WO2006110939A1

WO2006110939A1 - Detection of endoparasites in herbivores

Info

Publication number: WO2006110939A1
Application number: PCT/AU2006/000396
Authority: WO
Inventors: Leo Frederick Le Jambre; Ian Gordon Colditz
Original assignee: Commonwealth Scientific And Industrial Research Organisation; The University Of New England; Australian Meat Processor Corporation Limited; The State Of New South Wales Through Its Department Of Primary Industries; The State Of Western Australia Through Its Department Of Agriculture; The State Of Queensland Through Its Department Of Primary Industries
Priority date: 2005-04-22
Filing date: 2006-03-24
Publication date: 2006-10-26

Abstract

The present invention relates to methods, systems and kits for quantifying and detecting parasite infection in herbivores by measuring the amount of blood in the faeces of said herbivores, and to related methods for the intensive management of herbivores.

Description

DETECTION OF ENDOPARASITES IN HERBIVORES

Technical Field

Background Art

Blood-sucking parasites, such as Haemonchus contortus, are a common source of infection among ruminants including sheep, goats and cattle. Adult worms live in the abomasum of host animals, with female worms depositing up to 10,000 eggs per day which pass out of the host in the faeces. Infection can lead to emaciation, anaemia and in severe cases death. Of these, the principal symptom is anaemia, due to the blood-letting activities of the parasite. Prior to the present invention, the only method that had been used to determine blood loss in these infections was isotopic labeling of red blood cells with the radioactive compound Cr⁵¹. Although this is a sensitive method of detecting blood in faeces, Cr⁵¹ is a gamma emitter and the waste products of sheep injected with it must be treated as radioactive waste. Faecal blood loss in humans has been measured by assaying the peroxidase activity of the heme portion of hemoglobin, which catalyzes the oxidation of alpha-guaiaconic acid by hydrogen peroxide to form a quinone compound. This reaction is the basis of several tests for faecal occult blood, such as the Colo-Rectal test, which has been used to screen people in Papua New Guinea with Necator americanus infection for the presence of blood in stools (Bauerfeind et ah, 1992; Allemann et ah, 1996). However, the test gave positive results in 17% of cases without infection, in 24% of cases with low levels of infection, in 57% of cases with moderate levels of infection and in 67% of cases with high levels of infection. Therefore, due to the large number of false positives in uninfected people and false negatives in people with even moderate and high levels of Necator americanus infections, the Colo-Rectal test was not useful as an instrument for general screening for such infections.

There also exist non-isotopic techniques for determining blood production, for example, by measuring red blood cell potassium levels (Albers and Le Jambre, 1983) and mean corpuscular volumes (Schalm et al, 1975). Using such methods, reticulocytosis is seen at approximately 1 week after the start of blood loss, being the time at which eggs start appearing in the faeces. Thus, the detection of increased blood production provides no earlier detection of infection than does faecal egg counts (FECs). However, during H. contortus outbreaks in sheep, infections can be so severe that lambs can become anemic concurrent with the commencement of egg counts. Packed cell volumes (PCVs), hemoglobulin levels and FAMACHA® are measures of pathology based on anaemia, being the main symptom caused by infection with H. contortus. The FAMACHA® system relies on a method of diagnosis relating to the colour of the eyelid of the animal which becomes paler as anaemia develops. A six point colour chart is used to determine when sheep or goats should be treated. However, since the colour changes are measures of pathology, a production loss must be incurred if treatment is deferred until anaemia is present. Furthermore, if diagnosis is made on the basis of anaemia, it is likely that the parasites would have been laying eggs for several weeks and thus the environment would be contaminated with infective larvae.

The present invention is predicated on the surprising and unexpected finding that the amount of blood present in the faeces of herbivores can be used to detect and quantitatively determine the level of parasite infection in said herbivores.

Summary of the Invention

According to a first aspect of the present invention, there is provided a method for quantifying parasite infection in a herbivore, wherein said method comprises measuring the amount of blood in the faeces of said herbivore.

The amount of blood in said faeces may be measured by:

(a) obtaining a faecal sample from said herbivore;

(b) pre-treating said faecal sample to inactivate peroxidase activity emanating from sources other than from haemoglobin of the herbivore;

(c) subjecting said faecal sample to a colourimetric assay; and

(d) analyzing said colourimetric assay to determine the level of parasite infection. The pre-treating of said faecal sample may comprise contacting said faecal sample with an alcohol. The alcohol may be selected from the group comprising methanol, butanol, propanol, methylated spirits or any combination thereof. The alcohol may be methylated spirits.

The alcohol may be diluted to a concentration in the range of from about 20 - 30%

(v/v) with water. The alcohol may be diluted to a concentration of about 25% (v/v) with water. The colouiϊmetric assay may measure the amount of at least one of haemoglobin, myoglobin or leukocytes. The colourimetric assay may measure the amount of haemoglobin.

The colourimetric assay may comprise the peroxidase activity of haemoglobin 5 catalyzing an oxidation reaction, thereby producing a change in colour. The oxidation reaction may comprise catalysis of the oxidation of diisopropylbenzene and 3,3 ',5,5'- tetramethylbenzidine, thereby producing a change in colour.

The analyzing may involve visual comparison of the result of said colourimetric assay against defined colour standards, wherein said colour standards correspond to levels o of parasite infection.

The analyzing may involve use of a spectrometer.

Additionally or alternatively, the analyzing may involve capturing an image of the result of said colourimetric assay. Said capturing an image may involve use of a camera.

The analyzing may further involve a computer program. The computer program s may quantify the amount of red, green and blue in each pixel of an image of the result of said colourimetric assay.

According to a second aspect of the present invention, there is provided a method for quantifying parasite infection in a herbivore, wherein said method comprises measuring the amount of blood in the faeces of said herbivore, wherein the amount of Q blood in said faeces is measured by:

(a) obtaining a faecal sample from said herbivore;

(b) subjecting said faecal sample to a colourimetric assay; and

(c) analyzing said colourimetric assay to determine the level of parasite infection. The method may further comprise pre-treating said faecal sample to inactivate ₅ peroxidase activity emanating from sources other than from haemoglobin of the herbivore. The pre-treating of said faecal sample may comprise contacting said faecal sample with an alcohol.

According to a third aspect of the present invention, there is provided a method for quantifying parasite infection in a herbivore, wherein said method comprises measuring o the amount of blood in the faeces of said herbivore, wherein the amount of blood in said faeces is measured by:

(a) obtaining a faecal sample from said herbivore;

(b) pre-treating said faecal sample to inactivate peroxidase activity emanating from sources other than from haemoglobin of the herbivore; 5 (c) subjecting said faecal sample to a colourimetric assay; and (d) analyzing said colourimetric assay to determine the level of parasite infection.

The pre-treating of said faecal sample may comprise contacting said faecal sample with an alcohol.

According to a fourth aspect of the present invention, there is provided a method for detecting infection of at least one blood-sucking parasite in a herbivore, wherein said method comprises detecting the presence of blood in the faeces of said herbivore.

The presence of blood in said faeces may be detected by:

(a) obtaining a faecal sample from said herbivore;

(c) subjecting said faecal sample to a colourimetric assay; and

(d) analyzing said colourimetric assay to detect parasite infection.

The pre-treating of said faecal sample may comprise contacting said faecal sample with an alcohol. The alcohol may be selected from the group comprising methanol, butanol, propanol, methylated spirits or any combination thereof. The alcohol may be methylated spirits.

The alcohol may be diluted to a concentration in the range of from about 20 - 30% (v/v) with water. The alcohol may be diluted to a concentration of about 25% (v/v) with water.

