Sources of Pseudomonas spp. in Bulk Tank Milk on Colorado Dairy Farms

R. Page Dinsmore, Scott Meyer, Frank Garry and Heather Hirst
Colorado State University
Fort Collins, Colorado, USA

Introduction

Bacteria in the family Pseudomonadaceae are among the most important spoilage bacteria originating in refrigerated raw milk (Jay 2000, Vela 1997).  They are considered psychrotrophs, growing well at common refrigeration temperatures (0-15C) (Jay 2000).  Pseudomonads such as Pseudomonas flourescens produce heat stable lipases and proteases (Frank 1997), which are responsible for milk defects such as bitterness, rancidity, fruity and cardboardy flavor, casein breakdown, and ropiness due to production of slime and coagulation of proteins (Jay 2000, Frank 1997, Ray 1996).

Pseudomonads, particularly Pseudomonas aeruginosa, can also cause outbreaks of intramammary infections.  Clinical mastitis is often severe with gangrene and death loss (Packer 1977, Malmo et al 1972, Erskine et al 1987).  Subclinical and recurrent mild clinical mastitis can also be seen (Kirk et al 1984, Erskine et al 1987).  Intramammary infections due to P. aeruginosa are generally quite refractory to treatment.  Kirk et al (1984) reported some success using spectinomycin, but found reinfection to be common.  Control of outbreaks of P. aeruginosa mastitis generally requires culling of chronically infected cows and identification and elimination of the source of the organisms.

Water and soil are the primary sources of Pseudomonas sp. (Erskine et al 1987, Frank 1977, Jay 2000).  Hose nozzles and milking equipment can become colonized by pseudomonads.  Under stressful conditions, such as the presence of low levels of iodine-based disinfectants, these organisms produce a slimy glycocalyx (Ombaka et al 1983).   This slime favors adherence to and colonization of pipe and hose surfaces, and offers increased resistance to surfactants, phagocytes, and certain antibiotics (reviewed by Erskine et al 1987).  Pseudomonas sp. contamination of hoses and water can be extremely difficult to eliminate (Erskine et al 1987), but Kirk et al (1984) and Erskine et al (1987) demonstrated that the discovery and elimination of Pseudomonas aeruginosa. contamination of udders and/or milking equipment were essential in the control of outbreaks of P. aeruginosa mastitis outbreaks.

For several years the Colorado State University Veterinary Diagnostic Laboratory (CSUVDL) has been performing bulk tank cultures for dairy producers in Colorado.  The primary purpose of this program is to identify contagious mastitis pathogens such as Streptococcus agalactiae and Mycoplasma bovis, but environmental bacteria are also identified and enumerated.  Some herds in the program have repeatedly isolated high levels of Pseudomonas in bulk tank samples, and in several instances high psychrophilic bacteria levels have been reported in the same herds by milk handler labs.  Limited herd investigations have revealed the occurrence of Pseudomonas in water on the affected dairies, as well as patterns of water use likely to contaminate milking equipment and teats.  The objective of this project was to survey farms with and without bulk tank Pseudomonas for the occurrence of Pseudomonas in water and on hose and milking equipment surfaces, and to evaluate the association between the presence of the organism in water and in bulk tank milk.

Materials and Methods

Thirty-nine dairy farms in central Colorado were enrolled in the project.  All dairies submitted monthly bulk tank milk samples to the CSUVDL, and were selected based on their willingness to participate.  Some dairies had a previous history of Pseudomonas spp. in the bulk tank culture while some did not.  A single milking-time visit was made to each dairy, at which time a questionnaire was administered and samples were collected.  Questions were asked regarding overall herd signalment and milk quality.  Milking procedures were observed, with particular attention paid to the use of water during milking.  Water samples were collected from representative drop hoses, other parlor hoses, and tank room sink water.  Milk samples were collected at the end of milking from the bulk tank.  Nozzles of all available hoses were swabbed with sterile Dacron swabs.

Milk samples and swabs were cultured at the CSUVDL.  Duplicate blood agar and MacConkey plates were streaked with swabs and with 100 ml milk; one set was incubated at room temperature, one at 37C.  All plates were read at 24 and 48 hours.  Water samples were cultured at the Colorado State Water Quality Lab.

Herds were subdivided into those with and those without Pseudomonas spp. in bulk tank milk samples collected on the day of the herd visit.  All water and swab samples were classified as positive or negative for Pseudomonas spp.  Chi-square or Fisher’s exact tests were used to compare herds with and without Pseudomonas spp. for proportion of water and swab samples positive for Pseudomonas spp as well as water usage.  

