Bacteriological quality of raw cow's milk from four dairy farms and a milk collection centre in and around Addis Ababa

Bacteriological quality of raw cow's milk taken at different sampling points from four dairy farms and a milk collection centre in and around Addis Ababa was evaluated. Milk samples were aseptically collected from udder, bucket, storage container before and after cooling and upon arrival at the processing plant. A high increase in the mean total aerobic plate count was observed in milk samples taken from the bucket (1.1 x 10(5) cfu/ml), storage container before cooling (4 x 10(6) cfu/ml) and upon arrival at the processing plant (1.9 x 10(8) cfu/ml). The mean coliform counts ranged from 1.3 x 10(4) cfu/ml (storage container before cooling) to 7.1 x 10(4) cfu/ml (upon arrival at the processing plant). The hygienic quality of raw milk from the collection centre was poor with a mean total bacterial count of 1.3 x 10(7) cfu/ml. Milk sampled from the udder contained mainly staphylococci and micrococci as udder-specific bacteria, while samples taken at later stages were additionally contaminated with bacteria of environmental origin (especially Enterobacteriaceae). Lack of knowledge about clean milk production, use of unclean milking equipment and lack of potable water for cleaning purposes were some of the factors which contributed to the poor hygienic quality of raw milk in the study farms.     

Mastitis in Camelus dromedarius in Saudi Arabia

The correlation between camels' milk samples collected from abnormal inflamed udders and samples positive in the California Mastitis Test (CMT) was +0.803 (P less than 0.01). The bacterial count ranges of milk samples differed significantly (P less than 0.05) for those with a negative CMT and those with a positive CMT. Infection with many but not all bacterial species was associated with positive CMT results. The highest percentage of camel milk samples was included in the bacterial count range of 3.0 x 10(2) to 3.0 x 10(3) cfu/ml rather than in the greater than 3.0 x 10(3) cfu/ml range for most of the bacterial species. The most predominant bacterial isolates were Micrococcus spp., Staphylococcus aureus, Streptococcus spp. and Corynebacterium spp. followed by eight other flora. Chloramphenicol was the most effective antimicrobial agent of six tested against 118 bacterial isolates. Preliminary observations are made on chemotherapy of mastitis cases in camels.     

Somatic cells count in cow's bulk tank milk

The objective of this study was therefore to present factors affecting somatic cell counts in bovine bulk milk as a result of intramammary infections as well as non-infectious factors. The paper presents also the impact of on-farm management practices on the level of bulk milk somatic cell counts and presents quality indicators in bulk tank milk. At the farm level bulk milk bacterial infection takes place through three main sources: bacterial contamination from the external surface of the udder and teats, from the surface of the milking equipment, and from mastitis microorganisms within the udder. The threshold of 200,000 cells/ml identifies bacteriological negative quarters of the udder. The counts of mammary pathogens in bulk tank milk are relatively low, on average not exceeding 1,000 cfu/ml. Environmental pathogens predominate in bulk tank milk samples with somatic cells count <300 × 10(3) ml.     

Effect of Sand and Shaking Duration on the Recovery of Aerobic Bacteria,Coliforms, and Escherichia coli from Prechill Broiler Whole Carcass Rinsates

Broilers entering a processing facility can be contaminated with bacteria internally, externally, or both, and additional contamination may occur during processing. Although processing generally reduces the bacterial load on a carcass, it does not eliminate all carcass bacterial contaminants. Processing plant personnel sample carcasses daily to determine overall carcass contamination using the whole carcass rinse procedure. The objective of this study was to evaluate the potential benefit of adding sand to the whole carcass rinse and extending shaking (rinsing) duration on the recovery of bacteria from broiler carcasses. Eviscerated broiler carcasses were obtained from a commercial processing plant before the prechill final wash. Carcasses were rinsed in peptone or peptone with sand for 1 and 4 min. Rinsates were analyzed for aerobic plate count, coliforms, and Escherichia coli. Bacterial levels recovered from rinsates with sand were significantly higher than levels recovered from the peptone-only rinsates, but the increase in recovery was relatively small at 0.6 log₁₀ cfu/mL of rinsate. There was no significant improvement in bacterial recovery when shaking duration was increased from 1 to 4 min for either rinse treatment.     

