Development of a cell culture-based method for detecting infectious tick-borne encephalitis virus (TBEV) in milk products.

Tick-borne encephalitis outbreaks have been reported in Europe after consumption of raw milk products from infected animals. While molecular methods are commonly used in viral foodborne outbreak investigations due to their sensitivity, specificity and rapidity, there are very few methods to detect infectious tick-borne encephalitis virus (TBEV) in milk products for routine use/analyses. To address this gap, we developed a cell culture-based method to detect infectious TBEV in artificially contaminated raw goat milk and raw goat cheese, and evaluated the sensitivity of TBEV infectivity assays. Raw goat milk samples were spiked with TBEV to achieve inoculation levels ranging from 106 to 100 TCID50/mL, and Faisselle and Tomme cheese samples were spiked so their TBEV concentrations ranged from 9.28 × 105 to 9.28 × 101 TCID50 per 2.5g. To detect infectious TBEV, Vero cells were infected by raw goat milk. For cheese samples, after homogenisation and membrane filtration, Vero cells were infected with samples adsorbed on the filter (method A) or with samples eluted from the filter (method B). After 5 days, cytopathic effects (CPEs) were observed and TBEV replication in Vero cells was confirmed by an increase in the number of genome copies/mL that were detected in cell supernatant. Infected Vero cells exhibited CPEs for both milk and cheese samples. Infectious TBEV was detected to 103 TCID50/mL in raw milk samples and to 9.28 × 101 TCID50 from Faisselle samples using both methods A and B. For Tomme samples, method A was able to detect TBEV to 9.28 × 102 TCID50/2.5g and method B to 9.28 × 103 TCID50/2.5g. The number of positive samples detected was slightly higher with method A than with method B. To conclude, this qualitative cell culture-based method can detect infectious TBEV artificially inoculated into raw milk and cheese; it should be further evaluated during foodborne outbreak investigations to detect infectious TBEV from naturally contaminated milk and cheese.

Physicochemical Parameters Affecting Norovirus Adhesion to Ready-To-Eat Foods.

The adhesion of noroviruses to strawberry, turkey slices, ham, and cheddar cheese was studied using murine norovirus 1 (MNV-1) as a surrogate for human norovirus (NoV). Based on plaque assay, the recovery and adhesion of MNV-1 depended on the food type (turkey versus strawberry), pH of the initial suspension buffer (pH 4 versus pH 7), and food fat composition (C8 versus C18). Recovery of infectious particles from turkey was 68% compared to 9.4% from strawberry. On turkey, adhesion of MNV-1 was lower at pH 7 (pH of fecal matter), and virus particles adhered to this pH were recovered more easily (33,875 PFU) than at pH 4 (pH of vomitus). The presence of fat and the composition of fatty acids seemed to increase MNV-1 recovery and adherent viral particles recovered but did not affect adhesion (68% on fat-free turkey and regular turkey). Adherent MNV-1 particles recovered from stainless steel coated with saturated fatty acid (C8, C14, C18) increased significantly with chain length (P < 0.05), but adhesion did not seem to change. Using liquid droplet contact angle to measure surface energy, it was deduced that hydrophobic interactions contribute considerably to the adhesion of MNV-1 to stainless steel, polyvinyl chloride, and high-density polyethylene. IMPORTANCE Ready-to-eat (RTE) foods are major vehicles of transmission of foodborne viral pathogens, including NoV. The high incidence of gastroenteritis caused by viruses is due largely to their persistence in the environment and adhesion to different kinds of surfaces in the food industry, including the foods themselves. Compared with bacteria, adhesion of viruses to surfaces is poorly understood. Better knowledge of the physicochemical parameters involved in the adhesion of NoV to ready-to-eat foods is essential to devising effective strategies for reducing the persistence and, thus, the transmission of this virus.

Viral metagenomic analysis of the cheese surface: A comparative study of rapid procedures for extracting viral particles.

