Hot topic: Bovine milk samples yielding negative or nonspecific results in bacterial culturing--the possible role of PCR-single strand conformation polymorphism in mastitis diagnosis

A large proportion of mastitis milk samples yield negative or nonspecific results (i.e., no mastitis pathogen can be identified) in bacterial culturing. Therefore, the culture-independent PCR-single strand conformation polymorphism method was applied to the investigation of bovine mastitis milk samples. In addition to the known mastitis pathogens, the method was suitable for the detection of fastidious bacteria such as Mycoplasma spp., which are often missed by conventional culturing methods. The detection of Helcococcus ovis in 4 samples might indicate an involvement of this species in pathogenesis of bovine mastitis. In conclusion, PCR-single-strand conformation polymorphism is a promising tool for gaining new insights into the bacteriological etiology of mastitis.     

Prevalence of minor udder pathogens after intramammary dry treatment

A total of 156 dairy cows was randomly assigned to one of four groups at drying off over an 18-mo period: untreated control or intramammary treatment of each mammary quarter with either 400 mg novobiocin, 300 mg cephapirin, or 1 g dihydrostreptomycin with 1 million units penicillin. Quarter foremilk samples were aseptically collected from each cow within 1 mo of drying off and within 1 mo after parturition for bacteriological analysis. Prior to drying off, 28.7% of quarters were positive of which 46.7% were Corynebacterium bovis and 45.5% were coagulase-negative staphylococci. Reductions in infection prevalence from drying off to postpartum samplings for control, novobiocin, cephapirin, and streptomycin-penicillin groups were 13.6, 60.5, 74.4, and 35.3% of quarters. Recovery rates for C. bovis infections were 47.6, 100, 100, and 94.1%; for coagulase-negative staphylococci infections they were 72.7, 86.4, 80.0, and 100%. The cephapirin group showed the lowest new infection rate (1.3%) with coagulase-negative staphylococci compared with control (6.9%). There were no significant differences in lactation milk production among groups following dry period therapy. Results suggest that dry treatment reduces the prevalence of infections by the minor mastitis pathogens.     

Evaluation of minor pathogen intramammary infection, susceptibility parameters, and somatic cell counts on the development of new intramammary infections with major mastitis pathogens

Major mastitis pathogens such as Staphylococcus aureus, Streptococcus uberis, Streptococcus dysgalactiae, and coliforms are usually considered more virulent and damaging to the udder than minor mastitis pathogens such as Corynebacterium spp. and coagulase-negative staphylococci (CNS). The current literature comprises several studies (n=38) detailing analyses with conflicting results as to whether intramammary infections (IMI) with the minor pathogens decrease, increase, or have no effect on the risk of a quarter acquiring a new IMI (NIMI) with a major pathogen. The Canadian Bovine Mastitis Research Network has a large mastitis database derived from a 2-yr data collection on a national cohort of dairy farms, and data from this initiative were used to further investigate the effect of IMI with minor pathogens on the acquisition of new major pathogen infections (defined as a culture-positive quarter sample in a quarter that had been free of that major pathogen in previous samples in the sampling period). Longitudinal milk samplings of clinically normal udders taken over several 6-wk periods as well as samples from cows pre-dry-off and postcalving were used to this end (n=80,397 quarter milk samples). The effects of CNS and Corynebacterium spp. on the major mastitis pathogens Staph. aureus, Strep. uberis, Strep. dysgalactiae, and coliform bacteria (Escherichia coli and Klebsiella spp.) were investigated using risk ratio analyses and multilevel logistic regression models. Quarter-, cow- and herd-level susceptibility parameters were also evaluated and were able to account for the increased susceptibility that exists within herds, cows and quarters, removing it from estimates for the effects of the minor pathogens. Increased quarter-level susceptibility was associated with increased risk of major pathogen NIMI for all pathogens except the coliforms. Increased somatic cell count was consistently associated with elevated risk of new major pathogen infections, but this was assumed to be a result of low sensitivity of bacteriology to diagnose major pathogen NIMI expediently and accurately. The presence of CNS in the sample 2 samplings before the occurrence of a NIMI increased the odds of experiencing a Staph. aureus NIMI 2.0 times, making the presence of CNS a risk factor for acquiring a Staph. aureus NIMI. Even with this extensive data set, power was insufficient to make a definitive statement about the effect of minor pathogen IMI on the acquisition of major pathogen NIMI. Definitively answering questions of this nature are likely to require an extremely large data set dedicated particularly to minor pathogen presence and NIMI with major pathogens.     

