Advances in diagnostic technology allow for more sensitive, specific, rapid and cost-effective diagnosis of diseases. New methods like PCR, real-time PCR, in situ hybridization, biosensors, infrared thermography, and ELISA have improved on classical diagnostic approaches by being able to detect minute amounts of pathogens, identify pathogens rapidly, and differentiate between field strains and vaccine strains. These advanced diagnostic techniques are important for disease control, treatment, and surveillance.

1. Advances in diagnostic
technology
Ritasree Sarma

2. Diagnosis
 Determination of the nature of a cause of a disease
 Improved, quality assured diagnostics are important for
disease control in animals
 They provide a basis for appropriate treatments of animal
patients for monitoring diseases and for the enhancement
of disease-surveillance capacity
 For this
Diagnostic methods should be sensitive, specific, rapid and
cost effective

3. Advanced Diagnostic approach ??
Classical methods
• Microscopic method: Organisms must be present in
sufficiently high concentration and use of appropriate set
stains and conditions make them visible
• Cultivation method: Certain microbes may require special
culture media and conditions and failure to consider these
microbes may yield negative culture results
• Cell culture can be used to detect some viruses and
intracellular microbes but the cost, labor, and time required
for this approach beg for better diagnostic method
• Serologic assays: May not be helpful for rapidly evolving
diseases, the host may succumb to infection before antibodies
can be produce

4. Advanced diagnostic technique
PCR based method
PCR is an enzyme-driven process for replicating DNA in vitro
• Using this technology, one is capable of turning a few molecules of DNA into large
quantities
Why used:
• The levels of microbial DNA present in clinical samples are frequently
too low for meaningful manipulation and measurement
 PCR can produce sufficient amounts of DNA so that microbes can be detected and
identified.
• Because each unique microbe has a unique complement of DNA (or RNA), DNA
can function as a molecular fingerprint to help identify microbes

7. Lumpy skin disease is an infectious, eruptive, occasionally fatal disease of cattle
• The clinical onset is characterized by fever, watery eyes, increased nasal secretions
and loss of appetite, severe in cows at the peak of lactation causes a sharp
reduction in milk production
• In July 2012 a massive LSD outbreak began in the northern part of Israel
• In order to strengthen the efforts to control the disease the Israeli Veterinary
Services initiated the use of a Neethling vaccine strain (LSDV)-based vaccine
• But typical LSD symptoms appeared in a few vaccinated animals and proved to be L
SDV positive
• It was of extreme importance to develop a differentiation approach that could
specifically identify the strain responsible for affecting the livestock (field or vaccine
strain)

8. • The approach was initiated following a BLAST
analysis between the vaccine strain and other
complete virus sequences to try to find mark
differences between them
• The alignment analysis identified a unique 27-
base deletion in the vaccine strain
• PCR enabled virus identification in case that
vaccinated animals demonstrate clinical
symptoms confirmed the presence of the
deletion only in the vaccine strain and not in the
virulent strains
• The finding raised the possibility that this
sequence could be important for virulence in the
field viruses, and that when this 28-base
fragment is missing the virus exists in an
attenuated state

9. Real time PCR technology
Better over general PCR
• Quantitative
• More rapid
• Sensitive
• Accurate
• Easy to perform

10. General procedure
• In real-time PCR assays, intercalating dyes or a target-specific probe
or primer (labelled with fluorescent dye) are used
• The measured fluorescent signal is proportional to the number of
specific DNA fragments produced
• Thus, during the real-time PCR, the accumulation of PCR products
can be monitored in each consecutive cycle as a change in the
degree of fluorescence
• So, the assay can be used for quantification of the DNA or RNA
content in a given sample

12. Uses
For detection of
Foot-and-mouth disease virus (FMDV)
• Classical swine fever virus (CSFV)
• Bluetongue virus (BTV)
• Avian influenza virus (AIV) and
• Newcastle disease virus (NDV)
• (Vet Sciences Tomorrow – Issue 1 - Jan 2001)
• Tick-borne diseases
• Lyme disease
• Ehrlichiosis
• Tick-borne encephalitis
• Feline coronavirus (FCoV)
• (B. Hoffmann et al. / Vet Microbl 139 (2009) 1–23

