JPC SYSTEMIC PATHOLOGY
DIGESTIVE SYSTEM
November 2021
D-V07 (NP)

 

SIGNALMENT (JPC #1902491):  9-day-old CD-1 mouse

 

HISTORY:  This mouse was inoculated with an infectious agent at 7 days of age.

 

HISTOPATHOLOGIC DESCRIPTION:  Small intestine:  Diffusely, the enterocytes of the superficial third to half of the distal villar tips are swollen/hypertrophied and contain multiple, variably sized, clear, discrete, cytoplasmic vacuoles or a single large vacuole measuring up to 40 microns in diameter that peripheralizes and compresses the enterocyte nucleus.  There is multifocal mild submucosal edema, characterized by mildly increased clear space and mildly dilated lymphatics. 

 

MORPHOLOGIC DIAGNOSIS:  Small intestine, villar tip enterocytes:  Vacuolar degeneration, diffuse, marked, with mild submucosal edema, CD-1 mouse, murine.

 

ETIOLOGY:  Murine group A rotavirus (RV-A)

 

ETIOLOGIC DIAGNOSIS:  Rotaviral enteritis

 

CONDITION:  Epizootic Diarrhea of Infant Mice (EDIM)

 

GENERAL DISCUSSION: 

• Rotavirus is a ubiquitous virus of the family Reoviridae, a 75nm diameter, non-enveloped, tri-layered (only the complete triple-layered virion is infective), double stranded, RNA virus that is host-specific and causes diarrhea and enteritis in infants
• Rotavirus is divided into 7 serogroups (A-G) by differences in genome codes for the VP6 intermediate capsid, and each is further divided into “G” and “P” genotypes by outer capsid proteins VP7 and VP4
• Group A is the most diverse and common of the serogroups
• Many group A rotaviruses have been isolated from mice, of which EDIM is a single strain; although EDIM has been accepted as inclusive of all mouse intestinal rotaviruses
• EDIM is a major cause of diarrhea in intensively reared animals
• Neonates (<2 weeks) are especially affected because of the high proportion of terminally differentiated enterocytes and the slow turnover rate; adults get infected and shed virus but typically are asymptomatic
  • Infection confers immunity; only mouse pups from non-immune dams develop clinical disease while pups from immune dams are protected by maternal antibodies in milk (lactogenic immunity)
• Virus-infected cells are most prevalent within 1 day of experimental infection and the numbers decrease rapidly, with only few cells with viral antigen present 3-4 days post infection
• A carrier state has not been identified, but subclinical infection is common, and virus can persist in the environment for extended periods

 

PATHOGENESIS: 

• Fecal-oral transmission à virus attaches and enters terminally differentiated enterocytes +/- goblet cells of the upper half to two-thirds of the small intestinal villi (predominantly jejunum and ileum) and large intestine (in mice) using viral attachment proteins VP4 and VP7 (which appear to attach to multiple host cell apical membrane proteins such as sialic acids, integrins, heat shock proteins, and gangliosides) à viral replication within host cell à cell lysis (cytolytic), virus release à degeneration and necrosis of terminally differentiated enterocytes (villar tips) à villous atrophy, blunting, and fusion (with compensatory crypt hyperplasia) à malabsorption and maldigestion à diarrhea, shedding of copious virus in feces
• Virus produces a secretory enterotoxin NSP4 (non-structural protein 4) that:
  • Increases chloride secretion via a calcium-dependent mechanism à secretory diarrhea
  • Activates enteric nervous system à increased peristalsis (intestinal hypermotility)
  • Increases intracellular calcium à disruption of cytoskeletal system and tight junctions à increased mucosal permeability
  • Blocks intestinal sodium/glucose transporter à secretory diarrhea
  • Blocks brush-border membrane disaccharidases à retention of lactose and other disaccharides in the intestinal lumen à osmotic diarrhea
• Infected enterocytes produce a vasoactive agent à villus ischemia and enteric nervous system activation à increased peristalsis, ischemic damage
• Antigenic shift via reassortment may result in emergence of more pathogenic strains
• Neonatal protection from infection is largely conferred via lactogenic immunity

 

TYPICAL CLINICAL FINDINGS:

• Mouse pups (<14 days): Abdominal bloating, pasting of unformed feces around anus; runting
• Affected pups continue to suckle
• Older mice: no signs
• Clinical signs are most prominent in naïve populations; once infection is endemic, disease is no longer apparent although the virus still persists
• BALB/c are relatively susceptible, B6 are relatively resistant, immunocompromised SCID mice follow the same age-related pattern of disease

 

TYPICAL GROSS FINDINGS: 

• Runting and pot-bellied appearance
• Stomach contains curdled milk
• Steatorrhea, oily hair
• Intestines may be flaccid and distended with mucoid yellow digesta and gas

