TECHNICAL FIELD

This invention relates to a method and device for measuring the infection status of mammary glands. In particular it relates to a method and device for measuring or detecting animal mastitis (particularly bovine mastitis).

BACKGROUND ART

Mastitis is an inflammatory disease of the mammary gland of a mammal.

The inflammation is the result of infection by any of a multitude of bacteria, mycoplasmas, yeast and fungi. The range of. different organisms that can cause mastitis, their varying susceptibilities to antibiotics or other treatments and the ability of the treatments to access the organisms present the greatest challenges in the treatment and prevention of mastitis. This is especially true in dairy cows, with mastitis accounting for the number one cost of production worldwide. Bovine mastitis may be caused by Gram negative bacteria such as Escherichia coli, Klebsiella species and Enterbacter species, or by Gram-positive bacteria such as Staphylococcus aureus, Enterococci species, and Streptococci such as Streptococcus uberus, Streptococcus agalactiae and Streptococcus dysgalactiae, and by Mycoplasma bovis. Bacterial infection via the teat canal is the most common cause of mastitis. The cause-effect spectrum of mastitis typically begins with a pathogen entering the mammary gland via the teat canal and colonising somewhere inside the mammary gland. Colonisation somewhere within the mammary gland leads to continued growth of pathogens internally, which in turn leads to cascades of both pathogenic effects and immune-related effects.

This cascade is known to start with increases in lactate levels. This is believed to be a result of the bacterial cells undergoing some levels of anaerobic metabolism.

Another early-stage effect is the release of immune defence proteins. This includes the acute phase proteins, such as milk amyloid A, lactoferrin, lactoperoxidase (Lp) and other proteins that have some form of antimicrobial effect. Concurrent with these stages, white blood cells in the form of neutrophils, leukocytes or macrophages, infiltrate the mammary gland, with the goal of engulfing and isolating the foreign bacteria. This leads to early increases in somatic cell count (SCC), a count used to measure the combined number of mammalian cells, including leukocytes or neutrophils and sloughed mammary secretory or epithelial cells. Other compounds, such as NAGase and lactate dehydrogenase, have also been shown to be present in early infection.

If the cow's immune system is not successful, bacterial growth continues and starts to cause internal damage to the gland. As mammary secretory cells start to die and slough, somatic cell counts (SCC) rise further. Left untreated, these effects can lead to internal swelling, tissue breakdown and milk clotting (the so-called clinical effects of mastitis). The tissue breakdown can lead to extracellular biochemicals breaching the blood-milk barrier, such as ions and red blood cells. This leads to conductivity changes and optical changes in the milk. Of course, this cascade can eventually lead to death of the animal if left untreated.

With such a complex cause-effect cascade of changes, one can appreciate that merely the presence or absence of any particular component on its own may not be sufficient to determine where a particular animal is positioned within the state of the mastitis progression.

The measurement of compounds in the milk has been used extensively for monitoring this cause-effect spectrum of mastitis. By far, the gold standard, worldwide is the measurement of somatic cell count (SCC) in milk. SCC is used as both an indicator of infection status and as a measure of milk quality. Traditionally, to measure SCC in milk, a sample has had to be removed and sent off to a laboratory for analysis.

More recently, on-line SCC devices have been commercialised. These devices are limited in commercial use due to their cost and complexity, which is primarily related to their need for external reagents. For decades, conductivity has been measured as means to monitor mastitis. While reagentless, its ability to detect early infection is limited, due to its placement in the cause-effect spectrum. Attempts to measure SCC optically, via absorbance and/or scattering, have also been tried; however, interference from fat globules in milk, which are a similar diameter to somatic cells and in high concentrations (up to 5% w/v), prohibit the accuracy required.

