THE ANALYSIS OF WEEKLY MILK BULK TANK COMPONENTS AS A ROUTINE INDICATOR OF THE HERD HEALTH STATUS

T. Zadnik, M. Klinkon, M. Nemec, M. Mesarič

University of Ljubljana, Veterinary Faculty, Clinic for Ruminants, Cesta v Mestni log 47, 1000 Ljubljana, Slovenia

Summary

In the future more and more guarantees that the production systems are animal friendly, healthy and safe will be required. Measuring milk parameters Na, K, Cl, urea, enzymes LDH, betahydroxybutyrate (BHB), acetone, SCC and antibodies for Enzootic Bovine Leukosis (EBL) by using milk samples taken from the bulk tank provides a simple low cost method of assessing the health status of dairy herds. In 1998, we analyzed weekly the bulk milk samples on 8 farms with 177 dairy cows. Investigated parameters: Na, K, Cl; enzyme activity LDH and SCC were sensitive indicators for subclinical mastitis. SCC over 400.000 cell/ml in bulk milk tank was associated with increased Na (>24 mmol/L), Cl (>35 mmol/L) and high activity of LDH enzyme (>80 U/L), K concentration was decreased (<38 mmol/L). Positive reaction for BHB and acetone in bulk milk samples were excellent parameters for identification of alimentary and production type ketosis. High urea concentration (>5,0 mmol/L) indicated an imbalance of energy, proteins and roughage in herd nutrition. With the bulk milk ELISA tests for EBL antibodies and individual sera AGID and ELISA tests we diagnosed a negative reaction. Conteporary research has shown that bulk milk analyses are becoming the basic diagnostic material used for herd health monitoring.

Introduction

By definition, milk bulk tank samples only reflect the status of cows whose milk is currently entering the tank and therefore do not include male animals, young stock, dry cows and those cows whose milk is being withheld for whatever reason. Samples will inevitably be affected by dilution and unequal contribution of individual cows to the tank (33).

Recent research has shown that milk analyses are becoming basic or support diagnostic material because the results are useful for the producers, milk industry, selectionists, and veterinarians, thus being of national significance (8,10,33,51).

For monitoring the production and health status of cows in herd the Milk Profile Test is implemented. Namely, milk sample examinations are performed on a regular basis, collection is simple and bulk tank milk samples can be monitored (10,33,47). Milk is a suitable diagnostic biological material for detection of sub-clinical metabolic disease or deficiency 1,11,9,14,15,22,23,25,26), infectious disease (27,33,45) and intoxication (3). Furthermore, changes in bulk milk composition (e.g. somatic cells, lactose, minerals and enzymes) can be attributed to a disease-combating response of the cows in the case of mastitis, reduced secretory activity and alteration of the blood-milk barrier (2,13,16,21,43,44,50).

An effective herd health program should include not only milk sampling but also regular farm visits to check animals, housing, feed stuffs, pastures and equipment (5,46). We therefore need other means to obtain information than the classical hands and eyes (10).

Markusfeld-Nir (24) concluded that this programme could become more widespread if the following points will be kept in mind:

- The nucleus of any program should be based on clinical services. Any other consultory services offered are of supportive nature.

- Health programs should be correctly balanced between the individual cow and herd medicine.

- Some form of "contract practice" should exist since a "one man practice" might be unable to deal with the new form of practice; and finally

- Palpation is not "herd medicine", and statistics not epidemiology.

Milk contains more than 300 different constituents. Changes in some milk variables are specifically related to the metabolic status of the cow, some others are associated with the udder health status and some milk variables indicate both the systemic and mammary gland condition (16). Measuring antibodies for some endemic infectious diseases by ELISA using milk samples taken from the bulk tank provides a simple method of assessing the health status of dairy herds (27,33).

Sample collection

The sample collected should be representative of the milking herd. Should be taken only after milking has finished and ensure that the contents of the tank are thoroughly mixed before collection. Collect sample from more than one tank if appropriate and either combine proportionately or test separately if this is likely to provide any useful information (e.g. heifers and younger cows,s milk collected in one tank). Samples can also be collected from different age cohorts, for example, when it is desirable to assess the status of first or second lactation homebred cows, or unvaccinated naive animals, as a guide to current/recent infection. For this, take an equal volume of milk from 6 - 10 cows in this category, mix well and submit an aliquot of the composite sample for testing (33).