The structure and functioning of microbial communities from fermented foods, including cheese, have been extensively studied during the past decade. However, there is still a lack of information about both the occurrence and the role of viruses in modulating the function of this type of spatially structured and solid ecosystems. Viral metagenomics was recently applied to a wide variety of environmental samples and standardized procedures for recovering viral particles from different type of materials has emerged. In this study, we adapted a procedure originally developed to extract viruses from fecal samples, in order to enable efficient virome analysis of cheese surface. We tested and validated the positive impact of both addition of a filtration step prior to virus concentration and substitution of purification by density gradient ultracentrifugation by a simple chloroform treatment to eliminate membrane vesicles. Viral DNA extracted from the several procedures, as well as a vesicle sample, were sequenced using Illumina paired-end MiSeq technology and the subsequent clusters assembled from the virome were analyzed to assess those belonging to putative phages, plasmid-derived DNA, or even from bacterial chromosomal DNA. The best procedure was then chosen, and used to describe the first cheese surface virome, using Epoisses cheese as example. This study provides the basis of future investigations regarding the ecological importance of viruses in cheese microbial ecosystems.

The Tape Measure Protein Is Involved in the Heat Stability of Lactococcus lactis Phages.

Virulent lactococcal phages are still a major risk for milk fermentation processes as they may lead to slowdowns and low-quality fermented dairy products, particularly cheeses. Some of the phage control strategies used by the industry rely on heat treatments. Recently, a few Lactococcus lactis phages were found to be highly thermo-resistant. To identify the genetic determinant(s) responsible for the thermal resistance of lactococcal phages, we used the virulent phage CB14 (of the Lactococcus lactis 936 [now Sk1virus] phage group) to select for phage mutants with increased heat stability. By treating phage CB14 to successive low and high temperatures, we were able to select two CB14 derivatives with increased heat stability. Sequencing of their genome revealed the same nucleotide sequences as the wild-type phage CB14, except for a same-sized deletion (120 bp) in the gene coding for the tape measure protein (TMP) of each phage mutant, but at a different position. The TMP protein sequences of these mutant phages were compared with their homologues in other wild-type L. lactis phages with a wide diversity in heat stability. Comparative analysis showed that the same nucleotide deletion appears to have also occurred in the gene coding for the TMP of highly thermo-resistant lactococcal phages P1532 and P680. We propose that the TMP is, in part, responsible for the heat stability of the highly predominant lactococcal phages of the Sk1virus group.IMPORTANCE Virulent lactococcal phages still represent a major risk for milk fermentation as they may lead to slowdowns and low-quality fermented dairy products. Heat treatment is one of the most commonly used methods to control these virulent phages in cheese by-products. Recently, a few Lactococcus lactis phages, members of the Sk1virus group, have emerged with high thermal stability. To our knowledge, the genetic determinant(s) responsible for this thermal resistance in lactococcal phages is unknown. A better understanding of the thermal stability of these emerging virulent lactococcal phages is needed to improve industrial control strategies. In this work, we report the identification of a phage structural protein that is involved in the heat stability of a virulent Sk1virus phage. Identifying such a genetic determinant for heat stability is a first step in understanding the emergence of this group of thermostable phages.

Complete genomic sequences of Propionibacterium freudenreichii phages from Swiss cheese reveal greater diversity than Cutibacterium (formerly Propionibacterium) acnes phages.

