Crypto - Screening for sub-clinical carriers
By Dr Mackie Hobson BSc(Agric),BVSc

1. Field Studies

Crypto - Screening for sub-clinical carriers

Cryptosporidium parvum, causes diarrhoea in young and immunocompromised animals and is usually associated with explosive outbreaks and significant mortalities in calves, lambs and kids.

It can be transmitted from man to animals (Zooanthroponosis) as well as from animals to man (Zoonosis). The initial outbreaks in South Africa were likely from human sewerage effluent entering our river and water systems and ending up on irrigated pastures or in livestock drinking water. However, the movement of animals has now become a reason for the infection spreading.

Biosecurity is critical in maintaining a disease-free environment on any Angora goat producer’s farm. Is it possible to screen healthy carrier adults to prevent the spread of crypto through the Mohair industry?

For more information on Cryptosporidiosis in Angora goats see:

https://www.angoras.co.za/article/cryptosporidiosis-in-angora-goats

What are the current testing methods used to diagnose Cryptosporidium?

There are a number of diagnostic tests available to test for Cryptosporidium parvum. These have a high sensitivity and specificity in the diagnosis of clinical cases.

Sensitivity- the ability of a test to correctly identify animals with crypto (True Positive).

Specificity- the ability of a test to correctly identify animals without crypto (True negative).

What is the ability of these tests to pick up SUBCLINICAL CARRIERS (ie. goats that are not showing any clinical signs but are shedding low numbers of Cryptosporidium oocytes)? To make testing for carriers even more difficult Cryptosporidium positive animals are known to shed oocytes intermittently so may test negative although still a carrier?

By testing a number of individuals from a flock it would be hoped that this would overcome the problem of detecting intermittent shedders to screen a flock or farm.

Field studies were conducted by Mohair SA to determine if there was any merit in using any of these tests to detect carrier animals.

1. Clocolan - Eastern Free State

13 healthy sheep, on a Crypto positive farm that was currently experiencing lamb deaths due to Crypto, were tested.

1. 5 rams that had been on the farm for over a year.
2. 3 rams that had been introduced 3 months prior to the current outbreak
3. 5 Ewes that had lost lambs due to Crypto

Results:

(1) BoviD-5 Ag Test Kit -Rapid Test Kit (Afrivet) ALL groups tested NEGATIVE

(2) PCR: ALL groups tested NEGATIVE (Vetdiagnostix)

(3) Microscopic staining - Modified Ziehl-Neelsen (mZN 7/13 tested,(54%) POSITIVE (Western Cape Labs Dr  J. Stroebel)

2. Graaff-Reinet - Eastern Cape.

We tested 5 healthy sheep ewes on a Crypto positive farm (histopath) -currently experiencing lamb deaths due to crypto.

Results:

(1) PetX Rapid Test Kit ALL ewes tested NEGATIVE

(2) PCR (Vetdiagnostix): ALL tested NEGATIVE

(3) Microscopic staining- Modified Ziehl-Neelsen (mZN): 1/5 tested (20%) POSITIVE

      (Western Cape Lab- Dr J, Stroebel)

SCREENING ANGORA GOAT FARMS:

The extent of the distribution of Cryptosporidium throughout South Africa is unknown.

The first explosive outbreaks in sheep occurred in the Eastern Free State in 2013 (Dr Liezel Wasserman).

In the Graaff-Reinet district (Karoo) the first incidental detection of Cryptosporidium was in 2013 Dr Hobson-Camdeboo Veterinary Clinic (Dr Rick last- VDX).

However, there were no reported cases of Crypto until 2021.

• Sheep introduced from the Fish River irrigation region resulted in a confirmed outbreak in lambs under 2 weeks of age. (Rapid antigen tests confirmed by histopathology VDX).
• A further positive case in 2021 was detected in young goats (year old) where diarrhoea and a few deaths in the Rietbron district, Both Crypto and Coccidiosis were implicated in the deaths. (Histopathology Liza du Plessis, Idexx Laboratories) The only new introductions to the farm had been goat rams over the years.

Prevalence of Cryptosporidium on intensive Angora goat farms in the Karoo?

In an attempt to determine the prevalence of Cryptosporidium on Angora goat farms in the Karoo region 8 farms (intensive farming conditions) were tested.

• 301 tests in total using special staining techniques (mZN). (Western Cape Labs- Dr Stroebel)

None of these farms had reported any clinical cases to suggest Cryptosporidium being present.

It is well known that Microscopic staining and detection of cryptosporidium requires a high level of expertise.

Were these false positives?

Without histopathology on intestinal samples (determine true positives/negatives) what can the data (statistics) tell us?

