Screening of intact yeasts and cell extracts to reduce Scrapie prions during biotransformation of food waste.

Yeasts can be used to convert organic food wastes to protein-rich animal feed in order to recapture nutrients. However, the reuse of animal-derived waste poses a risk for the transmission of infectious prions that can cause neurodegeneration and fatality in humans and animals. The aim of this study was to investigate the ability of yeasts to reduce prion activity during the biotransformation of waste substrates-thereby becoming a biosafety hurdle in such a circular food system. During pre-screening, 30 yeast isolates were spiked with Classical Scrapie prions and incubated for 72 h in casein substrate, as a waste substitute. Based on reduced Scrapie seeding activity, waste biotransformation and protease activities, intact cells and cell extracts of 10 yeasts were further tested. Prion analysis showed that five yeast species reduced Scrapie seeding activity by approximately 1 log10 or 90%. Cryptococcus laurentii showed the most potential to reduce prion activity since both intact and extracted cells reduced Scrapie by 1 log10 and achieved the highest protease activity. These results show that select forms of yeast can act as a prion hurdle during the biotransformation of waste. However, the limited ability of yeasts to reduce prion activity warrants caution as a sole barrier to transmission as higher log reductions are needed before using waste-cultured yeast in circular food systems.

An unusual presentation of pseudocowpox associated with an outbreak of pustular ulcerative vulvovaginitis in a Swedish dairy herd.

Species Pseudocowpox virus (PCPV; family Poxviridae) is known to cause pustular cutaneous disease in cattle. We describe an outbreak of pseudocowpox with an unusual clinical picture in a free-stall dairy herd of ~80 cows. Approximately 90% of the cows had vesicles, erosions, papules, and scabs on the vulva and vaginal mucosa. Histologic analysis of biopsy tissues indicated a primary, although not specified, viral infection. Transmission electron microscopy revealed parapoxvirus particles in both tissue and vesicular materials. Deep sequencing analysis of extracted DNA from swabbed vesicle areas gave a contig of nearly 120,000 nucleotides, matching the PCPV strain VR 634 with 100% identity. Analyses confirmed the absence of other potential causes of pustular vulvovaginitis such as bovine herpesvirus 1 and Ureaplasma diversum. A rolling cow brush was suspected to be the fomite.

A serological study of canine herpesvirus-1 infection in a population of breeding bitches in Norway.

BACKGROUND: Canine herpesvirus-1 (CHV1) causes a fatal hemorrhagic disease in neonatal puppies and is associated with infertility in female dogs. This study was conducted to assess the status of CHV1 infection in bitches in proestrus or estrus and to investigate possible risk factors by a detailed questionnaire. Blood samples were collected from healthy bitches (n = 193) not vaccinated against CHV1, aged one year or older and admitted for estrus control to the Canine Reproductive Clinical Unit, Norwegian School of Veterinary Science. The serum samples were analysed by immunoperoxidase monolayer assay and serum titers were recorded as the reciprocal value of the highest dilution producing specific cell staining. RESULTS: Altogether, 85.5% of the dogs had CHV1 titers ≥ 80 and were classified as positive. Mean age for dogs included in the study was 4.2 years (95% CI 4.0-4.5), and there was no difference in age between seronegative dogs vs seropositive dogs. When grouping the seropositive dogs into three categories according to the magnitude of the titer, a total of 38.8% of the bitches displayed a weakly positive titer of 80, 44.8% had moderately positive titers of 160 or 320 and 16.4% of the dogs fell into the strongly positive category with titer of ≥640. No association was demonstrated when comparing CHV1 antibody titers to fertility parameters such as previous matings, pregnancies, whelpings, puppies born or condition of puppies. Further, there was no difference in seroprevalence between bitches that had been abroad for a period of time and dogs only living within a Norwegian environment. Samples from dogs collected in summer and fall displayed moderate to high antibody titers indicating recent infection with CHV1. Season, previous birth, and participation in competitions/shows explained 67-78% of the variation in antibody titer. CONCLUSIONS: This study demonstrates that CHV1 infection is common in breeding bitches in the eastern part of Norway. Associations with putative risk factors were not identified. However, season, previous whelping, and participation in competitions/shows explained 67-78% of the variation in antibody titer.