Relationship between the immune response of sheep and the population dynamics of bacteria isolated from fleecerot lesions.

In sheep wetted by rain, proliferation of bacteria in the skin-fleece microenvironment invariably discolours the fleece and causes a dermatitic condition known as fleecerot. The changes in population dynamics of fleece bacteria were analysed by carrying out skin washings at randomly selected sites on the back of sheep before, and at 48 h and 96 h after exposure to rain. Gram-positive rods belonging to Bacillus species (10(2)-10(4) cfu/cm2) predominated in dry fleece. Gram-positive cocci (e.g. Micrococcus and Staphylococcus species) as well as Gram-negative rods (pseudomonads) were also present but in lower abundance (less than 10(2) cfu/cm2). Fleece bacterial populations generally increased in numbers during the first 24-48 h of wetting. By 96 h however, skin washings showed a preponderance of Pseudomonas aeruginosa (10(4)-10(6) cfu/cm2) and to a lesser extent, pigmented Micrococcus species. Growth of fleece bacteria was associated with a characteristic green or yellow/orange staining of fleece. Fewer species of bacteria were isolated from sheep showing green staining while those animals with yellow/orange discolourations appeared to have a more mixed microflora composition. The predominance of P. aeruginosa in the wet fleece of sheep displaying either green or yellow/orange bacterial stain, was accompanied by a significant serological response against this species. Since skin bacteria have never been observed to penetrate cutaneously in skin sections biopsied from fleecerot sites, it must be concluded that the sheep skin is sensitized by continuous exposure to antigens that are associated with or released by P. aeruginosa.     

Effects of management practices on yield and quality of milk from smallholder dairy units in urban and peri-urban Morogoro,Tanzania

A longitudinal study design was used to assess the management, chemical composition of cows’ milk and quantify the microbial load of raw milk produced at farm level. Data were collected between December 2010 and September 2011 in Morogoro municipality. Milk samples were collected once every month and analysed for butter fat (BF), crude protein (CP), total solids (TS) and solids non-fat (SNF). Total bacterial count (TBC) and coliform counts (CC) were normalized by log transformation. The average milk yield was 7.0 l/day and was not influenced by feeding systems and breeds. Dairy cows owned by people who had no regular income produced more milk than government employees and retired officers. Means of BF, TS, SNF and CP were similar in different feeding systems. Wet season had significantly higher TBC (5.9 log₁₀ cfu/ml) and CC (2.4 log₁₀ cfu/ml) but feeding systems had no effect. Stocking density influenced TBC but not CC. It can be concluded that dairy cows produced low milk yield and its quality was poor.     

Optimization of an in vitro assay to detect Streptococcus equi subsp. equi

Streptococcus equi is the etiologic agent of a highly infectious upper respiratory disease of horses known as strangles. Bacterial culture methods and polymerase chain reaction (PCR) of nasopharyngeal washes and guttural pouch lavages are used routinely to test clinical and carrier animals for the presence of S. equi but no definitive or gold standard test method has been shown to be optimal. We hypothesized that (i) a flocked swab submerged in ten-fold serial dilution suspensions of S. equi prepared in 0.9% NaCl would detect more colony forming units (CFU) than a rayon swab when used to inoculate a blood agar plate, (ii) centrifugation of a 1ml aliquot of each suspension would improve the limit of detection (LOD) by bacterial culture and PCR compared to the culture or PCR of submerged swab samples, (iii) PCR of the centrifuged samples from each suspension would be more sensitive than aerobic culture alone, and (iv) PCR of a 1ml aliquot directly from a sample would be more sensitive than PCR of a sample following submersion of a flocked swab in 1ml saline. Using 7 ten-fold serial dilutions of S. equi in 0.9% NaCl, the LOD for 4 bacterial culture methods and 3 PCR methods were compared. The LOD of direct PCR and flocked swab culture was determined at 1cfu/ml. All PCR methods were equivalent to each other and were more sensitive than any of the culture methods at the lower dilutions. At higher cell densities (>100cfu/ml) flocked swab culture was not statistically better than rayon swab culture, but it was superior to all other methods tested.     

