Isolation, whole genome sequencing and application of a broad-spectrum Salmonella phage.

Salmonella is considered as one of the most common zoonotic /foodborne pathogens in the world. The application of bacteriophages as novel antibacterial agents in food substrates has become an emerging strategy. Bacteriophages have the potential to control Salmonella contamination.We have isolated and characterized a broad-spectrum Salmonella phage, SP154, which can lyse 9 serotypes, including S. Enteritidis, S. Typhimurium, S. Pullorum, S. Arizonae, S. Dublin, S. Cholerasuis, S. Chester, S. 1, 4, [5], 12: i: -, and S. Derby, accounting for 81.9% of 144 isolates. SP154 showed a short latent period (40 min) and a high burst size (with the first rapid burst size at 107 PFUs/cell and the second rapid burst size at approximately 40 PFUs/cell). Furthermore, SP154 activity has higher survival rates across various environmental conditions, including pH 4.0-12.0 and temperatures ranging from 4 to 50 °C for 60 min, making it suitable for diverse food processing and storage applications. Significant reductions in live Salmonella were observed in different foods matrices such as milk and chicken meat, with a decrease of up to 1.9 log10 CFU/mL in milk contamination and a 1 log10 CFU/mL reduction in chicken meat. Whole genome sequencing analysis revealed that SP154 belongs to the genus Ithacavirus, subfamily Humphriesvirinae, within the family Schitoviridae. Phylogenetic analysis based on the terminase large subunit supported this classification, although an alternate tree using the tail spike protein gene suggested affiliation with the genus Kuttervirus, underscoring the limitations of relying on a single gene for phylogenetic inference. Importantly, no virulence or antibiotic resistance genes were detected in SP154. Our research highlights the potential of using SP154 for biocontrol of Salmonella in the food industry.

First detection of tick-borne encephalitis virus (TBEV) in raw milk samples in North-Western Iran.

Tick-borne encephalitis virus (TBEV) is a significant cause of flaviviral infections affecting the human central nervous system, primarily transmitted through tick bites and the consumption of unpasteurized milk. This study aimed to assess the prevalence of TBEV and identify new natural foci of TBEV in livestock milk. In this cross-sectional study, unpasteurized milk samples were collected from livestock reared on farms and analysed for the presence and subtyping of TBEV using nested reverse transcription-polymerase chain reaction , alongside the detection of anti-TBEV total IgG antibodies using ELISA. The findings revealed that the highest prevalence of TBEV was observed in goat and sheep milk combined, whereas no TBEV was detected in cow milk samples. All identified strains were of the Siberian subtype. Moreover, the highest prevalence of anti-TBEV antibodies was detected in sheep milk. These results uncover new foci of TBEV in Iran, underscoring the importance of thermal processing (pasteurization) of milk prior to consumption to mitigate the risk of TBEV infection.

Does pasteurization inactivate bird flu virus in milk?

Recently, an outbreak of highly pathogenic avian influenza A (H5N1), which carries the clade 2.3.4.4b hemagglutinin (HA) gene and has been prevalent among North American bird populations since the winter of 2021, was reported in dairy cows in the United States. As of 24 May 2024, the virus has affected 63 dairy herds across nine states and has resulted in two human infections. The virus causes unusual symptoms in dairy cows, including an unexpected drop in milk production, and thick colostrum-like milk. Notably, The US Food and Drug Administration reported that around 20% of tested retail milk samples contained H5N1 viruses, with a higher percentage of positive results from regions with infected cattle herds. Data are scant regarding how effectively pasteurization inactivates the H5N1 virus in milk. Therefore, in this study, we evaluated the thermal stability of the H5 clade 2.3.4.4b viruses, along with one human H3N2 virus and other influenza subtype viruses, including H1, H3, H7, H9, and H10 subtype viruses. We also assessed the effectiveness of pasteurization in inactivating these viruses. We found that the avian H3 virus exhibits the highest thermal stability, whereas the H5N1 viruses that belong to clade 2.3.4.4b display moderate thermal stability. Importantly, our data provide direct evidence that the standard pasteurization methods used by dairy companies are effective in inactivating all tested subtypes of influenza viruses in raw milk. Our findings indicate that thermally pasteurized milk products do not pose a safety risk to consumers.