Characterization and analysis of the full-length genome of a strain of the European QX-like genotype of infectious bronchitis virus.

In recent years, strains of infectious bronchitis virus belonging to the QX-like genotype have been causing huge economic losses in commercial chicken flocks in different countries in Europe. In order to expand the knowledge of the molecular features of these viruses, we have sequenced and characterized the complete genome of European QX-like IBV strain CK/SWE/0658946/10, which was isolated in 2010 in Sweden. The genome is 27664 nucleotides in length, comprising six genes and 5' and 3' untranslated regions. The ORF1a, spike and nucleocapsid genes were under strong positive selective pressure that resulted in genetic diversity in relation to classical IBV isolates. The full-length genome of the CK/SWE/0658946/10 strain has the highest nucleotide sequence identity (93.18%) to ITA/90254/2005 and the lowest nucleotide identity (89.10%) to strain CQ04-1. Phylogenetic analysis of partial S1 gene sequences of IBV strains showed that the European QX-like genotype comprises strains that have been predominantly circulating in this continent for the past decade.

Emergence of novel strains of avian infectious bronchitis virus in Sweden.

Infectious bronchitis virus (IBV) causes avian infectious bronchitis, an important disease that produces severe economic losses in the poultry industry worldwide. Recent IBV infections in Sweden have been associated with poor growth in broilers, drop in egg production and thin egg shells in layers. The complete spike gene of selected isolates from IBV cases was amplified and sequenced using conventional RT-PCR. Nucleotide and amino acid sequence comparisons have shown that the recent isolates bear 98.97% genetic similarity with strains of the QX-like genotype. The phylogenetic analysis revealed that strains predominant in the nineties, which were of the Massachusetts type, have been replaced by D388/QX-like strains, however the evolutionary link could not be established. The homology between the two genotypes was 79 and 81%. Remarkably, a strong positive selection pressure was determined, mostly involving the S1 subunit of the S gene. This strong selective pressure resulted in recombination events, insertions and deletions in the S gene. Two new isolates generated from recombination were found with nucleotide sequence diverging 1.7-2.4% from the D388/QX-like branch, indicating the emergence of a new lineage. The study demonstrates a constant evolution of IBV that might be in relation to increased poultry farming, trade and vaccine pressure. The findings underscore the importance of continuous monitoring to control spread of infections, as well as to timely adjust diagnostic methods, molecular epidemiological studies, development and use of vaccines that are adapted to the changing disease scenario.

Whole genome sequencing and characterization of a virulent Newcastle disease virus isolated from an outbreak in Sweden.

In this study, the complete genome sequence of a Newcastle disease virus (NDV) isolate collected from an outbreak in 1995 in chickens was fully characterized and compared with other NDV sequences. The genome was found to be 15,192 nucleotides long and to consist of six genes in the order 3'-NP-P-M-F-HN-L-5', similar to other avian paramyxoviruses type-I. However, a six-nucleotide insertion was observed in the 5' non-coding regions of the nucleoprotein (NP) gene, a feature that is unique to some NDV isolates. The isolate shows the amino acid sequence (112)RRQKRF(117) at the cleavage site of the F protein, which is identical to a known motif for virulent pathotypes of NDV. The phylogenetic analysis of the coding region of the F gene indicated that this isolate belongs to genotype VI, more specifically to genotype VId, along with isolates from the other European countries (Denmark, Switzerland and Austria). The same genotype caused outbreaks in the Middle East and Greece in the late 1960s, and in Hungary, in the early 1980s, suggesting a common source for these outbreaks.

Complete genome characterisation of a Newcastle disease virus isolated during an outbreak in Sweden in 1997.

The complete genome sequence of a Newcastle disease virus (NDV) isolated from a chicken in Sweden was determined and compared with other NDV sequences. The isolate was shown to belong to genotype VIIb, which arose in the Far East and spread around the world during the 1990s. It had a length of 15,192 bases and consisted of six genes in the order 3'-NP-P-M-F-HN-L-5'. The F protein cleavage site was 112-RRQRRF-117, corresponding to that of a virulent pathotype.

The first Swedish H1N2 swine influenza virus isolate represents an uncommon reassortant.