All references, including any patents or patent applications cited in this specification are hereby incorporated by reference. No admission is made that any reference constitutes prior art. The discussion of the references states what their authors assert, and the applicants reserve the right to challenge the accuracy and pertinency of the cited documents. It will be clearly understood that, although a number of prior art publications are referred to herein, this reference does not constitute an admission that any of these documents form part of the common general knowledge in the art, in New Zealand or in any other country. It is acknowledged that the term 'comprise' may, under varying jurisdictions, be attributed with either an exclusive or an inclusive meaning. For the purpose of this specification, and unless otherwise noted, the term 'comprise' shall have an inclusive meaning - i.e. that it will be taken to mean an inclusion of not only the listed components it directly references, but also other non-specified components or elements. This rationale will also be used when the term 'comprised' or 'comprising' is used in relation to one or more steps in a method or process.

It is an object of the present invention to address the foregoing problems or at least to provide the public with a useful choice.

Further aspects and advantages of the present invention will become apparent from the ensuing description which is given by way of example only.

DISCLOSURE OF INVENTION

According to one aspect of the invention there is provided a method of determining whether a milking animal has a mastitic condition characterised by the step of measuring on-line the level of oxygen within the milk.

It should be appreciated that a milking animal may be any appropriate species including sheep and goats. However, reference throughout the specification shall be made to bovines as this is a significant dairy industry. The mastitic condition can include all stages of mastitis as well as preclinical. Indeed, in preferred embodiments of the present invention, the condition detected is that of preclinical mastitis as early intervention can be conducted by the farmer without significant damage occurring to the cow. The inventor considers that the choice of oxygen levels in milk is one that is highly indicative of mastitis and justification for this follows.

Milk is secreted into the mammary gland with an oxygen content that is close to that of plasma, but there is no system to replenish oxygen once the milk has been secreted into the mammary gland. This is one of the few places in the body that does not have an oxygen supply adequate for its volume, making it quite unique.

Another unique feature is that the mammary gland contains no glucose, the main source of energy for cells in all other parts of the body.

Literature and clinical research by the inventors suggest that neutrophils within a mammary gland consume oxygen in significant amounts when combating bacterial infections. Further, the Lactoperoxidase (Lp) system that provides a defence mechanism producing a natural bactericidal agent also requires oxygen as a substrate.

The closest discussion that the inventors have found in relation to this area was in Mayer et al. as discussed below. Mayer et al. (1988) measured oxygen levels in milk from normal cows and mastitic cows. They used a Clark-type electrode (Corning 168, Corning Ltd, Halstead. Essex, UK). This study concluded that the oxygen in milk from normal cows was similar to that measured in venous plasma (1.3 μg/ml). However, inflammation of the mammary gland led to a dramatic drop in oxygen concentration. This effect occurred in both normal and mastitic quarters of the mastitic cows (0.68 μ9ΛηΙ and 0.07 μο/ιηΙ), respectively.

It should be noted that this study did not disclose methods for how "normal" and "mastitic" were defined. Consequently, the study makes no attempt to define the severity of the mastitis; rather, it simply labels some samples "normal" and others "mastitic." While this study showed a correlation between infection and inflammation as determined by whatever method the authors used, and oxygen levels, no attempt was made to correlate oxygen levels with SCC. Rather, the authors were searching for a reason why mammary glands with lower oxygen resulted in neutrophils being less effective against different types of pathogens. In other words, this study had a physiological, rather than a diagnostic, rationale.

It should also be noted that all measurements were off-line. This was likely due to limitations of using a Clark-type electrode, which is prone to fouling, uses up oxygen when being used and would not withstand the environment of a milking machine or milking parlour.

The detection of a mastitic condition on-line is very important. This means that the detection is made while the animal is being milked or very shortly thereafter while the milk is still in the environs of a milking system and the cow is still within the milking premises One of the advantages of being able to conduct measurements on-line is that the animals can subsequently be drafted immediately after milking for treatment or other purposes.

Further, there is understandably considerable less labour content. As noted above, previous measures of oxygen in milk has been offline and in a laboratory situation. Not only is this tedious and time consuming, but also adds significant additional steps to recording data against individual cow's history - which is a common requirement with current dairy management systems.

The inventors have determined that there are a number of requirements to enable the measurement of oxygen in milk on the line.