Influence of dry period bacterial intramammary infection on clinical mastitis in dairy cows

Milk samples were taken from 1920 quarters (480 cows, six herds) on four occasions to examine the relationship between quarter level intramammary infection (IMI) during the dry period and clinical mastitis in the next lactation. All quarters were sampled at drying off and within 1 wk of calving, and two quarters from each cow were sampled both 0 to 7 and 8 to 14 d before calving. Milk samples were collected from all cases of clinical mastitis during the following lactation. Logistic regression models were developed to investigate the associations between IMI present during the sampling period and clinical mastitis. The probability of a quarter succumbing to clinical mastitis increased when Streptococcus dysgalactiae, Streptococcus faecalis, Escherichia coli, or Enterobacter spp. were cultured at drying off and when Escherichia coli, coagulase-positive staphylococcus, Serratia spp., or Streptococcus faecalis were cultured in two out of three late dry and post-calving samples. Quarters from which Corynebacterium spp. were isolated at drying off were at an increased risk of clinical mastitis, whereas the presence of Corynebacterium spp. in the late dry and post-calving samples was associated with a reduction in the risk of clinical mastitis. The risk of mastitis for specific pathogens increased if the same species of bacteria that had caused mastitis was isolated at least twice in the late dry and post-calving samples. Kaplan-Meier survival plots indicated that clinical mastitis associated with dry period infections was more likely to occur earlier in lactation than clinical mastitis not associated with dry period infections. There was evidence of quarter susceptibility to IMI or the possibility that infection with one organism led to clinical mastitis with another.     

Diagnosis of Staphylococcus aureus intramammary infection by detection of specific antibody titer in milk.

The diagnostic value of the determination of Staphylococcus aureus antibody titer in milk as a method for identification of mammary quarters with S. aureus IMI was evaluated. Ten cows with a history of S. aureus IMI and 9 cows with no history of S. aureus IMI were sampled daily for 10 d. Quarter and composite milk samples were collected and processed by standard methods for concentration of S. aureus, S. aureus antibody titer (percentage of positive laboratory control), and SCC (cells per milliliter). Microbiologic culture identified 13 S. aureus-infected quarters from the 10 cows with a history of S. aureus IMI. Only 2 of the 130 samples (1.5%) from these infected quarters had undetectable concentrations of S. aureus. Antibody titers in milk from infected quarters of infected cows were below the previously established level considered to be indicative of IMI in 6 of 130 samples (4.6%). Four samples from infected quarters of infected cows had titers considered to be in the suspect range. The sensitivity of the antibody test was 83% (13% SE) when the suspect samples were included and 86% (12% SE) when they were not. Milk from uninfected quarters of cows with S. aureus IMI tended to have S. aureus antibody titers greater than the test's positive control, which would suggest that the quarters were infected with S. aureus. Antibody titer was below the infection threshold level in all pooled samples of uninfected cows, suggesting that the test correctly identified all cows free of S. aureus IMI.(ABSTRACT TRUNCATED AT 250 WORDS)     

Elimination of experimentally induced bovine intramammary infection assessed by multiplex real-time PCR and bacterial culture

Diagnosis of bovine intramammary infection (IMI) has traditionally been based on bacterial culture, but currently IMI can also be detected with DNA based methods, such as multiplex real-time PCR. The aim of this study was to describe the elimination of bacteria in experimentally induced IMI on the quarter level, using conventional bacterial culture (BC) and multiplex real-time PCR. Two coagulase-negative staphylococcal species, Staphylococcus epidermidis and Staphylococcus simulans, were experimentally inoculated into 14 healthy quarters of 8 dairy cows during 4 consecutive study periods. Intramammary infections were followed with 20 milk samplings per each quarter. Milk somatic cell count was monitored to evaluate the inflammation process in the quarters. Four quarters cured spontaneously during the study period according to the culture. The PCR detected staphylococcal DNA from these quarters for several days after they were defined as cured in BC. Agreement between BC and PCR results varied from substantial to almost perfect agreement for the first 36 h postchallenge, decreasing to moderate levels toward the end of the sampling period. Based on this study, we recommend collecting possible follow-up samples to assess the bacteriological cure from IMI not until 2 to 3 wk after the onset of mastitis or after the quarter milk somatic cell count has normalized when PCR is used.     