Reference Material Production and Milk Protein Concentration as Elements to Improve Bluetongue Serological Diagnosis in Bulk Tank Milk.

The serological surveillance of bluetongue in bulk tank milk is an efficient and cost-effective method for the early detection of bluetongue virus incursions in unvaccinated free areas of the disease. In addition, the availability of standardized and reliable reagents and refined diagnostic procedures with high sensitivity and specificity are essential for surveillance purposes. However, no available reference materials for bluetongue virus serological surveillance in bulk tank milk exist. This study shows the production and characterization of reference material for the implementation of a commercially available bluetongue milk ELISA test in official laboratories, as well as the evaluation of a procedure to increase the sensitivity in samples with low levels of antibodies. This procedure, based on milk protein concentration, allowed us to notably increase the ELISA test's analytical sensitivity, which is useful for milk samples from farms with low within-herd prevalence or pools of bulk tank milk samples. The standardized milk reference material produced here, together with the evaluated procedure to improve analytical sensitivity, could be applied as tools to ensure an accurate diagnosis by official laboratories in bluetongue unvaccinated free areas.

Characterization of vB_SalS_PSa2, a T5-like Demerecviridae bacteriophage, and its potential use in food matrices.

This study characterizes a newly isolated Demerecviridae phage, named vB_SalS_PSa2, belonging to the phage T5 group. The main variations between vB_SalS_PSa2 and T5 concern structural proteins related to morphology and host recognition. vB_SalS_PSa2 is infective to 19 out of the 25 tested Salmonella enterica (including the rare "Sendai" and "Equine" serotypes) and Escherichia coli isolates, most of them being multidrug resistant. vB_SalS_PSa2 displayed good thermal stability (4-60 °C) and broad pH stability (4.0-12.0). It also exhibited antibacterial activity against S. enterica sv. Paratyphi A Enb50 at 4 °C in milk during the whole tested period (5 d), and for 3-6 h at both 25 and 37 °C. Furthermore, vB_SalS_PSa2 was able to inhibit biofilm formation and to show degradation activity on mature biofilms of E. coli K12 and S. enterica sv. Paratyphi Enb50 in both LB and milk. Altogether, these results indicate that phage vB_SalS_PSa2 is a valuable candidate for controlling foodborne S. enterica and E. coli pathogens.

Bird Flu Outbreak in Dairy Cows Is Widespread, Raising Public Health Concerns.

This Medical News article discusses the current H5N1 avian influenza outbreak in US dairy cattle and its implications for occupational and public health.

Hepatitis E Virus in Domestic Ruminants and Virus Excretion in Milk-A Potential Source of Zoonotic HEV Infection.

The hepatitis E virus is a serious health concern worldwide, with 20 million cases each year. Growing numbers of autochthonous HEV infections in industrialized nations are brought on via the zoonotic transmission of HEV genotypes 3 and 4. Pigs and wild boars are the main animal reservoirs of HEV and play the primary role in HEV transmission. Consumption of raw or undercooked pork meat and close contact with infected animals are the most common causes of hepatitis E infection in industrialized countries. However, during the past few years, mounting data describing HEV distribution has led experts to believe that additional animals, particularly domestic ruminant species (cow, goat, sheep, deer, buffalo, and yak), may also play a role in the spreading of HEV. Up to now, there have not been enough studies focused on HEV infections associated with animal milk and the impact that they could have on the epidemiology of HEV. This critical analysis discusses the role of domestic ruminants in zoonotic HEV transmissions. More specifically, we focus on concerns related to milk safety, the role of mixed farming in cross-species HEV infections, and what potential consequences these may have on public health.

U.S. government in hot seat for cow flu outbreak response.

Veterinarians and researchers say it has taken too long to share data on viral changes, spread, and milk safety.

