Background to the Invention

Facial eczema is a fungal disease that affects ruminants across New Zealand. The disease is induced by the consumption of Pithomyces chartarum spores which reside at the base of ryegrasses and multiply in warm, wet weather. These spores, once ingested by ruminants, produce the toxin sporidesmin in the gut. The cytotoxic effects of sporidesmin lead to irreversible degradation of the liver tissues and build-up of liver breakdown products, including phytoporphyrin and gamma-glutamyltransferase (GGT). Phytoporphyrin photosensitises the skin, leading to lesions and injury upon exposure of the animal to sunlight.

The history of the disease is well-described by the literature (M. E. Di Menna , B. L. Smith & C. O. Miles (2009) A history of facial eczema (pithomycotoxicosis) research, New Zealand Journal of Agricultural Research, 52:4, 345-376) including the devastating effect it has on the agricultural sector of New Zealand (R. Thomson (1986) Facial Eczema - A Strategic Approach, Proceedings of the New Zealand Grassland Association, vol. 47, pp. 129-134). The impact of facial eczema is forecasted to increase due to a changing climate, with more of New Zealand’s pastures becoming suitable growth habitats for the causative fungus.

The GGT test utilises the enzyme’s capacity to catalyse a broad range of non-specific substrates. A spectrometer is used to correlate the rate of production of a coloured substrate by the GGT enzyme. To carry out this test in animals with a potential facial eczema infection, a blood sample is taken to a veterinary lab and analysed using photospectrometry equipment. Variations of this test are described in US Pat. No. 3769173, US Pat. No.

3703441 , US Pat. No. 3892631 , US Pat. No. 3773626, US Pat. No. 3979447, US Pat. No. 4087331 , and US Pat. No. 4511651 , all of which utilise similar nitroanilide substrates to measure total GGT present.

A significant barrier to both the correct timing of facial eczema interventions and the application of resistance breeding techniques is the slow and labour-intensive nature of photospectrometric GGT testing. Opportunities are lost to detect the onset of the disease and to separate healthy from clinical and subclinical animals, causing management methods such as selective grazing and the toxic administration of zinc to be applied across a herd for a broad span of time. This results in lost income and excess labour and stress for farmers.

The primary current use for the GGT test is by farmers on a small sample of their herd to measure the effectiveness of their zinc application, long after the disease has already become an issue. Zinc confers a significant protective advantage to animals afflicted with facial eczema, and is administered either through feed and water, or through boluses, multiple of which have been described in NZ Pat. No. 249598A, NZ Pat. No. 572035A, NZ Pat. No. 526939A, Aus Pat. No. 2002010015, Aus Pat. No. 2004292397, and Aus Pat. No. 2009245843. Zinc is toxic, particularly in higher quantities, and more precise administration of zinc would reduce the effects currently caused by overdosing of zinc in herds. A second reason for the development of more efficient facial eczema diagnosis tools is the potential gain for livestock improvement breeding. A key focus of practical research into facial eczema is in breeding for facial eczema tolerance due to the high heritability of disease resistance (approximately 0.4). GGT testing is key in resistance breeding in order to recognise which animals are least affected by the disease, but the cost and labour involved in implementing this test in its current form prevents farmers from utilising this key resource.

The nature of the current GGT test is such that it measures total GGT within a sample, without taking the specific isozymes into account. Thus, insufficient data exists on the levels of specific GGT isozymes present in relation to particular diseases. Proprietary research indicates that the GGT 1 isozyme of GGT is capable of distinguishing between positive and negative facial eczema specimens. Antibodies for GGT1 and GGT7 were incorporated into a plate-based ELISA and used to measure their respective levels of isozymes in blood samples sourced from sheep that were exposed to R chartarum and had been previously measured using the gold standard nitroanilide GGT. The GGT7 ELISA showed no response to varied total GGT levels across positive and negative facial eczema samples, while the GGT 1 ELISA showed a correlation between total GGT and GGT 1 levels in this experiment. GGT1

This discovery introduces the possibility of using antibodies in an assay for facial eczema, which was previously not possible due to the variation in structures across the isozymes of GGT. Antibodies raised against GGT have previously been patented for use in human anti-cancer therapy (US. Pat. No 5854006), but no such patents have been filed for animal use, or for specific isozymes of GGT. Antibodies are an attractive prospect for on-site and rapid testing for veterinary diseases due to their versatility and well-quantified properties.

Furthermore, facial eczema diagnosis using GGT has previously only been performed in serum, as the nitroanilide method was only known to be effective in this medium. However, the presence of GGT in the saliva, mucus, and urine of mammals, including ruminants, has been well-explored in literature (Van den Berg, JS. (1990) The activity of gamma glutamyltransferase in the urine of sheep. Journal of the South African Veterinary Association, vol. 61 , no. 2, pp. 46-49; G. Rico, J. P. Braun, P. Benard, and J. P. Thouvenot (1977) Blood and Tissue Distribution of Gamma Glutamyl Transferase in the Cow. Journal of Dairy Science Vol. 60, No. 8, pp 1283-1287; M. F. Ghazali, H. H. C. Koh-Tan, M. McLaughlin, P. Montague, N. N. Jonsson, and P. D. Eckersall, (2014) Alkaline phosphatase in nasal secretion of cattle: biochemical and molecular characterisation, BMC Veterinary Research, vol. 10, p. 204), and reports in a broad variety of species indicates that these GGT levels are elevated over the course of disease, particularly liver disease (J. Jimenez Alonso, L. Jaimez, L. Barrios, F. Perez Jimenez, G. Costan, and J. A. Jimenez Pereperez (1984) Salivary y-Glutamyl Transferase Activity in Internal Diseases, Archive of Internal Medicine, vol. 144, no. 9, pp. 1804-1806). The GGT in these mediums is accessible by antibody-based tests, making them appropriate mediums for facial eczema testing.

