Efficacy of Ultraviolet (UV-C) Light in a Thin-Film Turbulent Flow for the Reduction of Milkborne Pathogens.

Nonthermal technologies are being investigated as viable alternatives to, or supplemental utilization, with thermal pasteurization in the food-processing industry. In this study, the effect of ultraviolet (UV)-C light on the inactivation of seven milkborne pathogens (Listeria monocytogenes, Serratia marcescens, Salmonella Senftenberg, Yersinia enterocolitica, Aeromonas hydrophila, Escherichia coli, and Staphylococcus aureus) was evaluated. The pathogens were suspended in ultra-high-temperature whole milk and treated at UV doses between 0 and 5000 J/L at a flow rate of 4300 L/h in a thin-film turbulent flow-through pilot system. Of the seven milkborne pathogens tested, L. monocytogenes was the most UV resistant, requiring 2000 J/L of UV-C exposure to reach a 5-log reduction. The most sensitive bacterium was S. aureus, requiring only 1450 J/L to reach a 5-log reduction. This study demonstrated that the survival curves were nonlinear. Sigmoidal inactivation curves were observed for all tested bacterial strains. Nonlinear modeling of the inactivation data was a better fit than the traditional log-linear approach. Results obtained from this study indicate that UV illumination has the potential to be used as a nonthermal method to reduce microorganism populations in milk.

Synergistic effects of lactic acid and sodium dodecyl sulfate to decontaminate Escherichia coli O157:H7 on cattle hide sections.

The objective of this study was to investigate the antibacterial properties of chitosan acetate (CA), sodium dodecyl sulfate (SDS), lactic acid (LA) and their synergism when combined against a nontoxigenic strain of Escherichia coli O157:H7. Treatments that significantly reduced the concentration of E. coli O157:H7 in vitro by more than two logs were further investigated using a cattle hide decontamination model. In vitro treatments included CA (1% chitosan in 1% acetic acid vol/vol), SDS (1% vol/vol), SDS (2% vol/vol), LA (1% vol/vol), CA-SDS combination (1% chitosan in 1% acetic acid vol/vol mixed with 1% SDS vol/vol), and LA-SDS combination in two different concentrations (1% LA mixed with 1% SDS vol/vol, and 1% LA mixed with 2% SDS vol/vol). Butterfield's Phosphate Buffer water was used as a control. The antibacterial effect of 1% CA solution alone and in combination with 1% SDS in vitro resulted in a 1.8 and 1.7 log colony-forming units (CFU)/mL reduction, respectively (p<0.05). Only 1% LA, 1% SDS, 2% SDS and their combinations resulted in a >2 log reduction in E. coli O157:H7. On hide sections, both 1% LA-1% SDS and 1% LA-2% SDS combinations significantly (p<0.05) reduced E. coli O157:H7 concentration by 4.6 and 4.7 log CFU/ cm(2) greater than the control, respectively. There was no significant difference in the antibacterial effect of 1% LA compared to the control, 2% SDS compared to the control, or 1% LA compared to 2% SDS. Hence, the antibacterial efficacy of 1% LA against E. coli O157:H7 on hide sections was significantly enhanced when combined with 1% SDS. Results of this study support the use of low concentration LA-SDS combination as a hide wash to reduce the risk of E. coli O157:H7 contamination.

A one health framework to advance food safety and security: An on-farm case study in the Rwandan dairy sector.