Modified-atmosphere storage under subatmospheric pressure and beef quality: I. Microbiological effects

The microflora was studied in beef stored in stainless steel containers kept under reduced pressure (20 to 30 kPa) in a modified atmosphere (70% N2 + 30% CO2 or pure CO2) at 3 to 4 degrees C and 0 to 1 degrees C at a headspace:meat volume ratio of 2:1. Samples were obtained at weekly intervals, 1 to 3 times. Total colony counts (TCC) for Pseudomonas spp. and Brochothrix thermosphacta were generally 1 to 2 log10 cfu greater than in the control group of vacuum-packaged beef cuts stored at the same temperatures. In containers with the 70% N2 + 30% CO2 atmosphere at 20 to 30 kPa and 3 to 4 degrees C, substantial growth of Pseudomonas sp. was observed (median of 6 log10 cfu/cm2 at d 21 of storage compared with 3 log10 cfu/cm2 for vacuum-packaged beef). Pseudomonas counts were lower when the container system was held at 0 to 1 degrees C, especially when combined with the pure CO2 atmosphere. As expected for CO2-enriched atmospheres, B. thermosphacta was the dominant spoilage bacterium, in the same log10 order as the TCC. Lowering the storage temperature and changing the atmosphere to pure CO2 resulted in a reduction of 1 log10 for TCC (median values after 2 wk of storage). Although pathogenic bacteria such as Campylobacter, Salmonella, and Listeria monocytogenes were not detected in any sample, further studies are necessary to evaluate potential growth risks. The results demonstrate that CO2-enriched and O2-depleted atmospheres under low pressure have a limited effect on reducing bacterial growth, probably because the antibacterial activity of CO2 is proportional to the effective concentration of this gas in the headspace. At pressures of 20 to 30 kPa, a headspace with pure CO2 would still contain only approximately 20 to 30% CO2.     

Effect of boiling water carcass immersion on aerobic bacteria counts of poultry skin and processed ground poultry meat

This study was conducted to determine the relationship between bacteria destruction on poultry carcass skin and bacteria in raw ground poultry meat from the same carcasses. Immersion time in boiling water of broiler chicken whole carcasses required for maximum reduction of naturally occurring aerobic bacterial count on skin was measured. Treatments for chicken carcasses consisted of immersion in boiling water (approximately 95 degrees C) for 0, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, and 4 min. Four skin samples taken following treatment and three taken from subsequently ground carcass meat were analyzed for total aerobic plate counts (APC). Analysis of the data indicated a linear increase in bacterial destruction on skin with increased boiling water immersion time from 0 to 4 min. Reduction of skin bacteria to less than 1 log10 occurred at 3 min carcass immersion or longer. The analysis also indicated that treatment with boiling water and removal of skin was effective in reducing bacterial counts in ground meat to similar levels at all treatment times from 0.5 to 4.0 min. Findings from this study indicated that a boiling water immersion intervention and removal of skin could reduce subsequent bacteria contamination of ground meat. This intervention could minimize the risk of pathogen-contaminated primary processed poultry carcasses used in further processing.     

Artifactual changes in PCV, hemoglobin concentration, and cell counts in bovine, caprine, and porcine blood stored at room and refrigerator temperatures

