Mycopathologia (2011) 171:177–182 DOI 10.1007/s11046-010-9365-7
Effect of Different Heat Treatments and Disinfectants on the Survival of Prototheca zopfii Henryka Lassa • Tomasz Jagielski Edward Malinowski
•
Received: 10 February 2010 / Accepted: 1 September 2010 / Published online: 18 September 2010 Ó Springer Science+Business Media B.V. 2010
Abstract Bovine mastitis caused by the yeast-like alga Prototheca zopfii represents a serious veterinary problem and may result in heavy economic losses to particular dairy farms. The purpose of this study was to evaluate the survival of 50 isolates of P. zopfii in milk subjected to different heat treatments and the survival of further 106 P. zopfii isolates after exposure to three classes of teat disinfectants: iodine (Dipal), quaternary ammonium compounds (Teat), and dodecylbenzenesulphonic acid (Blu-gard). Of the 50 isolates tested for thermal tolerance, 29 (58%) survived heat treatment at 62°C for 30 s and 13 (26% of all isolates) of those survived after heat treatment at 72°C for 15 s. None of the 106 isolates were able to withstand the in-use concentrations of the three disinfectants tested. The highest disinfectant concentrations that permitted survival of at least one isolate were dilutions: 1:1,000 for Dipal (survival rate of 52.8–57.5%), 1:100 for Teat (88.7–90.6%), and 1:10 for Blu-gard (100%). No differences in the survival
H. Lassa (&) E. Malinowski Department of Pathophysiology of Reproduction and Mammary Gland, National Veterinary Research Institute, Powstan´co´w Wlkp. 10, 85-090 Bydgoszcz, Poland e-mail:
[email protected] T. Jagielski Department of Applied Microbiology, Institute of Microbiology, Faculty of Biology, University of Warsaw, Warsaw, Poland
rates of P. zopfii were observed with respect to the duration of exposure to disinfectant. The results of this study support the previous findings that P. zopfii may resist high-temperature treatments, including that applied in the high-temperature, short-time (HTST) pasteurization process. The obtained data also demonstrate the efficacy of the three classes of teat disinfectants against P. zopfii, with the efficacy of iodine being most pronounced. The study emphasizes the necessity of using higher temperatures in the pasteurization of raw milk to kill the Prototheca algae, as well as the particular suitability of the iodine for the control procedures of protothecal mastitis. Keywords Bovine mastitis Disinfectants Milk Prototheca zopfii Protothecosis Thermal resistance
Introduction The genus Prototheca comprises unicellular, colorless, yeast-like algae that reproduce asexually by endosporulation [1]. These organisms, generally accepted as apochlorotic relatives of the green algae of the genus Chlorella, are saprophytic and ubiquitously distributed in the environment, with a particular affinity for wet areas with high organic matter content [2]. Two species, namely P. wickerhamii and P. zopfii, have been implicated as having pathogenic potential in both humans and animals. While P. wickerhamii is the predominant cause of
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human infections, P. zopfii is most often associated with animal disease. The latter manifests primarily as clinical and subclinical bovine mastitis. Bovine mammary protothecosis has been described in many countries across the world, including the Unites States [3, 4], New Zealand [5], the United Kingdom [6], Canada [7], Japan [8, 9] Belgium [10], Denmark [11], Germany [12], Italy [13], and Brazil [14]. In Poland, the occurrence of bovine mastitis due to P. zopfii was evidenced for the first time in the early 2000s [15]. Since then, cases of protothecal mastitis are still being reported from different parts of the country. Although protothecal mastitis appears increasingly to be endemic worldwide, its actual incidence remains uncertain. It is believed, however, to be much higher than that recorded, since P. zopfii can be misclassified as yeast or fail to grow under routine microbiological conditions [16]. Protothecal mastitis affects high-producing, machine-milked dairy cows, maintained under poor zoohygienic conditions [17]. The disease is usually associated with significantly reduced milk production and a sharp increase in somatic cell count (above 1,000,000 per mL). Among other symptoms, swelling and hardness of the mammary glands are demonstrated upon palpation, as the result of granulomatous inflammation developing within the udder tissue. The milk of infected cows is often of watery consistency and contains flakes, clots, and lumps [4, 16, 17]. Since the teat canal is the portal of entry for P. zopfii, cleaning and disinfecting the teats, milking machines, and animal housing facilities with a proper germicidal agent are among the most important preventive measures against protothecal mastitis. The presence of P. zopfii cells in milk poses a potential hazard to human health. Thermal processing of milk typically used to inactivate microorganisms associated with food poisoning or spoilage is also believed to kill the Prototheca algae. The purpose of this study was to evaluate the survival of P. zopfii in milk subjected to different heat treatments, as well as the survival of these algae after exposure to selected teat disinfectants.