In Rwanda, cattle and milk hold a cultural and historical significance, providing an opportunity for pro-dairy governmental policies aimed to alleviate food insecurity, malnutrition, and improve livelihoods. The government of Rwanda has identified strategies to grow the dairy sector through strategic investment to achieve these goals. It is estimated two-thirds of lactating cows in Rwanda have clinical or subclinical mastitis, which reduces milk production and increases the risk of milk as a source for zoonotic disease if the milk is consumed undercooked or unpasteurized. This case study outlines the implementation of a One Health framework that integrates education, research, and outreach in Rwanda to improve food safety and food security, for the social, economic, and health benefit of Rwandans and their livestock. Twenty-five Rwandan Extension Specialists participated in the Dairy Dynamic Management education, research, and outreach program. Once trained, the extension specialists supported 30 small holder dairy farmers in performing proper husbandry and animal health practices for mastitis control and reduction of bacterial counts in the udder. Over the 16-week program, 30 small holder dairy farmers and 100 dairy cows were surveyed weekly for animal husbandry, animal health, and mastitis indicators. Outcomes were evaluated by monitoring animal health, foodborne pathogens in milk, and compliance to animal husbandry protocols. Quarter milk samples were collected weekly and evaluated for the presence of bacteria that are common causes of mastitis. We found a statistically significant reduction of mean total bacterial counts and prevalence of bacterial species in quarters over the 16-week training (P ≤ .01). Smallholders were monitored through observing farmers performing hygienic milking protocols. Farmers conducted the protocol correctly greater than 90% of the time by the end of the 16-week program for 5 of 7 steps for proper hygienic milking procedures, indicating farmers were eager to learn and adopt the procedures. However, follow-up and retraining with Extension Specialists is vital to continued success. We demonstrate that an integrative One Health education, research, and outreach program can be successful in improving animal health, food safety, and food security and this framework can be applied to other agricultural sectors and geographic regions.

Preparing students for careers in food-supply veterinary medicine: a review of educational programs in the United States.

The real and/or perceived shortage of veterinarians serving food-supply veterinary medicine has been a topic of considerable discussion for decades. Regardless of this debate, there are issues still facing colleges of veterinary medicine (CVMs) about the best process of educating future food-supply veterinarians. Over the past several years, there have been increasing concerns by some that the needs of food-supply veterinary medicine have not adequately been met through veterinary educational institutions. The food-supply veterinary medical curriculum offered by individual CVMs varies depending on individual curricular design, available resident animal population, available food-animal caseload, faculty, and individual teaching efforts of faculty. All of the institutional members of the Association of American Veterinary Medical Colleges (AAVMC) were requested to share their Food Animal Veterinary Career Incentives Programs. The AAVMC asked all member institutions what incentives they used to attract and educate students interested in, or possibly considering, a career in food-supply veterinary medicine (FSVM). The problem arises as to how we continue to educate veterinary students with ever shrinking budgets and how to recruit and retain faculty with expertise to address the needs of society. Several CVMs use innovative training initiatives to help build successful FSVM programs. This article focuses on dairy, beef, and swine food-animal education and does not characterize colleges' educational efforts in poultry and aquaculture. This review highlights the individual strategies used by the CVMs in the United States.

Toxoplasma in animals, food, and humans: an old parasite of new concern.

All hosts, including humans, can be infected by any one of the three forms of the parasite Toxoplasma gondii that correspond to three morphological stages: tachyzoite, bradyzoite, and sporozoite form. Felids are definitive hosts for T. gondii, which is an intracellular pathogen that infects a wide range of warm-blooded intermediate hosts. Toxoplasmosis is a disease where the interest of the diverse medical and veterinary specialties converge. Awareness needs to be increased that toxoplasmosis can induce clinical disease not only in immunocompromised patients or through congenital infections, but also in healthy patients. This is a review article that aims at illustrating why toxoplasmosis should be regarded a veterinary public health issue and how veterinary practitioners can contribute in controlling the infection.

Developing a Global One Health Workforce: The "Rx One Health Summer Institute" Approach.

The One Health approach has gained support across a range of disciplines; however, training opportunities for professionals seeking to operationalize the interdisciplinary approach are limited. Academic institutions, through the development of high-quality, experiential training programs that focus on the application of professional competencies, can increase accessibility to One Health education. The Rx One Health Summer Institute, jointly led by US and East African partners, provides a model for such a program. In 2017, 21 participants representing five countries completed the Rx One Health program in East Africa. Participants worked collaboratively with communities neighboring wildlife areas to better understand issues impacting human and animal health and welfare, livelihoods, and conservation. One Health topics were explored through community engagement and role-playing exercises, field-based health surveillance activities, laboratories, and discussions with local experts. Educational assessments reflected improvements in participants' ability to apply the One Health approach to health and disease problem solving, as well as anticipate cross-sectoral challenges to its implementation. The experiential learning method, specifically the opportunity to engage with local communities, proved to be impactful on participants' cultural awareness. The Rx One Health Summer Institute training model may provide an effective and implementable strategy by which to contribute to the development of a global One Health workforce.