Highly Pathogenic Avian Influenza A(H5N1) Clade 2.3.4.4b Virus Infection in Domestic Dairy Cattle and Cats, United States, 2024.

We report highly pathogenic avian influenza A(H5N1) virus in dairy cattle and cats in Kansas and Texas, United States, which reflects the continued spread of clade 2.3.4.4b viruses that entered the country in late 2021. Infected cattle experienced nonspecific illness, reduced feed intake and rumination, and an abrupt drop in milk production, but fatal systemic influenza infection developed in domestic cats fed raw (unpasteurized) colostrum and milk from affected cows. Cow-to-cow transmission appears to have occurred because infections were observed in cattle on Michigan, Idaho, and Ohio farms where avian influenza virus-infected cows were transported. Although the US Food and Drug Administration has indicated the commercial milk supply remains safe, the detection of influenza virus in unpasteurized bovine milk is a concern because of potential cross-species transmission. Continued surveillance of highly pathogenic avian influenza viruses in domestic production animals is needed to prevent cross-species and mammal-to-mammal transmission.

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.

Breast milk transmission and involvement of mammary glands in tick-borne flavivirus infected mice.

Tick-borne flaviviruses (TBFs) are transmitted to humans through milk and tick bites. Although a case of possible mother-to-child transmission of tick-borne encephalitis virus (TBEV) through breast milk has been reported, this route has not been confirmed in experimental models. Therefore, in this study, using type I interferon receptor-deficient A129 mice infected with Langat virus (LGTV), we aimed to demonstrate the presence of infectious virus in the milk and mammary glands of infected mice. Our results showed viral RNA of LGTV in the pup's stomach milk clots (SMCs) and blood, indicating that the virus can be transmitted from dam to pup through breast milk. In addition, we observed that LGTV infection causes tissue lesions in the mammary gland, and viral particles were present in mammary gland epithelial cells. Furthermore, we found that milk from infected mice could infect adult mice via the intragastric route, which has a milder infection process, longer infection time, and a lower rate of weight loss than other modes of infection. Specifically, we developed a nano-luciferase-LGTV reporter virus system to monitor the dynamics of different infection routes and observed dam-to-pup infection using in vivo bioluminescence imaging. This study provides comprehensive evidence to support breast milk transmission of TBF in mice and has helped provide useful data for studying TBF transmission routes.IMPORTANCETo date, no experimental models have confirmed mother-to-child transmission of tick-borne flavivirus (TBF) through breastfeeding. In this study, we used a mouse model to demonstrate the presence of infectious viruses in mouse breast milk and mammary gland epithelial cells. Our results showed that pups could become infected through the gastrointestinal route by suckling milk, and the infection dynamics could be monitored using a reporter virus system during breastfeeding in vivo. We believe our findings have provided substantial evidence to understand the underlying mechanism of breast milk transmission of TBF in mice, which has important implications for understanding and preventing TBF transmission in humans.

Quasispecies Composition of Small Ruminant Lentiviruses Found in Blood Leukocytes and Milk Epithelial Cells.

Small ruminant lentiviruses (SRLVs) exist as populations of closely related genetic variants, known as quasispecies, within an individual host. The privileged way of SRLVs transmission in goats is through the ingestion of colostrum and milk of infected does. Thus, characterization of SRLV variants transmitted through the milk, including milk epithelial cells (MEC), may provide useful information about the transmission and evolution of SRLVs. Therefore, the aim of this study was to detect SRLVs in peripheral blood leukocytes (PBLs) and milk epithelial cells of goats naturally infected with SRLVs and perform single nucleotide variations analysis to characterize the extent of genetic heterogeneity of detected SRLVs through comparison of their gag gene sequences. Blood and milk samples from 24 seropositive goats were tested in this study. The double immunolabeling against p28 and cytokeratin demonstrated that milk epithelial cells originated from naturally infected goats were infected by SRLVs. Moreover, PCR confirmed the presence of the integrated SRLVs proviral genome indicating that MECs may have a role as a reservoir of SRLVs and can transmit the virus through milk. The blood and MEC derived sequences from 7 goats were successfully sequenced using NGS and revealed that these sequences were genetically similar. The MEC and blood-derived sequences contained from 3 to 30 (mean, 10.8) and from 1 to 10 (mean, 5.4) unique SNVs, respectively. In five out of seven goats, SNVs occurred more frequent in MEC derived sequences. Non-synonymous SNVs were found in both, PBLs and MEC-derived sequences of analyzed goats and their total number differed between animals. The results of this study add to our understanding of SRLVs genomic variability. Our data provides evidence for the existence of SRLVs quasispecies and to our knowledge, this is the first study that showed quasispecies composition and minority variants of SRLVs present milk epithelial cells.

