Fomites and reservoirs of Staphylococcus aureus causing intramammary infections as determined by phage typing: the effect of milking time hygiene practices

We studied the association of milkers' hands and milking unit liners as fomites, and teat skin as a reservoir, with S. aureus intramammary infections (IMI). Samples were collected from 40 commercial herds and S. aureus isolates were phage typed. Only 10 of 257 isolates were not typable. Of the milk samples, 8.4% had typable S. aureus; 4.5% and 9.2% of the skin and liner swabbings had typable S. aureus. Twenty-three different phage types were identified, 1 type was found in nearly 75% of the herds. Herds in which milking unit backflush was used were less likely to have the same phage type on the liners and the teat skin, and on the liners and milk samples, than herds that did not backflush. A greater percentage of liners had S. aureus of the same type as those causing S. aureus IMI, than skin swabbing solutions with the same type as that associated with IMI. Herds which did not use post-milking teat asepsis (teat dip) did not have a greater percentage of S. aureus isolates on the teat skin, nor were the S. aureus test skin isolates more likely to be of the same type as those causing intramammary infection. Results would suggest that the liner appears to be a significant fomite, that backflushing reduces its significance, and that teat skin is a less significant reservoir for S. aureus intramammary infection.     

Biofilm production by Staphylococcus aureus associated with intramammary infection

Biofilm production by 221 Staphylococcus aureus isolates from 45 dairy herds was evaluated. Isolates were from composite milk of 117 cows, from teat skin of 70 cows, and from 34 milking machine unit liners. Of S. aureus from milk samples, 41.4% were biofilm producers, as compared to 24.7 and 14.7% of the isolates collected from skin and liners. Pulsed field gel electrophoresis (PFGE) best categorized S. aureus biofilm producers as compared to phage typing and binary typing. PFGE types that were significantly associated with isolation from milk as opposed to teat skin or liners, had isolates that were more likely to produce biofilm than PFGE types that were isolated from milk, skin and liners at similar frequencies. By contrast, PFGE type A was significantly associated with isolation from teat skin and had few biofilm producers. PFGE type Q, which is exclusively a milk, isolate produced more biofilm as evidenced by absorbance values. Given S. aureus that are associated with milk are more likely to produce biofilm as compared to extramammary sources (teat skin and milking unit liners), suggests that biofilm production is a risk factor for infection.     

Apparatus for pasteurising teat cup liners between cows in a herringbone parlour.

In a herringbone milking parlour, teat cup liners were deliberately contaminated in turn with Staphylococcus aureus, Escherichia coli, Streptococcus agalactiae and Sterp uberis. Contamination was achieved by filling the liners with milk that contained 10(6) test organisms per ml. After the clusters had been back-flushed with water at 85 degrees C for five seconds, normal swabbing methods failed to recover any contaminating organisms from the teat liners in 56 tests out of 64. After 10 seconds back-flushing no recoveries were made in the same number of tests. The apparatus developed to effect this back-flushing for a particular herringbone parlour is described, with details of its routine use during milking. For a 100-cow herd, the running cost of such equipment using a five-second back-flush is estimated at no more than 4 pounds per week and, in its present form, would not add more than 10 seconds to the total milking time for each cow. Improvements in design of the apparatus, and in milking techniques arising from the routine use of the device, are also considered.     

Detection of Staphylococcus aureus in bulk tank milk using modified Baird-Parker culture media.

The purpose of this project was to evaluate the use of 2 selective/differential culture media for detecting Staphylococcus aureus in bulk tank milk. One medium was Baird-Parker agar base supplemented with egg york tellurite emulsion and acriflavine. The other medium was Baird-Parker agar base supplemented with rabbit plasma/bovine fibrinogen and acriflavine. An increased inoculum of bulk tank milk (0.3 mL) was used to enhance the detection of S. aureus in samples containing low numbers of organisms. The sensitivity and specificity for detecting S. aureus in bulk tank milk were 94.8% and 100%, respectively, using Baird-Parker agar base supplemented with egg yolk tellurite emulsion and acriflavine, and 89.7% and 100%, respectively, using Baird-Parker agar base supplemented with rabbit plasma/bovine fibrinogen and acriflavine. Both media are practical for detecting S. aureus in bulk tank milk and monitoring its spread in lactating dairy herds in Alberta.     