Quantitative microbial risk assessment for Staphylococcus aureus and Staphylococcus enterotoxin A in raw milk.

A quantitative microbial risk assessment was constructed to determine consumer risk from Staphylococcus aureus and staphylococcal enterotoxin in raw milk. A Monte Carlo simulation model was developed to assess the risk from raw milk consumption using data on levels of S. aureus in milk collected by the University of California-Davis Dairy Food Safety Laboratory from 2,336 California dairies from 2005 to 2008 and using U.S. milk consumption data from the National Health and Nutrition Examination Survey of 2003 and 2004. Four modules were constructed to simulate pathogen growth and staphylococcal enterotoxin A production scenarios to quantify consumer risk levels under various time and temperature storage conditions. The three growth modules predicted that S. aureus levels could surpass the 10(5) CFU/ml level of concern at the 99.9th or 99.99th percentile of servings and therefore may represent a potential consumer risk. Results obtained from the staphylococcal enterotoxin A production module predicted that exposure at the 99.99th percentile could represent a dose capable of eliciting staphylococcal enterotoxin intoxication in all consumer age groups. This study illustrates the utility of quantitative microbial risk assessments for identifying potential food safety issues.

A one health perspective on dairy production and dairy food safety.

As the global population approaches 9.7 billion inhabitants by the year 2050, humanity faces enormous challenges to feed, house, and provide basic living requirements for the growing population while preserving the health of wildlife and the ecosystem. Dairy source foods play an important part in providing nutrient and energy dense sources of calories and establishing Bifidobacterium as a keystone species in the gut for positive health outcomes in infants and children. In developed countries, dairy products have a high food safety record when pasteurized and properly processed. However, when milk is consumed unpasteurized, as often occurs in developing countries where regulation and oversight of the dairy industry is lacking, dairy can serve as a vector for zoonotic transmission of disease and can contain adulterants such as antibiotic residues. Here we provide an overview for the importance of dairy source foods for nutrition and with a One Health perspective and discuss the historical events that have resulted in a high standard of dairy food safety in the United States. This review article covers the Origins of One Health, the role of milk in transmission of disease, management practices and regulations to ensure safe dairy products reach consumers, current challenges facing the dairy industry and impacts on public health, and how these standards can be employed in low and middle income countries to improve public health, nutrition and economic benefits to farmers.

Corrigendum: Use of UV Treated Milk Powder to Increase Vaccine Efficacy in the Elderly.

[This corrects the article DOI: 10.3389/fimmu.2018.02254.].

Isolation of Mycobacterium avium subsp. paratuberculosis from waste milk delivered to California calf ranches.

The objective of this study was to determine if viable Mycobacterium avium subsp. paratuberculosis (MAP) was present in waste milk delivered and fed to calves on California calf ranches. Four calf-raising facilities in the Central Valley of California that fed pasteurized waste milk to calves were enrolled. Pre- and post-pasteurization waste milk samples were cultured for MAP using liquid and solid media over a 5-day period during each of four seasons. Aerobic cultures were performed simultaneously to enumerate total bacteria count and evaluate the efficiency of pasteurization which was estimated by the log-reduction of the total number of bacteria. Viable MAP was cultured from 2% of the waste milk samples. Of the three culture-positive samples, two were from pre-pasteurized and one was from post-pasteurized milk samples. The mean total bacterial count for pre- and post-pasteurized waste milk varied from 1.8 x 10(8) to 5.5 x 10(8) colony-forming units (CFU)/mL and 4.9 x 10(5) to 1.1 x 10(8) CFU/mL, respectively, and on average ranches 1, 2, 3, and 4 had, respectively, 3.5-, 3-, 4.7-, and 2.6-log reduction in the number of total bacteria in their waste milk. This is the first study to document results from on-farm pasteurization under field conditions and it indicates the lack of uniformity and adequate controls of the process which could allow the survival of MAP and other pathogens. Calf-raising facilities could benefit from the implementation of standard operating procedures and farm worker training for pasteurization of waste milk. Dairy herds should be aware that placing calves in specialized off-site calf-raising facilities might not eliminate all possible routes of infection of calves with MAP.

Use of UV Treated Milk Powder to Increase Vaccine Efficacy in the Elderly.

