Immunohistochemistry for the Study of the Pathogenesis and the Diagnosis of
Infectious Diseases in Cattle and Small Ruminants
Fabio Del Piero, DVM, PhD, Dipl. ACVP
Professor of Pathology
Louisiana State University
School of Veterinary Medicine
Department of Pathobiological Sciences
Baton Rouge – LA - 70806
Indirect immunohistochemistry (IHC) is widely used for the detection of viruses
in fixed tissue for the study of the pathogenesis and the diagnosis of viral diseases. In
addition, IHC is used for the detection of other agents such as bacteria, protozoa and
fungi and for the detection of cell proteins. The technique is rapid, inexpensive, sensitive,
specific, safe, and characterized by permanent staining because infectious agents, with
the exception of prions, are inactivated by common fixatives. In addition, IHC also
allows the simultaneous visualization of histologic and histopathologic features and their
correlation to localization of the target. Monoclonal and polyclonal antibodies can be
produced against virtually any infectious agent and used for their detection. Dextran
polymers and enzyme polymerization are used to increase sensitivity and increase the
quality of the staining. The selective or combined use of IHC, PCR, in situ hybridization
and conventional isolation of agents will give the best results, since the techniques are
often complementary. Here we briefly describe tissue localization and lesions during
natural infection in cattle and small ruminants by several agents that are diagnosed and
studied by veterinary pathologists.
Viruses can be classified in polyspecific (tropism for multiple species), species
specific with similar lesions and distribution and species specific with variation of lesions
and distribution. The following viruses are polyspecific. Rabies rhabdovirus causes
polioencephalomyelitis with Negri bodies and ganglionitis and abundant intracytoplasmic
virus can be detected within neurons, fibers, particularly within the CNS gray matter, but
also within retinal cells and less frequently within the corneal epithelium and in some
carnivores within the skin adnexa. Intensity of inflammation does not correlate with viral
distribution. West Nile flavivirus (WNV) is a mosquito transmitted arbovirus that causes
polioencephalomyelitis in mammals with rhombencephalic tropism, sparing the
neocortex. Intracytoplasmic WNV can be detected within neurons, nerve fibers and glial
cells, particularly in glial nodules. It rarely causes a productive infection in dogs, where it
has also been identified within the cytoplasm of renal tubular epithelial cells. Birds are a
natural host of the virus and any cell type of receptive avian species can be colonized.
Lesions in birds may be mild to severe and may include acute hemorrhage, encephalitis,
and enteritis with gland crypt necrosis. Virus distribution does not correlate with lesions.
WNV has been identified in a sheep but there are no reports of this disease in cattle.
Eastern equine encephalitis alphavirus (EEEV) geographic distribution follows the
presence of the carrier Culiseta melanura. EEEV and the related viruses (WEE, VEE)
cause severe polioencephalomyelitis with neuronal necrosis and neutrophils in mammals.
In horses, deer and humans, lesions are severe and correlate with abundant virus
distribution, whereas in camelids, lesions are less intense and do not necessarily correlate.
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Viral targets are neurons, fibers, glial cells, but also cardiomyocytes, smooth muscle cells
and renal dendritic interstitium. The extraneural locations contain small viral quantities.
Horses and pheasants present very similar viral localization but lesions are more severe in
horses, which also present smooth muscle and myocardium necrosis. EEEV has been
very rarely identified in cattle. Foot and mouth disease aphthovirus infects numerous
cloven foot species, and a few others, and can be localized within the cytoplasm of
rapidly replicating squamous epithelia where it is associated with the formation of
vesicles. In cases of myocarditis in calves, the virus is present in cardiomyocytes and
interstitial cells. Vesicular stomatitis rhabdovirus is characterized by similar lesions and
localization. The followings are species specific viruses with similar pathogenesis and
distribution. Papillomaviruses are common epithelial pathogens and often associated
with normal and neoplastic squamous epithelia. They can be detected within the nucleus
of squamous cells in a process of apical progressive maturation and condensation.