Isolation of Staphylococcus aureus from raw fish in relation to culture methods

Five hundred and fifty fish samples from various stages in the course of distribution in Hyogo Prefecture (209 retailed in super markets, 173 obtained from fishery cooperatives at a harbor, 91 caught by trawling and 77 caught by rod fishing) were examined for contamination with Staphylococcus aureus (S. aureus). S. aureus was detected in 41 (19.6%) of the retail fish samples and 46 (26.6%) of the samples from the fishery cooperatives. No S. aureus was isolated from the live fish (91 trawled and 77 fished by rod). With regard to the retail fish, the contamination rate of processed fish (26.0%) was significantly higher than that of unprocessed fish (14.2%). For 88 samples, the efficacy of the selective medium was compared using Baird-Parker agar and mannitol salt agar supplemented with egg yolk (MSEY agar) by the direct plate and enrichment culture methods. Using the direct culture method, the S. aureus positive rate with the Baird-Parker agar (30.7%) was significantly higher (P<0.01) than that with the MSEY agar (6.8%). The enrichment culture method remarkably raised the S. aureus detection rate. Seventy-eight (85.7%) of 91 isolates belonged to the human ecovar. Sixty-two (68.1%) of the 91 isolates had some enterotoxin genes, including 44 (48.4%) with the sea gene. These data showed that the fish were contaminated with S. aureus after landing and that Baird-Parker agar had an advantage in detecting S. aureus with a direct plate culture.     

Raised herd somatic cell count due to Staphylococcus aureus following the failure of an automatic teat spraying system

This study describes the failure of a single jet exit race automatic teat spray (ATS) system resulting in the spread of Staphylococcus aureus infection in a 135-cow dairy herd, which showed an increased herd somatic cell count from 91,000/ml to 554,000/ml over a nine-month period. S aureus was isolated from 34 of 46 high cell count cows. The milking procedures were modified and manual teat spraying was restarted. Bacteriology was used to identify S aureus positive high cell count cows, and first and second lactation cows were treated during lactation. If their cell counts were not reduced, these were then culled. High cell count S aureus cows in lactation three or above were culled. The three-month geometric mean cell count fell to below 150,000/ml within five months. As all replacements were home-bred, S aureus infection must have spread from within the herd itself. All other causes have been eliminated, and this spread is attributed to the failure of the ATS to carry out effective postmilking teat disinfection. The advantages and disadvantages of ATS systems are discussed, especially in relation to robotic or voluntary milking systems.     

Effects of postmilking teat treatment on the colonization of Staphylococcus aureus on chapped teat skin

Sixteen Holstein cows were used to test the effect of postmilking teat treatment on colonization and intramammary infection by Staphylococcus aureus on chapped teats. Treatments were (1) chapping the teat and using 1% I2/10% glycerin postdip solution, (2) 1% I2/10% glycerin postdip solution on nonchapped teats, (3) chapping the teat and using 10% glycerin postdip solution, (4) chapping the teat and not using a postdip solution. All mammary glands were free of S aureus teat skin colonization and intramammary infection at the start of the study. Teats selected for chapping were dipped in 1N NaOH prior to 3 applications of S aureus broth culture; cultures were applied at 12-hour intervals on all teats. Treatments were applied after each milking for 30 days and were initiated after the second broth dip. Teat skin swab specimens and milk samples were collected before treatment application. Teat skin condition was scored daily. Nonchapped teats (treatment 2) did not support skin or orifice colonization by S aureus. Treatment-1 teats healed most rapidly and supported less colonization in skin and orifice than did treatment-3 and -4 teats. Teat skin scores and skin colonization were lower for treatment-3 than treatment-4 teats. A correlation between teat skin colonization and teat skin conditions was found. Two intramammary infections were found in treatment-4 quarters and 1 in a treatment-3 quarter.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of preculture freezing and incubation on bacteriological isolation from subclinical mastitis samples