Identifying the major bacteria causing intramammary infections in individual milk samples of sheep and goats using traditional bacteria culturing and real-time polymerase chain reaction

Use of DNA-based methods, such as real-time PCR, has increased the sensitivity and shortened the time for bacterial identification, compared with traditional bacteriology; however, results should be interpreted carefully because a positive PCR result does not necessarily mean that an infection exists. One hundred eight lactating dairy ewes (56 Manchega and 52 Lacaune) and 24 Murciano-Granadina dairy goats were used for identifying the main bacteria causing intramammary infections (IMI) using traditional bacterial culturing and real-time PCR and their effects on milk performance. Udder-half milk samples were taken for bacterial culturing and somatic cell count (SCC) 3 times throughout lactation. Intramammary infections were assessed based on bacteria isolated in ≥2 samplings accompanied by increased SCC. Prevalence of subclinical IMI was 42.9% in Manchega and 50.0% in Lacaune ewes and 41.7% in goats, with the estimated milk yield loss being 13.1, 17.9, and 18.0%, respectively. According to bacteriology results, 87% of the identified single bacteria species (with more than 3 colonies/plate) or culture-negative growth were identical throughout samplings, which agreed 98.9% with the PCR results. Nevertheless, the study emphasized that 1 sampling may not be sufficient to determine IMI and, therefore, other inflammatory responses such as increased SCC should be monitored to identify true infections. Moreover, when PCR methodology is used, aseptic and precise milk sampling procedures are key for avoiding false-positive amplifications. In conclusion, both PCR and bacterial culture methods proved to have similar accuracy for identifying infective bacteria in sheep and goats. The final choice will depend on their response time and cost analysis, according to the requirements and farm management strategy.     

Real-time polymerase chain reaction-based identification of bacteria in milk samples from bovine clinical mastitis with no growth in conventional culturing

In more than 30% of milk samples from clinical and subclinical bovine mastitis, bacteria fail to grow even after 48 h of conventional culture. The “no-growth” samples are problematic for mastitis laboratories, veterinarians, and dairy producers. This study provides the first investigation of the bacteriological etiology of such samples, using a real-time PCR-based commercial reagent kit. The assay targets the DNA of the 11 most common bacterial species or groups in mastitis and the staphylococcal blaZ gene (responsible for penicillin resistance) and can identify and quantify bacterial cells even if dead or growth-inhibited. A study was made of 79 mastitic milk samples with no-growth bacteria in conventional culture, originating from cows with clinical mastitis. Of the 79 samples, 34 (43%) were positive for 1 (32 samples) or 2 (2 samples) of the target bacteria. The positive findings included 11 Staphylococcus spp. (staphylococci other than Staphylococcus aureus), 10 Streptococcus uberis, 2 Streptococcus dysgalactiae, 6 Corynebacterium bovis, 3 Staph. aureus, 1 Escherichia coli, 1 Enterococcus, and 1 Arcanobacterium pyogenes. The positive samples contained as many as 10³ to 10⁷ bacterial genome copies per milliliter of milk. This study demonstrates that in nearly half of the clinical mastitis cases in which conventional culture failed to detect bacteria, mastitis pathogens were still present, often in substantial quantities. The clearly elevated N-acetyl-β-d-glucosaminidase activity values of the milk samples, together with clinical signs of the infected cows and quarters, confirmed the diagnosis of clinical mastitis and indicated that real-time, PCR-based bacterial findings are able to reveal bacteriological etiology. We conclude that all common mastitis bacteria can occur in large quantities in clinical mastitis samples that exhibit no growth in conventional culture, and that the real-time PCR assay is a useful tool for bacteriological diagnosis of such milk samples. Low bacterial concentration is commonly speculated to explain the no-growth milk samples. This hypothesis is not supported by the results of the current study.     

Microbiological results from milk samples obtained premilking and postmilking for the diagnosis of bovine intramammary infections.