Aging populations experience a decline in adaptive immune system function also known as immunosenesence. Protein nutrition has been shown to stimulate and strengthen the immune system, and such approaches are needed for this growing segment of the population. A controlled, randomized, double blind pilot study was conducted to compare two different protein sources (soy and dairy) as nutritional supplementation to enhance vaccine response. Our objective was to examine the immune stimulating effects of dairy protein subjected to ultraviolet radiation (UV-C) radiation treatment process instead of pasteurization. Participants were 21 healthy individuals over 60 years of age who consumed 6 g of the dairy protein or a comparison, soy isoflavone protein, twice a day for 8 weeks. DTaP vaccine administered at week 4. Non-parametric t-tests revealed a significant increase in Tetanus antibodies in the dairy group compared to the soy group at week 8. These findings suggest additional benefits of UV-C treated unheated dairy protein as a solution to counteract immunosenescence, but warrant further study in elderly and other populations that might benefit from immune system stimulation.

Antimicrobial properties of the chelating agent EDTA on Streptococcal bovine mastitis isolates.

To determine the efficacy of the chelating agent EDTA on microbial growth, separate cultures of two streptococcal bovine mastitis isolates, Streptococcus agalactiae and Streptococcus uberis, were exposed to known concentrations of EDTA. Bacterial cultures of 10(8) CFU/ml were exposed to concentrations of EDTA ranging from 30 to 100 mM in an in-vitro-milk environment. Multiple replications of cultures exposed to EDTA were plated during a two-hour time course. A concentration of 100 mM EDTA resulted in a 90% reduction of S. agalactiae and a 99% reduction of S. uberis. Under these experimental conditions, EDTA treatments in cultures of both isolates exhibited from 1 to 2 log reductions suggesting that EDTA is a potentially effective antimicrobial against streptococcal isolates implicated in causing bovine mastitis.

A multiplex PCR for simultaneous detection and differentiation of North American serotypes of bluetongue and epizootic hemorrhagic disease viruses.

In the present study, a multiplex RT-PCR-based assay for simultaneous detection and differentiation of North American serotypes of bluetongue (BT) virus (BTV) and epizootic hemorrhagic disease (EHD) virus (EHDV) in cell culture and clinical samples was developed. Two pairs of primers (B1 and B4) and (E1 and E4) were designed to hybridize to non-structural protein 1 (NS1) genomes of (BTV-11) and (EHDV-1), respectively. The multiplex PCR-based assay utilized a single tube-PCR amplification in which EHDV and BTV primers were used simultaneously in a multiplex format. The BTV primers generated a 790 base pair (bp) specific PCR product from RNA samples of North American BTV serotypes 2, 10, 11, 13 and 17; whereas EHDV serotypes 1 and 2 or total nucleic acid extract from non-infected baby hamster kidney (BHK) cells failed to demonstrate the 790bp specific BTV PCR product. Likewise, the EHDV primers produced a 387bp specific PCR product from RNA samples of EHDV serotypes 1 and 2, but not from BTV serotypes 2, 10, 11, 13, 17 or from total nucleic acid extract of BHK cell controls. Two pairs of nested primers (B2 and B3) and (E2 and E3), internal to the annealing sites of primers (B1and B4) and primers (E1 and E4), produced a 520bp specific BTV and a 224bp specific EHDV PCR product from BTV and EHDV first amplification products, respectively. These nested amplifications increased the sensitivity of the PCR assay and confirmed the specificity of the first amplified EHDV or BTV PCR products. The described multiplex RT-PCR-based assay could be used to facilitate rapid detection and differentiation of North American BTV and EHDV serotypes and to provide a valuable tool to study the epidemiology of these orbivirus infections in susceptible animal populations.

Use of pulsed ultraviolet laser light for the cold pasteurization of bovine milk.

Because of concerns that some potentially dangerous microorganisms may survive conventional heat pasteurization of milk and because the heat needed to sterilize milk affects marketability, the ability to efficiently cold pasteurized milk may become more desirable. In this pilot study, we investigated the use of pulsed ultraviolet (PUV) laser light to nonthermally (cold) pasteurized bovine milk. Dairy bulk tank milk was treated with UV light (248 nm) emitted from a pulsed excimer laser. The samples were then analyzed for surviving bacteria by spiral plate counting and subculturing in Trypticase soy broth. Other bulk tank milk samples were inoculated with one of eight relevant milk bacterial species before being exposed to laser light. There was no growth observed for any of the plated or subcultured samples exposed to 25 J/cm2. One bacterial isolate was then used to inoculate milk to further investigate bactericidal laser light doses. Growth was observed for samples treated with an average of 0.3 to 6.6 J/cm2 but not for those treated with 12.6 J/cm2. The results indicate that in principle, the bacterial content of milk can be adequately controlled by exposure to PUV laser light.

