Acute phase proteins are continuing to gain importance particularly as diagnostic tools in veterinary medicine [1]. With the discovery of the major APP in bovine milk and its correlation with mastitis [2], there has been a growing interest of their exploitation as biomarkers of bovine mastitis which can be adapted to a rapid, on farm measuring format. Given that bovine mastitis is one of the most prevalent and costly conditions affecting dairy cows which greatly impacts on the economics, welfare and public health aspects of dairy farming, the substantiation of APP for mastitis diagnosis is on-going and methods to apply their measurement to a rapid format has been explored [3, 4].

Acute phase proteins are proteins that are synthesized in the liver and other organs in response to inflammatory stimuli and released into body fluids such as serum, milk [1], ovarian fluid [5] among others. These samples are therefore collected and assayed for APP in order to make clinical diagnosis of inflammatory and other disease conditions.

Samples for diagnostic procedures often require some form of preparation, preservation or safe handling for transportation prior to analysis in the laboratory. This is especially true for milk samples which frequently need the use of preservatives to retain milk composition as close to that found in freshly collected samples as possible. Chemical preservation is used to avoid cellular degradation prior to somatic cell counts (SCC) or progesterone analysis (pregnancy test). Storage at freezing temperatures of between −20 °C and −80 °C is also a common way of preserving milk samples.

In addition, as a requirement for the safe transfer of biological samples across international borders, some national regulations demand heat treatment of liquid samples in order to inactivate harmful pathogens in the samples have been put in place (for example; Council directive 82/894/EEC of 21 December 1982, under the European Communities Act 1972).

However, high temperature is known to denature proteins and to influence the concentration and immunologic activities of some serum proteins [6]. Heat treatment has also been shown to result in irreversible changes in the structure of milk proteins [7]. In addition, heating of milk has been shown to affect various characteristics of milk ranging from allergenicity of milk proteins [8, 9] to the concentration of water soluble vitamins, as well as concentration and activity of some milk proteins [10–12]. Furthermore, it was observed that heating colostrum to 60 °C for 60 min resulted in inactivation of pathogens and loss (although minimal) in colostral IgG [11]. A number of other studies have also focused on the effect of heat on residues of drugs (quninolones, aminoglycosides) in milk [13, 14].

In the handling of milk samples for analysis, the use of preservatives has become a requirement for some tests due to distances of laboratories from the farms where samples are obtained. Potassium dichromate (K₂Cr₂O₇) as in Lactab Marks III® is a corrosive and toxic biocide which is often used in the preservation of milk samples in order to maintain milk composition from time of collection to analysis. It is known to be effective as a milk preservative [15], and is used to maintain progesterone levels in milk for pregnancy diagnosis. Another broad spectrum milk preservative, bronopol (C₃H₆BrNO₄; 2-bromo-2-nitro-1, 3 propanediol), which unlike potassium dichromate is reported to have no toxic effects, is often used particularly for preserving milk somatic cells prior to analysis through its action as an antimicrobial agent [16, 17]. Other preservatives that have been used in milk samples include Mercury chloride (HgCl₂), sodium azide [18], Azidiol [19] and formalin [20].

Although the efficacy of these milk preservatives in maintaining milks’ natural composition including protein, fat and SCC has been proven [21–23], there has also been early reports [24–26] of alterations in analyte concentrations following the use of these preservatives. For example potassium dichromate was shown to lead to a decrease in protein concentration within 24 h after milk collection [27]. In addition, bronopol based preservatives were shown to alter the mid infrared measurements of protein contents of milk samples [15]. In another study however, the use of the preservatives potassium dichromate, bronopol and sodium azide did not significantly change fats, proteins, lactose and milk solids content of milk when samples were analyzed within 7 days of preservative use, but were reported to lead to a decline in these components when measured in samples stored for over 7 days after preservation [17]. Use of bronopol has also been reported to yield higher values of % proteins and fat compared to values measured in aliquots of the samples following the use of potassium dichromate [25]. Differences in effects on milk microflora between use of different preservatives have also been reported [28]. Other parameters that have been reported to be affected by chemical preservative use include the electrical conductivity (EC) of milk by potassium dichromate [29].

From these reports, it can be inferred that there is a possibility that the use of chemical preservatives may affect analytes in milk. Indeed it has been recommended that where the preservative used in milk interferes with the analyte being tested; a suitable correction factor should be used [30].

It is not known if the use of chemical preservatives in milk meant for analysis or heat treatment of milk samples in order to inactivate pathogens before international transit, has any effect on the concentration of milk APP. It is important to be aware of such effects if they exist, if such treated milk samples are to be used for analysis of APP.