In this study the sensitivity of three methods of isolation of udder pathogens from milk samples from subclinical mastitis cases was compared. For analysis 1827 quarter milk samples were selected. Milk was cultured using a standard culture technique (0.01 ml of fresh milk streaked on a sheep blood agar plate and on Edward's medium). In addition, an inoculum of 0.01 ml of the original milk sample was incubated for 24h at 37 degrees C in broth, followed by culture using the standard culture technique. In the third method, the whole milk sample was frozen for 24h, and then incubated for 24h at 37 degrees C, followed by culture using standard culture technique. The isolation percentage of Staphylococcus aureus was 4.7% for standard culture technique, 14.2% for incubation in broth, and 21.5% for the combination of freezing plus incubation. Isolation percentage of Streptococcus dysgalactiae and Streptococcus agalactiae was highest using the standard culture technique, while isolation rate of Streptococcus uberis was not different among the three methods used. With increasing somatic cell count, the likelihood of S. aureus, S. dysgalactiae and S. uberis isolation increased.Based on the relative sensitivity, defined as the isolation rate using a single technique compared to the isolation rate of the three techniques together, a combination of standard culture technique and freezing plus incubation was most attractive for achieving a high isolation rate of S. agalactiae and S. dysgalactiae. Relative sensitivity of S. uberis isolation was highest using the standard culture technique and incubation in broth, while S. aureus was most often isolated using a combination of incubation in broth and freezing plus incubation. A combination of the three methods increased the isolation rate for S. dysgalactiae, S. uberis and S. aureus. The standard culture technique, together with the combination of freezing plus incubation, can be recommended for isolating major udder pathogens. If S. aureus is the pathogen of main interest, using incubation in broth together with the combination of freezing plus incubation performed best.     

Fluorescent amplified fragment length polymorphism genotyping of human and animal Staphylococcus aureus isolates from dairy farms with manual milking

Fluorescence amplified fragment length polymorphism (fAFLP) was used to assess the genetic relatedness of 40 Staphylococcus aureus strains isolated from human and animal skin samples in seven dairy farms with manual milking. S. aureus was isolated from 11 out of 30 (36%) human skin samples and from 29 out of 100 (29%) teat skin samples from apparently healthy cows. Genomic DNA from each isolate was double-digested with EcoRI and MseI and complementary oligonucleotide adaptors were ligated to the restriction fragments. Pre-selective and selective amplification reactions were performed, the amplified fragments were separated by electrophoresis in an ABI377 sequencer and analysed using GeneScan 3.1 and Genotyper 2.5. Three single isolates (a-c), a predominant cluster with 35 isolates (d) and another cluster with two isolates (e) were identified. Both clusters d and e included human and animal isolates genetically related, because the profiles had 90-100% homology. Since no cluster was comprised uniquely of human or animal isolates and given the close genetic relatedness among human and animal samples in the farms, the present findings support the hypothesis that dairy workers can spread S. aureus through manual milking.     

On the role of sawdust in the development of mastitis in cows

Sawdust was aspirated into one of the teatcups of the milking unit by induced liner slip during milking of 10 cows, which were then slaughtered. Sawdust particles were recovered from the interior of the quarters, the teat skin and the inside of the liners corresponding to both treated and untreated teats, and from the milk claw and the bucket as well. Sterile sawdust was introduced into the teat cisterns of three quarters of one cow. Clinical mastitis developed. The investigation was initiated by an outbreak of Serratia-mastitis in a dairy herd and supports the view that the outbreak was caused by contaminated sawdust used as litter in combination with improper detachment of the milking units. Widening of the impact concept is propsosed.     