An amount of blood in faeces in excess of 10ml / day may be indicative of parasite infestation.

An amount of blood in faeces producing a colour change in excess of grade 2 on a Hemastix panel maybe indicative of parasite infestation. According to a fifth aspect of the present invention, there is provided a method for detecting infection of at least one blood-sucking parasite in a herbivore, wherein said method comprises detecting the presence of blood in the faeces of said herbivore, wherein the presence of blood in said faeces is detected by:

(a) obtaining a faecal sample from said herbivore; (b) subjecting said faecal sample to a colourimetric assay; and

(c) analyzing said colourimetric assay to detect parasite infection. The method may further comprise pre-treating said faecal sample to inactivate peroxidase activity emanating from sources other than from haemoglobin of the herbivore. The pre-treating of said faecal sample may comprise contacting said faecal sample with an alcohol. The alcohol may be selected from the group comprising methanol, butanol, propanol, methylated spirits or any combination thereof. The alcohol may be methylated spirits.

According to a sixth aspect of the present invention, there is provided a method for detecting infection of at least one blood-sucking parasite in a herbivore, wherein said method comprises detecting the presence of blood in the faeces of said herbivore, wherein the presence of blood in said faeces is detected by:

(a) obtaining a faecal sample from said herbivore;

(c) subjecting said faecal sample to a colourimetric assay; and (d) analyzing said colourimetric assay to detect parasite infection.

The alcohol may be selected from the group comprising methanol, butanol, propanol, methylated spirits or any combination thereof. The alcohol may be methylated spirits.

According to a seventh aspect of the present invention, there is provided a system for quantifying parasite infection in a herbivore, wherein said system comprises a means for measuring the amount of blood in the faeces of said herbivore.

Said means for measuring the amount of blood in said faeces may comprise:

(a) means for obtaining a faecal sample from said individual herbivore;

(b) means for pre-treating said faecal sample to inactivate peroxidase activity emanating from sources other than from haemoglobin of the herbivore;

(c) a colourimetric assay for testing the amount of blood in said faecal sample; and

(d) means for analyzing said colourimetric assay to determine the level of parasite infection. The colourimetric assay may measure the amount of at least one of haemoglobin, myoglobin or leukocytes. The colourimetric assay may measure the amount of haemoglobin.

The colourimetric assay may comprise the peroxidase activity of haemoglobin catalyzing an oxidation reaction, thereby producing a change in colour. The oxidation reaction may comprise catalysis of the oxidation of diisopropylbenzene and 3,3', 5, 5'- tetramethylbenzidine, thereby producing a change in colour.

The means for pre-treating said faecal sample may involve contacting said faecal sample with an alcohol. The alcohol may be selected from the group comprising methanol, butanol, propanol, methylated spirits or any combination thereof. The alcohol may be methylated spirits.

The means for analyzing may comprise visual comparison of the result of said colourimetric assay against defined colour standards, wherein said colour standards correspond to levels of parasite infection.

The means for analyzing may comprise a spectrometer. Additionally or alternatively, the means for analyzing may comprise a means for capturing an image of the result of said colourimetric assay. Said means for capturing an image may comprise a camera. Said camera may comprise a digital camera.

The means for analyzing may further comprise a computer program. The computer program may quantify the amount of red, green and blue in each pixel of an image of the result of said colourimetric assay.

According to an eighth aspect of the present invention, there is provided a system for quantifying parasite infection in a herbivore, wherein said system comprises means for measuring the amount of blood in the faeces of said herbivore, wherein said means for measuring the amount of blood in said faeces comprises: (a) means for obtaining a faecal sample from said individual herbivore;

(b) a colourimetric assay for testing the amount of blood in said faecal sample; and

(c) means for analyzing said colourimetric assay to determine the level of parasite infection. The system may further comprise means for pre-treating said faecal sample to inactivate peroxidase activity emanating from sources other than from haemoglobin of the herbivore. The means for pre-treating of said faecal sample may involve contacting said faecal sample with an alcohol. According to a ninth aspect of the present invention, there is provided a system for quantifying parasite infection in a herbivore, wherein said system comprises means for measuring the amount of blood in the faeces of said herbivore, wherein said means for measuring the amount of blood in said faeces comprises:

(a) means for obtaining a faecal sample from said individual herbivore; (b) means for pre-treating said faecal sample to inactivate peroxidase activity emanating from sources other than from haemoglobin of the herbivore;

(d) means for analyzing said colourimetric assay to determine the level of parasite infection.

The means for pre-treating of said faecal sample may involve contacting said faecal sample with an alcohol.

According to a tenth aspect of the present invention, there is provided a system for the intensive management of at least one herbivore, wherein said system comprises: (a) means for identifying an individual herbivore;

(b) means for quantifying parasite infection in said individual herbivore, comprising means for measuring the amount of blood in the faeces of said individual herbivore, comprising:

(i) means for obtaining a faecal sample from said individual herbivore; (ii) means for pre-treating said faecal sample to inactivate peroxidase activity emanating from sources other than from haemoglobin of the herbivore;

(iii) a colourimetric assay for testing the amount of blood in said faecal sample; and

(iv) means for analyzing said colourimetric assay to determine the level of parasite infection;

(c) means for processing information obtained by said means for quantifying parasite infection; and

(d) means for implementing strategies for the intensive management of said individual herbivore based on parasite infection status. The means for pre-treating said faecal sample may involve contacting said faecal sample with an alcohol.

According to an eleventh aspect of the present invention, there is provided a system for the intensive management of at least one herbivore, wherein said system comprises:

(a) means for identifying an individual herbivore;

(b) means for quantifying parasite infection in said individual herbivore, comprising means for measuring the amount of blood in the faeces of said individual herbivore, comprising: (i) means for obtaining a faecal sample from said individual herbivore;

(ii) a colourimetric assay for testing the amount of blood in said faecal sample; and

(iii) means for analyzing said colourimetric assay to determine the level of parasite infection; (c) means for processing information obtained by said means for quantifying parasite infection; and

(d) means for implementing strategies for the intensive management of said individual herbivore based on parasite infection status.

The means for quantifying parasite infection in said individual herbivore may further comprise means for pre-treating said faecal sample to inactivate peroxidase activity emanating from sources other than from haemoglobin of the herbivore. The means for pre-treating said faecal sample may involve contacting said faecal sample with an alcohol.

According to a twelfth aspect of the present invention, there is provided a kit for quantifying parasite infection in a herbivore, wherein said kit comprises means for measuring the amount of blood in the faeces of said herbivore.

The means for measuring the amount of blood in the faeces of said herbivore may comprise:

(a) means for obtaining a faecal sample from said herbivore; (b) means for pre-treating said faecal sample to inactivate peroxidase activity emanating from sources other than from haemoglobin of the herbivore;

(c) a colourimetric assay for testing the amount of blood in said faecal sample; and (d) means for analyzing said colourimetric assay to determine the level of parasite infection.

The means for pre-treating said faecal sample may involve contacting said faecal sample with an alcohol.

The alcohol may be diluted to a concentration in the range of from about 20 - 30% (v/v) with water. The alcohol may be diluted to a concentration of about 25% (v/v) with water. According to a thirteenth aspect of the present invention, there is provided a kit for quantifying parasite infection in a herbivore, wherein said kit comprises means for measuring the amount of blood in the faeces of said herbivore, wherein said means for measuring the amount of blood in the faeces of said herbivore comprises:

(a) means for obtaining a faecal sample from said herbivore; (b) a colourimetric assay for testing the amount of blood in said faecal sample; and

(c) means for analyzing said colourimetric assay to determine the level of parasite infection.