Materials and Methods The experimental material comprised of 156 isolates of P. zopfii, collected over a 2-year period
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(2000–2001) in the laboratory of Bydgoszcz Division of the National Veterinary Research Institute. The isolates were recovered from cows with clinical and subclinical mastitis from dairy herds located in five provinces in Poland: Kuyavian-Pomeranian, Lublin, Lubusz, Opole, and Greater Poland. They were revived by subculturing onto Sabouraud-dextrose agar (SDA; Difco) at 37°C for 48 h. Species identification was based on morphological features examined macro- and microscopically (Gram stain or methylene blue stain) and by distinctive carbohydrate assimilation profiles with the API 20C AUX system (BioMe´rieux). All the isolates investigated could be assigned to biotype 2, showing assimilation of glycerol and glucose but not galactose [18, 19]. The sensitivity to heat treatment was determined for a set of 50 of P. zopfii. For this experiment, a cell suspension equivalent to a 2.0 McFarland density standard was made in a sterile saline solution. A test sample was prepared by transferring 0.5 mL of the cell suspension into a sterile glass tube containing 3 mL of UHT milk (0.5% fat content), submerged in a water bath of 62, 72, or 80°C. The milk temperature was measured in a separate control tube. The samples were kept at increasing temperatures for decreasing periods of time (30, 15, or 10 s, respectively), then immediately cooled in ice water, and cultured on SDA plates (0.5 mL onto each). After a 48-h incubation at 37°C, the number of colony forming units (c. f. u.) was counted. Prototheca-free and Prototheca-containing milk not subjected to heat treatment were inoculated onto separate agar plates, in order to provide a negative and positive control, respectively. Each isolate was tested in triplicate in each experiment (i. e. under three different temperature/ time regimes). Thus, a total of 150 tests were performed under each of the regimes. An isolate was defined as resistant to a particular temperature/time ratio if it showed growth of C 2 c. f. u./1 mL of milk on SDA in at least two repetitions. One hundred and six isolates of P. zopfii, independent of those used for thermal sensitivity analysis, were examined for the in vitro efficacy of three commonly used teat disinfectants, namely Dipal (DeLavalÒ; www.delaval.pl), Teat (IodexÒ), and Blu-gard (Henkel-EcolabÒ; www.ecolab.com.pl), containing as active ingredients 1% available iodine, 3% didecyldimethylammonium chloride (DDAC), a quaternary ammonium compound (QAC), and 2%
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dodecylbenzenesulphonic acid (DBSA), respectively. The working solutions of the disinfectants were prepared according to the manufacturers’ label directions. Apart from the working (in-use) concentrations recommended by the manufacturer, three additional concentrations made by diluting the stock solution (i. e. undiluted disinfectant) 10-, 100-, and 1,000-fold in distilled water, were tested for their effect on P. zopfii viability (Table 1). For preparation of the challenge suspension, each P. zopfii isolate was adjusted to a 2.0 McFarland standard, and then diluted with water to obtain a concentration of 1.0–5.0 9 103 c. f. u./mL. So prepared algal suspensions were transferred into appropriate disinfectant solutions (in volumes equivalent to those of the algal suspensions) and after incubation at room temperature for 5 min, 6, and 12 h were plated in 0.5 mL amounts on SDA. Plates were incubated at 37°C for 48 h, and the number of colonies (c. f. u./1 mL of the original culture suspension) was counted. The cell suspensions that had been treated with distilled water, instead of disinfectant, in the amount equivalent to that of the disinfectant, served as a placebo control. Each isolate was tested in duplicate in each experiment (i. e. with four different disinfectant concentrations and three different times of exposure). This resulted in a total of 2,544 tests for each of the disinfectants.
An isolate was defined as tolerant to a particular disinfectant solution if it showed growth of C 2 c. f. u./mL on SDA in both repetitions.
Results The results of the heat treatment tests are shown in Fig. 1. Of the 50 isolates analyzed, 29 (58%) were resistant to 62°C, with a holding time of 30 s. The algae showed growth of 2–32 c. f. u./1 mL of milk. Holding the Prototheca at a temperature of 72°C for 15 s reduced substantially the survival rate of the algae. Only 13 (26%) of the P. zopfii isolates
Fig. 1 Resistance of P. zopfii isolates in milk samples subjected to different temperature/time ratios (n = 50)
Table 1 Effect of three commercially available teat disinfectants on the growth of P. zopfii isolates (n = 106) Disinfectant (Active ingredient)
Dipal (iodine)
Dilution rate (Percent concentration of active ingredient) 1:4 (0.25)
*
1:10 (0.1) 1:100 (0.01) 1:1,000 (0.001) Teat (didecyldimethylammonium chloride, DDAC)
*
5 min.