32 Angora goat rams were tested twice at time intervals of 1-3 months

(Blind tests- The laboratory unaware the same goats were tested and no ID was provided)

What is the probability of the results matching by chance?

Using bionomial theory to calculate the probability of the chance of matching 29 out of 32 samples.  1.2 in a million*5

Using the data to determine the accuracy of the operator: in 93.75% of the cases the same result was obtained.*6

It is also interesting to note that 2 of the 3 unmatched results went from Negative to Positive which does not rule out the fact that these goats may potentially have picked up Crypto in the 1 to 3 months after the previous test meaning they were correctly detected at the subsequent screening!

Conclusion

The results suggest that using a highly skilled experienced examiner of microscopic stains at high power can detect carrier animals carrying Crypto that do not show clinical signs. Dr Jacob Stroebel, Western Cape labs, was able to detect low numbers of Oocytes (on average less than 1 per 400x field)

Histopathology on intestinal samples would have been conclusive but ethically not an option.

A reproducible test may not necessary be an accurate test but the data does suggest that cryptosporidium may already be on many Angora goat farms but up to this point has had no significant impact.

Dr Mackie Hobson (Mohair SA)

GENERAL INFORMATION ON THE DIFFERENT TESTS FOR CRYPTOSPORIDIOSIS.                                                        

1. Direct microscopic examination

As the cysts of Cryptosporidium are tiny, a staining procedure is required to identify them. Conventional methods of stool examination (ie, routine "stool for ova and parasites" testing) are unreliable.

A number of different staining techniques have been developed.

• Ziehl-Neelsen (ZN) is an Acid-fast procedure -staining is a bacteriological stain used to identify acid-fast organisms. Acid Fast positive cells are stained pink/red and Acid Fast negative cells are stained the light blue colour of methylene blue.

• Modified Ziehl-Neelsen (mZN) -with this method, the dyeing time is shortened and heating is not required.
• Auramine Phenol (AP) The parasites appear greenish-yellow against a dark background. The method is more rapid and sensitive than the ZN technique.
• Immunofluorescence Microscopy (IFM) is a widely used example of immunostaining and is a form of immunohistochemistry. Specific antibodies bind to the protein of interest. Fluorescent dyes are coupled to these immune complexes in order to visualize the protein of interest using microscopy. IF microscopy can be used in several microscope designs for the analysis of immunofluorescence samples.

2. Immunology:

Detect soluble protozoan antigens in faecal samples, thus removing the need for microscopic examination.

Enzyme immunoassay (EIA) and enzyme-linked immunosorbent assay (ELISA) technologies have been used to develop commercial products for the detection of Cryptosporidium

EIA, ELISA has the advantage of good specificity, and a large number of samples can be processed in a short time.

Immunochromatographic lateral-flow (IC) or (ICLF) 'dipstick' tests have also been developed for antigen detection in stool samples. These tests are sensitive and specific, easy to use and provide a result in 10 minutes.

Immunochromatography is a test format that uses tagged particles as a colour signal rather than an enzyme-catalyzed colour change reaction as in ELISA. The test uses biologics and tagged microparticles coated with specific Crypto antibodies.

These ICLF tests are the Rapid tests used in the field.

3. Molecular biology

PCR or real-time PCR (RT-PCR) is a molecular biology-based diagnostic detection method developed to target specific sequences in the protozoan genome. These methods offer highly specific and sensitive assays but are expensive and time-consuming.

• PCR is the GOLD STANDARD test- are more sensitive with the detection range from 1 to 10⁶oocysts, are relatively rapid, and have the major advantage of speciation which is very important from an epidemiological point. This comes with costs and time disadvantages.

 REFERENCES

• Dr Liezel Wasserman (Clocolan Veterinary Clinic)

• Dr Jacob Stroebel (Western Cape Laboratories)

• Dr James Hill (Vetdiagnostix)
• *2 Comparison of PCR and Microscopy for Detection of Cryptosporidium parvum in Human Fecal Specimens: Clinical Trial U. M. MORGAN, L. PALLANT,B. W. DWYER,D. A. FORBES, G. RICH, AND R. C. A. THOMPSON
• *1 Comparison of diagnostic sensitivity and specificity of seven Cryptosporidium assays used in the UK Free Rachel M. Chalmers, Brian M. Campbell, Nigel Crouch, André Charlett, Angharad P. Davies,
• *3 Zoetis- Veterinary Diagnostic Laboratory, University of Minnesota, St. Paul, Minnesota.
• *4 BioNote Clinical Evaluation: Anigen Rapid Rota Ag Test Kit Diagnostic,Sensitivity and Specificity, Zoetis LLC.
• *5 Luke Hobson (University of Cape Town)
• *6 Dr Gretha Snyman (GADI –Grootfontein)