This study was carried out to evaluate the effects of heat treatment and chemical preservative use on milk APP concentration. This consisted of the assessment of milk Hp, M-SAA3 and CRP following the use of the milk preservatives; potassium dichromate (Lactab Marks III®) and bronopol (broad spectrum Microtab® II) for milk preservation after 24 h and heating samples to pathogen-inactivation temperature (56 °C) for 30 min.

In this study, three pre-analytical treatments regimes were carried out on milk samples with the aim of comparing the APP values in treated and untreated samples.

Studies have shown that some protocols of heat treatment could have significant reduction effects on milk (colostral) proteins especially IgG [32, 33] and on lactoferrin [34]. The observed lowering of M-SAA3 and CRP concentrations in heated samples and changes in CRP values of chemically preserved milk in the present study, lends further credence to these observations. It is generally known that high temperatures can result in the irreversible change in milk proteins structure [7], moreover it has been shown that aggregation of whey proteins can result upon heating of milk [35], this may explain some of the changes observed in the concentration of protein analytes in milk (which are mainly contained in the whey fraction).

It has been shown that high temperatures, can result in dissociation of CRP pentameres into its monomeric forms in blood [36], and although this may not affect the total concentration of serum CRP, it signals possible protein structural changes that are possible upon heating. There has been no report of the effect of heating on the structure of bovine milk CRP or M-SAA3. It can be inferred that a denaturation effect of heat on the protein structure or moiety of these compounds as reported by Hausen et al. [6] causes a loss of functional ability of these proteins to bind antibodies.

This finding should therefore be taken into consideration when assaying for M-SAA3 and CRP in samples which have undergone heating as a result of international transfer regulations involving heat treatment of biological samples. In such studies, control samples should be treated in the same manner as experimental samples or a correction factor based on the reduction in concentration should be applied. Structural changes in Haptoglobin have been associated with increased body temeprature (heat shock conditions) [37]. However, no significant change in milk Hp concentration due to heating was observed in this study. Studies have shown good correlation between milk Hp and SCC [31, 38], and in the study of Hachana et al. [39], heating milk samples to 60 °C prior to analysis did not alter the Fossomatic SCC, although the duration for which samples were held at that temperature before analysis was not ascertained.

Only CRP concentrations were affected by the use of chemical preservatives in milk, with the concentration showing a significant increase following treatment with both potassium dichromate and Bronopol but the reason for this is not clear. Therefore samples being collected for SCC or for progesterone analysis can also be used for determination of Hp and M-SAA3 concentrations but should be cautiously used for CRP determination.

In this study, a minimum time of 24 h was used to assess the possible preservative effects of potassium dichromate and Bronopol and it is possible that samples preserved for longer (beyond 24 h) may develop changes that could significantly alter milk concentration of Hp and M-SAA3 as observed for some other proteins by Chalermsan et all [17]. The milk used in this study were residual samples collected for either clinical investigation or prior experimental study and selected to give a range of APP concentrations. The effect of chemical preservatives was assessed after 24 h mixing and incubation at 4–8 °C in order replicate to some degree the likely treatment of samples between collection addition of preservative and analysis. While it would be ideal to assess the effect of heat and chemical preservation on fresh samples treated immediately, this was beyond the scope of the present investigation. In future investigation of APP in milk, where preservatives are being used to allow SCC or progesterone analysis, it would be of value to assess the effects on APP under a variety of conditions. Nevertheless, it is recommended that samples needing to be transported over long distances without any means of cold-chain storage, can be preserved using either potassium dichromate (Lactabs Mark III) or Bronopol (Microtabs), but should be analyzed as soon as possible and a correction factor should be applied in cases of CRP assay. It would be interesting to examine the effect of prolonged storage of milk with preservatives on milk APP.

Heating milk samples and preservative use in milk can affect the measurement of APP concentration. However, if this is recognized and taken into consideration when assaying milk samples which have undergone heat treatment, as maybe required to conform with international transport regulations, or when assaying samples needing chemical preservation, then APP analysis will give valid results especially when control samples are treated with identical procedures.

APP, acute phase proteins; APR, acute phase response; CRP, C-reactive protein; CV, coefficient of variation; ELISA, enzyme linked immunosorbent assay; Hp, haptoglobin; IMI, intramammary infection/inflammation; LOD, limit of detection; M-SAA3, mammary associated serum amyloid A3; SAA, serum amyloid A; SCC, somatic cell counts; SPSS, statistical package for social sciences; x, times