The shift from slow, spectrophotometric methods for facial eczema testing that require specialised equipment to a quicker, cheaper, and more accessible method that can be carried out by farmers with minimal training or investment is key to future management of this disease. The most attractive candidate format for a point-of-care, cheap, and accessible test for disease is the lateral flow test. Lateral flow tests are industry standard apparatuses for diagnosis, and can utilise a broad variety of mediums, including the four biological fluids highlighted in this description (serum, saliva, mucus, and urine). They use antibodies as the affinity reagent for recognising the analyte, which is also consistent with our approach. It is for these reasons that we are targeting lateral flow tests as the best medium with which to utilise our research.

Statement of Invention

This invention pertains to the use of an isozyme for testing the urine, serum, saliva, or mucus of ruminants to identify animals affected by facial eczema (pithomycotoxicosis), further to a test that uses a lateral flow device containing an antibody to detect isozyme concentrations in urine, serum, saliva, or mucus in ruminants, and including where the isozyme is type I gamma-glutamyltransferase (GGT1) and the antibody is generated against this isozyme.

Detailed Description of the Invention

The assay comprises an antibody and a means for bringing this antibody into contact with bodily fluids. In this instance the bodily fluids to be contacted are the serum, mucus, saliva, and urine of ruminant animals, which are analysed by this assay for facial eczema infection.

Suitable means for contacting the antibody with the fluids includes a lateral flow test. In general, a lateral flow test comprises a test strip with a detection agent mounted on it (in this case an antibody) and a signal system to inform the user of the presence of the analyte.

This assay is mounted on a backing pad and features a wicking pad at the end of the assay opposite to the sample pad to promote flow of sample. The sample to be analysed is placed upon a sample pad at the opposite end of the assay from the wicking pad. Suitable backing, wicking, and sample pad materials include porous paper, nitrocellulose membrane, and manufactured polymers.

The sample pad commonly features a filtration element to alter the sample for assay compatibility. In specific, the sample pad commonly removes particulates from the sample and changes the consistency and chemical composition of the sample to make it more appropriate for the assay conditions. This is appropriate for the biological fluids that this assay is performed in.

An antibody is used as the detection agent in the assay, which is conjoined with a reporter molecule. Suitable reporters include the commonly used gold nanoparticles and latex particulates, which are coloured and can be assessed visually.

The antibody used in this assay is generated against gamma-glutamyltransferase (GGT). More specifically, the antibody is generated against the GGT1 isozyme of GGT. Suitable antibodies include, but are not limited to, commercial antibodies such as Invitrogen’s MA5-11815 antibody, Sigma’s Anti-MGC96963 antibody, and Sigma’s SAB2701966 antibody.

The detection antibody is present on a conjugation pad next to the sample pad, contacting the sample with the detection antibody as the sample wicks down the pad through capillary action. The detection antibody is kept in a protective state to ensure assay longevity during transportation and storage. Suitable methods of preservation of the detection antibody can include freeze-drying, coating in sugar or another desiccant, or chemical preservation.

The detecting antibody attaches to the target if present in the sample as it passes through the conjugation pad. This is the first instance in which the antibody contacts the fluid sample. The second instance occurs as the sample-antibody mixture passes the test line.

The test line consists of immobilised antibodies which capture the analyte if present. In this example, the test line binds to exposed epitopes on the analyte within the analyte-antibody conjugate, and their presence becomes visible due to the reporter molecule being bound to the antibody. The test line can consist of the same or different antibodies to the detection antibodies, depending on epitope availability on the target molecule. Suitable antibodies include, but are not limited to, commercial antibodies such as Invitrogen’s MA5-11815 antibody, Sigma’s Anti-MGC96963 antibody, and Sigma’s SAB2701966 antibody.

The control line consists of species-specific anti-IgG antibodies that bind to the detection antibody and provide visual evidence that the wicking and detection agent aspect of the assay have worked as intended. The presence of the reporter molecule conjugated to the detection antibody allows for visual assessment that the assay has been effective. Suitable control line antibodies include but are not limited to Sigma-Aldrich’s A6154 anti-rabbit anti-IgG antibody produced in goat and Jackson ImmunoResearch’s 315-035-045 anti-mouse anti-IgG antibody produced in rabbit.

Examples

1 . A farmer brings in their herd of heifers. During milking, when other metrics of cow health and performance are taken, the farmer captures some mucus from the cow’s nostril and places it on the sample pad, noting the ear tag number. Before milking is complete, the GGT1 level in the mucus has been assayed by this device and returns a positive (two bands visible) or negative (second band visible only) result. This information is noted with other health statistics that inform grazing management.

2. A stud farm for Corriedale rams wants to measure facial eczema resistance in their rams in order to market some rams as being able to increase facial eczema resistance in the lambs they produce. Over the course of the facial eczema season, the rams are tested with this device by drawing blood with a sterile pin and dripping this blood onto the sample pad. The results are noted along with their ear tag number; this allows the stud farm to note which rams go through the season without exhibiting facial eczema.