13. Non PCR based method
In situ hybridization
• It relies on the principle that specific sequences of single-
stranded cell- and tissue-bound RNA and DNA will hybridize
with single-stranded labeled probes of complementary
sequence
• Every infectious organism has unique segment of DNA or
RNA that are not found in other organisms, cells or tissues
• ISH can localize single-copy genes and mRNA transcripts in
samples with fewer than 10 copies per cell present

15. •
Collecting samples
Careful sample collection is critical, especially if RNA sequences are
tested.
This activity is more intense after the animal’s death or after the tissue has been
removed from a living body (enzymatic activity is still present but no new RNA is
formed. Therefore, the sooner the tissue is collected and preserved is the better
Preserving the tissues
Formalin adequately preserves tissue morphology and provides good retention of the
nucleic Acids
Processing the samples
 A nucleic acid template (DNA or RNA) within the tissue to be tested
 A labeled nucleic acid probe (usually an oligo nucleotide which is a short chain of
specifically ordered nucleotide bases 8 or segments of RNA or double-stranded
DNA specific for the organism to be detected)
 A detection system to demonstrate the hybridization reaction
The duration of this test varies, but usually is 30–90 minutes

16. Detection systems
At first, ISH was performed using radioactive probes as the detection system.
Although radioactive probes are usually more sensitive and still in use,
nonradioactive probes are common today
Nonradioactive probes minimize health hazards and circumvent governmental laws
that regulate work with radio nucleides
Interpretation of results
The presence of hybrids indicates a positive reaction
Diseases for which ISH is being used include
• porcine circovirus
• porcine reproductive and respiratory syndrome virus
• respiratory coronavirus
• foot and mouth disease virus
• swine vesicular disease virus

18. Biosensors
• A biosensor is an analytical device which involve the use of a biological
element on a solid-state surface enabling a reversible bio specific interaction
with the analyte and a signal transducer
• The biological element used are like enzymes, receptors, peptides, single
stranded DNA even living cells
There are two different types of biosensors:
• Biocatalytic and Bioaffinity-based biosensors
• The biocatalytic biosensor uses mainly enzymes as the biological compound,
catalyzing a signaling biochemical reaction
• The bio affinity-based biosensor designed to monitor the binding event of
lectins, receptors, nucleic acids, membranes, whole cells, antibodies or
antibody related substances for bio molecular recognition

20. The development of biosensor technologies will enable rapid and specific
disease diagnosis on-site so that a clinician can quickly determine whether
treatment is needed
In 1998, immunoassay was developed for the detection of African Swine
Fever virus (Uttenthaler et al., 1998)
Su et al. (2000) reported a immunosensor for porcine reproductive and
respiratory syndrome virus (PRRSV). The proposed biosensor was used to
screen pigs suspected to have been infected with the virus and to provide
positive or negative results in a few minutes
Kumar (2000) developed a method for diagnosis of tuberculosis and other
infections caused by myco bacteria.
Biosensor technology could also be applied to detect mastitis infection by
sensing markers such as enzyme N-acetylglucosaminidase (NAGase) in milk
This enzyme is released into milk as a result of tissue damage when the cow is
resisting a clinical intra-mammary infection

21. Veterinary Infrared Thermography
• It enables professionals to diagnose and monitor injuries,
diseases and illnesses in large and small animals, like horses,
dogs, cats, birds, livestock, zoo animals and marine animals
• Animals emit infrared heat from their bodies, which can be
visualized by thermography through an infrared camera
• Heat patterns show if the animal’s blood circulation is normal or
abnormal
Both increased or decreased blood flow are symptoms of health
problems, injuries, diseases or illnesses
• General inflammations and injuries are visible as warmer areas
or “hot spots” in the thermal image, since they cause an increase
in blood circulation
• On the other hand swelling, nerve damage and scar tissue show
up as colder areas or “cold spots” and indicate a decreased blood
flow

22. Veterinary Thermal Imaging allows
identification and treatment of orthopedic
pathologies at their early onset, while
they are still in the acute phase, avoiding
the possible catastrophic consequences
that can arise from untreated conditions
Positive aspects of Veterinary
Thermography are that:
it provides rapid and quick
assessments;
it is a non-contact / non-invasive
diagnostic tool
it can be repeated as frequently as
necessary;
it provides valuable information
without the need to sedate the animals