Results and Discussion

Of the 39 herds enrolled in the trial, 15 had no detectable Pseudomonas in bulk tank milk. All herds, regardless of the presence or absence of Pseudomonas spp. in the bulk tank, had positive Pseudomonas cultures of water, swabs, or both.  Table 1 gives the proportion of water and swab samples positive for Pseudomonas in the two groups of herds.  Table 2 shows for the two herd groups the proportion using water in the parlor such a way that milk or teats could be contaminated.  One would expect higher Pseudomonas contamination in herds with a combination of excessive water use and positive cultures for Pseudomonas in the same water; these proportions appear in Table 3. There were no significant differences in any comparisons between herds positive and negative for Pseudomonas.

Previous work has shown that outbreaks of mastitis due to Pseudomonas aeruginosa were strongly associated with the use of water contaminated with this organism (Erskine et al 1987, Kirk et al 1984).  Eliminating this source of P. aeruginosa arrested the spread of new infections.  Our results do not identify a clear association between a water-associated source of Pseudomonas spp. and the occurrence of Pseudomonas spp. in bulk milk; rather, we have confirmed the ubiquitous nature of these bacteria in the dairy environment.  It may be that we have neglected to identify all potential sources or modes of contamination of bulk milk.  One must also consider the possibility that species differences may exist:  while Pseudomonas aeruginosa is associated with mastitis and may be less common, other pseudomonads (e.g. P. flourescens) are ubiquitous and other unidentified sources may be more important.

The finding that Pseudomonas spp. may frequently be found in herds without Pseudomonas in bulk tank milk has interesting implications to consider when troubleshooting herds with high levels of Pseudomonas in bulk milk.  Finding the organism in a water source does not guarantee the solution of the problem is in hand.  One must proceed in a careful, step-wise fashion while eliminating all possible sources of Pseudomonas, and even so a solution may never be found.

Table 2.  Herds using water in such a way that milk or teats could be contaminated.

Procedure	Positive Herds*	Negative Herds*
Chase milk at end of milking with water	8/24 (33%)	3/15 (20%)
Water used to wash teats	5/24 (21%)	2/15 (13%)
Water used to wash units/udders during milking	6/24 (25%)	6/15 (40%)
Water used on floors during milking	14/24 (58%)	12/15 (80%)
Water used to rinse units	13/24 (54%)	13/15 (87%)

*Herds positive or negative for Pseudomonas in bulk tank milk.

Table 3.  Herds using Pseudomonas-containing water in such a way that milk or teats could be contaminated.

Procedure	Positive Herds*	Negative Herds*
Chase milk at end of milking with water	6/24 (25%)	2/15 (13%)
Water used to wash teats	4/24 (17%)	2/15 (13%)
Water used to wash units/udders during milking	5/24 (21%)	6/15 (40%)
Water used to rinse units	8/24 (33%)	7/15 (47%)

*Herds positive or negative for Pseudomonas in bulk tank milk.

References

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Jay, J.M. 2000. Taxonomy, role, and significance of microorganisms in food. In Modern Food Microbiology, Aspen Publishers, Gaithersburg MD, p. 13.

Kirk, J.H., P.C. Bartlett.  1984.  Nonclinical Pseudomonas aeruginosa mastitis in a dairy herd.  J. Am. Vet. Med. Assoc. 184:671.

Malmo, J., B. Robinson, R.S. Morris.  1972.  An outbreak of mastitis due to Pseudomonas aeruginosa in a dairy herd.  Aust. Vet. J. 48:137.

Ombaka, E.A., R.M. Cozens, M.R.W. Brown.  1983.  Influence of nutrient limitation of growth on stability and production of virulence.  Factors of mucoid and nonmucoid strains of Pseudomonas aeruginosa.  Rev. Infec. Dis. 5:880.

Packer, R.A. 1977.  Bovine mastitis caused by Pseudomonas aeruginosa.  J. Am. Vet. Med. Assoc. 170:1166.

Ray, B.  1996.  Spoilage of Specific food groups.  In Fundamental Food Microbiology, CRC Press, Boca Raton FL, p. 220.

Vela, G.R.  1997.  Microbiology of milk.  In Applied Food Microbiology, Star Publishing, Belmont, CA,  p. 325.