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79 Laboratory Diagnosis of Infectious Diseases 843

immitis is based on detection of a circulating secretory therefore is less commonly used in clinical practice for
VetBooks.ir antigen of the adult female worm. parasites and arthropod‐borne pathogens. This tech-
nique is employed in research studies because it permits
the identification and maintenance of pathogens. It is
Molecular Diagnostics
important to highlight that some organisms, such as
Polymerase chain reaction is a sensitive and specific Babesia species, are difficult to culture. The culture
diagnostic technique frequently employed for the diag- method is pathogen specific and usually requires special
nosis of protozoal and arthropod‐borne diseases. It is media and temperature conditions. Special culture
used for diagnostic purposes, monitoring during and medium is used for the isolation of some protozoal
after treatment, research studies, and for screening of pathogens, such as L. infantum.
blood donors. Moreover, it is particularly useful for
detection of infection in animals with low parasitemia
levels and for speciation of pathogens. PCR can be Viral Infections
carried out on DNA extracted from tissues, blood, body
fluids, conjunctival and oral swabs, or even cytologic Microscopic Examination
preparations or histopathologic specimens.
A large number of PCR assays and protocols using a Most viral infections are not diagnosed by microscopy.
variety of gene targets have been described for the detec- Viruses are too small to be detectable by light micros-
tion of protozoal and arthropod‐borne infections of dogs copy, but typical cytoplasmatic or intranuclear inclusion
and cats. It is important to highlight that for diagnostic bodies caused by viruses may be detected in blood or
purposes, the best DNA target will often be the locus or tissue cells. Electron microscopy may be used for the
gene with the largest number of copies per organism. For detection of certain viruses such as canine parvovirus in
instance, the kinetoplast DNA (kDNA) of L. infantum is feces of dogs shedding the virus, although this is a
an excellent target as it has about 10 000 copies in each cumbersome, time‐consuming technique which is often
Leishmania amastigote. Other targets are more useful unable to specify the exact species involved in infection,
for distinguishing between species such as the leishma- as it relies on morphology.
nial ribosomal internal transcribed spacer 1 (ITS1). Due
to the fact that in some endemic areas, more than one Serologic Testing (Antibody and Antigen Detection)
species co‐exist and infect animals, molecular tech-
niques have been developed to discriminate DNA from Serologic tests are frequently used for the detection of
different species of the same genus or related genera. viral infections in cats and dogs. There are a number of
These techniques include semi‐nested PCR, reverse line basic serologic techniques such as ELISA, IFAT, virus
blotting, PCR restriction fragment length polymorphism neutralization, complement fixation, agglutination, agar
(RFLP), and high‐resolution melting curve quantitative gel immunodiffusion, and western blotting. Serology can
fluorescence resonance energy transfer PCR. Sequencing be aimed at detecting specific antibodies produced
may also reveal infections with novel organisms that against a certain virus in the infected animal, as in the
have not been described before. most common tests for feline immunodeficiency virus
It is important to highlight that negative results by (FIV) which detect antibodies to the p24 core protein.
molecular techniques only indicate that specific DNA It can also be aimed at the actual detection of a certain
was not detected under the assay conditions and should viral antigen in a body fluid such as the blood, as in the
not be interpreted as absolute evidence for the absence common feline leukemia virus (FeLV) antigen test based
of infection. In addition, false‐positive results are possi- on the detection of the viral p27 capsid protein.
ble due to DNA contamination or the amplification of Rapid commercial tests are available for many of the
DNA from other sources which may not be noticed if common viral infections, and they are also frequently
sequencing is not performed. Controls should be based on the detection of either antibodies against the
included in each step of the assay to ensure that DNA virus or virus antigen. Rapid commercial tests are often
contamination has not occurred. based on immunochromatographic devices which pro-
duce a color response when antibodies against a virus are
detected, or when the actual viral antigens are detected,
Isolation if the assay is geared to the detection of antigen. These
Diagnosis may also be established by culture of the infec- types of assays are often used for in‐house rapid diagno-
tious agent. However, this technique is usually tedious sis in veterinary clinics and hospitals. Several manufac-
and time‐consuming. It sometimes requires special turers have produced such rapid assays for the detection
conditions and a long duration for obtaining results and of antibodies against FIV, antigenemia with FeLV, or the