The document discusses diagnostic advances for haemoprotozoan infections in livestock. It covers conventional parasitological diagnosis using blood smears as well as molecular diagnosis techniques like PCR, LAMP, and probes. Immunological diagnosis methods such as ELISA, IFAT, CATT, ICT are also summarized. Molecular tools allow specific and sensitive detection of parasites while serological assays are suitable for chronic infections when parasitemia is low. Advanced diagnostics combined with effective treatment can help control haemoprotozoan infections and drug resistance.

1. Maharashtra Animal and Fishery Sciences University
KNP COLLEGE OF VETERINARY
SCIENCE, SHIRWAL
Credit Seminar
DIAGNOSTIC ADVANCES IN HAEMOPROTOZOAN
INFECTIONS OF LIVESTOCK
PRESENTED BY:
VRUSHALI SARAWADE
M.V.Sc. Scholar
Dept. of Vet. Parasitology
1

2. OUTLINE
• INTRODUCTION
• CONVENTIONAL PARASITOLOGICAL DIAGNOSIS
• MOLECULAR DIAGNOSIS
• IMMUNOLOGICAL DIAGNOSIS
• CONCLUSIONS
• REFEREENCES
2

3. INTRODUCTION
 Protozoan parasites are responsible for causing severe infections
both in humans and animals worldwide.
 Transmitted by arthropod vectors : R.appendiculatus, R.
sanguineus, Hyalomma anatolicum, Glossina spp, Haemaphysalis
etc .
 Trypanosomosis, Theileriosis, Babesiosis.
(Biswas Ranjan et al., 2017)
3

4. 4
Distribution of livestock Trypanosomes spp
Giordani et al., 2016)
Distribution of Trypanosoma evansi in the world.
(Marc Desquesnes et al., 2013)
Distribution of Theileria oreintalis
(Sivakumar et al., 2014)
Geographical distribution of Theileria annulata
(Wang et al., 2014)
Geographical Distribution

5. Diagnosis of
Haemoprotozoan
Parasite
DIRECT
METHOD
Conventional
Parasitological
Diagnosis
Molecular
Diagnosis
INDIRECT
METHOD
Immunological
diagnosis
5

6. CONVENTIONAL PARASITOLOGICAL
DIAGNOSIS6
The Microscopic method
for diagnosis of tick-borne
diseases are still
considered as “gold
standard” techniques.
Thin and Thick
Blood Smears
(Biswa Ranjan et al 2016)

7. THEILERIA
 In the field, diagnosis is usually achieved by finding Theileria
parasites in Giemsa-stained blood smears and lymph node needle
biopsy smears.
7

8. BABESIA
 In acute cases Giemsa stained Thin and Thick blood smear.
(Mosqueda et al., 2012)
 Gold standard for acute cases. (O.I.E, 2010)
8

9. TRYPANOSOMA
 Thin and thick blood smears, wet blood films
 QBC (Qualitative Buffy cote ) technique.
 Anion exchange (OIE ,2008)
9

10. Molecular diagnosis
 Commonly used molecular tools are polymerase chain reaction
(PCR), real-time PCR, loop-mediated isothermal amplification
(LAMP).
 The Molecular test allow to direct specific and sensitive detection
of parasite.
(Parida et al.,2008)
10

11. Molecular diagnostic techniques
PCR-(Polymerase Chain Reaction)
LAMP-Loop-mediated Isothermal Amplification
DNA PROBES
Real-time Polymerase Chain Reaction (RT-PCR)
RFLP-Restriction Fragment Length Polymorphism
MICROARRAY
11

12. Polymerase chain reaction
 PCR is an in vitro technique for the amplification of a region of
DNA which lies between two regions of known sequence.
12
(D'Oliveira et al. 1995)
Denaturation 93 to
95°C 1min
Annealing 50 to 55°C
45 secElongation 70 to 75°C
1-2 min

13. Real-Time Polymerase Chain Reaction
(RT-PCR)
 RT-PCR , allow for the
quantification of the original
template’s concentration
through the use of various
fluorescent chemistries, such as
Sybergreen, Taqman probes.
 The concentration is measured
through comparison to standard
curves.
13

14. LAMP(Loop –Mediated Isothermal
Amplification)
 It having High specificity and sensitivity able to discriminate
between a single nucleotide difference.
 The LAMP method requires a set of four specific primers
 Recently, parasitologists have adapted the LAMP approach for the
detection of several parasitic diseases including the Trypanosoma,
Theileria and Babesia and even to the identification of vector
mosquitoes carrying Plasmodium
(Notomi et al., 2000).
14

15. LAMP15
(Notomi et al. 2000)

16. Cont..
16
PCR LAMP
1 Requires temperature cycling Isothermal –single temperature
2 Requires 2 primers Requires 6 primers
3 Typical yield ~ 0.2 µg Typical yield ~ 10–20 µg
4 Slow: Typically <1hr Rapid: Typically >30 min
5 Not amenable to visual
detection
Amenable to visual detection
based on turbidity etc.