Determination of the median toxic dose of type C botulinum toxin in lactating dairy cows.

Because of the difficulty in identifying botulinum toxin in cattle, it is hypothesized that cattle are sensitive to levels of toxin below the detection limits of current diagnostic techniques (the mouse protection bioassay and the immunostick enzyme-linked immunosorbent assay [ELISA] for type C botulinum toxin). Using an up-down method for toxicologic testing, the median toxic dose (MTD50) for cattle was determined. Four lactating Holstein cows were dosed at 0.125 or 0.25 ng/kg with Clostridium botulinum type C toxin and failed to develop clinical signs of botulism during the 7-day observation period. Three cows given 0.50 ng/kg of toxin developed clinical signs of botulism. From these results, the MTD50 was calculated at 0.388 ng/kg (3.88 mouse lethal doses/kg) using the trim-logit method. These results suggest that cattle are 12.88 times more sensitive to type C botulinum toxin than a mouse on a per kilogram weight basis. The mouse protection bioassay and the immunostick ELISA for type C botulinum toxin failed to identify the presence of the toxin in the serum, blood, and milk samples taken from all 7 animals.

Seroprevalence of antibodies specific for gram-negative core antigens in chickens on the basis of an Escherichia coli J5 enzyme-linked immunosorbent assay.

Antibodies directed toward gram-negative core antigens (GNCAs) have been demonstrated in many mammalian species but to date are unexamined in any avian species. An enzyme-linked immunosorbent assay with phenol-killed whole cell Escherichia coli J5 was used to assess the presence of serum antibodies directed toward GNCAs in chickens. The first experiment consisted of collecting blood samples from randomly selected hens at egg laying ranches in northern California. The ages ranged from several days of age to 77 wk of age. Birds were classified into age groups (hatchling [1 day-4 wk], pullet [4-18 wk], pullet cycle [18-60 wk], and postmolt [>60 wk]) and husbandry style for titer comparison. The geometric mean titer (GMT) for all adult hens regardless of age was 2147. The geometric mean titers were 220, 5691, 2304, and 1776 for hatchlings, pullets, pullet cycle hens, and postmolt hens, respectively. The age group titer trends were similar to those of humans rather than those of farm animals in that the highest titers occurred during "adolescence" (pullets) and titers decreased slightly with maturity. The GMTs were 2870 for hens housed intensively and 1872 for those housed extensively. The second experiment looked at the progression of GNCA titers within individual birds over a 1-yr period. Individual titers increased slightly throughout the study time of the second experiment.

Comparison of the use of regulatory assays and high-performance liquid chromatography for detection of residues of ceftiofur sodium metabolites in tissue specimens of culled dairy cattle.

OBJECTIVE: To compare the results of regulatory screening and confirmation assays with those of high-performance liquid chromatography (HPLC) in the detection of ceftiofur metabolites in the tissues of culled dairy cattle. ANIMALS: 17 lactating Holstein dairy cows. PROCEDURE: Daily IM injections of ceftiofur sodium were administered at a dose of 2.2 mg of ceftiofur equivalents/kg (n = 6) or 1.0 mg of ceftiofur equivalents/kg (10) for 5 days. Following withdrawal times of 12 hours (high-dose ceftiofur) and either 5 or 10 days (low-dose ceftiofur), cows were slaughtered and liver, kidney, and diaphragmatic muscle specimens were harvested and analyzed by HPLC and standard regulatory methods that included the following assays: the swab test on premises, the fast antimicrobial screen test, the calf antibiotic and sulfa test, and the 7-plate bioassay confirmation test. RESULTS: In all tissue specimens, residues of ceftiofur and desfuroylceftiofur-related metabolites, as measured by HPLC, were less than regulatory tolerance, as defined by the FDA. False-positive screening assay results were more likely for tissue specimens that had been frozen for shipment to a federal laboratory, compared with fresh tissue specimens that were assayed at the slaughter establishment (23% vs 3% false-positive results, respectively). CONCLUSIONS AND CLINICAL RELEVANCE: The observation that fresh tissues had negative results on screening assays, whereas subsets of the same tissue specimens had false-positive results on screening assays following freezing, suggests that freezing and thawing interferes with microbial inhibition-based regulatory screening assays.

