Bovine acute phase proteins in milk

The composition and quality of the raw milk is essential for the dairy industry and since only healthy cows produce milk of high quality, it is important with prospering cows. The most important and common disease among dairy cows is mastitis (inflammation of the udder). Mastitis is not only an animal welfare problem, but also results in impaired milk quality, reduced product yield, higher production costs and consequently a higher price for the consumer. The most common way to detect subclinical mastitis is by measuring the somatic cell count (SCC). The SCC is also an important parameter in milk payment systems, highly affecting the price to the producer. Since SCC is influenced by other factors than mastitis, e.g. lactation number, stage of lactation, stress etc., there is a need for new biomarkers for detection of subclinical mastitis as well as for raw milk quality. The aim of this thesis was to obtain further knowledge about the occurrence of the two major acute phase proteins (APP) in bovine milk, haptoglobin (Hp) and serum amyloid A (SAA), and to evaluate their potential as biomarkers for raw milk quality. For the first time, a method for analysis of Hp using an optical biosensor based on surface plasmon resonance (SPR) technology was developed. The method is fully automated with no need for sample preparation before analysis and each sample requires approximately 8 minutes. The occurrence of Hp and SAA in quarter, cow composite and bulk tank milk samples were investigated and the results showed that APP could be detected in all types of samples. In general, detectable levels of APP in milk were related to high SCC, probably originating from cows with subclinical mastitis. In general, samples containing APP had lower casein content, casein number (casein in relation to total protein) and lactose but also increased whey protein content or increased proteolysis. Hp and SAA were suggested to be useful biomarkers for milk quality, especially the protein quality. To our knowledge, this thesis is the first describing Hp and SAA as potential biomarkers for raw milk quality

Haptoglobin and serum amyloid A in relation to the somatic cell count in quarter, cow composite and bulk tank milk samples

Milk somatic cell count (SCC) is the gold standard in diagnosis of subclinical mastitis, and is also an important parameter in quality programmes of dairy cooperatives. As routine SCC analysis is usually restricted to central laboratories, much effort has been invested in the search for alternative biomarkers of mastitis and milk quality, including the presence in the milk of the acute phase proteins (APP), haptoglobin (Hp) and serum amyloid A (SAA). The aim of this study was to investigate relationships between Hp, SAA and SCC in quarter, cow composite, and bulk tank milk samples. Cows (n=165), without any clinical signs of disease or abnormalities in the milk or udder, from three different dairy farms, were used. Cow composite milk samples from all cows delivering milk at the sampling occasion were taken once in each herd. In one of the farms, representative quarter milk samples (n=103) from 26 cows were also collected. In addition, bulk tank milk samples from 96 dairy farms were included in the study. Samples were analysed for Hp, SAA and SCC, and relationships between the parameters were evaluated at quarter, cow and tank milk levels using Chi-square analysis. Milk samples were categorized according to their SCC, and the presence, or no presence, of SAA and Hp, based on the detection limits of the screening methods (0.3 mg/l and 1.0 mg/l for SAA and Hp, respectively). Hp and SAA were found in milk at quarter, cow composite and bulk tank levels. A large proportion (53%) of the animals had detectable milk concentrations of APP, and SAA was detected more frequently, and at higher concentrations than Hp, regardless of sample type. SAA was detected in as many as 82% of the bulk tank milk samples. Significant relationships were found between Hp, SAA and SCC at quarter and cow composite milk levels, but only between SAA and SCC at bulk tank milk level. Detectable levels of APP were more common at high SCC