Parvoviruses cause enteric glandular crypt necrosis, lymphoid tissue necrosis, conceptus
loss and malformations. They can be detected within the nucleus and cytoplasm of
enterocytes and other epithelia, white cells and in young animals in several cells types
including cardiomyocytes. Bovine parvovirus is a sporadic pathogen of cattle, able to
induce enteritis and abortion. It colonizes the nuclei of enterocytes and the rapidly
replicating epithelial fetal cells. Rotaviruses colonize the proximal part of the small
intestinal villi epithelium in young animals producing enteric disease. Coronaviruses are
able to colonize the large intestine as well as the small intestine, and may be detected
within the cytoplasm of epithelium and in a few mucosal macrophages. They have been
associated with interstitial pneumonia, but reports generally lack convincing morphologic
evidence of lung localization. Adenoviruses are able to cause enteritis, bronchiolitis,
rhinitis, laryngopharyngitis, and necrotizing hepatitis. They colonize the cell nucleus
only. Lentiviruses responsible for pneumonia, mastitis, polyarthritis and
myeloencephalitis can be found in the cytoplasm of macrophages of lung, bone marrow,
mammary gland, lymph node, spleen, synovium, brain, and spinal cord, frequently in
association with lymphocyte infiltrates.
The followings are species specific viruses with some variations in viral
distribution and induced lesions. Bluetongue orbiviruses targets the cytoplasm of
endothelial cells, monocytes, macrophages and dendritic cells. Lesions include epithelial
erosions, ulcers and necrosis of cardiac papillary muscles. Bovine diarrhea virus, border
disease virus of small ruminants and classical swine fever virus are pestiviruses able to
cause conceptus loss, malformations, natimortality, enterotyphlocolitis, pneumonia,
lymphoid tissue necrosis and persistent infection. They are pantropic viruses and, in
persistently infected animals, they are able to colonize any cell type, with the possible
exception of skeletal muscle. In non-persistently infected animals, cell targets are
epithelia, endothelia and white cells. IHC on skin biopsy is a very good diagnostic
technique to detect persistently infected (PI) cattle. Persistently infected animals also
harbor pestivirus within the encephalic pericytes and neurons, allowing the post mortem
immunohistochemical differentiation between PI animals and naïve animals with acute
infection. Bovine herpesvirus 1 (BHV-1) causes abortion, laryngotracheitis and
bronchopneumonia, lymphoid necrosis, vulvovaginitis and likely meningoencephalitis.
Like in other herpesviral infections, intranuclear and intracytoplasmic virus can be
detected within epithelia, endothelia and white cells. The fetal lesions and viral
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distribution are very similar to the other alfa-herpesviruses and include multifocal
necrosis in several organs (in particular lung, liver, lymphoid tissue, thymus and adrenal
glands) with colonization of nucleus and cytoplasm of epithelia, endothelia and white
cells. BHV-1 heavily infects the chorionic endothelia and for this reason, the placental
chorion is an excellent diagnostic tissue. Bovine respiratory syncytial pneumovirus
(BRSV) causes bronchointerstitial pneumonia with inclusion bodies and syncytia. BRSV
can be detected within pneumocytes and macrophages. Parainfluenza 3 paramyxovirus
has a similar distribution, but lesions are less severe. Rinderpest morbillivirus causes
enterotyphlocolitis with lymphoid necrosis and syncytia with intranuclear and
intracytoplasmic epithelio- and lympho-tropism. Pest des petite ruminants morbillivirus
causes similar lesions and virus distribution in small ruminants; in addition
bronchointerstitial pneumonia with syncytia can be observed. Rift Valley fever
bunyavirus phlebovirus and Wesselbron’s disease flavivirus are African zoonotic
arboviruses able to cause abortion, natimortality, malformations and hepatic necrosis with
non-viral inclusion bodies and encephalitis. Viruses can be detected within the cytoplasm
of hepatocytes and neurons. Spleen, lymph node, lungs and kidneys may contain a few
infected cells. Malignant catarrhal fever viruses, alcelaphine herpesvirus 1 and 2 and
ovine herpevirus 2 are lymphotropic and cause mucosal erosion and ulcers, vasculitis and
lymphoid necrosis and hyperplasia. They can be visualized within lymphocytes,
macrophages and dendritic cells of lymphoid organs.
Prion diseases affect ruminants in the form of bovine spongiform encephalopathy
(BSE), scrapie and chronic wasting disease. Histologically there is vacuolation of the
grey matter neuropil with involvement of corpus geniculatum medialis, thalamus, gyrus
dentatus of the hippocampus, corpus striatum, and deep layers of the cerebral and
cerebellar cortex as well as brain stem. Diffuse glial reaction involving astrocytes and
microglia and intraneuronal vacuolation in brain stem neurons can be observed.
Abundant PrPSc antigen can be detected in brain, retina, optic nerve, pars nervosa of the
pituitary gland, trigeminal ganglia and small amounts in the myenteric plexus of the small
intestine, in the medulla of the adrenal gland, spleen, tonsils, lymph nodes. There are
some significant differences of the distribution of the lesions and PrPSc in the various
ruminant species and in other species affected by this disease.