Infrared thermography and ultrasonography to indirectly monitor the influence of liner type and overmilking on teat tissue recovery

Eight Danish Holstein cows were milked with a 1-mm thick specially designed soft liner on their right rear teat and a standard liner mounted under extra high tension on their left rear teat. Four of the animals were overmilked for 5 min. Rear teats were subjected to ultrasound examination on the first day and to infrared thermography on the second day. Teats were submersed in ethanol 20 min post-milking on the second day. Ultrasonography measurements showed that teat canal length increased by 30-41% during milking. Twenty minutes after milking, teats milked with modified standard liners still had elongated teat canals while teats milked with the soft liner were normalized. Overmilking tended to increase teat wall thickness. Approximately 80% of variability in teat canal length, from before teat preparation to after milking, could be explained by changes during teat preparation. Thermography indicated a general drop in teat temperature during teat preparation. Teat temperature increased during milking and continued to increase until the ethanol challenge induced a significant drop. Temperatures approached pre-challenge rather than pre-milking temperatures within 10 minutes after challenge. Teat temperatures were dependent on type of liner. Mid-teat temperatures post-challenge relative to pre-teat preparation were dependent on overmilking. Thermography and ultrasound were considered useful methods to indirectly and non invasively evaluate teat tissue integrity.     

Herd management and prevalence of mastitis in dairy herds with high and low somatic cell counts

Thirty-two dairy herds, 16 with low somatic cell counts (LSCC; Dairy Herd Improvement Association 12-month mean herd SCC less than or equal to 150,000 cells/ml) and 16 with high somatic cell counts (HSCC; Dairy Herd Improvement Association 12-month mean herd SCC greater than or equal to 700,000 cells/ml) were evaluated to determine the relationship between the prevalence of mastitis in each herd and each herd's mastitis control and management practices. Once for each herd, duplicate quarter milk samples were collected from the lactating cows, a survey of herd mastitis control, milking hygiene, and management practices of each herd was performed, and milking-machine function was evaluated. Of the 16 herds with LSCC, 2 (12.5%) had Streptococcus agalactiae isolated and 7 (44%) had Staphylococcus aureus isolated. Both organisms were found in all of the herds with HSCC. In herds with LSCC, the mean percentage of quarters infected with Str agalactiae was 0.1%, the mean percentage infected with streptococci other than Str agalactiae was 1.9%, and the mean infected with S aureus was 0.7%. In herds with HSCC, 25.7% of the quarters were infected with Str agalactiae, 3.7% were infected with streptococci other than Str agalactiae, and 7.6% were infected with S aureus. A program of postmilking teat dipping and treatment of all cows at the beginning of the nonlactating period was practiced more frequently in the herds with LSCC (81.3%) than in the herds with HSCC (37.5%). Major differences were not found between the 2 groups of herds in the use of the more common milking hygiene techniques or in the maintenance and functional characteristics of the milking equipment.     

Enrichment for isolating Salmonella Choleraesuis and other Salmonella spp. from pigs

The growth of Salmonella Choleraesuis was examined in Rappaport Vassiliadis broth (RV) and Hajna-tetrathionate broth (HTT) at 37 and 42 degrees C. As the enrichment in RV at 37 degrees C was satisfactory for isolating S. Choleraesuis, we used this enrichment for isolation from the samples collected from 15 asymptomatic pigs reared on a S. Choleraesuis contaminated farm. S. Choleraesuis was frequently isolated from six pigs (40.0%) under field conditions. The isolation of other Salmonella serovars than S. Choleraesuis was attempted by using both RV enrichment at 37 degrees C and HTT enrichment at 42 degrees C. Salmonella organisms were isolated from 156 (44.8%) of 348 fecal samples and more frequently with HTT at 42 degrees C (43.4%) than with RV at 37 degrees C (20.9%). If other serovars in addition to S. Choleraesuis are to be surveyed, HTT enrichment should be used in combination with RV enrichment.     