Bacteriological culture results were compared between 336 pairs of quarter milk samples collected premilking and postmilking. Using a positive result on either premilking or postmilking samples as the definitive diagnosis, premilking sampling sensitivity was 91% for Staphylococcus aureus, 91% for coagulase-negative staphylococci, and 97% for Streptococcus other than agalactiae. Postmilking sampling sensitivities were 81, 45, and 58%, respectively, for the same pathogens. Requiring both premilking and postmilking samples for the definitive diagnosis, specificities were 92, 86, and 95% for premilking sampling alone and 96, 98, and 99% for postmilking sampling alone. Such differences in specificity would result in major differences in predictive value of a positive culture for herds with a low prevalence. Multiple isolates were significantly more common from premilking samples.     

Effects of a selective regimen of dry cow therapy on intramammary infection and on antibiotic sensitivity of surviving pathogens

Selective dry cow therapy with 1 million U of procaine penicillin G and 1 g of dihydrostreptomycin in a slow-release base was administered to all quarters of cows currently infected or treated for mastitis in the previous lactation. All other cows in the herd that were dried off during the experimental period were left untreated. Milk samples were cultured for detection of intramammary infection at drying off, 1 wk later (at time of treatment), and after next calving from 227 treated and 143 untreated cows. Isolants were tested for sensitivity to 12 antibiotics including penicillin and streptomycin. Although treatment resulted in clearance of Staphylococcus aureus, Staphylococcus epidermidis, streptococci other than agalactiae, and coliforms from 78, 82, 88, and 87% of infected glands, the net effect on udder health of the herd was a gain of 1.4% in infected quarters. Incidences of new infection in the early dry period (1 wk) were 18.1 and 12.3% among treated and untreated cows. Therapy failed to control new infection in the late dry and peripartum periods; incidence was about 18% of quarters among both long and short dry periods. Among 46 instances of apparent survival of an intramammary pathogen through a treated dry period, 7 of 10 strains previously sensitive to streptomycin became resistant and 8 of 10 strains previously sensitive to penicillin became resistant. The surviving pathogen population was not large enough to be considered a threat to herd udder health in the next lactation.     

Evaluation of test-day milk somatic cell count information to predict intramammary infection with major pathogens in dairy cattle at drying off

The objectives of this study were (1) to determine the test characteristics and predictive values of cow-level milk somatic cell count (SCC) information from (multiple) test-day recordings before drying off to identify major-pathogen-infected cows at drying off; and (2) to explore to what extent (an estimate of) the herd prevalence of subclinical mastitis, milk yield level, and parity of the cows affects the estimates. In total, 550 cows from 15 commercial dairy herds with overall good udder health management were dried-off during a study period of 6 mo. Test-day SCC were available through milk recording and within 5 d before drying off cows were sampled for quarter-level bacteriological culturing serving as the gold standard. Sensitivity (Se), specificity (Sp), positive predictive value (PPV), and negative predictive value (NPV) were calculated at different threshold values of SCC, ranging between 50,000 and 500,000 cells/mL, to detect major-pathogen-infected cows. At a commonly used threshold of 200,000 cells/mL, the Se and Sp of (a combination of) test-day SCC before drying off ranged between 37.6 and 57.6% and between 66.7 and 79.3%, respectively. Still, estimates were modified by the herd level prevalence of subclinical mastitis and the cow's milk yield and parity. For instance, at the 200,000 cells/mL threshold using the geometric mean SCC of the 3 last test-days, the overall Se, Sp, PPV, and NPV were 37.6, 79.3, 30.8, and 83.9%, respectively, whereas these were 27.8, 87.5, 21.7, and 90.6%, respectively, for heifers and 40.3, 73.5, 33.3, and 78.9%, respectively, for multiparous cows. In conclusion, test-day SCC records obtained via milk recording are reliable to detect dairy cows at drying off that are not infected with major pathogens as determined by bacteriological culture and could eventually facilitate implementation of selective dry cow therapy in commercial dairy herds. Because estimates of the herd-level prevalence of subclinical mastitis, milk yield level, and parity of the cows affect the estimates of the test characteristics and predictive values to some extent, one should consider taking these parameters into account when differentiating infected from uninfected cows based on SCC data.     