The European swine influenza viruses (SIVs) show considerable diversity comprising different types of H1N1, H3N2, and H1N2 strains. The intensifying full genome sequencing efforts reveal further reassortants within these subtypes. Here we report the identification of an uncommon reassortant variant of H1N2 subtype influenza virus isolated from a pig in a multisite herd where H1N2 swine influenza was diagnosed for the first time in Sweden during the winter of 2008-2009. The majority of the European H1N2 swine influenza viruses described so far possess haemagglutinin (HA) of the human-like H1N2 SIV viruses and the neuraminidase (NA) of either the European H1N2 or H3N2 SIV-like viruses. The Swedish isolate has an avian-like SIV HA and a H3N2 SIV-like NA, which is phylogenetically more closely related to H3N2 SIV NAs from isolates collected in the early '80s than to the NA of H3N2 origin of the H1N2 viruses isolated during the last decade, as depicted by some German strains, indicative of independent acquisition of the NA genes for these two types of reassortants. The internal genes proved to be entirely of avian-like SIV H1N1 origin. The prevalence of this SIV variant in pig populations needs to be determined, as well as the suitability of the routinely used laboratory reagents to analyze this strain.The description of this H1N2 SIV adds further information to influenza epidemiology and supports the necessity of surveillance for influenza viruses in pigs.

Bovine spongiform encephalopathy in Sweden: an H-type variant.

Bovine spongiform encephalopathy (BSE) had never been detected in Sweden until 2006, when the active surveillance identified a case in a 12-year-old cow. The case was an unusual form, because several molecular features of the protease-resistant prion protein (PrP(res)) were different from classical BSE. The differences included higher susceptibility for proteinase K, higher molecular weight of the PrP(res) bands, affinity to the N-terminus-specific antibodies 12B2 and P4, and peculiar banding pattern with antibody SAF84 showing an additional band at the 14 kDa position. The molecular characteristics were in accordance to previous descriptions of H-type BSE. This report shows that a range of Western blot techniques and antibodies can be applied to confirm H-type BSE and further describes that the ratio of the amounts of PrPres#1 and PrPres#2, after deglycosylation, depends on the antibody used during processing. Immunohistochemistry on sections of medulla at the level of the obex applying antibodies with epitopes covering a broad range of the PrP sequence showed accumulation of disease-specific PrP (PrP(d)) in the gray matter. Fine punctate deposition in the neuropil was the most predominant type and was more severe in BSE target nuclei. The types of PrP(d) deposition are described in comparison with classical BSE. PrP-gene sequencing showed 6 copy octarepeat alleles and no abnormalities. It is postulated that the disease had a spontaneous origin, rather than having had been acquired in the BSE epidemic.

Evaluation of a commercial Erns-capture ELISA for detection of BVDV in routine diagnostic cattle serum samples.

BACKGROUND: Bovine viral diarrhoea virus (BVDV) is an important pathogen in cattle. The ability of the virus to cross the placenta during early pregnancy can result in the birth of persistently infected (PI) calves. These calves shed the virus during their entire lifespan and are the key transmitters of infection. Consequently, identification (and subsequent removal) of PI animals is necessary to rapidly clear infected herds from the virus. The objective of this study was to evaluate the suitability of a commercial Erns-capture ELISA, in comparison to the indirect immunoperoxidase test (IPX), for routine diagnostic detection of BVDV within a control programme. In addition, the effect of passive immunity and heat-inactivation of the samples on the performance of the ELISA was studied. METHODS: In the process of virus clearance within the Swedish BVDV control programme, all calves born in infected herds are tested for virus and antibodies. From such samples, sent in for routine diagnostics to SVA, we selected 220 sera collected from 32 beef herds and 29 dairy herds. All sera were tested for BVDV antigen using the Erns ELISA, and the results were compared to the results from the IPX used within the routine diagnostics. RESULTS: All 130 samples categorized as virus negative by IPX were tested negative in the ELISA, and all 90 samples categorized as virus positive were tested positive, i.e. the relative sensitivity and specificity of the ELISA was 100% in relation to IPX, and the agreement between the tests was perfect. CONCLUSION: We can conclude that the Erns ELISA is a valid alternative that has several advantages compared to IPX. Our results clearly demonstrate that it performs well under Swedish conditions, and that its performance is comparable with the IPX test. It is highly sensitive and specific, can be used for testing of heat-inactivated samples, precolostral testing, and probably to detect PI animals at an earlier age than the IPX.