Differential levels of mRNA transcripts encoding immunologic mediators in mammary gland secretions from dairy cows with subclinical environmental Streptococci infections.

Dry-off, and the period around parturition, are associated with increased susceptibility to intramammary infections in dairy cows. The immunological profiles of mammary gland secretions during these periods are not well described. The objective of the present study was to better characterize association(s) between chronic subclinical Environmental Streptococci infections at dry-off and relative levels of mRNA transcripts encoding multiple immunologic mediators present in cells derived from mammary gland secretions at dry-off and continuing through parturition. The chronic subclinical bacterial infections in the present study were characterized by multiple isolations of Streptococcus species and elevated SSC for a minimum of three weeks prior to dry-off. The majority of differences between principal and control quarters were identified at dry-off. Transcript levels of IL-17, IL2Rα and iNOS were increased while pro-inflammatory cytokine IL-6, and the regulatory cytokine IL-10, were reduced. Following antibiotic treatment of mammary glands, IL-17 transcripts remained elevated over the course of the study, indicative of a persistent insult. IL-4 transcript levels were modestly elevated at 7 days following dry-off and significantly elevated at 14 days, consistent with activated T(H)1 and T(H)2 lymphocytes in the principal quarters, respectively. From a temporal perspective, transcript levels of IL-8 decreased in all animals through the dry-off period animals and returned to pre-dry-off levels at parturition; levels of iNOS peaked at parturition. Five of the six principal cows experienced recurrent bacterial mastitis during the subsequent lactation; four were in the same quarter as was initially infected with Streptococcus and three of these four were due to coliforms. Taken together, this apparent chronic susceptibility of select mammary glands to bacterial infection would suggest a physiologic and/or immunologic dysfunction. Identification of factor(s) that contribute to the predisposition of mammary glands to developing mastitis should facilitate development of new control strategies.

Survey of ground beef for the detection of Mycobacterium avium paratuberculosis.

Mycobacterium avium paratuberculosis (MAP) is thought to be associated with Crohn's disease in humans. Since Johne's disease affects dairy and beef cattle, meat may be a possible route of transmission of MAP to humans. In this study, we compared a rapid multiplex real time PCR assay and conventional culture to detect MAP in ground beef. The real time PCR assay amplifies both an internal sequence of the IS900 gene and an internal control targeting the ruminant-specific mt-cyt-b gene, in order to control for any false negative results. The sensitivity of this multiplex real time PCR assay on ground beef is 10(1) CFU/g and the sensitivity of conventional culture at 10(3) CFU/g. Furthermore, we conducted a survey of 200 retail ground beef samples using this system and did not detect the presence of MAP.

Development and evaluation of a real-time FRET probe based multiplex PCR assay for the detection of prohibited meat and bone meal in cattle feed and feed ingredients.

A novel real-time fluorescent multiplex polymerase chain reaction (PCR) assay for detecting and discriminating between bovine, ovine, and caprine contaminates in cattle feed was developed that simultaneously performs quality control monitoring on both the DNA extraction process and the level of PCR inhibition in the final DNA extract in a single PCR run. The assay used a single set of primers and two sets of FRET probes targeting the ruminant-specific mitochondrial cytochrome b gene. An internal control PCR reaction targeting a region of the chloroplast RNA polymerase beta-subunit (rpobeta) gene, which is conserved among plants, was incorporated into the ruminant multiplex PCR reaction in order to both monitor the DNA extraction method and to test for the presence of PCR inhibitors. The detection limit for bovine and ovine contaminates was evaluated over a period of two sets of six trials on 15 different types of cattle feed and feed ingredients spiked with known concentrations of bovine meat and bone meal (BMBM) and lamb meat and bone meal (LMBM). The assay was able to detect 0.05% w/w BMBM contamination and 0.1% w/w LMBM contamination in all samples of cattle feed and feed ingredients tested.