Evolution of Listeria populations in food samples undergoing enrichment culturing

The isolation of Listeria monocytogenes from food is carried out using a double enrichment. It is believed that the double enrichment can allow the overgrowth of Listeria innocua in samples where both species are present. In this study, we have evaluated the impact of overgrowth between Listeria species and strains during each step of the enrichment process. The effect of factors minimizing interactions between strains or phage inhibitory effects has also been estimated. In an artificially contaminated food undergoing enrichment, overgrowth could result from competitive interactions between Listeria spp. resulting from the production of bacteriocins and bacteriophage at high initial contamination levels (>10(4) cfu/g), but not at lower levels (50-100 cfu/g) as generally found in contaminated foods. At high levels of inoculation, the competitive effect could be reduced by solidification of the selective broths, to limit the diffusion of the inhibitors. Overgrowth resulting from differences in growth rate occurred independent of the initial contamination level. However, in naturally contaminated foods undergoing enrichment, there were no absolute correlations between growth rates or inhibitory profiles in terms of strain evolution during enrichment. In fact, Listeria strains which were predominant in the original sample in most cases remained the dominant strains at the end of the enrichment, although the relative proportion of any given strain could change significantly over the enrichment process. Additional factors which have yet to be identified impact on the evolution of Listeria in the two-step enrichment process. Analysis of strain evolution in eight naturally contaminated foods has indicated that the second enrichment step in Fraser broth can be reduced from 48 to 24 h without impacting on the recovery of L. monocytogenes. Our limited survey of naturally contaminated foods also demonstrated that maximum recovery of L. monocytogenes and other Listeria strains was found following 24 h incubation in 1/2 Fraser Broth. This finding suggests that it may be possible to shorten the current two-step isolation method further without reducing method sensitivity.     

Diagnosing intramammary infections: comparison of multiple versus single quarter milk samples for the identification of intramammary infections in lactating dairy cows

The objective was to examine the potential benefits of using different combinations of multiple quarter milk samples compared with a single sample for diagnosing intramammary infections (IMI) in dairy cattle. Data used in the analyses were derived from 7,076 samples from 667 quarters in 176 cows in 8 herds in 4 locations (Minnesota/Wisconsin, n = 4; Prince Edward Island, n = 2; Ontario, n = 1; New York, n = 1). Duplicate quarter milk samples were collected at morning milking for 5 consecutive days. Cows were evenly distributed between early postparturient and mid- to late-lactation cows. All samples were frozen for shipping and storage, thawed once, and cultured in university laboratories using standardized procedures consistent with National Mastitis Council guidelines. The presence of specific pathogens was confirmed and identified using the API identification system (bioMerieux, Marcy l’Etoile, France) in each laboratory. A previously developed gold standard was applied to the first sample from d 1, 3, and 5 to classify infected quarters. The data were analyzed separately for coagulase-negative staphylococci (CNS) and Streptococcus spp. Various combinations of test results from d 2 and 4 were used in the test evaluation. These consisted of single samples (n = 4), 2 sets of duplicate samples (2 samples collected on the same day), 2 sets of consecutive samples (2 samples collected 2 d apart), and 2 sets of triplicate samples (2 samples on the same day and a third sample 2 d apart). Series interpretation of duplicate or consecutive samples (i.e., positive = same pathogen isolated from both samples) resulted in the highest specificity (Sp; CNS Sp = 92.1-98.1%; Streptococcus spp. Sp = 98.7-99.6%), but lowest sensitivity (Se; CNS Se = 41.9-53.3%; Streptococcus spp. Se = 7.7-22.2%). Parallel interpretation of duplicate or consecutive samples (i.e., positive = pathogen isolated from either) resulted in the highest Se (CNS Se = 70.8-80.6%; Streptococcus spp. Se = 31.6-48.1%), but lowest Sp (CNS Sp = 72.0-77.3%; Streptococcus spp. Sp = 89.5-93.3%). The difference in estimates between single and duplicate samples was larger than between single and consecutive samples. Overall, triplicate samples provided the best combination of Se and Sp, but compared with a single sample, provided only a modest gain in Sp and little or no gain in Se.     