BACKGROUND: Blood samples collected from farm animals for hematology testing may not reach the laboratory or be examined immediately upon collection, and in some cases may need to be transported for hours before reaching a laboratory. OBJECTIVE: The objective of this study was to investigate the artifactual changes that may occur in PCV, hemoglobin (Hgb) concentration, and cell counts in bovine, caprine, and porcine blood samples stored at room (30 degrees C) or refrigerator (5 degrees C) temperature. METHODS: Baseline values for PCV, Hgb concentration, and RBC and WBC counts were determined immediately after blood collection from 36 cattle, 32 goats, and 48 pigs using manual techniques. Blood samples were split into 2 aliquots and stored at 30 degrees C or 5 degrees C. Hematologic analyses were carried out at specified intervals during 120 hours of storage. Results were analyzed by repeated measure ANOVA; results at different temperatures were compared by paired t-tests. RESULTS: Compared to baseline values, there were no significant changes in Hgb concentration, RBC count, or WBC count in samples from cattle; in Hgb concentration and RBC count in samples from goats; and in Hgb concentration and WBC count in samples from pigs throughout the 120 hours of storage at both 30 degrees C and 5 degrees C. Significant changes (P <.05) from baseline occurred in PCV after 14 hours of storage at 30 degrees C and after 19 hours of storage at 5 degrees C in cattle and goats; and after 10 hours of storage at 30 degrees C and 14 hours of storage at 5 degrees C in pigs. Significant changes also were observed in Hgb concentration at 96 hours at 30 degrees C and 5 degrees C, and in RBC counts at 48 hours at 30 degrees C and 96 hours at 5 degrees C in porcine samples; and in total WBC counts at 120 hours at 30 degrees C and 5 degrees C in caprine samples. Artifactual changes were more pronounced in the samples stored at 30 degrees C. CONCLUSIONS: At both 30 degrees C and 5 degrees C, blood samples from cattle and goats can be stored for up to 12 hours, while blood samples from pigs can be stored for up to 8 hours without any significant changes in PCV. Blood samples from all 3 species can be stored for more than 24 hours without significant changes in Hgb concentration, RBC count, and total WBC count.     

Comparison of host water wash and trimming of pork carcasses for reducing the level of bacterial contamination

This study was carried out to evaluate the microbial contamination on pork carcasses after they had fallen on the floor in the cooler and also to evaluate the effectiveness of trimming and hot, high-pressure water washing (55 degrees C). A bacteriological analysis was done on 2 groups of 40 carcasses before and after trimming or washing, along with a group of 10 control carcasses. Results showed that the bacterial total count was higher (P = 0.01) on carcasses after they had fallen, but, in this study, no significant difference (P = 0.76) was found for total coliform contamination. Also, no significant difference was observed between total count for aerobic bacteria, total coliforms, and Escherichia coli before and after decontamination, no matter which technique was used. Neither trimming nor washing carcasses showed, in this study, a significant difference (P = 0.37) in the reduction of the total aerobic bacterial count on the pork carcasses analyzed (P = 0.65).     

Risk factors for bulk milk somatic cell counts and total bacterial counts in smallholder dairy farms in the 10th region of Chile

We investigated the principal management factors that influenced bulk milk somatic cell count (BMSCC) and total bacterial count (TBC) of smallholder dairy farms in the 10th region of Chile. One hundred and fifty smallholder milk producers were selected randomly from 42 milk collection centres (MCCs). In April and May of 2002, all farms were visited and a detailed interview questionnaire on dairy-cow management related to milk quality was conducted. In addition, the BMSCC and TBC results from the previous 2 months' fortnightly tests were obtained from the MCCs. The mean BMSCC and TBC were used as the dependent variables in the analyses and were normalised by a natural-logarithm transformation (LN). All independent management variables were categorised into binary outcomes and present (=1) was compared with absent (=0). Biserial correlations were calculated between the LNBMSCC or LNTBC and the management factors of the smallholder farms. Management factors with correlations with P0.05) factors. A random MCC effect was included in the models to investigate the importance of clustering of herds within MCC. In the null model for mean LNTBC, the random effect of MCCs was highly significant. It was explained by: milk collected once a day or less compared with collection twice a day, not cleaning the bucket after milking mastitic cows versus cleaning the bucket and cooling milk in a vat of water versus not cooling milk or using ice or a bulk tank to cool milk. Other factors that increased the LNTBC were a waiting yard with a soil or gravel floor versus concrete, use of plastic buckets for milking instead of metal, not feeding California mastitis test (CMT)-positive milk to calves and cows of dual-purpose breed. The final model explained 35% of the variance. The model predicted that a herd that complied with all the management practices had a mean predicted TBC of 105 colony forming units (cfu)/ml, whereas a herd that did not comply with any of these management factors had a predicted TBC of 59 x 10(9)cfu/ml. The model of mean LNBMSCC explained 18% of the variance; the random effect of MCC was not significant. Management factors that decreased the mean LNBMSCC were: using the CMT for 1 year versus using the test for more than 1 year or not at all, absence of a concrete waiting yard, not filtering the milk or using filters other than a plastic sieve to filter the milk, milking cows with mastitis last, and sometimes or always examining the udder before milking. A herd that complied with all of these management factors had a BMSCC of approximately 46,166 cells/ml, whereas a herd that did not comply with any of the management practices above had a mean BMSCC of 2 x 10(6)cells/ml.     