BACKGROUND: A remarkable exception to the large genetic diversity often observed for bacteriophages infecting a specific bacterial host was found for the Cutibacterium acnes (formerly Propionibacterium acnes) phages, which are highly homogeneous. Phages infecting the related species, which is also a member of the Propionibacteriaceae family, Propionibacterium freudenreichii, a bacterium used in production of Swiss-type cheeses, have also been described and are common contaminants of the cheese manufacturing process. However, little is known about their genetic composition and diversity. RESULTS: We obtained seven independently isolated bacteriophages that infect P. freudenreichii from Swiss-type cheese samples, and determined their complete genome sequences. These data revealed that all seven phage isolates are of similar genomic length and GC% content, but their genomes are highly diverse, including genes encoding the capsid, tape measure, and tail proteins. In contrast to C. acnes phages, all P. freudenreichii phage genomes encode a putative integrase protein, suggesting they are capable of lysogenic growth. This is supported by the finding of related prophages in some P. freudenreichii strains. The seven phages could further be distinguished as belonging to two distinct genomic types, or 'clusters', based on nucleotide sequences, and host range analyses conducted on a collection of P. freudenreichii strains show a higher degree of host specificity than is observed for the C. acnes phages. CONCLUSIONS: Overall, our data demonstrate P. freudenreichii bacteriophages are distinct from C. acnes phages, as evidenced by their higher genetic diversity, potential for lysogenic growth, and more restricted host ranges. This suggests substantial differences in the evolution of these related species from the Propionibacteriaceae family and their phages, which is potentially related to their distinct environmental niches.

Characterization of airborne viromes in cheese production plants.

AIMS: To characterize airborne virus-like particles isolated from two cheese production plants in order to reveal their complexity in terms of viral communities and microbial genes potentially mobilized by viruses. METHODS AND RESULTS: Airborne virus-like particles have been isolated from Grana Padano and Gorgonzola PDO cheese production plants and ripening cellars. A shotgun metagenomics analysis of the isolated viromes highlighted a high complexity of the viral communities both in terms of viral taxonomy and phage-host associations. Bacterial reads in each of the viromes were confirmed to be abundant and their taxonomy appeared to be associated with the environmental parameters and the technological processes that characterize the sampling area. Antibiotic resistance genes have been identified in each virome thus confirming that phages could be involved in the mobilization of antimicrobial resistances among bacterial populations. Interestingly human viruses were also identified even if the contamination source was not revealed. CONCLUSIONS: The environmental conditions, which are imposed by the technology of the dairy process, seam to shape the viral populations as a consequence of the adaptation of microbial taxa to those environments. The identification of sequences belonging to Legionella pneumophila and to the human papillomavirus, raised some considerations about the safety of cheese-ripening cellars. SIGNIFICANCE AND IMPACT OF THE STUDY: In conclusion, the analysis of the dairy airborne viromes, has revealed a high complexity of the viral communities even if the environments where the samples were collected were confined environments. Metagenomics of airborne viral population could be a promising monitoring tool for the biological characterization of dairy environments.

A cluster of two human cases of tick-borne encephalitis (TBE) transmitted by unpasteurised goat milk and cheese in Germany, May 2016.

In May 2016, two cases of tick-borne encephalitis (TBE) were confirmed by serology (positive IgM and IgG antibodies against TBE virus (TBEV) in serum), with a possible link to raw milk and cheese from a goat farm in a region in Baden-Württemberg, Germany not previously known as TBE-endemic. The outbreak investigation identified 32 consumers of goat dairy products (29 consumers, one farm employee, two owners) of whom none had IgM antibodies against TBEV 3-8 weeks after consumption. Of the 27 notified TBE cases in the State, none reported consumption of raw goat milk or cheese from the suspected farm. Five of 22 cheese samples from 18 different batches were RT-qPCR-positive for TBEV -genome, and two of the five samples were confirmed by virus isolation, indicating viability of TBEV in the cheese. Nine of the 45 goats had neutralising TBEV antibodies, two of them with a high titre indicating recent infection. One of 412 Ixodes ricinus was RT-qPCR-positive, and sequencing of the E gene from nucleic acid extracted from the tick confirmed TBEV. Phylogenetic analyses of tick and cheese isolates showed 100% amino acid homology in the E gene and a close relation to TBEV strains from Switzerland and Austria.

Novel Variants of Streptococcus thermophilus Bacteriophages Are Indicative of Genetic Recombination among Phages from Different Bacterial Species.