Effect of intramammary infection on milk electrical conductivity in Murciano-Granadina goats

Measurements of electrical conductivity (EC) of milk are used in mastitis detection in cows due to the low cost, possibility of automation, and rapid diagnosis, but the literature about EC measurement in goats is scarce. In this study, we studied the effect of the establishment of intramammary infection (IMI) on EC of goat milk by gland using daily measurements. Additionally, the effects on milk yield, somatic cell count (SCC), and mineral content were analyzed. Eight primiparous and 10 multiparous Murciano-Granadina goats free from IMI were included in the study. Health conditions of the participating animals were monitored for 16 d and then various unfavorable health situations that may arise on commercial farms were simulated to increase the chances of IMI. Once the IMI was confirmed, the experiment continued for another 16 d. Statistical analysis was conducted using a linear mixed model considering several periods regarding the establishment of the infection and whether it affected one or both glands in the animal. The establishment of IMI caused a significant increase of EC, SCC, and chlorides in the infected glands, whereas the sodium:potassium ratio and the ratio of EC between collateral glands showed significant increases only in bilaterally infected animals. The microorganisms that caused greater increases of EC were Staphylococcus aureus and a gram-negative bacterium. Changes due to other isolated microorganisms (coagulase-negative staphylococci and streptococci) were small. No significant differences in milk yield were determined. The significant effect of infection on EC in the affected glands suggests that the use of a system based on daily readings of EC could be useful in IMI detection of goats.     

Fate of foodborne bacterial pathogens in pesticide products

The effects of diluting concentrated pesticide products with water containing Escherichia coli O157:H7 (human, bovine and ground meat strains), Salmonella typhimurium, Salmonella enteritidis, Shigella sonnei, Shigella flexneri and Listeria monocytogenes were investigated. Individual cultures were inoculated at about 10² colony‐forming units (cfu) ml^?1 into the following commercial pesticide products—Roundup, Poast & Merge, Gramoxone, Afolan, 2,4‐D amine, Dithane M45, Benlate, Bravo 500, Ridomil 240EC, Thiram 75WP, Sevin XLR+, Lorsban 4E, Diazinon 500, Ambush 500EC and Lagon 480E—following dilution with sterile tap water to recommended spray application concentrations (pesticide solution). Following incubation at 22 °C for 1 h, survivor levels for all three E coli O157:H7 strains in pesticide solutions of Roundup, Poast & Merge, Gramoxone, Dithane M45, Ridomil 240EC and Lagon 480E were lower (P ≤ 0.05) compared to their survival in water. In several pesticide solutions (Afolan, Bravo 500, Lorsban 4E and Ambush 500EC), survivor levels were no different (P≥0.05) from the control after 1 h; however, by 24 h, population levels of all strains in these four pesticide solutions were higher (P ≤ 0.05) than those observed at 1 h. Overall, similar survivor or growth patterns were observed with the remaining bacteria, with the exception of S flexneri. For this bacterium, survival or growth was poor, especially in Bravo 500, which consistently supported substantial growth of the other micro‐organisms. Time course growth studies over 96 h at 22 °C were conducted with Salmonella, Shigella, Listeria and E coli O157:H7 in solutions of Afolan, Bravo 500, Lorsban 4E and Ambush 500EC. Overall, a 100 to 1060‐fold increase in population was usually obtained in these pesticide solutions by 96 h. Changes in either the initial inoculum level of E coli O157:H7 from about 10² to 10⁴ cfu ml^?1 or temperature (from 20–22 to 31 °C) and concentration (from 0.5 to 1.5 times the recommended spray concentration) were shown to affect its growth in the pesticide solutions. The findings of this study indicate that some pesticide products, when diluted using contaminated water, may have the potential to promote growth of pathogens which could increase the risk of more widespread contamination of standing crops, including fruits. © 2001 Society of Chemical Industry     