Avoid collecting milk from the top of the tank to minimize the amount of cream present since this can significantly interfere with the result. Use a clean ladle or similar receptacle for sample collection and do not allow preservative to get into the bulk tank. Preliminary findings suggest that it is not essential that the bronopol preservative (MMB* preservative solution) used in the tubes is added immediately. Depending on environmental temperature, it could be added at the laboratory provided the sample is received within 48 hours of collection. Once preservative has been added, the sample is stable for several months provided it is kept refrigerated. For long term storage the samples can be frozen. The amount of preservative used (currently 150 µL/25 ml of milk) has no effect on optical density value (33,35).

Bulk milk components as indicators of metabolic cow status

A significant proportion of dairy cows that are culled after only 1 - 4 lactations are very thin and/or chronically lame and it is difficult to escape the conclusion that such animals are "worn out" by continuous hard work (6,46).

During the last two decades the following milk parameters which affect metabolic disorders of the herd were determined in clinical laboratories worldwide in milk bulk tank samples:

Acetone and beta-hydroxybutric acid - negative energy balance, hepatic lipidosis (1,40)

Urea - protein deficiency (22)

Urea - protein oversupply (22)

Sodium - calcium/phosphorus deficiency (9)

Sodium, potassium - alkalosis (26)

Potassium - acidosis (25)

Sodium - sodium deficiency (32)

Selenium – selenium deficiency (23)

Effects of udder health on bulk milk composition

For the assessment  of health status of the udder in bulk milk samples we have established the following parameters:

Somatic cell count - disease combating animal response (44)

Activity of enzyme lactate dehydrogenase (LDH) - disease combating animal response (40)

Lactose - secretory activity (50)

Potassium - alteration of blood-milk barrier (43)

Sodium - alteration of blood-milk barrier (21)

Chlorine - alteration of blood-milk barrier (2)

Infectious diseases and bulk milk antibody status

Performance indices that are routinely monitored programs, such as abortions, reproductive failure, diseased animals, mortality, early culling or retarded growth may point to impending problems with regard to specific diseases (5,46).

Researchers focus specifically on infections in dairy cattle herds. These are most important endemic infection diseases and distributed worldwide (5,33,46).

The recent introduction by the Veterinary Laboratories Agency of bulk milk antibody testing by enzyme-linked immunosorbent assay (ELISA) for BVD, IBR and Leptospira  hardjo now offers similar opportunities to veterinary surgeons in Britain (33). Bulk milk antibody testing is used in some countries, in conjunction with individual animal blood or milk antibody testing, as a diagnostic tool for the investigation of disease outbreaks, including abortions (30,33). Such samples could thus be used to screen for positive herds, in which all cows could be sampled subsequently. Examples of viruses that can be detected by analyzing bulk milk samples for antibodies are infectious bovine rhinotracheitis (IBR) virus (18,30,4,5,45), bovine leukemia virus (BLV) (12,37), bovine virus diarrhoea virus (BVDV) (27,30), and bovine corona virus (BCV) (10,30).

The brucellosis, leptospirosis and salmonellosis status of a herd can be determined by the detection of antibodies in bulk milk (7,17,19). Research workers have developed sensitive assays for detection of antibodies against M. paratuberculosis in milk (42). Unfortunately these tests are not yet suitable for bulk milk screening.

Advantages

The main advantages of a bulk milk diagnostic test are that the sample is easy to obtain and the test is reasonably cheap, and can therefore be used as a routine indicator of subclinical diseases. Such use also yields material for use in epidemiological studies. Large bulk of data is  necessary when studying the genetic background of diseases and pertinent parts of the milk profile outlined above have been used to study the heritability of ketosis and mastitis (10). The bulk milk tests provide a good starting point for differential diagnosis, in additition to their value for blood serological monitoring. It is envisaged that veterinary practices could record the information from bulk milk tests and incorporate it into preventive medicine programmes, particularly in relation to purchased animals (34). Regular testing of bulk milk samples  every few months provides a simple low cost method of confirming continuing freedom from infection in known disease-free herds; including those at potential risk of introducing new infection, and thereby allows opportunity for prompt action if indicated. For example, a positive result in a previously negative herd could herald an upsurge in infertility, abortions or milk drop (33).