Bacteriophages are the main cause of fermentation failures in dairy plants. The majority of Streptococcus thermophilus phages can be divided into either cos- or pac-type phages and are additionally characterized by examining the V2 region of their antireceptors. We screened a large number of S. thermophilus phages from the Chr. Hansen A/S collection, using PCR specific for the cos- or pac-type phages, as well as for the V2 antireceptor region. Three phages did not produce positive results with the assays. Analysis of phage morphologies indicated that two of these phages, CHPC577 and CHPC926, had shorter tails than the traditional S. thermophilus phages. The third phage, CHPC1151, had a tail size similar to those of the cos- or pac-type phages, but it displayed a different baseplate structure. Sequencing analysis revealed the genetic similarity of CHPC577 and CHPC926 with a subgroup of Lactococcus lactis P335 phages. Phage CHPC1151 was closely related to the atypical S. thermophilus phage 5093, homologous with a nondairy streptococcal prophage. By testing adsorption of the related streptococcal and lactococcal phages to the surface of S. thermophilus and L. lactis strains, we revealed the possibility of cross-interactions. Our data indicated that the use of S. thermophilus together with L. lactis, extensively applied for dairy fermentations, triggered the recombination between phages infecting different bacterial species. A notable diversity among S. thermophilus phage populations requires that a new classification of the group be proposed.IMPORTANCEStreptococcus thermophilus is a component of thermophilic starter cultures commonly used for cheese and yogurt production. Characterizing streptococcal phages, understanding their genetic relationships, and studying their interactions with various hosts are the necessary steps for preventing and controlling phage attacks that occur during dairy fermentations.

Short communication: Survival of Vaccinia virus in inoculated cheeses during 60-day ripening.

Bovine vaccinia is a neglected zoonosis caused by Vaccinia virus (VACV) and has a major economic and public health effect in Brazil. Previous studies showed infectious VACV particles in milk from either experimentally or naturally infected cows and in fresh cheeses prepared with experimentally contaminated milk. Ripening is a process that leads to major changes in the physical and chemical characteristics of cheese, reducing contamination by spoilage, pathogenic microorganisms, or both. However, it is not known if VACV infectious particles persist after the ripening process. To investigate this issue, viral infectivity at different ripening times was studied in cheeses manufactured with milk experimentally contaminated with VACV strain Guarani P2 (GP2). Cheeses were analyzed at 1, 7, 14, 21, 45, and 60 d of ripening at 25°C. Viral DNA was quantified by real-time PCR, and VACV isolation and titration were performed in Vero cells. The whole experiment was repeated 4 times. Analysis of the mean viral DNA quantification and infectivity indicated a reduction of approximately 2 logs along the ripening process; however, infectious viral particles (1.7 × 102 pfu/mL) could still be recovered at d 60 of ripening. These findings indicate that the ripening process reduces VACV infectivity, but it was not able to inactivate completely the viral particles after 60 d.

An outbreak of tick-borne encephalitis associated with raw goat milk and cheese consumption, Croatia, 2015.

The aim of this report is to emphasize the risk of acquiring TBE by the consumption of raw milk and dairy products. In April-May 2015, we registered the first outbreak of tick-borne encephalitis in Croatia in seven members out of ten exposed persons who consumed raw goat milk or cheese from the same supplier. Infection was confirmed by TBEV enzyme-linked immunosorbent assay (ELISA) in all patients. None had been vaccinated nor had observed a tick bite.

Strain diversity and phage resistance in complex dairy starter cultures.