Controlling highly prevalent Staphylococcus aureus mastitis from the dairy farm

In 57 Holstein cows where the dairy farm uses a milking parlor system, the somatic cell count (SCC) increased persistently in the bulk milk (monthly mean 52.3 x 10(4) cells/ml; range 21 to 94 x 10(4) cells/ml). We detected S. aureus in 24 (41.2%) of the 54 lactating cows and in 29 (12.8%) of 227 quarters of the 57 milking cows in the herd. A control program was implemented in an effort to eradicate S. aureus mastitis from this dairy farm. The control plan established improved handling of the lactating cows, improved milking procedures, dry-cow therapy, and culling of infected cows. The program was monitored for 3.5 years by frequent checkups on the rate of S. aureus infection, the SCC, and the changes in milk composition. Eighteen months after the control program was started, the rate of S. aureus infection in the quarter milk decreased dramatically, and no S. aureus isolates were found in the milk of the remaining cows. The SCC in the bulk milk of the herd dropped to a monthly mean of <20 x 10(4) cells/ml. In conclusion, the control program was effective for controlling persistent S. aureus mastitis in this dairy herd.     

The association between teat end hyperkeratosis and teat canal microbial load in lactating dairy cattle

Most pathogens that cause bovine mastitis invade the udder lumen through the teat canal. Amino acids and intercellular lipids may support microbial colonisation of the teat canal epithelium by pathogenic microorganisms. The aim of this study was to investigate the association between teat end hyperkeratosis, which is induced by machine milking, and teat canal microbial load. Contralateral teats, which differed in teat end hyperkeratosis scores, were identified in a split-udder experiment. The teat canal's microbial load was evaluated using the wet and dry swab technique. Staphylococcus (S.) aureus, Streptococcus (Sc.) uberis, Escherichia (E.) coli and other coliforms were detected by agar plate cultures. The positive detection of E. coli and the log(10)-transformed E. coli load of a teat canal were significantly associated with the teat end hyperkeratosis score (P<0.05). There were significant differences with respect to positive findings for E. coli, as well as the microbial load of E. coli and Sc. uberis, between the less-calloused and the more-calloused teat of a pair. For S. aureus, no significant associations between hyperkeratosis score and teat canal microbial load were detected. In general, a teat with a highly calloused teat end had an increased teat canal microbial load compared with the contralateral teat, characterised by a lower callosity. The results of the present study indicate that the environmental pathogen load is associated with teat end hyperkeratosis. Further research is needed to identify factors that may affect teat canal microbial load in lactating dairy cattle.     

In vitro and in vivo evaluation of a 0.5% chlorhexidine gluconate teat dip

The activity of a 0.5% chlorhexidine gluconate postmilking teat germicide in reducing the numbers of Staphylococcus aureus and Streptococcus agalactiae on the skin of excised teats from cows was determined. The product yielded logarithmic reductions of 4.09 and 4.10 against S aureus and Str agalactiae, respectively, compared with 3.80 and 3.81 reductions, using a 1% iodophor dip. Germicide tolerance to an organic load containing Serratia marcescens or Pseudomonas spp was also determined. Organisms were not recovered from the product 8 hours after introduction of a simulated organic load containing either species of bacteria. The germicide was further evaluated against S aureus and Str agalactiae, using experimental challenge-exposure procedures in a research dairy herd. Efficacy was 73.4% (P less than 0.001) against S aureus and 68.1% (P less than 0.005) against Str agalactiae.     

Isolation of L-form variants after antibiotic treatment in Staphylococcus aureus bovine mastitis

An unstable L-form of Staphylococcus aureus was identified in milk samples from 3 quarters of 2 cows after treatment with cloxacillin. Milk samples incubated on standard 5% blood agar plates were culture-negative for 7 to 30 days after treatment, but S aureus was reisolated in 80% of 66 samples by additional culturing on enriched L-form media when incubated in 10% CO2 at 37 C. The organism was identified at various phases of reversion of L-form agar and was confirmed on transfer to blood agar plates.     