The means for measuring the amount of blood in the faeces of said herbivore may further comprise means for pre-treating said faecal sample to inactivate peroxidase activity emanating from sources other than from haemoglobin of the herbivore. The means for pre-treating said faecal sample may involve contacting said faecal sample with an alcohol.

According to a fourteenth aspect of the present invention, there is provided a kit for quantifying parasite infection in a herbivore, wherein said kit comprises means for measuring the amount of blood in the faeces of said herbivore, wherein said means for measuring the amount of blood in the faeces of said herbivore comprises:

(a) means for obtaining a faecal sample from said herbivore;

(b) means for pre-treating said faecal sample to inactivate peroxidase activity emanating from sources other than from haemoglobin of the herbivore; (c) a colourimetric assay for testing the amount of blood in said faecal sample; and

(d) means for analyzing said colourimetric assay to determine the level of parasite infection. The means for pre-treating said faecal sample may involve contacting said faecal sample with an alcohol.

According to a fifteenth aspect of the present invention, there is provided a method for the intensive management of at least one herbivore, wherein said method comprises:

(a) identifying an individual herbivore; (b) quantifying the level of parasite infection in said individual herbivore, wherein said quantifying comprises measuring the amount of blood in the faeces of said individual herbivore;

(c) processing information obtained by said testing; and

(d) implementing strategies for the intensive management of said individual herbivore based upon said processed information.

Brief Description of the Drawings

The present invention will now be described, by way of example only, with reference to the following drawings. Figure 1 : The relationship between H. contortus log FEC plotted against log worm numbers.

Figure 2 : The relationship between log worm number and haemoquant values. Figure 3: The ratio of green to blue in Hemastix indicator patches exposed to sheep faeces diluted in water plotted against their log mean worm counts. Figure 4: Hemoquant values from faeces collected 13 days post infectionplotted against worm counts from the same sheep at day 36-post infection.

Figure 5: Hemastix ratio of green to red from faeces collected 13 days post infection plotted against worm counts from the same sheep at day 36 post infection.

Figure 6: Predicting FEC from dipstick score: graphical representation of the visual assessment of colour change in the indicator substrate converted to a numerical value and then plotted against faecal egg counts.

Figure 7: Average daily weight gain of uninfected weaner lambs compared to the decrease in weight gain in weaner lambs infected with H. contortus with egg counts shown on the X-axis. Blood loss (ml/day) for given egg counts is also shown. Figure 8: Effect of methylated spirits on dipstick score. Definitions

As used herein, the term "comprising" means "including principally, but not necessarily solely". Furthermore, variations of the word "comprising", such as "comprise" and "comprises", have correspondingly varied meanings.

As used herein, the term "real-time", when used in relation to the method of quantifying parasite infection, means that the method can be performed and deliver quantitative information as to parasite infection within a short and predictable time frame. In particular, "real-time" means that the method can deliver quantitative information as to parasite infection substantially immediately after it has been performed for any particular animal, without significant lag time and without the animals needing to be herded into yards to implement requisite management procedures.

As used herein, the term "on-site", when used in relation to the method of quantifying parasite infection, means that the method can deliver quantitative information as to parasite infection at the same place in which the method is performed. In particular,

"on-site" means that the method can deliver information as to parasite infection without the animals needing to be herded into yards to implement requisite management procedures, or without performance of the method or generation of information arising from execution of the method requiring to be done at a different location to that of the herbivores, for example, in a diagnostic laboratory.

As used herein, the term "herbivore" means an animal that feeds mainly or exclusively on plant material.

As used herein, the term "ruminant" includes any of various hoofed, even-toed, usually horned mammals of the suborder Ruminantia, such as cattle, sheep, goats, buffalo, deer, and giraffes, characteristically having a stomach divided into four compartments and chewing a cud consisting of regurgitated, partially digested food.

As used herein, the term "bovid" includes any animal belonging to the family Bovidae, which includes hoofed, hollow-horned ruminants such as cattle, sheep, goats, and buffaloes. As used herein, the term "ovine" refers to sheep.

As used herein, the term "equidae" refers to the family of horse-like animals of the order Perissodactyla.

As used herein the term "spectrometer" includes any device capable of measuring either wavelengths or indices of refraction of light. In particular, "spectrometer" may include a handheld device capable of digitally reading the precise colour produced by a colourimetric assay.

Best Mode of Performing the Invention As there is significant blood loss in sheep infected with H. contortus by day 11 of infection, the present invention can provide a much earlier indicator of H. contortus infection than worm eggs, which first appear in faeces on day 18. The present invention can therefore provide a non-isotopic means of detecting and quantifying H. contortus infections at least one week before eggs appear in the faeces. A number of haematological parameters can be measured in sheep with H. contortus infections. Blood loss caused by infection can be estimated by the Hemoquant technique to measure faecal heme and heme breakdown products such as porphyrin, and by test strips or diffusion assays to measure faecal haemoglobin.

The present invention therefore contemplates methods for quantifying parasite infection in herbivores by measuring the amount of blood in the faeces of said herbivores. Accordingly, the present invention provides methods for quantifying parasite infection in a herbivore, wherein said methods comprise measuring the amount of blood in the faeces of said herbivore.

The amount of blood in said faeces may be measured by: (a) obtaining a faecal sample from said herbivore;

(c) subjecting said faecal sample to a colourimetric assay; and

(d) analyzing said colourimetric assay to determine the level of parasite infection. The pre-treating of said faecal sample may comprise contacting said faecal sample with an alcohol.

The alcohol may be selected from the group comprising methanol, butanol, propanol, methylated spirits or any combination thereof. The alcohol may be methylated spirits. The alcohol may be diluted to form an alcohol diluent at a concentration in the range of from about 5 - 50% (v/v) with water, or from about 10 - 40% (v/v) with water, or from about 15 - 35% (v/v) with water, or from about 20 - 30% (v/v) with water. The alcohol may be diluted to form an alcohol diluent at a concentration of about 25% (v/v) with water. The alcohol diluent may then be contacted with faeces at a ratio of from about 100 -

5000 parts diluent to 1 part faeces, or from about 200 - 4000 parts diluent to 1 part faeces, or from about 300 - 3000 parts diluent to 1 part faeces, or from about 400 - 2000 parts diluent to 1 part faeces, or from about 500 - 1800 parts diluent to 1 part faeces, or from about 600 - 1600 parts diluent to 1 part faeces, or from about 700 - 1400 parts diluent to 1 part faeces, or from about 800 - 1200 parts diluent to 1 part faeces, or from about 900 -

1100 parts diluent to 1 part faeces, or about 1000 parts diluent to 1 part faeces.

Alternatively, the alcohol diluent may be contacted with faeces at a ratio of about 300 parts diluent to 1 part faeces, or 2000 parts diluent to 1 part faeces. The use of this diluent is directed to ensuring that no peroxidase activity by contaminating plant or microbial peroxidases contributes to catalysis of the oxidation reaction, thereby compromising the assessment of parasite load.

In one preferred embodiment, the alcohol used is methylated spirits diluted 25%

(v/v) with water to form an alcohol diluent, and contacted with faeces at a ratio of about 1000 parts alcohol diluent to about 1 part faeces.

In another preferred embodiment, a sodium chloride (NaCl) solution may be used to dilute the alcohol rather than water. The working NaCl concentration may be in the range of from about 70% to 100%. The working NaCl concentration may be about 4M - 6M

NaCl. In another preferred embodiment, a two-step dilution process may be made, wherein the faeces may be firstly diluted 1:30 with water or NaCl, and then secondly further diluted in alcohol diluent (1 part alcohol to 33 parts water or NaCl), giving a total ratio of about 999 parts alcohol to about 1 part faeces.