Firstly, the inventors have determined that oxygenation of milk can readily occur in a milking system. This oxygenation process can understandably affect considerably the measurements and hence any correlation with a systematic cell count. Therefore, an important consideration in the implementation of the present invention is to provide an oxygen controlled environment within which the measurement of oxygen in the milk is conducted.

Alternatively, ambient oxygen levels within the milking system could be measured and used as an on-line calibration routine.

Additionally, since it is suspected that oxygen is used up by components within the milk itself, a simple routine of adding or controlling oxygen levels within the system and monitoring the oxygen depletion rate by the milk could also be a strategy employed on-line.

In some embodiments the oxygen level is measured in milk within the milking system as close to the animal as possible so as to avoid any potential contamination of the milk sample with oxygen as it passes through the milking system.

Thus, in one embodiment of the present invention the milk being sampled is being channeled individually or diverted from close to an individual teat cup. An example of such a sampling device can be found in New Zealand Patent No. 525350.

In the some embodiments, the milk may not be sampled as such, but the sensor may be fully inline within the milking system. As can be appreciated, the regulations surrounding the milking industry are very strict in terms of avoiding contamination. Therefore any sensor that is used within the main milk flow would have to be readily cleaned. Thus, the choice and placement of the sensor is important.

Further, the sensor must be robust, capable of taking multiple readings without requiring manual intervention (such as cleaning or adjustment) and relatively inexpensive.

Preferably, the placement of the sensor ensures that measurement is taking from each teat quarter. This is because mastitis can be confined to one teat. A comparison between the teat quarters is thought to be able to give more useful data in terms of analysing an individual cow for comparative infections between each teat. This is a technique that has been employed for online systems which monitor electrical conductivity of milk. By using ratios between individual quarters, effects of stage of lactation, age of cow, environmental conditions, breed, etc. can be averaged out and more indicative baselines of individual cows can be established. Also, the actual dynamic ranges of components within an udder quarter can vary by cow. Therefore, monitoring individual quarters can also help to correct for errors due to all of these potential situations.

The inventors have determined that a suitable sensor for use in the present invention can be a Ruthenium electrode. This electrode fluoresces when exposed to oxygen and therefore an optical reading can be made.

The advantage of using a Ruthenium electrode is that it is not prone to fouling as the sensor does not contain a membrane through which the oxygen must diffuse. Of course, other membrane-less sensors could also be used as alternatives to a Ruthenium electrode.

One aspect of the present invention is using historical data to determine when a mastitic condition has been detected.

The inventors found that the decrease of oxygen levels and the increase of SCC levels are exponential with infection which is probably due to the neutrophils using oxygen and not the mammary secretary cells. Once the SCC level rises above a certain threshold (which is given by the oxygen reading) the inventors consider that most oxygen will be used up the neutrophils. Thus, by using this information and historical data, it will be possible to determine the threshold at which an infection (mastitic condition) occurs. This is of considerable advantage from a management of herd treatment.

The traditional methods of treating mastitis are intramammary injection of antibiotic remedies. There is a growing area of non-antibiotic, natural treatments for mastitis that are more 'preventative' than 'curative.' Such treatments require earlier detection of potential problems in order to be effective. Some measurement of oxygen use by a key component of the cause-effect spectrum, such as neutrophils, would provide such an early indication.

Thus is can be seen that the present invention provides an on-line system that meets the following criteria:

• can monitor the cow's milk during milking for mastitis and/or SCC • reagentless - doesn't require the use of any reagents

• low cost - has the potential to be a low-cost device

• robust - can withstand clean-in-place temperatures, pressures and chemicals. BEST MODES FOR CARRYING OUT THE INVENTION

The following, discussion illustrates trials conducted showing correlation between SCC and oxygen levels.

Methods

A herd of cows was screened to find cows with varying degrees of mastitis infection. Cows were screened using a standard Rapid Mastitis Test (RMT) to determine whether the SCC was low (score 0), moderate (score 1-2) or high (score 3). Cows were selected as follows: one cow with four low quarters (normal cow with normal quarters), one cow with at least one moderate quarter but no high quarters (mastitic cow with both normal and mastitic quarters) and one cow with at least one high quarter (mastitic cow with both normal and mastitic quarters).