Biosensor assay for determination of haptoglobin in bovine milk

Despite more than 30 years of research into mastitis diagnostics, there are few alternatives to the somatic cell count (SCC) in practical use for identification of cows with subclinical mastitis. Mastitis is not only an animal welfare problem, but also affects the yield, composition and technological properties of milk. Hence, dairy cooperatives give farmers a premium quality payment to encourage low SCC although there is no clear scientific data defining the level of SCC in bulk tank milk that is associated with additional benefits in terms of milk quality. Recent research on alternative markers for inflammatory reactions in the lactating cow, e.g. in mastitis, includes investigations of the acute phase protein, haptoglobin (Hp). So far, the content of Hp in milk has mainly been studied in relation to mastitis diagnostics, with little attention given to its importance for milk composition and technological properties. At present, Hp in milk is measured using ELISA, but this technique is not suitable for routine large-scale analysis. In recent years, optical biosensor technology has been used for automated and rapid quantitative analysis of different components in milk, but so far not for analysis of acute phase proteins. The aim of the present study was to develop a rapid and sensitive biosensor method to determine Hp in milk. An affinity sensor assay based on the interaction between Hp and haemoglobin was developed using surface plasmon resonance (SPR) biosensor technology. The assay was used to analyse Hp in composite milk samples from cows without any clinical signs of mastitis and quarter milk samples with a weak to strong reaction in the California Mastitis Test (CMT). A commercial ELISA for determination of Hp in milk was used for comparison. The limit of detection (LOD) of the biosensor assay was determined as 1.1 mg/l. Within-assay and betweenday variations were determined both with bulk tank milk spiked with human Hp and with composite milk samples containing bovine Hp. Coefficients of variation varied between 3.6 and 8.6% at concentrations between 4.0 and 12 mg/l, respectively. Agreement between the results obtained by the biosensor assay and the ELISA was satisfactory ; however, the results obtained by the biosensor were generally lower than the results obtained by the ELISA. Possible explanations for this observation are discussed

Relationship between haptoglobin and serum amyloid A in milk and milk quality

The objective of this study was to evaluate relationships between the presence in milk of the major bovine acute phase proteins, haptoglobin (Hp) and serum amyloid A (SAA), and milk quality parameters. Composite milk samples were collected from 89 clinically healthy dairy cows and analysed for Hp and SAA, total protein, casein, and whey protein levels, casein number, proteolysis, total fat and lactose levels, and somatic cell count (SCC). Milk samples with detectable levels of Hp showed lower total protein and casein levels, whereas milk samples with detectable levels of SAA had lower casein number and lactose level. Samples with detectable levels of acute phase proteins also showed an elevated SCC. The results suggest that the presence of Hp and SAA in milk might indicate unfavourable changes in milk composition, especially in relation to protein quality

Förändras mjölkens proteinsammansättning i separata juverdelar i samband med höga celltal (SCC)?