The colourimetric assay may measure the amount of at least one of haemoglobin, myoglobin or leukocytes, preferably haemoglobin. The colourimetric assay may comprise the peroxidase activity of haemoglobin catalyzing an oxidation reaction, thereby producing a change in colour.

In a preferred embodiment, the substrate being oxidized can be affixed to a strip to provide a testing strip, or alternatively affixed to filter paper to provide a diffusion assay. In a further preferred embodiment, the oxidation reaction comprises catalysis of the oxidation of diisopropylbenzene and 3,3',5,5'-tetramethylbenzidine, thereby producing a change in colour.

The colourimetric assay can be analyzed by visual comparison of the result of the assay against a set of defined colour standards, wherein the colour standards correspond to levels of parasite infection. The colourimetric assay may involve the use of a commercially available reagent testing strip and/or filter paper. The commercially available reagent testing strip may be a Hemastix®, available from Bayer Australia Ltd, PO Box 903875 Pacific Highway Pymble NSW 2073Australia. Hemastix® comprise a strip to which is affixed a reagent area that tests for occult blood. The reagent test strip is ready to use upon removal from the bottle and the entire reagent test strip is disposable. No additional laboratory equipment is necessary for testing. The strip is suitable for the methods, systems and kits described herein.

Additionally or alternatively, the colourimetric assay can be analyzed by use of a spectrometer, preferably adapted for portable use.

Additionally or alternatively, the analysis may comprise a means for capturing an image of the result of said colourimetric assay, such as with a camera.

The analysis may further comprise use of a computer program. The computer program may quantify the amount of red, green and blue in each pixel of an image of the result of said colourimetric assay.

The present invention also provides methods for quantifying parasite infection in a herbivore, wherein said methods comprise measuring the amount of blood in the faeces of said herbivore, wherein the amount of blood in said faeces is measured by:

(c) analyzing said colourimetric assay to determine the level of parasite infection.

The methods may further comprise pre-treating said faecal sample to inactivate peroxidase activity emanating from sources other than from haemoglobin of the herbivore. The pre-treating of said faecal sample may comprise contacting said faecal sample with an alcohol.

The present invention additionally provides methods for quantifying parasite infection in a herbivore, wherein said methods comprise measuring the amount of blood in the faeces of said herbivore, wherein the amount of blood in said faeces is measured by: (a) obtaining a faecal sample from said herbivore;

(c) subjecting said faecal sample to a colourimetric assay; and

The present invention further provides methods for detecting infection of at least one blood-sucking parasite in a herbivore, wherein said methods comprise detecting the presence of blood in the faeces of said herbivore.

The presence of blood in said faeces may be detected by:

(a) obtaining a faecal sample from said herbivore;

(b) pre-treating said faecal sample to inactivate peroxidase activity emanating from sources other than from haemoglobin of the herbivore; (c) subjecting said faecal sample to a colourimetric assay; and

(d) analyzing said colourimetric assay to detect parasite infection. The pre-treating of said faecal sample may comprise contacting said faecal sample with an alcohol.

The type of alcohol, its diluent, dilution and ratio for contacting with the faecal sample may be selected as described above.

An amount of blood in faeces producing a colour change in excess of grade 2 on a Hemastix panel may be indicative of parasite infestation. This colour change may be produced when faeces is diluted 1 in 1000 in an alcohol diluent, wherein the colours of a 5 point reaction scale depicted on the Hemastix colour chart are given numbers wherein 1 = no change, 2 = trace amount of infestation, 3 = small amount of infestation, 4 = moderate amount of infestation and 5 = large amount of infestation.

The present invention moreover provides methods for detecting infection of at least one blood-sucking parasite in a herbivore, wherein said methods comprise detecting the presence of blood in the faeces of said herbivore, wherein the presence of blood in said faeces is detected by:

(a) obtaining a faecal sample from said herbivore;

(b) subjecting said faecal sample to a colourimetric assay; and (c) analyzing said colourimetric assay to detect parasite infection.

The methods may further comprise pre-treating said faecal sample to inactivate peroxidase activity emanating from sources other than from haemoglobin of the herbivore. The pre-treating of said faecal sample may comprise contacting said faecal sample with an alcohol. The type of alcohol, its diluent, dilution and ratio for contacting with the faecal sample may be selected as described above.

The present invention also provides methods for detecting infection of at least one blood-sucking parasite in a herbivore, wherein said methods comprise detecting the presence of blood in the faeces of said herbivore, wherein the presence of blood in said faeces is detected by:

(a) obtaining a faecal sample from said herbivore;

The type of alcohol, its diluent, dilution and ratio for contacting with the faecal sample may be selected as described above. hi addition, faecal porphyrin values as determined by the present invention can also be correlated with faecal egg counts and with worm burdens. Digital photographs of colour changes in an indicator substrate dipped in an appropriate dilution of faeces to water (e.g. 1:300) can be quantified using computer software. Colour change values as determined by computer software can be expressed as the ratio of green to red units within a defined area on an indicator substrate patch. For example, ER mapping software quantifies on a scale of 0-255 the amount of red, green and blue in each pixel within a defined area of an indicator substrate square on a test strip. In doing so, there is higher correlation with FEC and worm burdens than with hemoquant values. Another advantage of the present invention is that faecal blood can be detected in sheep with larval H. contortus post infection before eggs are present in the faeces. Visual assessment of colour change in the substrate can also identify sheep with low and high egg counts. As test strip assays are quick, semi-quantative, inexpensive and blood loss can be detected before infections become apparent, the present invention is a particularly useful means of estimating H. contortus burdens.

Although the test strip readings can be converted to numerical values for statistical analysis in the present study, they can also be read visually. For example, the colour of indicator patches is yellow in the absence of haemoglobin, which changes to pale green, and then dark green and then blue as the level of haemoglobin increases. Farmers in the field can easily assess these differences within minutes of collecting a faecal sample. The colour change provides an instant measure of the damage being done.

Utilizing the peroxidase-like activity of haemoglobin to catalyze the reaction of diisoproppylbenzene dihydroperoxide and 3,3 ',5,5'- tetramethylbenzidine and thereby producing a colour change relative to the amount of heme present in the sample appears more sensitive than catalyzing the oxidation of alpha-guaiaconic acid to heme levels in faeces. Consequently, the present invention is sensitive enough to detect heme levels in faecal suspension to allow it to be used to diagnose H. contortus infections in sheep.

Accordingly, in another embodiment, the methods for quantifying and/or detecting parasite infection in a herbivore further comprise the prior addition of at least one protease enzyme. The use of this enzyme is directed to ensuring that no peroxidase activity by contaminating microbial peroxidases contributes to catalysis of the oxidation reaction, thereby compromising the assessment of parasite load.

In a preferred embodiment, the infection being quantified results from Haemonchus contortus, and the herbivore is a ruminant or equidae. More preferably, the ruminant is a bovid or the equidae is a horse. More preferably still, the bovid is an ovine.

In a further preferred embodiment, the methods for quantifying and/or detecting parasite infection in a herbivore are performed in real-time and more preferably in addition on-site. The present invention also provides systems for quantifying parasite infection in a herbivore, wherein said systems comprise means for measuring the amount of blood in the faeces of said herbivore.