Milk was collected prior to the morning milking, using the following set-up in order to keep the milk from becoming oxygenated during collection. A 10 ml syringe body was retrofitted with a syringe needle facing into the body. On the outside of the body a small piece of tubing was connected to a canula that could be inserted through the teat canal. Test tubes that were capped with a penetrable septum and evacuated were used to receive the milk directly from the teat cistern (i.e., without oxygenation). Further samples were collected from each quarter for analysis of SCC and bacteriology. These samples were collected normally without the canula.

Example 1 - Effect of Ambient Oxygen:

Two of the quarters were measured immediately for oxygen content using a Clark- type electrode. While not ideal for commercial on-line measurement, off-line usage was possible. Prior to measurement, the milk samples were poured into containers open to ambient air, thus allowing them to start to become oxygenated. The same samples were then taken to an oxygen-controlled environment, opened and re- tested to assess the effect of the ambient conditions on decreasing the oxygen in the samples.

Example 2 - Effect of SCC:

All samples were taken to an oxygen-controlled environment, opened and tested using the Clark-type electrode. The levels of oxygen were then matched against the SCC levels in the samples. Bacteriology results were used only for assurance that the quarters were infected.

Results

Example 1 - Effect of Ambient Oxygen:

Table 1 shows the effect of measurement of oxygen in milk samples in ambient conditions versus lower oxygen controlled environment. From these results, one can clearly see that, by controlling the ambient levels of oxygen exposed to the sample during measurement, one can affect the actual levels of oxygen in the milk. This phenomenon implies that oxygen levels in the environment exposed to extracted milk will need to be either controlled or repeatable, if oxygen levels can be repeatably correlated to SCC.

Table 1. Effect of measurement of oxygen in milk samples in ambient conditions versus lower oxygen controlled environment. All measurements in μg/ml.

Example 2 - Effect of SCC: Table 2 shows the effect of SCC on oxygen. This clearly shows that there is a distinct correlation between SCC and oxygen that holds true for both normal and mastitic cows. The no growth quarters of the mastitic cows clearly show lower oxygen than the normal quarters of the normal cow, which is consistent with the results of Mayer et al. (1988). From these results, it appears that a method of measuring oxygen in a controlled fashion has the ability to (1) determine the mastitic state of a quarter and (2) predict the SCC of the quarter. Additionally, these results also clearly show the advantages of measuring all four quarters separately and comparing the results within cow as well as between cows.

Table 2. Effect of SCC on oxygen levels in milk.

Moderate Quarter 7 CNS (low) 395 0.62

Moderate Quarter 8 no growth 1237 0.59

Moderate Quarter 9 no growth 2390 0.32

High Quarter 10 S aureus (mod) 5368 0.94

Moderate Quarter 11 CNS (low) 6439 0.46

High Quarter 12 S uberis (high) 23795 0.34

Aspects of the present invention have been described by way of example only and it should be appreciated that modifications and additions may be made thereto without departing from the scope of the appended claims.

References:

Goldberg, J.J., Bramley, A.J. Sjogren, R.E. and J.W. Pankey, 1994. Effects of temperature and oxygen tension on growth of E coli in milk, Journal of Dairy Science, 77:3338-3346.

Fahtone, J.C. and P.A. Ward, 1982. Role of oxygen-derived free radicals and metabolites in leukocyte-dependent inflammatory reactions, American Journal of Pathology, 107(3):397-418.

Heyneman, R., Burvenich, C. and R. Vercauteren, 1990. Interaction between the respiratory burst activity of neutrophil leukocytes and experimentally induced Escherichia coli mastitis in cows, Journal of Dairy Science, 73:985-994.

Mayer, S.J., Waterman, P.M., Keen, P.M., Craven, N. and F.J. Bourne, 1988. Oxygen concentration in milk of healthy and mastitic cows and implications of low oxygen tension for the killing of S. aureus by bovine neutrophils, Journal of Dairy Research, 55:513.