SEPTEMBER 2023

P-P03

Signalment (JPC #2131712): Three‑week‑old broiler cockerel.

HISTORY: Unknown

HISTOPATHOLOGIC DESCRIPTION: Lung: Focally extensively affecting over 60% of the section, parabronchial and atrial lumina are variably occluded by numerous lymphocytes and histiocytes, abundant necrotic debris, few viable and necrotic heterophils, hemorrhage, fibrin, edema, and sloughed epithelial cells. Multifocally, the parabronchial and atrial respiratory epithelium is either hyperplastic or attenuated. The bronchial respiratory epithelium is hyperplastic, piling up to 3 layers thick, with occasional mitotic figures (regeneration) and occasional loss of cilia. Numerous basophilic, round, 2‑5µm cryptosporidia either line the ciliated surface of the bronchial epithelium or are within parabronchial lumina. The affected interstitium is expanded by numerous macrophages, lymphocytes, and plasma cells that also obscure air capillary profiles. The lamina propria of the mesobronchus, and to a lesser extent an adjacent secondary bronchus, is multifocally expanded by increased numbers of lymphocytes and plasma cells that form lymphoid aggregates (hyperplastic bronchial-associated lymphoid tissue).

MORPHOLOGIC DIAGNOSES: 1. Lung: Bronchitis, proliferative and lymphohistiocytic, subacute, focally extensive, moderate, with BALT hyperplasia and numerous epithelial apical-associated protozoa, chicken, avian.

2. Lung: Pneumonia, interstitial, lymphohistiocytic, subacute, focally extensive, moderate.

ETIOLOGIC DIAGNOSES: Cryptosporidial bronchopneumonia

CAUSE: Cryptosporidium baileyi

GENERAL DISCUSSION:

Note: As Cryptosporidium baileyi is not typically associated with interstitial pneumonia, there is likely a second disease process occurring in this bird; unfortunately, no additional clinical or diagnostic information is available to determine what that might be
Cryptosporidiosis: Respiratory disease is most common in birds, while enteric disease is more common in other species
Cryptosporidium spp. develops intracellularly at an extracytoplasmic location within the brush border membrane of host epithelial cells (parasitophorous vacuole)
Although it can be a primary pathogen, Cryptosporidium is usually opportunistic, infecting young and immunocompromised animals; zoonotic pathogen
Avian Cryptosporidium spp. do not infect mammals

PATHOGENESIS:

C. baileyi infects the digestive tract (cloaca, bursa of Fabricius), the upper and lower respiratory tracts, and the eyelids
Broiler chickens may develop resistance to reinfection with C. baileyi; there is evidence that cell mediated immunity (via T-cell interactions) is more important in resistance than humoral immunity (i.e. circulating antibody), though chicks may derive some protection through maternal (yolk) antibodies that hamper zoite invasion
Enterocyte tropism mediated by ligand-receptor interactions (Cryptosporidium sporozoite ligand) and GP900 adherence factor, and other glycoproteins used in adhesion
Cytolysis due to parasite or T cell and macrophage mediated products

LIFE CYCLE:

Sporulated oocysts are inhaled or ingested > sporozoites are released by excystation > attach to epithelial brush border > merogony- asexual multiplication within epithelial cells forming first and second generation merozoites > schizonts formed by: 1) asexual multiplication; 2) gametogony - sexual formation of male (microgamonts) and female (macrogamonts) gametes > oocyst wall formed> sporogony – formation of infective sporozoites within oocyst wall > oocyst infective when released in feces or discharge from upper respiratory tract
There are two kinds of oocysts formed: thin-walled and thick-walled; both sporulate within the host cell
Autoinfection occurs via thin walled oocysts; thick walled oocysts are shed in nasal secretions and feces; autoinfection of host allows for large biotic potential and heavy colonization of the gut/airway from a small infective dose

TYPICAL CLINICAL FINDINGS:

Nasal and ocular discharges; swollen sinuses
Coughing, sneezing, dyspnea, rales
Morbidity and mortality may be high

TYPICAL GROSS FINDINGS:

Swelling of the nasal sinus mucosa
Respiratory tract mucosal congestion
Gray or white mucoid exudate on mucosal surface
Lungs may be congested and edematous, and may have extensive consolidation
Moderate to severe tracheitis, pneumonia, and air sacculitis

TYPICAL MICROSCOPIC FINDINGS:

