J. Dairy Sci. 88:3079-3083
© American Dairy Science Association, 2005.
Short Communication: Pasteurization of Milk Abolishes Bovine Herpesvirus 4 Infectivity
C. Bona1,
B. Dewals1,
L. Wiggers1,
K. Coudijzer2,
A. Vanderplasschen1 and
L. Gillet1
1 Immunology-Vaccinology (B43b), Faculty of Veterinary Medicine, University of Liège, B-4000 Liège, Belgium
2 Department of Animal Product Quality and Transformation Technology, Brusselsesteenweg 370, B-9090 Melle, Belgium
Corresponding author: Alain Vanderplasschen; e-mail: A.vdplasschen{at}ulg.ac.be.
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ABSTRACT
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Bovine herpesvirus 4 (BoHV-4) is a gammaherpesvirus highly prevalent in the cattle population that has been isolated from the milk and the serum of healthy infected cows. Several studies reported the sensitivity and the permissiveness of some human cells to BoHV-4 infection. Moreover, our recent study demonstrated that some human cells sensitive but not permissive to BoHV-4 support a persistent infection protecting them from tumor necrosis factor-
–induced apoptosis. Together, these observations suggested that BoHV-4 could represent a danger for public health. To evaluate the risk of human infection by BoHV-4 through milk or serum derivatives, we investigated the resistance of BoHV-4 to the mildest thermal treatments usually applied to these products. The results demonstrated that milk pasteurization and thermal decomplementation of serum abolish BoHV-4 infectivity by inactivation of its property to enter permissive cells. Consequently, our results demonstrate that these treatments drastically reduce the risk of human infection by BoHV-4 through treated milk or serum derivatives.
Key Words: bovine herpesvirus 4 • milk • serum • pasteurization
Abbreviation key: BoHV-4 = bovine herpesvirus 4, EGFP = enhanced green fluorescent protein, MDBK = Madin-Darby bovine kidney, pfu = plaque-forming unit.
Bovine herpesvirus 4 (BoHV-4) belongs to the herpesviridae family, gammaherpesvirinae subfamily, rhadinovirus genus (Zimmermann et al., 2001). Bovine herpesvirus 4 has been isolated throughout the world from healthy cattle as well as from those exhibiting a variety of diseases (Thiry et al., 1997). In contrast to most gammaherpesviruses, BoHV-4 is able to replicate in a broad range of host species both in vitro and in vivo (Thiry et al., 1997). Several in vitro studies demonstrated that some human cell lines are sensitive (i.e., support viral entry) or permissive (i.e., support viral replication) to BoHV-4 (Egyed, 1998; Donofrio et al., 2002; Gillet et al., 2004). These observations led to the hypothesis that BoHV-4 could represent a danger for human health (Egyed, 1998; Gillet et al., 2004).
Our recent study suggested that BoHV-4 could be harmful for humans either by replicating in permissive cells or by protecting nonpermissive, persistently infected cells from apoptosis (Gillet et al., 2004). In vivo, the latter phenomenon could allow the infected cells to accumulate mutations leading eventually to transformation (Gillet et al., 2004). Importantly, the latter property has been shown to play an important role in the oncogenesis induced by several gammaherpesviruses (Hunt et al., 1972; Fickenscher and Fleckenstein, 2001).
The risk of viral cross-species transmission relies on several factors such as the prevalence of the virus in the environment, the existence of events permitting the transmission of the virus, and the capacity of the virus to infect the non-natural host. In relation to these factors, several observations support the existence of a risk of BoHV-4 transmission to humans. Firstly, BoHV-4 is highly prevalent in the cattle population (Thiry et al., 1997) and no eradication scheme is directed against this virus. Secondly, many factors enable the exposure of the virus from cattle to humans: 1) infected animals excrete BoHV-4 in nasal and vaginal discharges both after primary infection and after reactivation (Thiry et al., 1997), making human contamination possible for people having contact with infected cattle; 2) BoHV-4 is frequently isolated from bovine serum (Bublot et al., 1991), which is abundantly used in food and pharmacological preparations (Asher, 1999; Brown et al., 1999; Studer et al., 2002), making human contamination by BoHV-4 possible by enteral or parenteral routes; and 3) BoHV-4 has been found in the milk of cows with mastitis as well as from apparently healthy cows, suggesting possible human contamination by oral route through milk ingestion (Donofrio et al., 2000; Wellenberg et al., 2000, 2001, 2002; Wellenberg, 2002).