Material and methods

1. Our observation called the Milk profile test was carried out during 1998 in 8 large-scale dairy herds  that included 177 Holstein-Friesian cows aged 5,23 ± 1,23 years on average with 6.822 ± 0,823 kg milk/cow/year. In 6 herds the cattle were loose-housed and in  2 herds the cows were tied-housed. All cows were fed home prepared hay, corn and grass silage, and in the summer they were fed fresh grass (n=6) or pastured (n=2). All through the year concentrates were fed individually by hand(6 herds) or by use of automatic Westfalia feeders (2 herds). Each cow received 150 g of  commercially mixed concentrate (corn meal 60%, 15% barley meal, 15% soya bean meal, 10%  molassed dried sugar beet pulp and mineral-vitamin premix) per every kg of produced milk. The blood samples were taken from clinically healthy animals, assigned from 3 various groups of cows: 1. One week after drying off (n=152),  2. One week after parturition (n=152) and 3. Three weeks after parturition (n=152). Individual milk samples were taken every month together with regular milk controle. The milk profile test is based not only on laboratory analysed weekly milk bulk samples (n=416) and monthly individual milk samples (n=1812) but on field examination as well. During field examination data were collected on feed quality, feeding regime, milk production, reproductive performance, health status of the herd and  body condition of the animals was scored 12 times every month. All data were written down in special recording books  called the milk profile test books. For  each farm two books were kept – one field and one laboratory book. Data were exchanged on regular  weekly visits to farms.

Milk parametrs measured in our project are not included in regular milk  control methods in Slovenia with the exception of fat, protein, lactose and SCC.

2. Bulk milk samples were collected weekly through March 5,  1998 to March 5, 1999 on 8 dairy farms with 177 cows and analyzed for concentration of fat, protein, urea, acetone, betahydroxy butirate (BHB), somatic cells (SCC), sodium (Na), potassium (K), chlorine (Cl) and enzyme lactate dehydrogenase activity (LDH). The obtained results of milk analyses were processed by the statistical package SAS (36).

The statistical model used was:

                                            Yijklmn= µ + Li + Kj+ Sk + SCCl + Acm + eijklmn

where:

Yijklmn= observation resp. measured property ijklmn

µ = mean value of observations

Li= influence of farm (i= 1..8)

Kj= influence of husbandry (j= 1,2)

Sk= influence of ration with regard to season (k= 1,2)

  k=1 from 1. to 17. and from 40. to 52. week of experiment

  k=2 from to 18. to 39. week of experiment

SCCl= effect of SCC class (l= 1... 5)

Acm= effect of milk acetone concentration (m=1,2)

eijklmn= residual random (error)

3. Two-times a year the bulk milk antibody tests used for enzootic bovine leukosis are based on the ELISA test (29). In this point we examinated one time blood sample of all cows (n=177) from observed farms with ELISA and Agar gell imunodiffusion test (29).

Results and discussion

During a one year observation study we established: 35,2 % incidence of hypocalcemia with 4,5 % incidence of milk fever, 12,56 % incidence of hypophosphatemia, 23,5 % incidence of hyperketolaktia, 21,38 % incidence of clinical mastitis and 56,24 % incidence of increased SCC (>400.000/ml) in individual milk samples. Via bacteriological analysis of milk samples from clinically (n=57) and subclinically (n=81) infected cows (>400.000/ml) 57% S. aureus, 32% other streptococci, 6% E. coli, 4% Str. agalactiae, 1% fungi and yeats  were detected. The interval from calving to first insemination was 103,5 + 20,2 days on average.  

Table 3 presents mean values of parameters analyzed in weekly bulk milk samples with regard to housing system, season, somatic cell count, acetone content and the highest and respectively lowest average in 8 herds.

A one-year monitoring of somatic cell count in weekly bulk milk revealed that the obtained average cell count 493.000  ±  122.124 sc/ml was too high. Namely, 44% of samples contained over 400.000 sc/ml. The results also demonstrated that from 8 monitored herds 2 exhibited weekly elevated cell count almost through observation time.

On the basis of these findings we recommend that producers keep the somatic cell count in bulk milk below 250.000/ml. It was concluded that the count exceeding 250.000 sc/ml indicated a greater number of animals with udder inflammation (39).

Table 3 also demonstrates that an elevated cell count in milk is closely related to a higher LDH, Na, Cl and urea level and that protein content decreases if somatic cell count increases. All these parameters increased statistically significantly with the exception of protein content which decreased statistically insignificantly (Table 4).

Within the framework of the research the acetone content in bulk milk was also determined on a weekly basis and 56 (13,46%) positive responses were established. A detailed analysis has revealed that the acetone presence was mainly a reflection of feeding grass silage with elevated butyric acid content - the so-called alimentary or false ketosis. About these findings we reported in 1993 (47).

With the statistical model of the analysis of variance with 5 entries we calculated F-values and established statistical significance of individual factors affecting milk parameters. In Table 4 F-values for individual influences and determination coefficients (R2) for milk content are presented.