2-8 µm, apicomplexan protozoa that colonize gastrointestinal and respiratory epithelium at the brush border surface in vertebrates
Epithelial hyperplasia
Thickened mucosa due to infiltration of mononuclear cells with some heterophils
Loss of cilia
Discharge of mucocellular exudate into the airways
Cryptosporidium found within the ciliated region of trachea and bronchi
Affected air sacs contain large numbers of protozoa within respiratory epithelium and similar changes
In the GI tract, there is villar blunting and loss, causing osmotic diarrhea

ULTRASTRUCTURAL FINDINGS:

Cryptosporidium spp. attach to host cell glycocalyx
Located within a parasitophorous vacuole (derived from host microvilli) lined by glycocalyx

Development is intracellular but extracytoplasmic

Attachment zone contains feeder organelle
Loss of microvilli at site of attachment; organisms are associated with loss of microvilli and formation of a pedestal

ADDITIONAL DIAGNOSTIC TESTS:

Cytology - Fecal smears stained with red carbon fuchsin, Ziehl-Neelsen, or Giemsa
Fecal flotation – Sheather’s sucrose solution, zinc sulfate, sodium chloride
Phase-contrast, interference phase-contrast microscopy – Touch impressions
Fluorescent staining with auramine O or fluorescein-labeled antibody
Acid fast stains
EM, TEM
Kinyoun acid-fast stain direct smear is cheap and effective (versus PCR, lateral flow immunoassay, and fluorescent antibody) though all methods detect cryptosporidium reliably and have a high degree of agreement among each other (Atwal et al., JVDI 2022)
Potassium dichromate stabilizes oocysts for later study

DIFFERENTIAL DIAGNOSIS:

Respiratory: Concurrent infection with bacterial, viral, or fungal organisms is common:

Infectious bronchitis (Coronavirus)
Infectious laryngotracheitis (Gallid herpes-1)
Newcastle disease (Paramyxovirus-1)
Avian influenza (Orthomyxovirus)
Reovirus
Adenovirus
Infectious coryza (Avibacterium paragallinarum, formerly Haemophilus paragallinarum)
Mycoplasmosis
Salmonellosis
Escherichia coli
Pasteurella multocida
Aspergillosis

Bursitis: Bursal cryptosporidiosis is commonly seen in chickens with Infectious Bursal Disease (Birnavirus) or chicken anemia virus (Circovirus).

COMPARATIVE PATHOLOGY:

Mammals:

Cryptosporidium parvum: Small intestine (all mammals); freely transmissible among mammals, but does not infect vertebrates in other Orders; usually clinically inapparent; intractable diarrhea (profuse, watery) in young or immunocompromised
C. muris: Gastric mucosa (ruminants, dogs, cats, rats, hamster, mice); more significant in nude/SCID mice
C. wrairi: Jejunum, ileum, cecum (guinea pigs); commonly seen in conventional colonies
C. cuniculus: Small intestine (rabbits); often incidental finding
C. suis and scrofarum: Mild enteritis and diarrhea (domestic pigs and wild boar)
Pulmonary cryptosporidiosis reported in SIV-infected Rhesus Macaques
Cryptosporidium skunk genotype (CSkG) also reported in raccoons co-infected with canine distemper or parvovirus; coinfection phenotype of cryptosporidium and distemper mimics classic GI parvovirus lesions (crypt necrosis) in mesocarnivores (Gonzalez-Astudillo et al., JVDI 2021)
C. muris-like infection reported in stomach of Cynomolgus Monkeys
C. andersoni infection reported to cause proliferative abomasitis in cattle
Severe fatal infections occur in Arabian foals with CID; non-human primates infected with SIV; cats with FeLV

Birds:

C. baileyi: Digestive tract (cloaca, bursa of Fabricius), upper and lower respiratory tracts; most common in chickens, but also infects turkeys, ducks, geese, gallinaceous birds, ratites, raptors, and psittacines; intestinal infection is asymptomatic in chickens and turkeys; associated with syndrome of cloacal prolapse in ostrich chicks, particularly males; necrosis and inflammation in the pancreatic ductal epithelium
C. galli: Proventriculus; mucosal hyperplasia with lymphoplasmacytic inflammation, luminal hemorrhage, and atrophy of adipose tissue (psittacines)
C. meleagridis: Small intestine; associated with diarrhea, unthriftiness, and moderate mortality (turkeys, quail)

Reptiles and Amphibians:

C. serpentis and C. crotali - Proliferative gastritis in snakes (D-P06A), atrophic gastritis in lizards
C. spp. associated with aural-pharyngeal polyps in iguanas
C. fragile – Weight loss and mild gastric epithelial hyperplasia without inflammation (blank spined toads)
C..spp. – Proliferative gastritis (African clawed frog)

Fish:

C. nasorum associated with severe wasting in stomach and intestine; apicomplexans rarely cause severe disease however in fish