Effect of liner design,pulsator setting,and vacuum level on bovine teat tissue changes and milking characteristics as measured by ultrasonography

: Friesian-type dairy cows were milked with different machine settings to determine the effect of these settings on teat tissue reaction and on milking characteristics. Three teat-cup liner designs were used with varying upper barrel dimensions (wide-bore WB = 31.6 mm; narrow-bore NB = 21.0 mm; narrow-bore NB1 = 25.0 mm). These liners were tested with alternate and simultaneous pulsation patterns, pulsator ratios (60:40 and 67:33) and three system vacuum levels (40, 44 and 50 kPa). Teat tissue was measured using ultrasonography, before milking and directly after milking. The measurements recorded were teat canal length (TCL), teat diameter (TD), cistern diameter (CD) and teat wall thickness (TWT).Teat tissue changes were similar with a system vacuum level of either 50 kPa (mid-level) or 40 kPa (low-level). Widening the liner upper barrel bore dimension from 21.0 mm (P < 0.01) or 25.0 mm (P < 0.001) to 31.6 mm increased the magnitude of changes in TD and TWT after machine milking. Milk yield per cow was significantly (P < 0.05) higher and cluster-on time was reduced (P < 0.01) with the WB cluster as compared to the NB1 cluster. Minimum changes in teat tissue parameters were achieved with system vacuum level of 40 kPa and 50 kPa using NB and WB clusters, respectively. Similar changes in teat tissue and milk yield per cow were observed with alternate and simultaneous pulsation patterns. Widening pulsator ratio from 60:40 to 67:33 did not have negative effects on changes in teat tissue and had a positive effect on milk yield and milking time. Milk liner design had a bigger effect on teat tissue changes and milking characteristics than pulsation settings.     

Effects of machine-milking on the bacterial flora of teat duct and mammary gland of ewes

In total, 308 paired-samples of teat duct material and milk, were collected before and 50-70 min after machine-milking, from 30 ewes. Samples were processed bacteriologically. For analysis of results, we compared changes in bacterial isolation following milking, for duct and milk samples; statistical significance was assessed by the Sign Test. Bacteria were isolated from 18 (6%) duct and 19 (6%) milk samples collected before the milking procedure; respective figures after it, were 81 (26%) and 33 (11%). In 77 (25%) cases, bacteriological findings in the two duct samples of each pair were different; in seven cases bacteria were isolated only before, whilst in 70 cases bacteria were isolated only after milking (P < 0.005); respective results for milk samples were 26 (8%): 6 and 20 cases (P = 0.693). The majority of bacterial isolates were staphylococci, accounting for 63% of 99 isolates. The milking procedure predisposes to entrance of bacteria into the teat duct; however, increased bacterial isolation from the teat did not result to increased mammary infections, likely as a consequence of defence mechanisms present in healthy teats.     

Effects of Machine‐milking on the Bacterial Flora of Teat Duct and Mammary Gland of Ewes

In total, 308 paired‐samples of teat duct material and milk, were collected before and 50–70 min after machine‐milking, from 30 ewes. Samples were processed bacteriologically. For analysis of results, we compared changes in bacterial isolation following milking, for duct and milk samples; statistical significance was assessed by the Sign Test. Bacteria were isolated from 18 (6%) duct and 19 (6%) milk samples collected before the milking procedure; respective figures after it, were 81 (26%) and 33 (11%). In 77 (25%) cases, bacteriological findings in the two duct samples of each pair were different; in seven cases bacteria were isolated only before, whilst in 70 cases bacteria were isolated only after milking (P < 0.005); respective results for milk samples were 26 (8%): 6 and 20 cases (P = 0.693). The majority of bacterial isolates were staphylococci, accounting for 63% of 99 isolates. The milking procedure predisposes to entrance of bacteria into the teat duct; however, increased bacterial isolation from the teat did not result to increased mammary infections, likely as a consequence of defence mechanisms present in healthy teats.