The compositional stability of the complex Gouda cheese starter culture Ur is thought to be influenced by diversity in phage resistance of highly related strains that co-exist together with bacteriophages. To analyze the role of bacteriophages in maintaining culture diversity at the level of genetic lineages, simple blends of Lactococcus lactis strains were made and subsequently propagated for 152 generations in the absence and presence of selected bacteriophages. We first screened 102 single-colony isolates (strains) from the complex cheese starter for resistance to bacteriophages isolated from this starter. The collection of isolates represents all lactococcal genetic lineages present in the culture. Large differences were found in bacteriophage resistance among strains belonging to the same genetic lineage and among strains from different lineages. The blends of strains were designed such that 3 genetic lineages were represented by strains with different levels of phage resistance. The relative abundance of the lineages in blends with phages was not stable throughout propagation, leading to continuous changes in composition up to 152 generations. The individual resistance of strains to phage predation was confirmed as one of the factors influencing starter culture diversity. Furthermore, loss of proteolytic activity of initially proteolytic strains was found. Reconstituted blends with only 4 strains with a variable degree of phage resistance showed complex behavior during prolonged propagation.

Cheese-related listeriosis outbreak, Portugal, March 2009 to February 2012.

In Portugal, listeriosis has been notifiable since April 2014, but there is no active surveillance programme for the disease. A retrospective study involving 25 national hospitals led to the detection of an outbreak that occurred between March 2009 and February 2012. The amount of time between the start of the outbreak and its detection was 16 months. Of the 30 cases of listeriosis reported, 27 were in the Lisbon and Vale do Tejo region. Two cases were maternal/neonatal infections and one resulted in fetal loss. The mean age of the non-maternal/neonatal cases was 59 years (standard deviation: 17); 13 cases were more than 65 years old. The case fatality rate was 36.7%. All cases were caused by molecular serogroup IVb isolates indistinguishable by pulsed-field gel electrophoresis and ribotype profiles. Collaborative investigations with the national health and food safety authorities identified cheese as the probable source of infection, traced to a processing plant. The magnitude of this outbreak, the first reported food-borne listeriosis outbreak in Portugal, highlights the importance of having an effective listeriosis surveillance system in place for early detection and resolution of outbreaks, as well as the need for a process for the prompt submission of Listeria monocytogenes isolates for routine laboratory typing.

Dynamics of the viral community on the surface of a French smear-ripened cheese during maturation and persistence across production years.

The surface of smear-ripened cheeses constitutes a dynamic microbial ecosystem resulting from the successive development of different microbial groups such as lactic acid bacteria, fungi, and ripening bacteria. Recent studies indicate that a viral community, mainly composed of bacteriophages, also represents a common and substantial part of the cheese microbiome. However, the composition of this community, its temporal variations, and associations between bacteriophages and their hosts remain poorly characterized. Here, we studied a French smear-ripened cheese by both viral metagenomics and 16S metabarcoding approaches to assess both the succession of phages and bacterial communities on the cheese surface during cheese ripening and their temporal variations in ready-to-eat cheeses over the years of production. We observed a clear transition of the phage community structure during ripening with a decreased relative abundance of viral species (vOTUs) associated with Lactococcus phages, which were replaced by vOTUs associated with phages infecting ripening bacteria such as Brevibacterium, Glutamicibacter, Pseudoalteromonas, and Vibrio. The dynamics of the phage community was strongly associated with bacterial successions observed on the cheese surface. Finally, while some variations in the distribution of phages were observed in ready-to-eat cheeses produced at different dates spanning more than 4 years of production, the most abundant phages were detected throughout. This result revealed the long-term persistence of the dominant phages in the cheese production environment. Together, these findings offer novel perspectives on the ecology of bacteriophages in smear-ripened cheese and emphasize the significance of incorporating bacteriophages in the microbial ecology studies of fermented foods.IMPORTANCEThe succession of diverse microbial populations is critical for ensuring the production of high-quality cheese. We observed a temporal succession of phages on the surface of a smear-ripened cheese, with new phage communities showing up when ripening bacteria start covering this surface. Interestingly, the final phage community of this cheese is also consistent over large periods of time, as the same bacteriophages were found in cheese products from the same manufacturer made over 4 years. This research highlights the importance of considering these bacteriophages when studying the microbial life of fermented foods like cheese.

Evaluation of Extraction Methods to Detect Noroviruses in Ready-to-Eat Raw Milk Minas Artisanal Cheese.