17. Restriction Fragment Length
Polymorphism (RFLP)
 This technique utilizes the specific restriction enzyme to digest the
genomic DNA sequence in small fragments followed by separation
on gel electrophoresis .
 The gel electrophoresis showed different patterns of nucleic acid
fragment.
 Probes are used for hybridization.
 It was also used for differentiation of Theileria lestoquardi,
T. ovis, in sheep (Zaeemi et al.,2011)
17

18. PROBE HYBRIDIZATION
 The Hybridization probe is a 100-1000
bases long fragment of DNA or RNA
used for detection of presence of
nucleotide sequences complementary
to probe sequence.
 The nucleotide sequence of probe allows
probe-target base pairing due to
nucleotide base complementarity
between the target and probe.
 A Dot-blot based probe hybridization
assay for detection of T. annulata was
developed in India
(Saravanan et al., 2011).
18

19. MICROARRAY
 The probes are allowed to
hybridize labelled target
nucleic acid i.e., cDNA or
cRNA (anti-sense RNA).
 DNA microarrays along
with PCR and gene
sequence analysis
technique have been used
for molecular
identification of Theileria
and Babesia in cattle.
19
(El-Ashker et al., 2015).

20. Immunological Diagnosis
 Serological assays are more suitable for the diagnosis of the disease
during the chronic phase of the infection, where the animals serve
as carrier.
 While the level of parasitemia is low and microscopically hardly
undetectable.
 ASSURED criteria
(Mabey et al., 2004).
20

21. IMMUNOLOGICAL DIAGNOSTIC TECHNIQUES
ELISA
(Enzyme-
Linked
Immunosor
bent assay)
IFAT
(Immunoflu
orescence
Antibody te
st)
CATT
(Card
Agglutinati
on Test)
(ICT)
Lateral
flow
Immunochr
omatograp
hic Test
CMT
(compleme
nt fixation
test)
21

22. ELISA
 In ELISA an unknown antigen/antibody is absorbed to a
surface(solid phase),and then a specific enzyme linked antibody is
allowed to react.
 In the final step a substrate is added that is oxidized by the enzyme
for some detectable signal like color development. The intensity of
color developed on quantity of enzyme present in the reaction and
thereby antigen/antibody concentration
22

23. DOT-ELISA
 This is a simple and field oriented test where, the plastic well are
replaced by a nitrocellulose or other paper membrane.
 The addition of a perceptible, chromogenic substrate causes the
formation of a colored dot on the membrane which can be visually
read
 Several studies have demonstrated the usefulness of the study in
detection of the parasitic infection caused by Theileria equi,
Trypanosoma cruzi, and Trypanosoma brucei in different livestock
species
.
(Ranjan et al., 2015)
23

24.  SVANOVIR Theileria annulata-Ab, the first commercial ELISA
kit introduced now.
 SVANOVIR Theileria annulata-Ab diagnostic assay detects
the infection in chronic and carrier animals.
 SVANOVIR Theileria annulata-Ab is high (76.56%) and in
comparison to 38.70% for the thin blood smear.
( AL- Hosary et al., 2015)
24

25. IMMUNO FLORESCENT
ANTIBODY TECHNIQUE (IFAT)
 Fluorescent dyes (fluorochromes) illuminated by UV lights are
used to show the specific combination of an antigen with its
antibody. The antigen-antibody complexes are seen fluorescing
against a dark background.
 The most widely used diagnostic test for Theileria species is the
indirect fluorescent antibody (IFA) test.
(OIE 2008)
25

26. Card Agglutination Test For
Trypanosomiasis
 Direct Card Agglutination Test for detection of
trypanosome specific antibodies in blood, serum or plasma,
 CATT T. evansi, the latter being one of the antibody
detection tests recommended by OIE
 Freeze dried suspension of purified, fixed and stained
trypanosomes of a cloned predominant Variable Antigen
Type (VAT LiTat 1.3) of T.b.gambiense .
(OIE,2012).
26