Today the milk production per cow is increasing but the milk delivered by the Swedish farmer contains less amounts of fat and protein than earlier. The contents have decreased since 1993. In average the milk contain 4,2 percent fat and 3,4 percent protein. Earlier the fat content in milk was important. Nowadays the dairy's attention has turned to the milk's valuable proteins, principally the caseins, which have a considerable nutritional value and are important for several dairy products like cheese and yoghurt. An inflammation in the udder which injuries the udder tissue is called mastitis. Mastitis is a big problem among dairy cows. The immune system become activated when the cow gets mastitis which cause an increased somatic cell count in the milk and also changes in the milk composition. Mastits cause economical losses for the farmer, health problem for the cow and also problem for the dairy industry. Knowledge about different factors affecting the composition of the milk is therefore very important. The purpose of this study was mainly to investigate how the protein composition in milk was affected in connection with an increased somatic cell count (SCC). Except the protein composition even parameters like fat, lactose and milk yield have been studied. Compared to many other investigations this study was based on milk samples from each separate udder quarter. During a subclinical mastitis, when milk is not visually affected, is it especially important to collect samples from each quarter to avoid the dilution from the other healthy quarters. The study was conducted on Kungsängen research station at the Department of Animal Nutrition and Management, Swedish University of Agricultural Sciences, Uppsala. The cows participating in the study were from the ordinary research stock. 24 cows participated in the study, 12 of these participated twice. 18 of the 24 cows were milked in an AMS (automatic milking system) while the rest of the cows were in conventional stables. 140 quarter milk samples were collected at seven occasions. To get a rough estimation about the cell count in the milk CMT (California Mastitis Test) was used, but all samples have also been analysed with electronic fluorescensbased cellcounting to get a more precise value of the cell count in the milk. This information was then used to decide which quarter that had an increased somatic cell count compared to the opposite quarter. To precipitate the casein the rennet method was used. Both the milk and the serum protein have then been analysed on the FTIR (Fourier Transform Infrared Spectroscopy) to get values for the fat- lactose- and protein content. To decide the casein content an indirect method has been used. The difference between the protein content in whole milk and in the protein content in the serum protein fraction have been calculated to get a value for the casein content in the milk. Most of the cows participating in this study had a very good udder health. Despite a low somatic cell count it was possible to detect several changes in the composition when a quarter had an increased SCC. The results from this study revealed that casein, casein number (casein: total protein), milk yield and lactose decrease significant with an increase in SCC. The whey protein increased significant. The fat content and the total protein content had some tendency to increase with an increased SCC, but these results were not significant. The amount of milk decreased with 23-24 %, lactose decreased with 4-5 % while the serum protein increased with 7-8 %. The casein number decreased with 2,5 % in a quarter with an increased SCC. Maybe 2.5 % sound negligble but because the casein number is a quotient between the casein content / total protein content such a decrease can have a considerable effect for the cheese making industry.Dagens kor mjölkar allt mer men mjölken som Sveriges bönder levererar till mejerierna innehåller allt lägre halter av fett och protein. I genomsnitt innehåller mjölken 4,2 procent fett och 3,4 procent protein. Information om innehållet av mjölkens proteiner, främst kaseinerna, har i dag fått större uppmärksamhet än tidigare då fetthalten var av stort intresse. Kaseinerna är viktiga på grund av deras näringsvärde samt deras betydelse vid framställning av mejeriprodukter såsom ost och yoghurt. En inflammation i juvret, som leder till att juvervävnaden skadas, kallas mastit. Mastit är ett stort problem hos mjölkkor i dag. Vid mastit aktiveras immunförsvaret, vilket leder till ett ökat celltal i mjölken och förändringar i mjölkens sammansättning. Detta innebär ekonomiska förluster för lantbrukaren, i vissa fall lidande för djuret, samt problem för mejeriindustrin. Kunskap om olika faktorer och hur dessa påverkar mjölkens sammansättning är därför mycket viktig. Syftet med denna studie var framförallt att undersöka hur mjölkens proteinsammansättning påverkas av ett förhöjt celltal. Förutom proteinsammansättningen har även parametrar såsom fett, laktos och mjölkmängd studerats. Till skillnad mot många andra studier har denna utförts på juverfjärdedelsnivå. Vid subkliniska mastiter, då mjölken visuellt inte är förändrad, är det speciellt viktigt att ta prover från varje juverdel för att undvika utspädning från de friska juverdelarna. Studien genomfördes vid Kungsängens forskningsstation, Institutionen för husdjurens utfodring och vård, Sveriges Lantbruksuniversitet, Uppsala. Korna som ingick i studien valdes ut från den ordinarie försöksbesättningen. 24 kor ingick i studien, 12 av dessa var med i två provtagningsomgångar. 18 av de 24 korna som ingick i studien var inhysta i stallet med automatiskt mjölkningssystem (AMS) medan de övriga var inhysta i konventionellt stall. Vid sju provtagningstillfällen har 140 juverdelsprover insamlats, vid fyra tillfällen ingick trespenta kor. För att få en grov uppskattning av celltalet i mjölken har CMT (California Mastitis Test) använts, men alla prover har även blivit analyserade med hjälp av elektronisk fluorescensbaserad cellräkning för att få ett noggrannare värde på mjölkens innehåll av celler. Detta för att kunna bestämma vilka juverdelar som hade ett förhöjt celltal. För att fälla ut kaseinet i mjölken har löpemetoden använts. Både mjölken och vasslen har därefter analyserats på FTIR (Fourier Transform Infrared Spectroscopy) för att erhålla värden på dess innehåll av fett, laktos och protein. För att bestämma kaseininnehållet i mjölken har en indirekt metod använts. Differensen mellan proteininnehållet i helmjölken och i vasslefraktionen har beräknats och på så sätt har man kunnat erhålla mjölkens innehåll av kasein. De flesta korna som ingick i denna studie hade en mycket god juverhälsa, men trots detta kunde man vid relativt låga celltal även upptäcka förändringar i mjölken. Resultaten från denna studie visar att kasein, kaseintalet, mjölkmängd och laktos minskar signifikant vid ett förhöjt celltal. Vassleinnehållet ökar däremot signifikant. Fett och totala proteininnehållet tenderar till att öka vid ett förhöjt celltal, men dessa resultat var dock ej signifikanta. Mjölkmängden minskade med 23-24 %, laktos minskade med 4-5 % medan vassle ökade med 8-9 %. Kaseintalet minskade med ca 2,5 % på juverdel som hade ett förhöjt celltal, vilket kan verka obetydligt, men i och med att kaseintalet är en kvot mellan kaseininnehåll / totala proteininnehållet kan en sådan minskning ha en betydande effekt för bl.a. ysterierna

