysis of tryptic peptides from the glycosylated molecule(s) identifies a single branched carbohydrate motif, with six N-acetylgalactosamines, and no sialic acid. The only glycan microheterogeneity seems to reside in the number of l-fucose residues (one to three). The lack of the C-terminal cluster of basic residues, and the presence of nonsialylated glycans, result in a very low net charge of TrIFN-gamma molecule. However, the 17-residue truncation does not affect the antiproliferative activity of TrIFN-gamma on different cells, among which is a porcine uterine epithelial cell line. It is suggested that these specific properties might confer on TrIFN-gamma a particular ability to invade the uterine mucosa and exert biological functions beyond the endometrial epithelium.

Novel serum replacement based on bovine ocular fluid: a useful tool for cultivation of different animal cells in vitro.

Different mammalian cells in culture have individual nutritional requirements, which are mainly fulfilled by the addition of foetal calf serum (FCS) to the basic medium. Collecting FCS is accompanied with severe animal welfare problems as conscient animals usually are bleeded to death by heart-punctuation without anaesthetics. There exists scientific problems too. Due to the batch-to-batch variability and the relatively high price, different types of serum replacements were introduced. Among them bovine colostrum as a serum substitute for the cultivation of hybridoma cells should be mentioned. The presented experiments were aimed to introduce the simple and effective serum replacement (SR) based on the bovine ocular fluid. Throughout the experiments the bovine ocular fluid alone and in the combination with the sheep's defibrinated plasma and human serum albumin was tested for the growth of different cells growing as a monolayer: (a) Cell lines: WISH (human amniotic cell lines) and VERO. (b) Primary culture: chicken embryonic fibroblasts, human bone-marrow fibroblasts. All growth experiments were performed in parallel with the Foetal Calf Serum (FCS) of three different sources. All types of cells were cultivated in Eagle's medium + antibiotics (Penicillin, Streptomycin, Gentamycin). The most effective was the SR containing approximately 35% of sheep's defibrinated plasma and 1.5% of serum albumin in the bovine ocular fluid. During the experiments 1 and 10% of SR-2.05 or FCS in Eagle's medium were used. After 1, 3 and 6 days of cultivation the cells were counted. The results show that the use of SR-2.05 gives a higher number of cells as compared to most batches of FCS. It is also important that practically no adaptation is needed, meaning that the cells could be grown in Eagle's medium + FCS and in the next passage in Eagle's medium + SR-2.05 and vice versa.