References:

Abee CR, Mansfield K, Tardif S, Morris T. Nonhuman Primates in Biomedical Research: Volume 2: Diseases. 2nd ed. San Diego, CA: Elsevier; 2012, 209-213.
Atwal HK, Zabek E, Bidulka J, DuCharme A, Pawlik M, Himsworth CG. A comparison of assays for the detection of Cryptosporidium parvum in the feces of scouring calves. J Vet Diagn Invest. 2022;34(2):284-287.
Barthold SW, Griffey SM, Percy DH. Pathology of Laboratory Rodents & Rabbits, 4th ed. Ames, IA: Blackwell Publishing Ltd; 2016:82,108,152,189,204-205,235-236,297.
Bowman DD. In: Bowman DD, ed. Georgis’ Parasitology for Veterinarians. 11th ed. St. Louis, MO: Saunders Elsevier; 2021:101-103.
Boulianne M. Avian Disease Manual. 8th ed., Atlanta, GA:American Association of Avian Pathologists; 2019:141-142.
Cheville NF. Ultrasructural Pathology, The Comparative Cellular Basis of Disease. 2^nd ed., Ames, IA:Wiley-Blackwell; 2009:555-9.
Gardiner CH, Fayer R, Dubey JP. An Atlas of Protozoan Parasites in Animal Tissues. 2nd ed., Washington, DC:Armed Forces Institute of Pathology, American Registry of Pathology; 1998:37-38.
Gelburg HB. Alimentary system and the peritoneum, omentum, mesentery, and peritoneal cavity. In: Zachary JF. Pathologic Basis of Veterinary Disease. 7th ed. St. Louis, MO: Mosby Elsevier; 2022:287-289,456.
Gonzalez-Astudillo V, Sheley MF, Uzal FA, Navarro MA. Pathology of cryptosporidiosis in raccoons: case series and retrospective analysis, 1990–2019. J Vet Diagn Invest. 2021 33:4, 721-727
Jimenez Martinez MA, Gasper DJ, Carmona Mucino MC, Terio KA. Suidae and Tayassuidae. In: Terio KA, McAloose D, St. Leger J, eds. Pathology of Wildlife and Zoo Animals, Cambridge, MA Academic Press; 2018: 221.
Origgi FC. Lacertilia. In: Terio KA, McAloose D, St. Leger J, eds. Pathology of Wildlife and Zoo Animals, Cambridge, MA Academic Press; 2018: 882-883.
Ossiboff FJ. Serpentes. In: Terio KA, McAloose D, St. Leger J, eds. Pathology of Wildlife and Zoo Animals, Cambridge, MA Academic Press; 2018: 908, 910.
Pessier AP. Amphibia. In: Terio KA, McAloose D, St. Leger J, eds. Pathology of Wildlife and Zoo Animals, Cambridge, MA Academic Press; 2018: 939.
Reavill DR, Dorrestein G. Passeriformes, Caprimulgiformes, Coraciiformes, Piciformes, Bucerotiformes, and Apodiformes. In: Terio KA, McAloose D, St. Leger J, eds. Pathology of Wildlife and Zoo Animals, Cambridge, MA Academic Press; 2018: 811-812.
Ritchie BW, Harrison GJ, Harrison LR. Avian Medicine: Principles and Application. Lake Worth, FL:Wingers Publishing, Inc.;1994:1016-1017.
Saif YM. Cryptosporidiosis. In: ed. Swayne DE, Diseases of Poultry. 14th ed., Hoboken, NJ: Wiley; 2020:1217-1222.
Smith DA. Palaeognathae: Apterygiformes, Casuariiformes, Rheiformes, Struthioniformes; Tinamiformes. In: Terio KA, McAloose D, St. Leger J, eds. Pathology of Wildlife and Zoo Animals, Cambridge, MA Academic Press; 2018: 644-645.
Taylor MA, Coop RL., Wall RL. Veterinary Parasitology. 4th ed., Cambridge, MA:Blackwell Science, Inc.; 2016:139,718-719.
Trees AJ. Parasitic disease. In: eds. Pattison M, McMullin PF, Bradbury JM, Alexander DJ, Poultry Disease. 6^th ed., Philadelphia, PA: Elsevier; 2008:457-8.
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; 2016:239-241.
Wunschmann A, Armien AG, Hofle U, Kinne J, Lowenstine LJ, Shivaprasad HL. Birds of Prey. In: Terio KA, McAloose D, St. Leger J, eds. Pathology of Wildlife and Zoo Animals, Cambridge, MA Academic Press; 2018: 734.