Heart rate variability (HRV) and muscular system activity (EMG) in cases of crash threat during simulated driving of a passenger car

Objectives: The aim of the study was to verify whether simultaneous responses from the muscular and circulatory system occur in the driver's body under simulated conditions of a crash threat. Materials and Methods: The study was carried out in a passenger car driving simulator. The crash was included in the driving test scenario developed in an urban setting. In the group of 22 young male subjects, two physiological signals - ECG and EMG were continuously recorded. The length of the RR interval in the ECG signal was assessed. A HRV analysis was performed in the time and frequency domains for 1-minute record segments at rest (seated position), during undisturbed driving as well as during and several minutes after the crash. For the left and right side muscles: m. trapezius (TR) and m. flexor digitorum superficialis (FDS), the EMG signal amplitude was determined. The percentage of maximal voluntary contraction (MVC) was compared during driving and during the crash. Results: As for the ECG signal, it was found that in most of the drivers changes occurred in the parameter values reflecting HRV in the time domain. Significant changes were noted in the mean length of RR intervals (mRR). As for the EMG signal, the changes in the amplitude concerned the signal recorded from the FDS muscle. The changes in ECG and EMG were simultaneous in half of the cases. Conclusion: Such parameters as mRR (ECG signal) and FDS-L amplitude (EMG signal) were the responses to accident risk. Under simulated conditions, responses from the circulatory and musculoskeletal systems are not always simultaneous. The results indicate that a more complete driver's response to a crash in road traffic is obtained based on parallel recording of two physiological signals (ECG and EMG)

Social support and sleep : Longitudinal relationships from the WOLF study

Aim: To investigate the relationship between two social support dimensions (network and emotional support) and sleep quality and between two social support sources (at and outside work) and sleep quality. Methods: The three-wave prospective Work Lipids and Fibrinogen (WOLF) study from Northern Sweden was used including 2420 participants who had filled out a questionnaire on working life, life style and health. Sleep quality was assessed by the Karolinska Sleep Questionnaire (KSQ). Structure and function of social support were measured as network support both at and outside work by Availability of Social Integration (AVSI) and emotional support both at and outside work by Availability of Attachment (AVAT). Logistic regression was used, utilizing variables created to assess development over time. Moreover, reversed causation was tested. Results: Improved network support at work decreased the risk of disturbed sleep (OR .65; 95% CI .47 - .90) as did improved emotional support outside work (OR .69; 95% CI .49 - .96). Reporting a constant poor network support at work increased the risk of disturbed sleep (OR 1.53, 95% CI 1.10 - 2.11) as did reporting a constant poor emotional support outside work (OR 1.46; 95% CI 1.02 - 2.05). In men constant good network at work decreased the risk of disturbed sleep (OR .49, 95% CI .34 - .71). Reversed causation analyses indicate some bi-directionality. Conclusion: Being able to perceive social support is a human strength promoting sleep. Both dimension (structure and function) and source (at and outside work) of support matters in sleep quality and seem to be related since the structural dimension was more likely to affect sleep when derived from work, whereas the functional dimension affected sleep quality if it was provided outside work. Men’s sleep seems to be more sensitive to network support at work. Disturbed sleep may also alter the perception of social support