In the present study, as bovine milk and bovine serum represent a possible source of human contamination by BoHV-4, we investigated the resistance of this virus to the mildest thermal treatments usually applied to these products. The data obtained demonstrated that milk pasteurization and thermal decomplementation of serum drastically reduce BoHV-4 infectivity.
Milk pasteurization was introduced in the late 19th century as a public health measure to destroy human bacterial pathogens such as Mycobacterium tuberculosis and Coxiella burnetii that are likely to be present in raw milk (Jay, 1992). Surprisingly, the ability of pasteurization to inactivate viruses has been poorly studied (Tomasula and Konstance, 2004); no study has addressed the effect of pasteurization on BoHV-4 infectivity or on that of the other bovine herpesviruses. Here, we addressed the ability of pasteurization to inactive BoHV-4 infectivity. A laboratory model for milk pasteurization was first developed using a PCR apparatus (T3 thermocycler, Biometra, Germany). After testing various conditions, the following thermal cycle was selected to mimic high temperature, short time (HTST) pasteurization protocols used in industry (Tomasula and Konstance, 2004). Milk samples were incubated at 4°C before and after thermal treatment. The temperature was increased at the rate of 2°C/s to reach a holding temperature of 72 or 75°C maintained for various times, then reduced to 4°C at the rate of 2°C/s. Pasteurization protocols were assayed by spectrophotometric analysis of raw milk alkaline phosphatase activity as described in the International Standard ISO/TS 6090–FIL/RM 82:2004 of the International Dairy Federation (International Dairy Federation, 2004). This standard requires that pasteurization protocols lead to complete inactivation of milk alkaline phosphatase activity. The results presented in Figure 1
revealed that, using our laboratory model, incubation of raw milk at 72°C for 15 s (panel A) or at 75°C for 6 s (panel B) reduced milk alkaline phosphatase activity by 89 and 96%, respectively. These data indicate that even if the laboratory model developed does not mimic perfectly the rate at which the temperature increases and decreases in the industry, it is valid for testing the effect of pasteurization on BoHV-4 infectivity.
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Figure 1. Effect of milk pasteurization on BoHV-4 infectivity. Samples of raw milk were incubated at 4°C before and after thermal treatment at 72 (A, B, and C) or 75°C (D, E, and F). The holding temperature was maintained for various periods of time, then reduced to 4°C at the rate of 2°C/s. Panels A and D: Milk alkaline phosphatase activity was assessed by measuring fluorogenic conversion of 4-nitrophenyl phosphate disodium added to raw milk (final concentration of 3.4 mM). Panels B, C, E, and F: Before thermal treatment, raw milk was contaminated with semipurified BoHV-4 EGFP XhoI [final concentration of 3 x 106 plaque-forming units (pfu)/mL] (cell-free virus) or with Madin-Darby bovine kidney (MDBK) cells (final concentration of 106 cells/mL) infected 48 h earlier with the BoHV-4 EGFP XhoI strain at a multiplicity of infection of 1 pfu/cell (cell-associated virus). After thermal treatments, the properties of BoHV-4 to replicate in permissive cells (panels B and E) or to enter cells as revealed by the expression of enhanced green fluorescent protein (EGFP) (panels C and F) were investigated on MDBK cells by plaque assay and by flow cytometry analysis of infected cells 24 h postinfection, respectively. The percentage of EGFP-expressing cells was determined using cells infected by untreated virus as control (CT). Representative flow cytometry EGFP/forward scatter dot plots are presented.