Said means for measuring the amount of blood in said faeces may comprise:

The colourimetric assay may measure the amount of at least one of haemoglobin, myoglobin or leukocytes. The colourimetric assay may measure the amount of haemoglobin.

The colourimetric assay may comprise the peroxidase activity of haemoglobin catalyzing an oxidation reaction, thereby producing a change in colour. The oxidation reaction may comprise catalysis of the oxidation of diisopropylbenzene and 3,3',5,5'- tetramethylbenzidine, thereby producing a change in colour.

The means for pre-treating said faecal sample may involve contacting said faecal sample with an alcohol. The type of alcohol, its diluent, dilution and ratio for contacting with the faecal sample may be selected as described above.

The means for analyzing may comprise visual comparison of the result of said colourimetric assay against defined colour standards, wherein said colour standards correspond to levels of parasite infection. The means for analyzing may comprise a spectrometer.

Additionally or alternatively, the means for analyzing may comprise a means for capturing an image of the result of said colourimetric assay. Said means for capturing an image may comprise a camera. Said camera may comprise a digital camera.

The present invention additionally provides systems for quantifying parasite infection in a herbivore, wherein said systems comprise means for measuring the amount of blood in the faeces of said herbivore, wherein said means for measuring the amount of blood in said faeces comprises:

(a) means for obtaining a faecal sample from said individual herbivore;

The systems may further comprise means for pre-treating said faecal sample to inactivate peroxidase activity emanating from sources other than from haemoglobin of the herbivore. The means for pre-treating of said faecal sample may involve contacting said faecal sample with an alcohol. The type of alcohol, its diluent, dilution and ratio for contacting with the faecal sample may be selected as described above.

The present invention further provides systems for quantifying parasite infection in a herbivore, wherein said systems comprise means for measuring the amount of blood in the faeces of said herbivore, wherein said means for measuring the amount of blood in said faeces comprises: (a) means for obtaining a faecal sample from said individual herbivore;

The means for pre-treating of said faecal sample may involve contacting said faecal sample with an alcohol. The type of alcohol, its diluent, dilution and ratio for contacting with the faecal sample may be selected as described above.

The present invention also provides systems for the intensive management of at least one herbivore, wherein said systems comprise:

(a) means for identifying an individual herbivore; (b) means for quantifying parasite infection in said individual herbivore, comprising means for measuring the amount of blood in the faeces of said individual herbivore, comprising:

(iv) means for analyzing said colourimetric assay to determine the level of parasite infection; (c) means for processing information obtained by said means for quantifying parasite infection; and

The present invention additionally provides systems for the intensive management of at least one herbivore, wherein said systems comprise: (a) means for identifying an individual herbivore; (b) means for quantifying parasite infection in said individual herbivore, comprising means for measuring the amount of blood in the faeces of said individual herbivore, comprising:

(i) means for obtaining a faecal sample from said individual herbivore; (ii) a colourimetric assay for testing the amount of blood in said faecal sample; and

(iii) means for analyzing said colourimetric assay to determine the level of parasite infection;

The type of alcohol, its diluent, dilution and ratio for contacting with the faecal sample may be selected as described above. Optionally, the system further comprises a database for storing, correlating and retrieving information.

In one embodiment, the means for processing information correlates processed information with external information to determine the intensive management strategy for the individual herbivore. Preferably, the external information comprises at least one of pasture information or industry benchmark information for animal health and performance. More preferably, the pasture information comprises at least one of pasture worm load information or pasture feed capacity information, and the industry benchmark information comprises treatment information for Haemonchus contortus infection. hi another embodiment, the implementing strategies comprise at least one of treatment for parasite infection, quarantine or culling. Preferably, the treatment comprises treatment for Haemonchus contortus infection.

The present invention further provides kits for quantifying parasite infection in a herbivore, wherein said kits comprise means for measuring the amount of blood in the faeces of said herbivore. The means for measuring the amount of blood in the faeces of said herbivore may comprise:

(a) means for obtaining a faecal sample from said herbivore;

The present invention moreover provides kits for quantifying parasite infection in a herbivore, wherein said kits comprise means for measuring the amount of blood in the faeces of said herbivore, wherein said means for measuring the amount of blood in the faeces of said herbivore comprises:

(a) means for obtaining a faecal sample from said herbivore;

The type of alcohol, its diluent, dilution and ratio for contacting with the faecal sample may be selected as described above. The present invention also provides kits for quantifying parasite infection in a herbivore, wherein said kits comprise means for measuring the amount of blood in the faeces of said herbivore, wherein said means for measuring the amount of blood in the faeces of said herbivore comprises:

The present invention additioanlly provides methods for the intensive management of at least one herbivore, wherein said methods comprise:

(a) identifying an individual herbivore;

(b) quantifying the level of parasite infection in said individual herbivore, wherein said quantifying comprises measuring the amount of blood in the faeces of said individual herbivore;

(c) processing information obtained by said testing; and

(d) implementing strategies for the intensive management of said individual herbivore based upon said processed information. In one embodiment, the processed information is correlated with external information to determine the intensive management strategy for the individual herbivore. Preferably, the external information comprises at least one of pasture information or industry benchmark information for animal health and performance. More preferably, the pasture information comprises at least one of pasture worm load information or pasture feed capacity information, and the industry benchmark information comprises treatment information for Haemonchus contortus infection.

In another embodiment, the implementing of strategies comprises at least one of treatment for parasite infection, quarantine or culling. Preferably, the treatment comprises treatment for Haemonchus contortus infection. The present invention will now be further described in greater detail by reference to the following specific examples, which should not be construed as in any way limiting the scope of the invention.

Examples Example 1 : General Methods

1.1 Faecal collection and egg counts

Faeces were not collected direct from the rectum to avoid the possibility that rectal 5 manipulation would damage the tissues and result in contamination of the sample with blood. Instead, sheep were fitted with faecal collection harnesses or held in clean pens and faeces collected from the floor. Faeces collected in this way were used in Haemoquant and test strip analyses for the presence of haemoglobin. Faecal egg counts were done using the modified McMaster technique, with a sensitivity of 100 eggs/gram. 0

1.2 HaemoQuant test

The Hemoquant test is a quantitative method for measuring blood in faeces (Schwartz et al. 1983). Using this technique, total haemoglobin in faeces can be reacted with a heated oxalic acid:FeSO₄ reagent, converting any remaining heme to porphyrin s without loss of the porphyrin pre-formed from heme in the herbivore intestine. The conversion of non-fluorescing heme to fluorescing porphyrin allows the amount of heme to be indirectly estimated by comparing the sample with standards using a spectrophotometer.

0 1.3 Testing for haemoglobin with Hemastix

The faeces to be tested for the presence of haemoglobin were diluted in water. In this procedure, one gram of sheep faeces was added to 49 g of water and mixed with a faecal mixing drill. The mixture was then diluted with water to make a final solution of 1 part faeces to 300 parts water. Bayer Hemastix for Urinalysis were used to test for the 5 presence of haemo globulin in the faecal solution. Hemastix have a single blood diagnostic patch that changes colour based on the peroxidase-like activity of haemoglobin catalyzing the reaction of diisoproppylbenzene dihydroperoxide and 3,3 ',5,5'- tetramethylbenzidine. The colour changes range from orange to green and with very high levels of haemoglobin the change continues to blue. The reagent strip was dipped into the faecal solution for 3 o seconds, then taken out and blotted briefly on its edge and then laid face up on a piece of paper for 1 minute, then rinsed in water prior to photographing. The strip was photographed with a 3.1 megapixel digital camera and saved as a Tiff file. The Tiff file was interrogated with ER Mapper 6.4 software, a desktop imaging and mapping tool for photo imagery. ER Mapper quantifies from 0 - 255 the amount of red, green and blue in 5 each pixel within a defined area of the blood reagent square on the test strip. The output was given as the average of each colour with the standard deviation and variance. Since the defined area may vary between samplings the ratio of green to red units was used in the statistical analysis of the data.