[BVD situation on farms with high serological values in the tank milk - individual animals have a greater influence than previously known]. / BVD-Situation auf Betrieben mit hohen serologischen Werten in der Tankmilch ­ Einzeltiere haben einen grösseren Einfluss als bisher bekannt.

INTRODUCTION: Bovine virus diarrhea (BVD) is an economically important disease in cattle. Switzerland started an eradication program in 2008. After the initial virological examination of all cattle followed by the examination of all newborn calves, the BVD prevalence in 2012 was low enough to start serological monitoring. An unusually high number of increased tank milk values were observed during this serological monitoring in autumn 2019. No seropositive animals were found on most farms in the follow-up cattle group testing. The present study was designed in the form of a multiple case report to better assess the BVD situation in a herd after a serological tank milk result above the cut-off value. The tank milk of 13 farms with serological values above the cut-off value from the analyses in autumn 2019 was examined again with two different ELISA tests in spring 2020. In addition, at the same time blood samples were taken to obtain serological values of all adult cattle on the farm. The results of the two tests that were used to examine the tank milk samples correlated well with each another. The results of the tank milk serology showed a low correlation with the seroprevalence in the lactating cows, but no correlation with the seroprevalence of all adult animals in the herd. A single seropositive animal had a major influence on the results of the tank milk serology in some herds. In addition, correct tank milk sampling must be ensured because the representativeness of the tank milk sample is decisive for a meaningful result for the investigated farm. If the result of the tank milk test is above the cut-off value, the examination of a group of cattle is still the best way to identify a current infection in a herd.

INTRODUCTION: La diarrhée virale bovine (BVD) est une maladie des bovins économiquement importante. En Suisse, cette maladie fait l'objet d'un programme d'éradication depuis 2008. Après l'examen virologique initial de tous les bovins, suivi de l'examen de tous les veaux nouveau-nés, la prévalence de la BVD en 2012 était suffisamment faible pour permettre d'adapter le programme de surveillance et de procéder à un suivi sérologique des exploitations laitières. Un nombre inhabituellement grand de valeurs élevées dans les examens de lait de citerne a été observé lors de cette surveillance sérologique en automne 2019. Dans de nombreuses exploitations, aucun animal séropositif n'a pu être découvert lors des tests de suivi des groupes de bovins. Cette étude a été conçue sous la forme d'un rapport de cas multiples pour mieux évaluer la situation de la BVD dans un troupeau après un résultat sérologique de lait de citerne supérieur à la valeur limite. Le lait de citerne de 13 exploitations avec des valeurs sérologiques supérieures à la valeur limite en automne 2019 a été analysé à nouveau avec deux tests ELISA différents au printemps 2020. De plus, des échantillons de sang ont été prélevés en même temps pour obtenir les valeurs sérologiques de tous les bovins adultes présents sur l'exploitation. Les résultats des deux tests utilisés pour examiner les échantillons de lait de citerne montraient une bonne corrélation entre les deux tests. Les résultats de la sérologie du lait de citerne ont montré une faible corrélation avec la séroprévalence chez les vaches en lactation mais pas avec la séroprévalence de tous les animaux adultes du troupeau. Un seul animal séropositif pouvait avoir à lui seul une forte influence sur les résultats de la sérologie du lait de citerne dans certains troupeaux. De plus, un prélèvement correct du lait de citerne doit être assuré car la représentativité de l'échantillon est décisive pour obtenir un résultat significatif pour l'exploitation étudiée. Si le résultat du test est supérieur à la valeur limite, l'examen d'un groupe de bovins reste le meilleur moyen d'identifier une infection dans un troupeau.