6h
12 h
0 (0)
0 (0)
0 (0)
0 (0)
0 (0)
0 (0)
0 (0) 57 (53.8)
0 (0) 56 (52.8)
1:2 (1.5)*
0 (0)
0 (0)
1:10 (0.3)
0 (0)
0 (0)
1:100 (0.03) Blu-gard (dodecylbenzenesulphonic acid, DBSA)
Number (%) of isolates showing growth on SDA after exposure to disinfectant solution for
0 (0) 61 (57.5) 0 (0) 0 (0)
94 (88.7)
96 (90.6)
96 (90.6)
1:1,000 (0.003) Not diluted (2)*
106 (100) 0 (0)
106 (100) 0 (0)
106 (100) 0 (0)
1:10 (0.2)
106 (100)
106 (100)
106 (100)
1:100 (0.02)
106 (100)
106 (100)
106 (100)
1:1,000 (0.002)
106 (100)
106 (100)
106 (100)
Working concentration as recommended by the producer
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demonstrated resistance to the 72°C/15 s ratio, giving a concentration of 5–29 c. f. u. per 1 mL of milk. All the isolates that were resistant to the 72°C/15 s ratio were also resistant to the 62°C/30 s regime. Neither of the isolates were able to grow on SDA plates, after being held at a temperature of 80°C for 10 s. A colony count of 3–5 9 105 c. f. u./1 mL was obtained for the positive control, whereas no c. f. u. was observed for the negative control. The efficacies of the three disinfectants tested against 106 P. zopfii isolates are summarized in Table 1. All disinfectants showed a sterilizing effect with a complete kill of all organisms (0.0 c. f. u./mL) on all isolates, irrespective of the time of exposure, when applied at in-use concentrations. However, with decreasing concentrations, the three disinfectants differed in their algicidal activity. Whereas Dipal exhibited its sterilizing effect on P. zopfii even when diluted 1:100 from the stock solution, Teat could do that only when diluted 1:10 from the stock solution. Blu-gard was effective only at in-use concentrations (not diluted). The highest disinfectant concentrations that permitted survival of at least one isolate were dilutions of the stock solutions: 1:1,000 for Dipal (survival rate of 52.8–57.5%), 1:100 for Teat (survival rate of 88.7–90.6%), and 1:10 for Blu-gard (survival rate of 100%). No differences in the survival rates of P. zopfii were observed with respect to the duration of exposure to disinfectant.
Discussion The isolation of P. zopfii from milk of lactating cows is one of the primary criteria indicative of mastitis caused by this pathogen. The Prototheca-containing milk constitutes an important source of environmental contamination and transmission to susceptible cattle population [20]. Given the propensity of P. zopfii to induce persistent infections, with intermittent shedding, and its unresponsiveness to most treatment regimens [9], culling is the only effective option for combating the disease [16, 21]. Although an association between the protothecal mastitis and human infections has not yet been proven, it cannot be ruled out. This is because the consumption of Prototheca-contaminated milk and dairy products provides a potential opportunity for infection to develop. However, only very few cases of
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human protothecosis due to P. zopfii have been reported so far, and none of those cases involved the oral infection route [22–24]. Furthermore, although P. zopfii has been repeatedly found in human feces, the etiological relationship of this organism to any gastrointestinal disorders, as originally suggested [25], was questioned by subsequent investigators [2, 26]. The Prototheca sp. were concluded to be part of the transient flora of human and animal alimentary tracts. Nevertheless, in a quite recent Brazilian study, Costa et al. argued for a link between human enteritis and the consumption of P. zopfii-contaminated cheese [27]. Hence, a question whether Prototheca sp. as transient colonizers of the gastrointestinal tract may, under certain conditions, exert pathogenic effects on humans is still open. In this study, an attempt was made to evaluate the susceptibility of P. zopfii mastitis isolates to different temperature/time combinations applied during the thermal processing of milk. The most important finding was that close to one-third (26%) of the P. zopfii isolates tested survived in milk after 15 s of heating at 72°C, a temperature/time combination conventionally employed in the high-temperature, short-time (HTST) pasteurization method. The results from this study corroborated those obtained previously by Melville et al. [27] and Lagneau and Jagielski [28]. The former authors demonstrated that 62.5% of P. zopfii isolates from milk of cows with mastitis and bulk-tank milk samples survived heat treatment at 72–75°C for 15 s and that 45% survived after heat treatment at 72–75°C for 20 s [27]. In the second study, Lagneau and Jagielski reported the survival rates of P. zopfii mastitis isolates to be 80.8% after exposure to 75°C for 20 s and 40% after exposure to 80°C for 10 s [28]. Interestingly, prior to the studies cited above, Costa et al. observed survival of P. zopfii in milk after heating at 98–100°C for 3 min [29]. The observed resistance of P. zopfii to high temperatures is probably related to the presence in the cell walls of these algae of sporopollenin, a complex polymer, which is extremely resistant to enzymatic and chemical degradation [30]. Likewise, the resistance of P. zopfii isolates to several antimicrobial drugs [13, 15, 31] has been ascribed to their sporopollenin-containing cell walls. However, the mechanisms responsible for displaying variable levels of resistance to high temperatures or antimicrobial agents have yet to be elucidated.