23. Enzyme-linked Immunosorbent Assay (ELISA)
This test works on the principle that an antibody or antigen can be linked to an
enzyme, which can facilitate a color reaction that indicates a positive result
The mainstay for measuring antibody response in infectious diseases and to
support pathogen identification of potential use in infectious disease outbreaks
and clinical care of individual patients
ELISA techniques use antibodies linked to an enzyme as horse radish
peroxidase or alkaline phosphatase
Ag-Ab reactions are detected by the enzyme-substrate reaction
A color change indicates an antigen-antibody reaction has occurred
Types
The direct ELISA- Sandwich or Capture Assays & Competitive ELISA -
Used to detect antigens
The indirect ELISA -Used to detect specific antibodies against antigen bound
in a test well

26. Advantages of ELISA
Sensitive: Nanogram levels or lower
Minimal reagents
Qualitative & Quantitative
Qualitative  E.g. Disease testing
Quantitative assays  E.g. Theraputic Drug Monitoring
Greater scope : Wells can be coated with Antigens or Antibodies
Suitable for automation  high speed
No radiation hazards

27. • An indirect enzyme-linked immunosorbent assay (ELISA) has been developed
for the rapid detection of antibodies to the porcine reproductive and respiratory
syndrome (PRRS) virus in pig sera
• Competitive enzyme-linked immunosorbent assay (cELISA) was developed to
detect antibodies to the group antigen of bluetongue virus (BTV)
(J Vet Diagn Invest 3:144-147 ,1991)
Potential in future
• Utilizing available 3D printing platforms, a ‘3D well’ was designed and developed to
have an increased surface area compared to those of 96well plates
• The ease and rapidity of the development of the 3D well prototype provided an
opportunity for its rapid validation through the diagnostic performance of ELISA in
infectious disease without modifying current laboratory practices for ELISA.
• The improved sensitivity of the 3D well of up to 2.25fold higher compared to the
96well
• ELISA provides a potential for the expansion of this technology towards
miniaturization and Lab On a Chip platforms to reduce time

28. Magnetic resonance imaging
• This technique is based upon the inherent magnetic properties of
certain nuclei
• The patient is placed in the strong magnetic field and the protons in
the body are excited into a high energy state by application of a radio
frequency pulse
• When the second field is stopped, the nuclei return to ground state and
emit the absorbed energy in the form of a radio signal
• This signal is received by a coil that generally surrounds the specimen
and converted to an anatomic image through a process of computer-
assisted reconstruction

30. MRI is the gold standard for
imaging soft tissues and
therefore use for examining
tumors, trauma of the brain and
spinal cords malformations
MRI is also helpful in examining
the skeleton, including bone,
tendons, ligaments, and joints
Magnetic Resonance Imaging
(MRI or MR) is the most
advanced diagnostic imaging tool
with non-invasive procedure for
complete viewing of the body
Fig: Brain tumor in dog and cat

31. Advanced diagnostic methods are
• Cost relative
• Speed relative
• Higher sensitivity
• Accurate
But which is the best diagnostic method??
• Diagnostic lab should continually evaluate traditional and advanced
technology in the light of cost, time, accuracy and sensitivity to get
the best diagnostic results

32. References
• Maria N. Velasco-Garcia; Toby Mottram. Biosensor Technology addressing
Agricultural Problems. Biosystems Engineering (2003) 84 (1), 1–12
• Belak S., Thoren P., Leblanc N. & Viljoen G .(2009).. Advances in viral
disease diagnostic and molecular epidemiological techniques. Expert Rev.
Mol. Diagn., 9 (4), 367–381
• Sophia M, Marisol Rubinstein-G, Anita K, Yevgeny E,Orly F, Ditza R,
Yevgeny K, Yehuda S. Development of an assay to differentiate between
virulent and vaccine strains of lumpy skin disease virus (LSDV). J
Virological Methods 199 (2014) 95–101
• Fredricks, David N. and Relman, David A.Application of Polymerase
Chain Reaction to the Diagnosis of Infectious Diseases.(1999). U.S.
Department of Veterans Affairs Staff Publications. Paper 4
• Segales J, Ramos-Vara JA, Duran CO, et al. Diagnosing infectious diseases
using in situ hybridization. Swine Health Prod. 1999;7(3):125–128.