This study aimed to assess two homogenization methods to recover norovirus from Minas artisanal cheese (MAC) made with raw bovine milk obtained from four microregions of the Minas Gerais state, Brazil, with different ripening times and geographical and abiotic characteristics. For this purpose, 33 fiscal samples were artificially contaminated with norovirus GI and GII, and Mengovirus (MgV), used as an internal process control (IPC). TRIzol® reagent and Proteinase K homogenization methods were evaluated for all samples were then subjected to RNA extraction using viral magnetic beads and RT-qPCR Taqman® for viral detection/quantification. Proteinase K method showed better efficiency results for both norovirus GI and GII, with means recovery efficiency of 45.7% (95% CI 34.3-57.2%) and 41.4% (95% CI 29.1-53.6%), respectively, when compared to TRIzol method (16.6% GI, 95% CI 8.4-24.9%, and 12.3% GII, 95% CI 7.0-17.6%). The limits of detection for norovirus GI and GII for this method were 101GC/g and 103GC/g, respectively, independent of cheese origin. MgV was detected and revealed in 100% success rate in all types of cheese, with mean recovery efficiency of 25.6% for Proteinase K, and 3.8% for the TRIzol method. According to cheese origin, Triangulo Mineiro MAC had the highest mean recovery rates for the three viral targets surveyed (89% GI, 87% GII, and 51% MgV), while Serro MAC showed the lowest rates (p < 0.001). Those results indicate that the proteinase K adapted method is suitable for norovirus GI and GII detection in MAC and corroborated MgV as an applicable IPC to be used during the process.

Biodiversity of lactococcal bacteriophages isolated from 3 Gouda-type cheese-producing plants.

This study reports on the identification and characterization of bacteriophages isolated from cheese-production facilities that use undefined, mixed starter cultures. Phage screening was carried out on whey samples isolated from 3 factories, 2 utilizing one particular undefined starter mixture and 1 utilizing another undefined starter mixture. Phage screening was carried out using 40 strains isolated from the 2 mixed starter cultures, and phages were profiled using host range, electron microscopy, multiplex PCR, and DNA restriction analysis. Twenty distinct lactococcal phages were identified based on host range and DNA restriction profiles, all belonging to the 936-type phage species. Nineteen of these phages were found to be able to infect both recognized subspecies of Lactococcus lactis. Restriction of phage DNA isolated using a newly developed guanidinium thiocyanate disruption method showed that the genomes of the 20 isolated phages were between 26 and 31 kb in size. It is evident from this study that the use of mixed starters creates an ideal environment for the proliferation of different phages with slightly varying host ranges. Furthermore, in this environment, members of the 936-type phage species clearly dominated the phage population.

Short communication: Characterization of Shiga toxin 2-carrying bacteriophages induced from Shiga-toxigenic Escherichia coli isolated from Italian dairy products.

Forty samples of raw milk cheese and 25 samples of raw milk itself were subjected to enrichment culture for Shiga-toxigenic Escherichia coli (STEC), and a single Shiga toxin 2- (Stx(2)) positive strain was obtained from one of the cheese samples. Thus, aged cheeses in which the curd is subsequently heat treated (48°C) cannot be presumed to be STEC free. Infective Stx(2) bacteriophages were induced from 3 STEC strains isolated elsewhere from raw milk and 1 STEC strain from aged cheese sourced in Italy. Data on E. coli host range, morphology, genome size, and genetic variation determined by restriction fragment length polymorphism and multi-locus genotyping are presented. Although all 4 bacteriophages were found to be short-tailed Podoviridae, they exhibited considerable variation in both genome size and content. This extended to the Stx(2) genes themselves, whose sequences contained several point mutations, but these did not translate to amino acid substitutions.

New Cytoplasmic Virus-Like Elements (VLEs) in the Yeast Debaryomyces hansenii.