Characterization of proteinase K-resistant N- and C-terminally truncated PrP in Nor98 atypical scrapie.

An increasing number of scrapie cases with atypical characteristics, designated Nor98, have recently been recognized. Here, the proteinase K (PK)-resistant prion protein (PrP) fragments from two Swedish cases of Nor98 atypical scrapie have been characterized. The prominent, fast-migrating band in the distinct Nor98 Western immunoblot electrophoretic profile was determined to be of 7 kDa in size and was accordingly designated Nor98-PrP7. The antigenic composition of Nor98-PrP7, as assayed by a panel of anti-PrP antibodies, revealed that this fragment comprised a mid-region of PrP from around aa 85 to 148. N- and C-terminally truncated fragments spanning the mid-region of PrP have only been observed in the genetic prion disorder Gerstmann-Sträussler-Scheinker disease. It is shown here that the long-term PK resistance of Nor98-PrP7 is reduced compared with that of PrP(res) in classical scrapie. Enzymic deglycosylation did not change the distinct electrophoretic profile of Nor98-PrP7. A previously unidentified, PK-resistant, C-terminal PrP fragment of around 24 kDa was detected and its PK resistance was investigated. After deglycosylation, this fragment migrated as a 14 kDa polypeptide and was designated PrP-CTF14. Antigenic determination and the size of 14 kDa suggested a fragment spanning approximately aa 120-233. The existence of two PK-resistant PrP fragments, Nor98-PrP7 and PrP-CTF14, that share an overlapping region suggests that at least two distinct PrP conformers with different PK-resistant cores are present in brain extracts from Nor98-affected sheep. The structural gene of PrP in three Nor98-affected sheep was analysed, but no mutations were found that could be correlated to the aberrant PK-resistant profile observed.

Prevalence of antibodies against feline coronavirus and Chlamydophila felis in Swedish cats.

Serum samples from 214 Swedish cats with no signs of infectious disease were analysed for the presence of antibodies against Chlamydophila felis (Cp felis), while 209 of these were also analysed for feline coronavirus (FCoV) antibodies. The prevalence of antibodies against Cp felis was 11%, with no significant difference between purebred and mixed breed cats. The overall prevalence of antibodies against FCoV was 31%, significantly higher among pure breed cats (65%) than among mixed breed cats (17%). A high proportion of cats with antibodies against FCoV had relatively high antibody titres, and was therefore likely to be shedding FCoV in faeces. For Cp felis, the majority of seropositive animals had relatively low antibody titres, and the risk of these animals infecting others is not known.

Molecular epizootiology of infectious bronchitis virus in Sweden indicating the involvement of a vaccine strain.

To improve the detection and molecular identification of infectious bronchitis virus (avian coronavirus), two reverse transcriptase-polymerase chain reaction (PCR) assays were developed. As 'diagnostic#10; PCR', a set of consensus nested primers was selected from highly conserved stretches of the nucleocapsid (N) gene. As 'phylogeny' PCR, a fragment of the spike protein gene (S1) was amplified and the PCR products were directly sequenced. To study the phylogenetic relationships of the viruses from various outbreaks, studies of molecular epizootiology were performed in Sweden, a Nordic region, where the occurrence of natural cases of the disease is relatively low and the occasional use of live vaccine(s) is well recorded and monitored. The disease appeared in the region in 1994, associated with production problems among layers of various ages. During outbreaks in 1995 and 1997, both layers and broilers were affected. To reduce losses, a live attenuated vaccine has been applied since 1997. By examining 12 cases between 1994 and 1998, molecular epizootiology revealed that, before 1997, the viruses had gene sequences very similar to strains of the Massachusetts serotype. However, comparative sequence analysis of the S1 gene revealed that the identity was not 100% to any of the strains of this serotype that we analysed. A virus related to the Dutch-type strain, D274, was also identified on one farm. Surprisingly, from 1997, the year that vaccination commenced with a live Massachusetts serotype vaccine, the majority of viruses detected had S1 sequences identical to the live Massachusetts vaccine strain. This genetic relation to the vaccine virus was also confirmed by N gene sequence analysis. The studies of molecular epizootiology reveal a strong probability that the vaccination had lead to the spread of the vaccine virus, causing various disease manifestations and a confusing epizootiological situation in the poultry population.