L.fermentum F-6对肉鸡屠宰性能及肌肉品质的影响

试验旨在研究添加不同剂量的L.fermentum F-6对肉鸡的屠宰性能及肌肉品质的影响,进一步探讨其作为一种新型饲料益生菌的可行性。试验以玉米和大豆粕为主要原料配制日粮,采用单因子完全随机试验设计方法,选择1日龄雌雄各半的健康AA肉鸡240只,称重后随机分为4个组,其中1个对照组和3个L.fermenti F-6组,每个组6个重复,每个重复10只鸡,各组鸡的初始体重经方差检验差异均不显著(P>0.05)。对照组肉鸡不饲喂L.fermentiF-6,L.fermentiF-6组饮水饲喂L.fermentiF-6,添加水平分别为2×105、2×106和1×107 cfu/ml,试验期42 d。研究结果表明,添加L.fermenti F-6可显著提高肉鸡全净膛率和胸肌率(P<0.05),提高肉鸡屠宰性能,其中以添加剂量为2×106 cfu/ml时效果较好。添加L.fermenti F-6对肉鸡肌肉pH值、系水力和肉色指标无显著影响,但与对照组相比,L.fermentum F-6对肌肉系水力和肉色有一定改善效果。     

Survival of spray‐dried and free‐cells of potential probiotic Lactobacillus plantarum 564 in soft goat cheese

A high viability of probiotics in food product, with a living cells threshold of 10⁷/cfu/g (colony‐forming units/g) is a challenge to achieve in food production. Spray drying is an efficient and economic industrial method for probiotic bacterial preservation and its application in food products. In this study, the survival of free and spray‐dried cells of potential probiotic strain Lactobacillus plantarum 564 after production and during 8 weeks of storage of soft acid coagulated goat cheese was investigated, as well as compositional and sensory quality of cheese. Total bacterial count of spray‐dried Lb. plantarum 564 cells were maintained at the high level of 8.82 log/cfu/g in cheese after 8 weeks of storage, while free‐cell number decreased to 6.9 log/cfu/g. However, the chemical composition, pH values and sensory evaluation between control cheese (C1 sample made with commercial starter culture) and treated cheese samples (C2 and C3, made with the same starter, with the addition of free and spray‐dried Lb. plantarum 564 cells, respectively) did not significantly differ. High viability of potential probiotic bacteria and acceptable sensory properties indicate that spray‐dried Lb. plantarum 564 strain could be successfully used in the production of soft acid coagulated goat cheeses.     

Development of a real-time PCR for detection of Mycoplasma bovis in bovine milk and lung samples.