1.4 Control procedure for contaminating microbial peroxidases

Microbial peroxidases can also catalyze the reaction of diisoproppylbenzene dihydroperoxide and 3,3',5,5'-tetramethylbenzidme in Hemastix. Consequently, there is a possibility that microbial peroxidases in the faecal suspension could produce false positives changes in Hemastix colour. To overcome the possibility of false positive colour changes a test was conducted to determine if digestion of the microbial peroxidases with proteinase K would improve the accuracy of the reagent strip results. A stock solution of 20mg/ml of proteinase K was diluted to 10μg/5ml of the faecal suspension and incubated for 30 minutes at 37⁰C. After incubation the suspension was allowed to cool to room temperature before the strip was dipped. The remainder of the procedure was the same as that for samples that did not have a protease digestion step. The colour changes in Hemastix dipped in proteinase K treated and non-treated faecal solutions were photographed and scanned by ER mapper. A visual assessment of the Hemastix reactive patch was carried out concurrently with the photographing. The colours of a 5 point reaction scale depicted on the Hemastix colour chart were given numbers where 1 = no change, 2 = trace, 3 = small, 4 = moderate and 5 = large. These values were recorded and compared with the sheep's FEC.

1.5 Control procedure for contaminating plant peroxidases

Plant peroxidases can also catalyze the reaction of diisoproppylbenzene dihydroperoxide and 3,3',5,5'-tetramethylbenzidine in Hemastix. Consequently, there is a possibility that plant peroxidases in the faecal suspension could produce false positives changes in Hemastix colour. To overcome the possibility of false positive colour changes a test was conducted to determine if denaturation of the plant peroxidases with methylated spirits would improve the accuracy of the reagent strip results. A faecal suspension containing 1 part faeces to 30 parts water was further diluted in a diluent containing 25 parts methylated spirits_and 75 parts water to yield a final dilution of 1 part faeces to 1000 parts diluent. The remainder of the procedure was the same as that for samples that did not have an alcohol denaturation step. The colour changes in Hemastix dipped in ethanol diluted and ethanol-free faecal solutions were scored visually. The colours of a 5 point reaction scale depicted on the Hemastix colour chart were given numbers where 1 = no change, 2 = trace, 3 = small, 4 = moderate and 5 = large. These values were recorded and compared with the sheep's FEC. Similar results were obtained with concentrations of ethanol, methanol and propanol between 25 and 30 %).

1.6 Parasite Infections

The H. contortus infected sheep that were slaughtered for worm counts came from 3 different treatment groups, hi one, 15 yearling wethers were infected with 15,000 H. contortus larvae and, prior to slaughter on day 36-post infection, faecal collection harnesses were used to collect faeces on days 13, 15, 17 and 35 post infection. On days 13, 15 and 17 nine faeces were collected from nine of the infected sheep and five non- infected sheep, which were controls. The second group consisted of nine sheep that were infected with H. contortus strains for routine larval production at the F. D. McMaster Laboratory. These infections were with 5,000 larvae and were in excess of 2 months old. The third group was 17 sheep from the H. contortus selection flocks that were kept at the University of New England sheep house and were infected with 10,000 larvae. Some of the sheep in each of the three groups had zero FEC and worm counts at the time of testing and therefore were also controls on the blood parameter measures.

1.7 Worm Counts Sheep were slaughtered for worm counts and their abomasum removed immediately and the contents were washed with water and scraped into two-litre graduated cylinders. After settling, the abomasal fluid was reduced to a volume of 600ml. Three 30ml sub- samples were frozen for counting and the remainder was discarded. The worm burdens in the abomasum were based on counts of three replicate 5% samples.

Example 2: Assessment of Parasite Load by Ηemoquant and Ηaemostick

The three different H. contortus infections had a similar correlation between FEC and worm burden so that the data was combined for analysis. The correlation between

FEC and worm burden for the combined data was r² = 0.7726; P<0.001 (Figure 1). The faecal porphyrin values as determined by hemoquant analysis were correlated with FEC

(r² = 0.2484; P<0.01) and with worm burdens (r² = 0.5215; P<0.001) (Figure 2).

Ηemastix values as determined by the ER mapper readings were also correlated with the parasite burden indicators of faecal egg counts, worm burdens and with the hemaquant values. Ηemastix readings were even more highly correlated with faecal egg count (r² = 0.5960, P<0.001) and worm burdens (r² = 0.68780, PO.001) (Figure 3) than were the hemoquant values.

Faecal blood was also detected in sheep with larval H. contortus 13 days post infection. The correlation between hemoquant coproporphyrins on day 13 and the FEC at day 36 of the infection was r² = 0.74, P<0.001 (Figure 4); while the correlation between the Hemastix green to red ratio and FEC was r² = 0.84, P<0.001 (Figure 5). The correlation between the two methods of estimating faecal blood in patent infections was r² = 0.2393, P = 0. 01.

Example 3: Use of Proteinase K in Assessment of Parasite Load

Faecal suspensions that were incubated with Proteinase K did not produce Hemastix test strip readings that were significantly different from the non-treated suspensions and consequently are not shown. This demonstrates that there was no peroxidase activity by contaminating microbial peroxidases in the assessments of parasite load.

Example 4: Use of Methylated Spirits in Assessment of Parasite Load

Faecal suspensions that were diluted in an aqueous medium containing 25% methylated spirits gave Hemastix test strip readings in uninfected sheep from a score of 2 to a score of 1. Faeces were collected from four sheep infected with Haemonchus contortus average 1135 eggs per gram of faeces and 4 sheep with very low Haemonchus contortus (8 eggs per gram). Faeces from each animal were diluted to 1 in 30 in water. A subsample from each preparation was further diluted 1 in 33 in water containing 25%, 30% or 35% methylated spirits yielding a final concentration of 1 part faeces to 998 parts diluent. Samples were tested with Hemastix and the colour change scored on a scale from 1 to 5. As shown in Figure 8, faeces from infected sheep, when tested in water gave a Hemastix score of 3.375. In methylated spirits of 25%, 30% and 35% (v/v), Hemastix scores were 2.375, 2.0 and 1.25 respectively. Faeces from sheep with very low infections of Haemonchus, when tested in water, gave a Hemastix score of 2.125. In methylated spirits of 25%, 30% and 35% (v/v), Hemastix scores were 1.25, 1.25 and 1.0 respectively. These results demonstrate that the peroxidase activity of plant-derived peroxidases that can contaminate faeces can be denatured by methylated spirits.

Example 5: Visual Assessment of Hemastix

Table 1 demonstrates the relationship between visual assessment of colour change and parasite burden. It can be seen that although some animals with apparent infections had a low FEC they were still assessed to have no colour change on the Hemastix. All of the animals with high FECs were assessed as positive for blood heme in the faecal suspension. In the Hemastix test for faecal blood in 13 day old infections all of the sheep that later developed FEC were assessed as positive while none of the uninfected controls were positive for faecal blood.

Table 1. The relationship between visual assessment of Hemastix colour changes depicted on Hemastix colour chart (where 1 = no change, 2 = trace, 3 = small, 4 = moderate and 5 = large) and the FEC. The Hemastix score at day 13 was compared with the FEC of the same sheep on

10 day 35 of their infection.