The investigated milk properties in weekly samples were statistically most significantly affected by the farm. This result is, we believe, quite objective because there are great differences among herds with regard to the management regime and health condition of dairy cows. Similar reports have been obtained also by other authors (10,16,38). Season (temperature, humidity, husbandry) had also a significant effect on the majority of parameters, especially on fat, proteins and urea levels as well as somatic cell count. Through July, August and September the cell count was highest and the highest frequency of mastitis was observed as well (39). The authors reported that the cell count was also affected, besides the various causative agents of mastitis by a whole range of factors, among them the time of the year, and consequently changes in the feeding and husbandry measures (31,39).

The obtained results confirm that the concentration of Na, Cl and LDH activity are closely related to somatic cell count. An increased cell count results in Na, Cl and LDH concentration  increases. This finding points out that the results of the MLP-test should be interpreted from various viewpoints. The most frequently established clinical state in cows is depressed milk secretion due to bacteriological inflammation of the mammary gland. In such cases an elevated cell count (>350.000 sc/ml), elevated Na content (>23,00 mmol/L), Cl (>34,00 mmol/l and LDH activity (>80,00 U/L) were observed. Metabolic disorders in bulk milk samples associated with acetone content (ketolactia) were less frequently detected. The concentration of protein and Na was in such milk markedly lower. Acetone in milk appears in various forms of ketosis resp. impaired metabolism of carbohydrates and lipids which is reflected at least at the initial stage in the compensated rumen resp. metabolic acidosis. The established acetone in bulk milk Na content below 22,5 mmol/L as well as suppressed milk secretion are sensitive predictors of metabolic disorders. Milk protein content is also decreased, whereas the somatic cell count is below 250.000 sc/ml. Similar findings have been reported by other authors who suggested the same milk parameters monitored for evaluation of the energy and fat metabolism in dairy cows (10,16,32,41,49).

Through our statistical model we managed to clarify a substantial part of variance. Determination coefficient (R2) was highest by the protein percentage in milk and as much as 53,7% of variance was explained. Determination coefficients were rather high also by other milk concentrations (above 22,5%) with the exception of K (5,5%). Thus, the model encompasses the majority of factors significantly affecting milk composition.

By the end of 1987 there were no positive EBL reactors to our knowledge in Slovenia (20). All bulk milk and individual blood samples were negative.

Conclusion

Losses from metabolic, mastitis and some endemic infectious diseases (IBR, BVD, Leptospirosis etc.) are often subclinical and many veterinary surgeons and farmers are thus unaware of the health status of their herds. Bulk milk tests offer an ideal opportunity to improve this situation and stimulate an interest in monitoring herd health status, adopting appropriate control measures including vaccination and screening new stock. Opportunities for the future include bulk milk antibody tests for Salmonella typhimurium and Neospora caninum. Knowledge of herd health status is particularly relevant at present when many previously closed herds are now purchasing replacements as a consequence of the cull for bovine spongiform encephalopathy. In some countries, bulk milk antibody test have provided a simple and convenient means of surveying the infectious disease status of their national herd. On the basis of recent  research and the present one we recommend for evaluation of the herd health status (metabolic, udder diseases) with bulk milk analysis with the following parameters: fat >3,85%, protein >3,17%, urea from 3,0 to 5,0 mmol/L, SCC <250.000/ml, Na >24,00 mmol/L, K >38,00 mmol/L, Cl <35,00 mmol/L and LDH <55 U/L as baselines.

Acknowledgement

This research work was made possible by the support of farmers and the Ministry of Science and Technology of  Slovenia (Project L4 – 5473 – 04505 – 97).

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Table 1: Investigated milk parameters and laboratory methods

Table 2: Analysis and distribution of weekly bulk milk tank results with regard to farm, housing system, season, somatic cell count and ketolactia

* = negative (-); ** = positive (±= 0,16 mmol/L; + 0,17 - 0,42 mmol/L; ++ = 0,43 - 1,72 mmol/L; +++ > 1,72 mmol/L)

Table 3: Association of mean weekly ( n = 416) values of bulk milk parameters with, housing system, feeding season, udder health status, ketolactia and lowest resp. highest mean concentration in 8 herds

* 1 = up to 100.000 SC/ml

   2 = 100.000 to 250.000 SC/m

   3 = 250.000 to 400.000 SC/ml

   4 = 400.000 to 750.000 SC/ml

   5 = above 750.000 SC/ml

**  = mean min./max. value in herds

Table 4: Demonstration of the results of the analysis of variance with 5 entries, determination coefficients (R²) and coefficient of variantion (CV)

*** = P < ,001

**   = P < 0,01

*     = P < 0,05