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The BoHV-4 EGFP XhoI recombinant strain was used throughout this study (Gillet et al., 2004). This recombinant strain carries an enhanced green fluorescent protein (EGFP) expression cassette under control of the human cytomegalovirus immediate-early promoter/enhancer. The BoHV-4 EGFP XhoI recombinant strain leads to EGFP expression in cells that are permissive or only sensitive to BoHV-4 (Gillet et al., 2004). Because BoHV-4 has been detected in noncellular and cellular fractions of milk (Donofrio et al., 2000), the effect of pasteurization on BoHV-4 infectivity was assayed on cell-free and cell-associated virions. Both were tested by addition to raw milk of semipurified BoHV-4 virions or BoHV-4–infected Madin-Darby bovine kidney (MDBK, ATCC CCL-22) cells, respectively (Figure 1
, panels B, C, E, and F). Both viral preparations were then submitted to the thermal cycles described above by varying the temperature and time of holding. The remaining infectivity was then determined by plaque assay on MDBK cells as described elsewhere (Vanderplasschen et al., 1995) and compared with the infectivity of identical samples maintained at 4°C (controls). Interestingly, both holding temperatures tested led to rapid inactivation of BoHV-4. Holding time as short as 1 s at 72 or 75°C was sufficient to completely inactivate the infectivity of 3.0 x 106 plaque-forming units (pfu)/mL. To determine if the latter effect relied on the inhibition of entry or a postentry event of BoHV-4 replication cycle, EGFP expression was analyzed by flow cytometry 24 h after infection with thermally treated or untreated BoHV-4 EGFP XhoI recombinant (Figure 1, panels C and F). As described for the previous experiment, both cell-free and cell-associated virions were tested. The results presented in Figure 1 (panels C and F) demonstrated that inactivation of BoHV-4 by the thermal treatments described above is due to inhibition of viral entry. Indeed, although cells infected with untreated virus led to a large proportion of EGFP-expressing cells (90 and 45% for cell-free and cell-associated virions, respectively), detection of positive cells after infection with thermally treated virus was below the background level. The results presented above suggest that industrial pasteurization of milk should lead to complete inactivation of BoHV-4 infectivity by suppression of its property to enter cells. To test this hypothesis, semipurified BoHV-4 EGFP XhoI recombinant was added to 50 L of raw milk (free of BoHV-4 as demonstrated by PCR analysis and viral isolation assay) to reach a final concentration of 104 pfu/mL. The milk was then pasteurized in a semi-industrial apparatus with a holding temperature of 72°C maintained for 15 s. Analysis of pasteurized milk revealed that the thermal treatment completely inactivated BoHV-4 infectivity; no particle able to replicate or transduce EGFP expression was detected in 100 mL of milk. This result demonstrated that industrial pasteurization of milk is sufficient to abolish BoHV-4 infectivity.
Bovine serum used in pharmacological preparations represents another possible source of human contamination by BoHV-4. In the second part of this study, we investigated the resistance of BoHV-4 to the mildest thermal treatment usually applied to serum, which is thermal inactivation of complement by incubation at 56°C for 30 min. Incubation of BoHV-4 at 56°C gradually reduced its infectivity (Figure 2, panel A). After an incubation period of 30 min, the infectivity of BoHV-4 was reduced by a factor of 104. Analysis of EGFP expression confirmed that thermal inactivation of BoHV-4 relies on the suppression of its property to enter cells (Figure 2, panel B).
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Figure 2. Effect of thermal decomplementation of serum on BoHV-4 infectivity. Fetal calf serum free of BoHV-4 was contaminated with 2.4 x 106 plaque-forming units (pfu) of BoHV-4 EGFP XhoI. The sample was then incubated at 56°C. At the indicated incubation times, viral titer (pfu/mL) was determined by plaque assay on Madin-Darby bovine kidney (MDBK) cells (panel A), and the ability of particles to transduce enhanced green fluorescent protein (EGFP) expression in MDBK cells was estimated by flow cytometry analysis of infected cells 24 h postinfection (panel B). The percentage of EGFP-expressing cells was determined using cells infected by untreated virus as control (CT). Representative flow cytometry EGFP/forward scatter dot plots are presented.
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The titer of BoHV-4 in milk and serum of infected animals is never higher than a few infectious particles per milliliter (Bublot et al., 1991; Donofrio et al., 2000). The results of the present study demonstrate that milk pasteurization and thermal decomplementation of serum reduced BoHV-4 infectivity by a factor of at least 106 and 104, respectively. These thermal treatments not only drastically reduce the ability of BoHV-4 to replicate in permissive cells but also suppress the ability of BoHV-4 to enter cells. Consequently, these results demonstrate that pasteurization of milk and thermal decomplementation of serum are sufficient to reduce the risk of human contamination through these products. The demonstration that thermal inactivation of BoHV-4 relies on the suppression of its ability to enter cells excludes all possible deleterious effects resulting from human contact with thermally inactivated BoHV-4 (Gillet et al., 2004). Our results suggest that BoHV-4 is sensitive to thermal inactivation, and are in agreement with a recent study demonstrating that pasteurization of breast milk inactivates human cytomegalovirus infectivity (Hamprecht et al., 2004). The sensitivity of herpesviruses to thermal treatments could be explained by the instability of their envelope to such treatments, as suggested by a study on herpes simplex virus (Yanagi, 1981).
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ACKNOWLEDGEMENTS
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A. Vanderplasschen is a senior research associate of the "Fonds National Belge de la Recherche Scientifique" (FNRS). B. Dewals and L. Gillet are research fellows of the FNRS. This work was supported by a grant of the Belgian "Service public et fédéral santépublique, sécurité de la chaîne alimentaire et environnement" (program no. S-6146).
Received for publication February 22, 2005.
Accepted for publication May 24, 2005.
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