A real-time polymerase chain reaction (PCR) assay using hybridization probes on a LightCycler platform was developed for detection of Mycoplasma bovis from individual bovine mastitis milk and pneumonic lung tissues. The detection limit was 550 colony forming units (cfu)/ml of milk and 650 cfu/25 mg of lung tissue. A panel of bovine Mycoplasma and of other bovine-origin bacteria were tested; only M. bovis strains were positive, with a melting peak of 66.6 degrees C. Mycoplasma agalactiae PG2 was also positive and could be distinguished because it had a melting peak of 63.1 degrees C. In validation testing of clinical samples, the relative sensitivity and specificity were 100% and 99.3% for individual milks and 96.6% and 100% for the lung tissue. Using M. bovis real-time PCR, the M. bovis culture-positive milk samples were estimated to contain between 5 x 10(4) and 7.7 x 10(8) cfu/ml and the M. bovis culture-positive lungs between 1 x 10(3) and 1 x 10(9) cfu/25 mg. Isolation, confirmed with the real-time PCR and colony fluorescent antibody test, showed that at the herd level, the proportion of samples positive for M. bovis isolation in mastitis milk samples submitted to the Mastitis Laboratory, Animal Health Laboratory, University of Guelph, Ontario, Canada, was 2.4% (5/201). We conclude that this probe-based real-time PCR assay is a sensitive, specific, and rapid method to identify M. bovis infection in bovine milk and pneumonic lungs.     

Sympathoadrenal and adrenal hormonal responses of newborn calves to hypothermia.

Seventeen newborn Holstein-Friesian bull calves were cold-stressed by total body immersion in water at 15 to 17 degrees C until the core body temperature was lowered by 10 degrees C. Nine additional calves (noncold-stressed) were immersed in water at 35 to 37 degrees C. Eight of the cold-stressed calves were euthanatized soon after removal from the water while the others (n=9) were allowed to recover in a cold room at 4 degrees C for 72 hours. Noncold-stressed calves were kept at 25 degrees C for 72 hours. Sympathoadrenal an adrenal hormonal responses of calves were determined by analysis of plasma for glucose, corticosteroids, and catecholamines. Plasma concentration of glucose and corticosteroids rapidly increased in cold-stressed calves soon after immersion and remained higher (P less than of equal to 0.05) than concentrations in noncold-stressed calves during immersion and most of recovery. There was a threefold increase (P less than or equal to 0.05) in concentration of catecholamines in plasma of cold-stressed calves and only a slight increase in noncold-stressed calves during immersion. Catecholamine concentrations remained elevated in cold-stressed calves during most of recovery. Results provide direct evidence that sympathoadrenal and adrenal hormonal responses to cold are well developed in newborn calves and that changes in concentrations of glucose, corticosteroids, and catecholamines in plasma of these animals are sensitive indicators of their ability to respond to cold stress.     

Food intake of the laying hen following crop loads of maize oil and other nutrients

The metabolisable energy (ME) of the diet of laying hens at an ambient temperature (Ta) of 20 degrees C was abruptly changed from 10.9 MJ/kg to 12.9 MJ/kg, or vice versa. Food intake during the next 14 d was significantly reduced by the low ME diet and was increased by the high ME diet, that is, the expected compensatory changes in food intake did not occur. Laying hens given the same change of diet as above but kept at 32 degrees C did not show any change in food intake within 14 d. Thus ME intake increased with the high ME diet and decreased with the low ME diet. Daily doses of 10 ml maize oil/kg body weight given directly into the crop of laying hens at a Ta of 20 degrees C, resulted in an immediate, significant, reduction of food intake such that total ME intake remained the same as with normal feeding. Daily doses of 3 ml maize oil/kg, given as before, resulted in an immediate, significant, reduction in food intake at a Ta of 20 degrees C but at a Ta of 32 degrees C food intake remained unchanged; consequently daily ME intake increased. Loading the crop with glucose or sucrose, at Ta 20 degrees C, in quantities which provided a similar ME as 3 ml maize oil/kg, reduced food intake but the adjustment was less precise and daily ME intake increased. Loading with glycerol or protein hydrolysate decreased both food and ME intake. Crop loads of starch were as effective as maize oil in bringing about a significant and compensatory reduction of food intake. Similar volumes of water or liquid paraffin placed in the crop did not affect food or ME intake. A similar weight or cellulose placed in the crop reduced food and ME intake.     