 

TYPICAL LIGHT MICROSCOPIC FINDINGS: 

• In mice, lesions may be minimal despite significant diarrhea
• Lesions are typically more prominent at the distal small intestine, and are nonsynchronous along the length
• Blunting (club-shaped), atrophy, +/- fusion of intestinal villi covered by low columnar to cuboidal to flattened epithelium with a poorly defined brush border; prior to sloughing, affected enterocytes are swollen with vacuolated apical cytoplasm, and rarely contain eosinophilic intracytoplasmic inclusion bodies
• +/- crypt hypertrophy
• +/- lamina propria edema and/or inflammatory infiltrate (mononuclear cells, eosinophils, or neutrophils)
• In species that are affected by both rotavirus and coronavirus, the microscopic lesions of the small intestine cannot be differentiated

 

ULTRASTRUCTURAL FINDINGS:

• Enterocytes contain crystalline arrays of virions both in cytoplasm and within membrane-bound vacuoles with necrotic debris, or granular “viroplasm” containing incomplete virions in the apical cytoplasm
• Virions consist of a central core (containing double stranded DNA) with protein spikes that project from the surface, appearing wheel-like (hence “rota”)
• Virions acquire their capsid after budding into the endoplasmic reticulum
• Virions accumulate in dilated cisternae of the endoplasmic reticulum
• Affected enterocytes are degenerate or necrotic, with cell swelling, loss of cytoplasmic electron density, irregular stunted microvilli, membrane blebbing, and swollen mitochondria +/- syncytia (syncytia noted in bovine, porcine, and lab animal rotaviral infections); these degenerate cells are fragile and shed readily so may not be seen

 

ADDITIONAL DIAGNOSTIC TESTS: 

• Definitive diagnosis is via electron microscopy of intestinal mucosa or feces
• Fecal ELISA, PCR, and ISH
• Serology for surveillance and retrospective confirmation of infection

 

DIFFERENTIAL DIAGNOSIS:

For diarrhea in young mice

• Enterotropic mouse hepatitis virus (MHV;LIVIM; coronavirus): Multinucleated syncytial cells and eosinophilic, intracytoplasmic, inclusions
• Mouse adenovirus (MAdV-2): Intranuclear inclusions in intestinal epithelium; does not typically cause clinical disease in healthy mice
• Reovirus: Foci of hepatic necrosis and CNS lesions, but also causes myocardial necrosis and pulmonary hemorrhage
• Salmonella: Paratyphoid nodules in liver, bacteria cultured from feces
• Tyzzer's disease (Clostridium piliforme): Intracytoplasmic bacilli in hepatocytes

 

For enterocyte swelling and vacuolation:

• Absorption vacuoles of the neonatal enterocytic apical tubular system occur physiologically in the distal small intestine and contain solitary eosinophilic globules

 

COMPARATIVE PATHOLOGY: 