Yeasts can have additional genetic information in the form of cytoplasmic linear dsDNA molecules called virus-like elements (VLEs). Some of them encode killer toxins. The aim of this work was to investigate the prevalence of such elements in D. hansenii killer yeast deposited in culture collections as well as in strains freshly isolated from blue cheeses. Possible benefits to the host from harboring such VLEs were analyzed. VLEs occurred frequently among fresh D. hansenii isolates (15/60 strains), as opposed to strains obtained from culture collections (0/75 strains). Eight new different systems were identified: four composed of two elements and four of three elements. Full sequences of three new VLE systems obtained by NGS revealed extremely high conservation among the largest molecules in these systems except for one ORF, probably encoding a protein resembling immunity determinant to killer toxins of VLE origin in other yeast species. ORFs that could be potentially involved in killer activity due to similarity to genes encoding proteins with domains of chitin-binding/digesting and deoxyribonuclease NucA/NucB activity, could be distinguished in smaller molecules. However, the discovered VLEs were not involved in the biocontrol of Yarrowia lipolytica and Penicillium roqueforti present in blue cheeses.

Vaccinia virus is not inactivated after thermal treatment and cheese production using experimentally contaminated milk.

Bovine vaccinia is an emergent zoonosis caused by the Vaccinia virus (VACV). The disease is characterized by the appearance of exanthematic lesions that occur in humans and dairy cows. Previous studies have revealed the presence of infectious viral particles in milk samples during an outbreak of bovine vaccinia in Brazil, indicating the possibility of disease transmission through raw milk. To assess the viability of the virus in milk after thermal treatment and processing procedures, milk samples were experimentally contaminated with 10(3) plaque forming units (PFU)/mL (group I) and 10(5) PFU/mL (group II) VACV Guarani P2 virus, and the third group was not contaminated and served as a control. The samples were submitted to storage temperatures in a cold chamber, freezer for 48 hours, and to low temperature long-time treatment. Moreover, the viral viability was evaluated in cheese produced with contaminated milk using 10(4) PFU/mL VACV Guarani P2. Notably, the virus remained viable in milk after storage for 48 hours in both the cold chamber and the freezer, with a reduction in viral titer of 14.49% and 25.86%, respectively. Group II showed a viral reduction in titer of 61.88% and 75.98%, respectively. Thermal treatment 65°C for 30 minutes showed a reduction of viral titer of 94.83% and 99.99%, respectively, in group I and group II, but still showed remaining viable virus particles. In addition, it was possible to recover infectious viral particles from both the solid curds and the whey of the cheese produced with experimentally contaminated milk. The cheese shows a reduction in viral titer of 84.87% after storage at 4°C for 24 hours. The presence of viable viral particles in milk after both thermal treatment and cheese production indicates a potential public health risk.

Survival of Tick-Borne Encephalitis Virus in Goat Cheese and Milk.

Survival of tick-borne encephalitis virus was studied from pasteurized and unpasteurized goat milk and from salted/unsalted and spiced/unspiced cheese made from goat milk inoculated with low and high litres of infective virus. Both soft (63 °C, 30 min) and fast (72 °C, 15 s) pasteurization conditions destroyed viable virus particles. A small amount of infective virus could be detected only for 5‒10 days from milk, and from unsalted cheese. From milk inoculated with a higher amount of virus, infectious viral particles were detectable for 20‒25 days and from unsalted cheese samples for 10‒15 days, independently of the use of spices. Pasteurization and salt treatment made goat milk and cheese safely consumable. These two methods must be used when making any human food from goat milk to avoid milk-borne human TBEV infections.

Tick-borne encephalitis from eating goat cheese in a mountain region of Austria.

We report transmission of tick-borne encephalitis virus (TBEV) in July 2008 through nonpasteurized goat milk to 6 humans and 4 domestic pigs in an alpine pasture 1,500 m above sea level. This outbreak indicates the emergence of ticks and TBEV at increasing altitudes in central Europe and the efficiency of oral transmission of TBEV.