Identification and Characterization of Circular Single-Stranded DNA Genomes in Sheep and Goat Milk.

In recent years, a variety of circular replicase-encoding single-stranded (CRESS) DNA viruses and unclassified virus-like DNA elements have been discovered in a broad range of animal species and environmental samples. Key questions to be answered concern their presence in the human diet and their potential impact on disease emergence. Especially DNA elements termed bovine meat and milk factors (BMMF) are suspected to act as co-factors in the development of colon and breast cancer. To expand our knowledge on the occurrence of these potential pathogens in human nutrition, a total of 73 sheep and 40 goat milk samples were assayed by combining rolling circle amplification (RCA), PCR and Sanger sequencing. The present study further includes retail milk from the aforementioned species. We recovered 15 single stranded (ss) circular genomes. Of those, nine belong to the family Genomoviridae and six are members of the unclassified group of BMMF. Thus, dairy sheep and goats add to dispersal of CRESS viruses and circular ssDNA elements, which enter the food chain via milk. The presence of these entities is therefore more widespread in Bovidae than initially assumed and seems to be part of the common human nutrition.

Bulk Milk Tank Samples Are Suitable to Assess Circulation of Tick-Borne Encephalitis Virus in High Endemic Areas.

A reliable surveillance strategy of tick-borne encephalitis virus (TBEV) is necessary to ensure adequate disease control measures. However, current approaches assessing geographical TBEV circulation are ineffective or have significant limitations. In this study we investigated a total of 1363 goat and 312 sheep bulk tank milk samples for the presence of TBEV. Samples were collected from systematically selected farms in Lithuania every 4-5 days from April to November in 2018 and 2019. To validate results, we additionally tested 2685 questing ticks collected in the vicinity of milk collection sites. We found 4.25% (95% CI 3.25-5.47) and 4.48% (95% CI 2.47-7.41) goat and sheep milk samples to be positive for TBEV, respectively. Furthermore, geographical distribution of TBEV in milk samples coincided with the known TBE endemic zone and was correlated with incidence of TBE in humans in 2019. When sampling time coincides, TBEV detection in milk samples is as good a method as via flagged ticks, however bulk milk samples can be easier to obtain more frequently and regularly than tick samples. The minimal infectious rate (MIR) in ticks was 0.34% (CI 95% 0.15-0.64). Therefore, our results confirm that testing milk serves as a valuable tool to investigate the spatial distribution of TBEV at higher resolution and lower cost.

Grape seed extract against Aichi virus infectivity in model foods and contact surfaces.

Aichi virus (AiV) is an enteric virus that affects humans and is prevalent in sewage waters. Effective strategies to control its spread need to be explored. This study evaluated grape seed extract (GSE) for: a) antiviral potential towards AiV infectivity at 37 °C and room temperature (RT); b) antiviral behavior in model foods (apple juice (AJ) and 2% fat milk) and also simulated gastric environments; and c) potential application as a wash solution on stainless steel surfaces. GSE at 0.5 mg/mL decreased AiV suspensions containing ~4.75 log PFU/mL to titer levels that were not detected after 30 s at both 37 °C and RT. Infectious AiV titers were not detected after 5 min treatment with 1 mg/mL GSE at 37 °C in AJ. GSE at 2 mg/mL and 4 mg/mL in 2% fat milk decreased AiV after 24 h by 1.18 and 1.57 log PFU/mL (4.75 log PFU/mL to 2.86 and 3.25 log PFU/mL), respectively. As a surface wash, GSE at 1 mg/mL after 30 s decreased AiV to undetectable levels under clean conditions. With organic load (mimicking unclean conditions), 2 and 4 mg/mL GSE reduced AiV after 5 min by 1.13 and 1.71 log PFU/mL, respectively. Overall, GSE seems to be a promising antiviral agent against AiV at low concentrations and short contact times.