27. CATT
27
Step 1
• On a test are of the card put 25 of diluted serum or plasma
• 1 drop of the well homogenized CATT antigen in each test area.
Step 2
• Using a stirring rod ,mix spread out the reaction mixture of about 1mm
from the edge of test area. Wipeoff the stirring rod after each use.
• Rotate the test card on a flat bed orbital rotator for 5 min at 70rpm.
Step 3
• After 5 min rotation ,read the results before removing the card from the
rotator.
(O.I.E,2010)

28. Immunochromatography Test (ICT)
 The Immunochromatographic test is a rapid diagnostic device.
 It detects antibodies against a specific antigen in a small amount
of serum by means of specific antibody and a recombinant
antigen both impregnated on a nitrocellulose membrane-based
test strip
 ICT has been developed for Babesia bovis, B. bigemina, T.evansi
B.caballi, B. gibsoni and B. microti . These tests use recombinant
antigens.
28
(Katarzyna M.et al 2015 )

29. Lateral flow dipstick29
(A) Schematic representation of the assay's mechanism. Top: the sample is deposited
on the sample pad and migrates towards the conjugate. Middle: the conjugated
antibodies bind the target analyte and (bottom) migrate to the test line, where the
bound target analyte is captured.
(Katarzyna M.et al 2015).

30.  An Immunochromatographic
test for serodiagnosis of
Trypanosoma evansi infection
in domestic animals,
 Surra Sero. K-SeT, has been
developed .
 Sero-K-SeT has high
sensitivity (98・5%) and
specificity (98・6%).
30
(Hadush Birhanua et al.,2015) Sero-K-SeT (Coris
BioConcept, Gembloux, Belgium)

31. COMPLEMENT FIXATION TEST
 Based on this test, a commercial kit (COFEB Kit) has been
developed for the diagnosis of Equine piroplasmosis.
 The CFT was found reproducible and reliable assay for
Trypanosoma equiperdum sera sample .
(OIE, 2013; Luciani et al., 2013). (Sengupta, 2004).
31

32. CONCLUSION
 Specific diagnosis of the etiological agent is most important for control of the
hemoprotozoan infections.
 Microscopy-based detection methods are still the cheapest and fastest methods for
diagnosis though the techniques suffer from the limitations of sensitivity and specificity.
 Blood smear examination is Gold standard test.
 The molecular and serological methods are also useful in epidemiological studies.
 The LAMP assay is a simple and convenient diagnostic tool.
 These advanced diagnostics complemented with well-controlled and efficient
chemotherapeutic usage will provide the best means of controlling the hemoprotozoan
infections and emergence of drug resistance.
32

33. REFRENCES
 Anne Harwood Peruski* and Leonard F. Peruski, Jr.* Immunological Methods for Detection and Identification of
Infectious Disease and Biological Warfare Agents
 1AmiraA.T.Al-Hosary,1 JabbarAhmed,2 AnnNordengrahn,3 andMalikMerza Assessment of the First Commercial
ELISA Kit for the Diagnosis of Theileria annulata A review of nucleic-acid-based diagnostic tests for Babesia and
Theileria, with emphasis on bovine piroplasms .
 .Abdo J, Kristersson T, Seitzer U, Renneker S, Merza M, Ahmed J (2010). Development and laboratory evaluation of
a lateral flow device (LFD) for the serodiagnosis of Theileria annulata infection. Parasitol. Res. 107(5):1241-1248.
http://dx.doi. org/10.1007/s00436-010-1994-8 .
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Sudhakar.Important hemoprotozoan diseases of livestock: Challenges in current diagnostics and therapeutics: An
update
 H.Iseki,A.Alhassan,N.Ohta,etal.,“Developmentofamultiplex loop-mediated isothermal amplification (mLAMP)
method for the
 simultaneous detection of bovine Babesia parasites,” Journal of Microbiological Methods, vol. 71, no. 3, pp. 281–
287, 20
 Koushlesh Ranjan1*, PRasad MinaKshi2, Gaya PRasad3 Application of Molecular and Serological Diagnostics in
Veterinary Parasitology
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34.  O.I.E. (2010) Trypanosoma evansi infections. In: Terrestrial Manual. Office International Des
Epizooties, World Health Organization for Animal Health, Paris, France, Vol. 1. Ch. 2.1.17. p352-
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 Mishra, A.K., Rao, J.R. and Tewari, A.K. (2007b) Seroprevalence of babesiosis in cattle and
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 Review Article DiagnosisofParasiticDiseases:OldandNewApproaches
 Roy, S.,Tiwari,A., Galdhar, C. N., Upadhyay, S. R., Ratre, H. K., Sahu, S. K., and Maiti, S. K.
(2004) Seasonal prevalence of haemoprotozoan diseases in cross- bred cattle andbuffaloes. 24:5-7
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