Example 6: Predicting FEC from dipstick score

I₅ hi order to demonstrate the technique of estimating faecal egg counts from the colour change observed visually in test strips, the mean egg counts were plotted against the indicator substrate as converted to numerical values. These values were: 1 = no change; 2 = trace; 3 = small; 4 = moderate; 5 = large. This relationship is shown in Figure 6.

20

Example 7: Detection and management of internal parasite infections

Two parasite groups that are of significant economic importance in sheep management and production are Haemonchus contortus and the "scour worm" group (main species: Trichostrongylus colubriformis, T. vitrinus, Ostertagia (Teladorsagia) circumdncta and Nematodirus species). H. contortus is a parasite of the abomasum and sucks blood from that part of the digestive tract. Severe infections with H. contortus result in severe blood loss, acute anemia and death. The development of severe haemonchosis is rapid and animals can die within weeks of a severe infection. Trichostrongylus and other "scour worms", on the other hand, are parasites of the gastrointestinal tract that cause decreased efficiency of feed utilisation, slightly reduced intake and sub-optimal performance over extended periods. Infections of "scour worms" are usually chronic and do not cause the same acute problems and risk of death associated with haemonchosis.

Example 7a: Diagnosis and treatment for haemonchosis

Sheep grazing pastures in an environment endemic for H. contortus pass through a remote individual animal management system (RIAM) on a daily basis as they move to and from water. The weight of each animal is recorded and there is scan of the facial tissue and eye using remote image analysis linked to the RIAM in order to monitor haematocrit (anemia) on a daily basis. The database in the computer linked to the RIAM contains information about the historical performance of each animal and updates information on weight change, rank in group for weight and weight change, as well as rank in group for haematocrit score. Animals that have a sudden change in their absolute reading and in their performance relative to other animals in the flock are drafted to the quarantine pen via an automatic drafting system and a message sent via CDMA modem to alert the manager and/or the veterinarian to the fact that there is a need to check animals in the quarantine pen. Information is transmitted on a regular basis in order to be able to monitor the number of animals requiring treatment. The manager or veterinarian is able to download information on the sheep drafted into the quarantine pen and use this information together with a physical examination to determine the appropriate treatment. Treatment options are to have animals that are starting to decline in body weight and haematocrit drafted to a pen with supplementary feeding available each day to assist the animal overcome the parasite burden through nutrition-enhanced activity of the immune system while being monitored closely. The second strategy is to drench the animal with an anthelmintic. AU records of treatment are stored in the database.

It has been demonstrated that H. contortus infection in weaners has a negative influence on their weight gains. The effect of H. contortus infection on productivity of weaner lambs at pasture can best be predicted by haemocrits. For each 0.01 proportional decrease in haematocrit there is a 0.03 reduction in live weight gain (Albers et al. 1990). Hematocrit decreases can be determined from the daily blood loss of sheep (Albers and LeJambre, 1983) and blood loss determined from faecal egg counts. This relationship is exemplified in Figure 7. It is therefore possible to determine the effect that a given faecal egg count will have on liveweight gain, and in this regard Figure 7 emphasises the value 5 of diagnosis of parasitism based on faecal blood loss in order to identify and remove the infection before production loss occurs. Figure 7 also indicates that such a correlation can be used in devising and implementing management strategies via radio frequency identification (RFID) tags to identify animals in combination with automated walkthrough weighing to allow measurement of individual animal weight change at relatively io low cost. The use of weight change data for diagnostic purposes is facilitated by use of algorithms for weight change analysis that enables comparison of weight change performance of each individual animal with its own previous performance, that of the cohort group, and its changing rank in the cohort.

i₅ Example 7b: Diagnosis and treatment for "Scour worm" infection

"Scour worm" infection is diagnosed by animals grazing under the same conditions as described above for H. contortus. Gradual loss of performance is seen through lower growth rate or faster weight loss, both of which are triggers for further analysis. The first test is to examine whether there is heme present in the faeces through the use of a test

2Q strip. If there is heme present in the faeces, the animal is diagnosed not to have H contortus and highly likely to have Trichostrongylosis or other "scour worm" infection. These animals can be drafted to a paddock containing medicated feed, where they will have one meal of feed containing anthelmintic. Subsequent performance is monitored and if there is no improvement in condition, animals are drafted into the quarantine pen and ₅ the manager alerted to examine the animals, with a view to additional treatment. Animals detected as infected may also be drafted into a group for specific nutritional supplementation to boost natural immunity to nematode infection. References

Albers, G. A. A., Gray, G. D., Le Jambre, L. F. Barger, I. A. and Barker, J. S. F. 1990.

The effect of Haemonchus contortus infection on haematological parameters in young Merino sheep and its significance for productivity. Animal Production 50;

99-109.

Albers, G. A. A., Le Jambre, L. F. 1983. Erythrocyte potassium concentration: a simple parameter for erythropoiesis in sheep infected with Haemonchus contortus. Res. Vet. Sci. 35, 273-276. Allemann, A., Bauerfeind, P., Gyr, N. 1994. Prevalence of hookworm infection, anaemia and faecal blood loss among the Yupno people of Papua New Guinea. P. N. G. Med. J. 37, 15 -22.

Bauerfeind, P., Allemann, A. and Gyr, N. 1992. Differential diagnostic value of occult blood in stool in hookworm infection in the Third World. Study from Papua New Guinea. J Gastroenterol. 30; 791-4.

Schlam, O. W., Jain, N. C. and Carroll, E. J. 1975. Veterinary Haemotology. 3^rd ed.

Philadelphia, Lea & Febiger.

Schwartz, S., Dahl, J., Ellefson, M., Ahlquist D. 1983. The "HemoQuant" Test: A specific and quantitative determination of heme (haemoglobin) in faeces and other materials. Clin. Chem. 29, 2061-2067.

Claims

1. A method for quantifying parasite infection in a herbivore, wherein said method comprises measuring the amount of blood in the faeces of said herbivore.

2. The method according to claim 1, wherein the amount of blood in said faeces 5 is measured by:

(a) obtaining a faecal sample from said herbivore;

(c) subjecting said faecal sample to a colourimetric assay; and io (d) analyzing said colourimetric assay to determine the level of parasite infection.

3. A method for quantifying parasite infection in a herbivore, wherein said method comprises measuring the amount of blood in the faeces of said herbivore, wherein the amount of blood in said faeces is measured by:

(a) obtaining a faecal sample from said herbivore; I₅ (b) subjecting said faecal sample to a colourimetric assay; and

4. The method according to claim 3, wherein said method further comprises pre- treating said faecal sample to inactivate peroxidase activity emanating from sources other than from haemoglobin of the herbivore.

20 5. A method for quantifying parasite infection in a herbivore, wherein said method comprises measuring the amount of blood in the faeces of said herbivore, wherein the amount of blood in said faeces is measured by:

(a) obtaining a faecal sample from said herbivore;

(b) pre-treating said faecal sample to inactivate peroxidase activity emanating ₅ from sources other than from haemoglobin of the herbivore;

(c) subjecting said faecal sample to a colourimetric assay; and

(d) analyzing said colourimetric assay to determine the level of parasite infection.

6. A method for detecting infection of at least one blood-sucking parasite in a herbivore, wherein said method comprises detecting the presence of blood in the faeces of 0 said herbivore.

7. The method according to claim 6, wherein the presence of blood in said faeces is detected by:

(a) obtaining a faecal sample from said herbivore;

(b) pre-treating said faecal sample to inactivate peroxidase activity emanating ₅ from sources other than from haemoglobin of the herbivore; (c) subjecting said faecal sample to a colourimetric assay; and

(d) analyzing said colourimetric assay to detect parasite infection.