Persistence of Mycoplasma bovis infection in the mammary glands of lactating cows inoculated experimentally

To study the course of clinical mycoplasma mastitis and investigate its potential for persistence, 10(8) colony-forming units (cfu) of an Irish isolate of Mycoplasma bovis was inoculated aseptically into the right fore teat canal of three lactating cows. M bovis rapidly colonised the infected quarters and grew exponentially to more than 10(10) cfu/ml within the first three days, and spread to other quarters of each of the three cows within five to 10 days. After periods of between 24 and 72 hours the infected quarters became distended and sensitive to touch, and their secretions changed from containing visible particles, to a seropurulent exudate, to an aqueous suspension of fine particles which formed a sediment after a sample was collected. M bovis-specific antibody levels increased to varying degrees in all three cows. Subsequently, the concentrations of mycoplasma decreased to less than 10(7) cfu/ml in two of the cows, but remained at more than 10(8) cfu/ml to the end of the lactation of the other cow. Apparently normal milk was secreted by one of the cows within a month of the challenge, and by the other two cows at the start of their next lactation. However, in two of the cows subclinical M bovis infection persisted through the dry periods and into their next lactations.     

Survival of Listeria monocytogenes in wilted and additive-treated grass silage

Grass was field-dried to 3 different dry matter (DM) levels (200, 430 and 540 g/kg) and inoculated with 10(6)-10(7) cfu/g of a Listeria monocytogenes strain sharing a phagovar occasionally involved in foodborne outbreaks of listeriosis. Formic acid (3 ml/kg) or lactic acid bacteria (8 x 10(5)/g) with cellulolytic enzymes were applied only to forages with low and intermediate DM levels. Forages were ensiled in laboratory silos (1700 ml) and were stored at 25 degrees C for 30 or 90 days. After 90 days of storage, L. monocytogenes could not be detected in any silo, except one with the high dry matter grass without additive. After 30 days of storage, between 10(2) and 10(6) cfu L. monocytogenes/g silage were isolated from the untreated silages. Increasing the DM content from 200 to 540 g/kg did not reduce listeria counts possibly because of the lower production of fermentation acids (higher pH). In silages treated with additives, counts of L. monocytogenes were always lower than in silages without additive. In wet silages (DM 200 g/kg) both additives were effective, but in the wilted silages (DM 430 g/kg) only the bacterial additive reduced listeria counts below detection level. Listeria counts were highly correlated to silage pH (r = 0.92), the concentration of lactic acid (r = -0.80) and the pooled amount of undissociated acids (r = -0.83).     

Characterization of a bacteriophage lysin (Ply700) from Streptococcus uberis

The antibacterial properties of bacteriophage lytic enzymes may be of importance in future mastitis control programs. A prophage was isolated from a strain of Streptococcus uberis (ATCC 700407) following exposure to mitomycin C. Partial sequencing of the phage DNA revealed a putative lysin based on sequence similarity to other streptococcal phage lysins. The putative lysin (Ply700) was recombinantly expressed in Escherichia coli, and chromatographically purified. Addition of the purified Ply700 to bacterial suspensions of S. uberis, Streptococcus pyogenes, and Streptococcus dysgalactiae caused a rapid, calcium-dependent lysis while there was little activity against Streptococcus agalactiae, Staphylococcus aureus, or E. coli. Killing of S. uberis in milk by Ply700 (50 microg/ml) was confirmed by plate count assay. Activity was related to the initial concentration of bacteria in that 31% killing (P<0.05) was observed with an inoculating dose of approximately 4500 cfu/ml, while 81% killing (P<0.01) was observed when the inoculum was reduced to approximately 600 cfu/ml. In contrast, complete sterilization was observed in parallel cultures suspended in assay buffer indicating that factors in milk are able to neutralize the lysin. Functional characterization of the C-terminal domain, as a component of a GFP fusion protein, revealed its calcium-dependent ability to bind to S. uberis. The C-terminal domain may have utility in targeting S. uberis while it remains to be determined if the lysin by itself has sufficient potency in milk for effective use in the control of S. uberis mastitis.