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At present, there are a large number of teat disinfectants available for the dairy industry. It is to the decision of dairy producers to select the best product that, dependently on their particular herd situation, would display high germicidal activity and relatively low toxicity to the animal tissue. The principle active ingredients used in the teat disinfectants include iodine, acid anionic compounds, quaternary ammonium compounds (QACs), chlorhexidine, and others. The bactericidal and fungicidal activities of the majority of these agents have been well documented in the literature [32–34]. However, to the best of our knowledge, no such data exist for the mastitis-causing algae of the genus Prototheca. This study is thus the first to provide the results of the in vitro susceptibility of P. zopfii to different classes of the routinely used teat dips. The only study that dealt specifically with disinfectants and their effect on P. zopfii cells was that by Melville et al. [35]. These authors observed important changes in the ultrastructure of P. zofii, including cell wall thickening, decomposition of the cell wall’s inner layer or degradation of the intra-cellular organelles after exposure of the algae to silver nitrate, copper sulphate, and chlorhexidine, respectively. In this study, the algicidal activities of the representatives of the three teat dip classes were evaluated on a set of 106 P. zopfii mastitis isolates. All of the teat disinfectants tested were efficient against all P. zopfii isolates. An important observation was that iodine displayed the highest algicidal activity, causing the sterilizing effect even at 100-fold dilutions from the stock solutions, followed by the QASs and the DBSA, with the latter being efficacious only in undiluted formulation. Such a high susceptibility of P. zopfii to disinfectants may be somewhat surprising, since these algae were shown to resist standard chlorination and other sewage treatment regimens [2]. A possible explanation for these contrasting findings could be that the resistance of Prototheca organisms to any adverse chemical conditions is much lower under laboratory conditions than in the field. Nevertheless, the results from this study fully justify the use of the three groups of teat disinfectants analyzed as a preventive measure for bovine mastitis of P. zopfii etiology. Furthermore, since teat dips might cause skin irritation, allergy, and toxicity in susceptible people, it would be beneficial to their health if the
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disinfectants were used at lower concentrations. The results of this study clearly indicate the possibility to employ iodine and to a lesser degree also QASs at lower concentrations than conventionally used, as far as their algicidal activities are concerned. However, to preserve sufficiently high bactericidal and fungicidal activities of the disinfectants, their use at lower dilutions may not be a good option. In favor of the latter view is experimental evidence that some common staphylococcal mastitis species do have the capacity to develop resistance to QACs [36, 37]. The microbial resistance to disinfectants may be more likely to develop if they are used at concentrations lower than those required for optimal biocidal activity. An interesting finding of the study was the lack of difference in the activity of the disinfectants dependent on time of exposure. One possible reason for this may be that the killing activity of the disinfectants is determined by the effect of concentration (concentration-dependence) rather than by the length of time of exposure (time-dependence). Hence, only the same pool of resistant isolates will exhibit growth after being treated with a given disinfectant at certain concentration, irrespectively of time of exposure. To conclude, this study supported earlier findings that P. zopfii may survive high-temperature treatments, including that applied in the HTST pasteurization procedure. Hence, the necessity of using higher temperatures in the pasteurization of raw milk should be considered. In order to better recognize the level and durability of thermal resistance in P. zopfii, further studies are required. The results from this study also proved the efficacy of the representatives of the three commonly used teat disinfectant classes (iodine, QACs, and DBSA) against P. zopfii. The iodine performed best against the algae, with a sterilizing effect at up to 100-fold dilutions from the stock solutions. This underlines the particular suitability of the iodine for the control procedures of bovine mastitis caused by P. zopfii.
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