Detecting intramammary infection at the end of lactation in dairy cows

To reduce antimicrobial use, infusion of antimicrobials into only infected cows at the end of lactation (selective dry cow therapy) is preferable to infusion of every cow with antimicrobials. Use of selective dry cow antimicrobial therapy requires differentiation of probably infected from uninfected cows to enable treatment allocation. Milk somatic cell count (SCC) has been used to distinguish between cows with and without intramammary infection (IMI). However, SCC may be influenced by milk yield, stage of lactation, breed, and herd-level variables such as prevalence of infection. Cut points for SCC, to distinguish between cows with and without an IMI, may need to differ between cow age groups and breeds, or among herds. This study evaluated associations between SCC and major pathogen IMI in one or more quarters of 2,606 cows from 36 herds in 4 regions of New Zealand. In the last week of lactation, cows selected at random had milk samples collected from each quarter, and the teat-end condition and hygiene of the udder were scored. Herd- and cow-level data including age, breed, milk volume, and SCC at each production were recorded, and bulk tank milk SCC and volume of milk shipped were collated. At cow level, the association between average, maximum, and last cow-composite SCC, and presence of a major pathogen IMI in one or more quarters of cows, was examined using receiver operator curves. Predictive logistic regression models were then developed that included potential effect modifiers such as age, milk yield, and bulk tank milk SCC. The population average prevalence of major pathogen IMI was 7.2% of cows (95% confidence interval = 5.9–8.6), and this varied significantly between herds. The average, maximum, and last cow-composite SCC of lactation were all predictive of presence of a major pathogen IMI and did not differ in their ability to discriminate infected from uninfected cows. However, the optimal cut points for the last SCC, the maximum SCC, and average SCC were 108, 152, and 105 × 1,000 cells/mL, respectively. Inclusion of age, bulk tank SCC, and history of clinical mastitis improved overall model fit. However, inclusion of these variables did not improve the discriminatory power of maximum cow-composite SCC used alone. We conclude that cow-composite SCC on its own resulted in sensitivities and specificities of between 0.76 and 0.86, and 0.71 to 0.80, respectively, for determination of presence of major pattern IMI, and the predictive value was not improved by addition of other predictor variables.     

Using lytic bacteriophages to eliminate or significantly reduce contamination of food by foodborne bacterial pathogens

Bacteriophages (also called ‘phages’) are viruses that kill bacteria. They are arguably the oldest (3 billion years old, by some estimates) and most ubiquitous (total number estimated to be 10³⁰–10³²) known organisms on Earth. Phages play a key role in maintaining microbial balance in every ecosystem where bacteria exist, and they are part of the normal microflora of all fresh, unprocessed foods. Interest in various practical applications of bacteriophages has been gaining momentum recently, with perhaps the most attention focused on using them to improve food safety. That approach, called ‘phage biocontrol’, typically includes three main types of applications: (i) using phages to treat domesticated livestock in order to reduce their intestinal colonization with, and shedding of, specific bacterial pathogens; (ii) treatments for decontaminating inanimate surfaces in food‐processing facilities and other food establishments, so that foods processed on those surfaces are not cross‐contaminated with the targeted pathogens; and (iii) post‐harvest treatments involving direct applications of phages onto the harvested foods. This mini‐review primarily focuses on the last type of intervention, which has been gaining the most momentum recently. Indeed, the results of recent studies dealing with improving food safety, and several recent regulatory approvals of various commercial phage preparations developed for post‐harvest food safety applications, strongly support the idea that lytic phages may provide a safe, environmentally‐friendly, and effective approach for significantly reducing contamination of various foods with foodborne bacterial pathogens. However, some important technical and nontechnical problems may need to be addressed before phage biocontrol protocols can become an integral part of routine food safety intervention strategies implemented by food industries in the USA. © 2013 Society of Chemical Industry     

Technical note: Common analytical errors yielding inaccurate results during analysis of fatty acids in feed and digesta samples

The basic rules governing the proper fatty acid analysis of feed and digesta samples are sometimes overlooked, leading to potential errors in reporting the fatty acid content or composition of feed and digesta samples. The direct transesterification procedure of Sukhija and Palmquist (1988, J. Agric. Food Chem. 36:1202-1206) has become popular in analyzing fatty acids in feed and digesta samples obtained from animal feeding trials. One shortcoming of the Sukhija and Palmquist transesterification procedure is inaccurate analysis of fatty acids with conjugated double bonds. Digesta and milk samples from ruminant species typically contain a multitude of conjugated linoleic acid (CLA) isomers that easily undergo isomerization and epimerization following prolonged exposure to methanolic HCl. Modifications to the Sukhija and Palmquist procedure are given in this paper that allow successful determination of CLA isomers. Errors in fatty acid analysis also occur from misuse of internal standards; use of an internal standard is recommended in the Sukhija and Palmquist procedure as the preferred method to quantify total fatty acid content. The choice of internal standard may sometimes be important for obtaining accurate results. As an example, applying the direct transesterification procedure to a fat supplement high in saturated fatty acids yielded 613 mg/g of total fatty acids when C17 was used as the internal standard compared with 930 mg/g total fatty acids when C19 was used as the internal standard. Fatty acid content further increased to 952 mg/g when a unique unsaturated fatty acid (C13:1) was used as the internal standard.     

