Crypto - Screening for sub-clinical carriers
By Dr Mackie Hobson

Tuesday, 8th June 2021

CRYTOSPORIDIOSIS -SCREENING of HEALTHY (carrier) animals: BIOSECURITY

 

There are a number of diagnostic tests available to test for Cryptosporidium parvum.

These have a high sensitivity and specificity (see section sensitivity, specificity in below section) on diagnosis clinical cases.

 

However, are the number of Oocytes being shed in a healthy carrier sufficient to be detected using these tests?

 

Biosecurity is critical in maintaining a disease-free environment on any Angora goat producer’s farm. The movement of sub-clinical carriers is of concern within the industry so we did a field study to determine if these tests could detect low Crypto oocyte levels in the faeces (

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 for the presence of carrier animals.

To check the viability of these tests Mohair SA conducted 2 field studies?

a. Clocolan - Eastern Free State

Testing with Dr Liezel Wasserman (Clocolan Veterinary Clinic) where we tested 13 healthy sheep on a Crypto positive farm that was currently having lamb deaths.

3 Groups were tested:

  • (a)5 rams that had been on the farm for over a year.
  • (b) 3 rams that had been introduced 3 months prior to which were present during the current outbreak
  • (c)5 Ewes that had lost lambs due to Crypto

Test results:

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

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

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

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b. Graaff-Reinet - Eastern Cape.

The farm had lambs currently infected by Crypto which was confirmed positive on histopathology and rapid test results.

5 healthy ewes that had lost lambs due to Crypto were tested.

Test results:

(1): PetX Rapid Test Kit ALL ewes tested NEGATIVE (A lamb with clinical signs tested positive)

(2) PCR (Vetdiagnostix): ALL tested Negative

(3) Microscopic staining- Modified Ziehl-Neelsen (mZN) (Western Cape Lab- Dr J, Stroebel): 1/5 tested POSITIVE

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CONCLUSION:

These results suggest that testing for sub-clinical carriers as a Biosecurity measure to screen a flock by Microscopic staining would be beneficial. Able to detect low numbers of Oocytes (on average less than 1 per 400x field)

The results suggest that the PCR probe is not sensitive enough to detect the healthy carriers of crypto.

The emphasis will be on individual producers/farms to prevent Crypto occurring by:

Maintain a strict BIOSECURITY POLICY

  • Purchase livestock from farmers who Producers know and can trust.
  • Producers must request an owner’s declaration signed by the owner’s vet stating that cryptosporidiosis has not been diagnosed on the farm when purchasing goats.
  • Purchased goats should be placed in quarantine and faecal samples obtained (from approx. 10 goats) for microscopic screening.
  • If ewes are purchased run them as separate flock after quarantine until after the first kidding

 

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 the 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 dark background. The method is more rapid and sensitive than ZN technique.

 

  • Immunoflourescence 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 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 color change reaction as in ELISA. The test uses biologics and tagged microparticles coated with specific Crypto anti bodies.

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 epidemiological point. This comes with costs and time disadvantages.

 

Comparing the SENSITIVITY and SPECIFICITY of the different Tests

What is sensitivity and specificity?

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

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

PCR is considered the gold standard to test for presence of Crytosporidium.

COMPARING the different tests in the table below:

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HOWEVER, these sensitivity and specificity are tests on samples from cases showing clinical signs.

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)?

Microscopic staining techniques, mZN (Dr Jacob Stroebel-Western Cape laboratory) were able to detect carriers with on average less than 1 per 400X field. PCR (Vetdiagnostix) and ICLF tests were not able to detect healthy carriers.

 

Definitive diagnosis is always required in suspected cases of Cryptospodiosis by Post Mortem Tests and Sampling.

A presumptive diagnosis can be made on history and clinical signs as well as a positive ‘Rapid Test’ but a definitive diagnosis must be made by:

  • Histopathology: 2 sample pieces of the duodenum, jejenum, and proximal colon should be taken placed in 10% formalin and sent to the laboratory.
  • A culture sample helps to determine if other pathogens are present (E,Coli, Salmonella or Clostridia). Important in calves for Rota, Salmonella, E.Coli.
  • Rapid Test Antigen swabs are taken from the ileo-caecal junction. This allows a quick on site presumptive diagnosis. Take the sample from the ileum side of the junction. The Rapid Tests (Antigen detection) include:
  • Zoetis WITNESS Bovid-5 Test
  • Afrivet Rapid Test kit Bovid-5 (below left)
  • Pet-X Rapid Test kit (Below right)

cp_1.pngcp_2.png

  • Modified Ziehl-Neelsen (mZN) Microscopic staining of faecal samples. Faecal samples are sent ‘cold chain’ to the laboratory.

 

THE RAPID Antigen Tests as a SCREENING TEST on rectal or faecal samples from suspected SUBCLINICAL CARRIERS IS NOT EFFECTIVE.

 

Collection and storage of samples

 

Faecal samples for Cryptosporidium should be tested as soon as possible. Alternatively, samples can be frozen or preserved with 10% formalin if testing can only be done at a later date, however, if PCR-based techniques are to be used, formalin can reduce the sensitivity of PCR. Freezing also not advised if PCR is to be done (Vetdiagnostix)

 

Since the shedding of cysts may be sporadic, at least three consecutive samples should be collected or possible taking a pooled sample from 5 + individuals to account for sporadic shedding. Take rectal faecal samples - not from the floor.

 

Dr Mackie Hobson

Mohair SA

 

REFERENCES

  • Dr Liezel Wasserman (Clocolan Veterinary Clinic)
  • Dr James Hill (Vetdiagnostix)
  • Dr Jacob Stroebel (Western Cape Laboratories)
  • *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.

 

 

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