8. A method for detecting infection of at least one blood-sucking parasite in a herbivore, wherein said method comprises detecting the presence of blood in the faeces of said herbivore, wherein the presence of blood in said faeces is detected by:

(a) obtaining a faecal sample from said herbivore;

(b) subjecting said faecal sample to a colourimetric assay; and

(c) analyzing said colourimetric assay to detect parasite infection.

9. The method according to claim 8, wherein said method further comprises pre- treating said faecal sample to inactivate peroxidase activity emanating from sources other than from haemoglobin of the herbivore.

10. A method for detecting infection of at least one blood-sucking parasite in a herbivore, wherein said method comprises detecting the presence of blood in the faeces of said herbivore, wherein the presence of blood in said faeces is detected by: (a) obtaining a faecal sample from said herbivore;

(c) subjecting said faecal sample to a colourimetric assay; and

(d) analyzing said colourimetric assay to detect parasite infection.

11. The method according to any one of claims 2, 4, 5, 7, 9 or 10, wherein said pre-treating of said faecal sample comprises contacting said faecal sample with an alcohol.

12. The method according to claim 11, wherein said alcohol is methylated spirits.

13. The method according to any one of claims 2 to 12, wherein the colourimetric assay measures the amount of at least one of haemoglobin, myoglobin or leukocytes.

14. The method according to claim 13, wherein the colourimetric assay comprises the peroxidase activity of haemoglobin catalyzing an oxidation reaction, thereby producing a change in colour.

15. The method according to claim 14, wherein the oxidation reaction comprises catalysis of the oxidation of diisopropylbenzene and 3,3',5,5'-tetramethylbenzidine, thereby producing a change in colour.

16. The method according to any one of claims 2 to 15, wherein the analyzing involves visual comparison of the result of said colourimetric assay against defined colour standards, wherein said colour standards correspond to levels of parasite infection.

17. The method according to any one of claims 12 to 18, wherein the analyzing involves use of a spectrometer.

18. A system for quantifying parasite infection in a herbivore, wherein said system comprises a means for measuring the amount of blood in the faeces of said

5 herbivore.

19. The system according to claim 18, wherein said means for measuring the amount of blood in said faeces comprises:

(a) means for obtaining a faecal sample from said individual herbivore;

(b) means for pre-treating said faecal sample to inactivate peroxidase activity o emanating from sources other than from haemoglobin of the herbivore;

(d) means for analyzing said colourimetric assay to determine the level of parasite infection. s

20. A system for quantifying parasite infection in a herbivore, wherein said system comprises means for measuring the amount of blood in the faeces of said herbivore, wherein said means for measuring the amount of blood in said faeces comprises:

(a) means for obtaining a faecal sample from said individual herbivore; o (b) a colourimetric assay for testing the amount of blood in said faecal sample; and

21. The system according to claim 20, wherein said system further comprises 5 means for pre-treating said faecal sample to inactivate peroxidase activity emanating from sources other than from haemoglobin of the herbivore.

22. A system for quantifying parasite infection in a herbivore, wherein said system comprises means for measuring the amount of blood in the faeces of said herbivore, wherein said means for measuring the amount of blood in said faeces o comprises:

(a) means for obtaining a faecal sample from said individual herbivore;

(c) a colourimetric assay for testing the amount of blood in said faecal sample; 5 and (d) means for analyzing said colourimetric assay to determine the level of parasite infection.

23. The system according to any one of claims 19, 21 or 22, wherein said pre- treating of said faecal sample comprises contacting said faecal sample with an alcohol.

24. The system according to claim 23, wherein said alcohol is methylated spirits.

25. The system according to any one of claims 19 to 24, wherein the colourimetric assay measures the amount of at least one of haemoglobin, myoglobin or leukocytes.

26. The system according to claim 25, wherein the colourimetric assay comprises the peroxidase activity of haemoglobin catalyzing an oxidation reaction, thereby producing a change in colour.

27. The system according to claim 26, wherein the oxidation reaction comprises catalysis of the oxidation of diisopropylbenzene and 3,3',5,5'-tetramethylbenzidine, thereby producing a change in colour.

28. The system according to any one of claims 19 to 27, wherein the means for analyzing comprises visual comparison of the result of said colourimetric assay against defined colour standards, wherein said colour standards correspond to levels of parasite infection.

29. The system according to any one of claims 19 to 27, wherein the means for analyzing comprises a spectrometer.

30. A system for the intensive management of at least one herbivore, wherein said system comprises:

(a) means for identifying an individual herbivore;

(i) means for obtaining a faecal sample from said individual herbivore; (ii) means for pre-treating said faecal sample to inactivate peroxidase activity emanating from sources other than from haemoglobin of the herbivore; (iii) a colourimetric assay for testing the amount of blood in said faecal sample; and

(c) means for processing information obtained by said means for quantifying parasite infection; and (d) means for implementing strategies for the intensive management of said individual herbivore based on parasite infection status.

31. A system for the intensive management of at least one herbivore, wherein said system comprises: (a) means for identifying an individual herbivore;

32. The system according to claim 31, wherein said means for quantifying parasite infection in said individual herbivore further comprises means for pre-treating said faecal sample to inactivate peroxidase activity emanating from sources other than from haemoglobin of the herbivore.

33. A kit for quantifying parasite infection in a herbivore, wherein said kit comprises means for measuring the amount of blood in the faeces of said herbivore.

34. The kit according to claim 33, wherein said means for measuring the amount of blood in the faeces of said herbivore comprises:

(a) means for obtaining a faecal sample from said herbivore;

35. A kit for quantifying parasite infection in a herbivore, wherein said kit comprises means for measuring the amount of blood in the faeces of said herbivore, wherein said means for measuring the amount of blood in the faeces of said herbivore comprises:

(a) means for obtaining a faecal sample from said herbivore;

36. The kit according to claim 35, wherein said means for measuring the amount of blood in the faeces of said herbivore further comprise means for pre-treating said faecal sample to inactivate peroxidase activity emanating from sources other than from haemoglobin of the herbivore.

37. A kit for quantifying parasite infection in a herbivore, wherein said kit comprises means for measuring the amount of blood in the faeces of said herbivore, wherein said means for measuring the amount of blood in the faeces of said herbivore comprises:

(a) means for obtaining a faecal sample from said herbivore;

38. The kit according to any one of claims 34, 36 or 37, wherein said pre-treating of said faecal sample comprises contacting said faecal sample with an alcohol.

39. The kit according to claim 38, wherein said alcohol is methylated spirits.

40. The kit according to any one of claims 34 to 39, wherein the colourimetric assay measures the amount of at least one of haemoglobin, myoglobin or leukocytes.

41. The kit according to claim 40, wherein the colourimetric assay comprises the peroxidase activity of haemoglobin catalyzing an oxidation reaction, thereby producing a change in colour.

42. The kit according to claim 41, wherein the oxidation reaction comprises catalysis of the oxidation of diisopropylbenzene and 3,3',5,5'-tetramethylbenzidine, thereby producing a change in colour.

43. The kit according to any one of claims 34 to 42, wherein the means for analyzing comprises visual comparison of the result of said colourimetric assay against defined colour standards, wherein said colour standards correspond to levels of parasite infection.

44. The kit according to any one of claims 34 to 42, wherein the means for analyzing comprises a spectrometer.

45. A method for the intensive management of at least one herbivore, wherein said method comprises:

(a) identifying an individual herbivore;

(c) processing information obtained by said testing; and