Evaluation of 4 predictive algorithms for intramammary infection status in late-lactation cows

The objective of this observational study was to compare 4 cow-level algorithms to predict cow-level intramammary infection (IMI) status (culture and MALDI-TOF) in late-lactation US dairy cows using standard measures of test performance. Secondary objectives were to estimate the likely effect of each algorithm, if used to guide selective dry cow therapy (SDCT), on dry cow antibiotic use in US dairy herds, and to investigate the importance of including clinical mastitis criteria in algorithm-guided SDCT. Cows (n = 1,594) from 56 US dairy herds were recruited as part of a previously published cross-sectional study of bedding management and IMI in late-lactation cows. Each herd was visited twice for sampling. At each farm visit, aseptic quarter-milk samples were collected from 20 cows approaching dry-off (>180 d pregnant), which were cultured using standard bacteriological methods and MALDI-TOF for identification of isolates. Quarter-level culture results were used to establish cow-level IMI status, which was considered the reference test in this study. Clinical mastitis records and Dairy Herd Improvement Association test-day somatic cell count data were extracted from herd records and used to perform cow-level risk assessments (low vs. high risk) using 4 algorithms that have been proposed for SDCT in New Zealand, the Netherlands, United Kingdom, and the United States. Agreement between aerobic culture (reference test; IMI vs. no-IMI) and algorithm status (high vs. low risk) was described using Cohen's kappa, test sensitivity, specificity, negative predictive value, and positive predictive value. The proportion of cows classified as high risk among the 4 algorithms ranged from 0.31 to 0.63, indicating that these approaches to SDCT could reduce antibiotic use at dry-off by 37 to 69% in the average US herd. All algorithms had poor agreement with IMI status, with kappa values ranging from 0.05 to 0.13. Sensitivity varied by pathogen, with higher values observed when detecting IMI caused by Streptococcus uberis, Streptococcus dysgalactiae, Staphylococcus aureus, and Lactococcus lactis. Negative predictive values were high for major pathogens among all algorithms (≥0.87), which may explain why algorithm-guided SDCT programs have been successfully implemented in field trials, despite poor agreement with overall IMI status. Removal of clinical mastitis criteria for each algorithm had little effect on the algorithm classification of cows, indicating that algorithms based on SCC alone may have similar performance to those based on SCC and clinical mastitis criteria. We recommend that producers implementing algorithm-guided SDCT use algorithm criteria that matches their relative aspirations for reducing antibiotic use (high specificity, positive predictive value) or minimizing untreated IMI at dry-off (high sensitivity, negative predictive value).     

Influence of milk flow rate on new intramammary infection in dairy cows

In a series of short-term experiments cows were subjected to exaggerated bacterial challenge and accentuated milking machine conditions known to predispose to new mammary infection. The incidence of new intramammary infection was significantly greater in quarters with peak flow rates greater than 1.6 kg/min whether they were exposed to impacts (P less than 0.05) or milking without pulsation (P less than 0.001). The infection rates were much lower (P less than 0.001) in quarters milked with 'pulsation and shields' to protect against these two machine factors. Despite this, quarters with peak flow greater than 1.6 kg/min still showed a 12-fold increase in mastitis incidence compared with quarters with peak flow less than 0.8 kg/min. Rates of milk flow have increased dramatically in the last 40 years through selection and breeding: whole udder peak flow rates in heifers have doubled from 1.9 to 3.8 kg/min. Increased emphasis, therefore, should be placed on hygiene, husbandry and milking techniques to minimize bacterial numbers at teat ends to control mastitis as the drive for higher flow rate and yield make cows increasingly more susceptible to infection. These results suggest that the benefits of reduced infection rate from mastitis control are significantly underestimated since animals are now considerably more susceptible than 40 years ago.