• Each species of animal (mammals and birds) has its specific group A rotavirus, and these are generally not cross-infective except at extremely high doses (e.g. experimental infection); these viruses typically cause disease (diarrhea) in young animals in association with other enteropathogens (e.g. coronavirus, Cryptosporidium, E. coli, coccidia); diagnosis is difficult due to the virus’ ubiquitous nature including presence in healthy animals as well as test negative status in diseased animals (infected cells previously shed in the feces)
• Group A rotaviruses are important diseases in:
  • Humans
  • Calves: diarrhea in neonatal beef and dairy calves (both suckled and artificially reared) less than 1 week of age, due to weaning à reduced ingestion of antirotaviral antibodies; clinical signs include dehydration, yellow watery diarrhea, weakness, depression; virus infects enterocytes of the apical half of the villus, mainly of jejunum and ileum; disease is mild compared to ETEC or bovine coronavirus, and coronavirus can cause lesions in the colon while rotavirus is restricted to the small intestine
  • Piglets: less than 7 weeks of age, due to weaning à reduced ingestion of antirotaviral antibodies (“postweaning scours”); subclinical infection is common, clinical signs include dehydration, yellow watery diarrhea, weakness, depression; virus infects enterocytes of the entire villus, mainly of jejunum and ileum; enterocyte vacuolation, villus atrophy, epithelial attenuation, and cell debris in the lamina propria are the most common microscopic findings; infection is widespread and enzootic in most swine herds; signs resemble transmissible gastroenteritis virus (coronavirus) but are typically less severe and vomiting is less consistently seen; commonly associated with other enteropathogens ( coli, coccidiosis, adenovirus, Strongyloides)
    • In a recent study of rotaviral diarrhea in piglets (Almeida, J Vet Diagn Invest. 2018), Kobuvirus was detected as a sole etiologic agent in 3 of 34 cases; these were interpreted as most likely false negative for rotavirus
  • Group A rotaviruses are reported but not commonly considered important diseases in:
    • Puppies: uncommon, affects puppies less than 1-2 weeks of age, generally nonfatal diarrhea when coinfected with coronavirus and various other viral agents (e.g. astroviruses, adenovirus, paramyxovirus, calicivirus, herpesvirus, and circovirus has recently been associated)
    • Cats: rotavirus and other viruses (astrovirus, enteric coronavirus, calicivirus) have been reported from cat feces and have rarely been associated with diarrhea and even less frequently with mortality
    • Lambs: may cause diarrhea alone or in combination with ETEC and/or Cryptosporidium, lactogenic immunity is important; disease is similar to other species except may infect the colon
    • Foals: affects foals less than 3-4 months of age, and is a major cause of diarrhea although mortality is rare; horses are often infected with more than one strain of rotavirus, including group G rotaviruses, with unknown clinical significance; coinfections are common (e.g. Salmonella, Cryptosporidium, equine coronavirus)
    • Old World and New World camelid neonates: More prevalent in NW camelids with gross and histological lesions similar to cattle.
    • Nonhuman primates, including macaques: despite their ubiquitous nature, relatively few simian rotavirus isolates have been well characterized; thought to cause mild, self-limiting diarrhea during infancy
    • Rabbits: infection is common, clinical signs are rare
  • Non-group A rotaviruses can infect pigs, ruminants, ferrets, and rats
    • Group C rotaviruses are reported as an important cause of diarrhea in piglets, and are the primary cause of rotaviral enteritis of ferrets (these are not detected with commercially available group A rotavirus ELISA tests), with only anecdotal reports of group A rotavirus; causes diarrhea in neonates 1-3 weeks of age and may be fatal due to rapid-onset dehydration
    • Group B rotaviruses are found only in humans and rats (infectious diarrhea of infant rats, IDIR, see D-V08, pathognomonic enterocyte syncytia +/- intracytoplasmic eosinophilic inclusions, thought to be human origin)
    • Group H rotaviruses have been reported in humans, swine, and bats

 

REFERENCES: 

1. Agnew D. Camelidae. In: Terio KA, McAloose D, St. Leger J, eds. Pathology of Wild Life and Zoo Animals. Cambrige, MA, Elseveir, 2018: 197-198.
2. Almeida PR, Lorenzetti E, Cruz RS, et. al. Diarrhea caused by rotavirus A, B, and C in suckling piglets from southern Brazil: molecular detection and histologic and immunohistochemical characterization.  J Vet Diag Invest.  2018;30(3):370-376.
3. Barthold SW, Griffey SM, Percy DH. Pathology of Laboratory Rodents and Rabbits. 4th ed. Ames, IA: Blackwell Publishing; 2016: 37-38, 129, 267-268.
4. Delaney MA, Treuting PM, Rothenburger JL. Lagomorpha. In: Terio KA, McAloose D, St. Leger J, eds. Pathology of Wild Life and Zoo Animals. Cambrige, MA, Elseveir, 2018: 491
5. Flores PS, Costa FB, Amorim AA, Mendes GS, Rojas M, Santos N. Rotavirus A, C, and H in Brazilian pigs: potential for zoonotic transmission of RVA. J Vet Diagn Invest. 2021;33(1):129-135.
6. Gelberg HB. Alimentary system and the peritoneum, omentum, mesentery, and peritoneal cavity. In: Zachary JF, ed. Pathologic Basis of Veterinary Disease. 6th ed. St. Louis, MO: Elsevier; 2017:374-375.
7. Kiupel M, Perpinan D. Viral diseases of ferrets.  In: Fox JG, Marini RP. Biology and diseases of the ferret.  3rd ed.  Ames, IA: Wiley; 2014: 471-474.
8. Uzal FA, Plattner BL, Hostetter JM. Alimentary system. In: Maxie MG, ed. Jubb, Kennedy and Palmer’s Pathology of Domestic Animals. Vol 2. 6th ed. Philadelphia, PA: Elsevier Saunders; 2016:112, 115-117, 151-153.
9. Resende TP, Marthaler D, Vannucci FA. In situ hybridization detection and subtyping of rotaviruses in swine samples. J Vet Diagn Invest. 2019;31(1):113-117.
10. Wachtman L, Mansfield K. Viral diseases of nonhuman primates.  In: Abee CR, Mansfield K, Tardiff S, Morris T, eds.  Nonhuman Primates in Biomedical Research: Diseases, Vol. 2.  2nd ed.  Waltham, MA: Academic Press; 2012:71.
11. Zachary JF. Mechanisms of microbial infections. In: Zachary JF, ed. Pathologic Basis of Veterinary Disease. 6th ed. St. Louis, MO: Elsevier; 2017:200.

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