Several bacteria are also commonly able to cause lesions in ruminants. Cross
reactivity of sera may limit the use of IHC for the detection of bacterial infections. A few
are routinely detected via IHC, in particular Listeria monocytogenes. L. monocytogenes
causes rhombencephalitis, abortion and natimortalitiy with sepsis, vasculitis and
suppurative hepatitis. The short rods can be detected free in the exudate of
microbascesses, near the edges of necrotic areas, within the cytoplasm of neutrophils,
macrophages, and blood vessel wall and lumina. Other parthogenic bacteria for ruminants
for which IHC may be used are Histophilus somnus (sepsis, pneumonia, embolic
neutrophilic meningoencephalitis); Mycobacterium bovis (multisystemic caseous
granulomatous disease with lymphadenitis and pneumonia; serositis, enteritis, hepatitis,
nephritis, osteomyelitits, meningoencephalitis, salpyngitis, metritis and cotyledonitis with
abortion); Mycobacterium avium paratuberculosis (granulomatous enterocolitis,
lymphangitis and lymphadenitis; hepatitis); Yersinia spp (necrotizing enterocolitis);
Brucella spp. (sepsis, vasculitis, endometritis with abortion, orchitis, epididymitis,
mastitis); Bacillus anthracis, (sepsis, splenic hemorrhagic necrosis); Salmonella enterica
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(catarrhal, fibrinonecrotizing enterocolitis, lymphadenitis, hepatitis, pneumonia,
tonsillitis, splenitis, meningoencephalitis, polyarthritis, osteomyelitis, and abortion);
Clostridium spp, (enterocolitis, sepsis, hepatitis, skeletal muscle necrosis, pericarditis);
Trueperella (Arcanobacterium) pyogenes (sepsis, dermatitis, splenitis, lymphadenitis,
placentitis, abortion, polyarthritis and osteomyelitis); Pasteurella multocida and
Mannheimia haemolytica (pneumonia and sepsis), Escherichia coli (sepsis, enteritis);
Chlamydophila spp. (cotyledonitis and abortion, lymphadenitis, nephritis), Anaplasma
spp. (splenic hyperplasia, anemia); Leptospira interrogans (hepatitis, nephritis,
cotyledonitis and abortion): Mycoplasma spp (pleuropneumonia, polyarthritis and
mastitis).
Regarding protozoa, Neospora caninum causes abortion with chorionic placentitis
and fetal anasarca, myocarditis, encephalitis, but also pneumonia, hepatitis, nephritis,
glossitis and myositis. A few intralesional cysts and zoites may be observed. Dual
Neospora and BVD pestivirus infection may be observed. Similar lesions and localization
may be observed with Sarcocystis spp. and with Toxoplasma gondi in sheep.
Tripanosoma evansi is a blood protozoa, but visceral forms of it have also been identified
and associated with myocarditis, hepatitis, nephritis, and encephalitis. The protozoa can
be identified within the lesions and in areas with no inflammation. Theileria parva and
annulata cause necrosis of lymphoid tissue and infiltration of immature large
lymphocytes within lymphoidtissue, lung, liver kidney and brain. Blood vessel lumina
may appear occluded by lymphoblasts. Multinucleate intralymphocytic and extracellular
schizonts can be detected.
Mucormycosis (infections caused by Aspergillus spp, Mucor spp, Absidia spp) are
associated with ruminal acidosis and BVD virus infection. These angiocentric fungi cause
thrombosis and necrohemorrhagic ruminitis and abomasitis, cotyledonitis with cupping,
fetal dermatitis and abortion. Examples of pathogenic dimorphic fungi include
Histoplasma spp., Blastomyces spp., Cryptococcus spp., Coccidioides spp., and
Paracoccidioides spp. They are responsible of systemic mycoses. The primary focus of
infection may be skin, mucosae or lung, but secondary infection may occur elsewhere in
the body. Lesions generally are pyogranulomatous and necrotizing. Sporothrix schenckii
induces pyogranulomatous and ulcerative dermatitis, lymphangitis, and lymphadenitis
with systemic disease. Also the oomycete Pythium insidiosum causes cutaneous,
subcutaneous, lymphatic and systemic pyogranulomatous lesions. Their intralesional
presence can be identified via IHC.
Prototheca spp. is a saprophytic achlorophyllous alga able to cause
pyogranulomatous dermatitis, systemic disease. P. zopfii can cause pyogranulomatous
mastitis and organisms can be identified via IHC.
IHC is an invaluable tool for the diagnosis and study of the pathogenesis of
infectious diseases.
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