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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
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
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
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)
• 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
Real time PCR technology
Better over general PCR• Quantitative• More rapid• Sensitive• Accurate• Easy to perform
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
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
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
•
Collecting samples
Careful sample collection is critical, especially if RNA sequences aretested. 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 tissuesFormalin 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
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
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
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 milkThis enzyme is released into milk as a result of tissue damage when the cow is resisting a clinical intra-mammary infection
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
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 toolit can be repeated as frequently as necessary;it provides valuable information without the need to sedate the animals
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
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
• 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
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
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
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
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.