History
A naïve 1-year-old female athymic nude rat (strain, Crl:NIH-Foxn1rnu) used in an institutional animal care and use committee-approved research protocol at Emory University (an institution accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International) was evaluated because of respiratory distress and emaciation. Rats of the strain Crl:NIH-Foxn1rnu have an immunodeficiency characterized by a lack of T cells. Consequently, the rat was housed in a sterile, microisolator cagea with paper beddingb and provided an autoclaved rodent dietc and sterile water ad libitum. The animal was housed with 1 other, unaffected nude rat. In the preceding year, sentinel testing involving immunocompetent rats (strain, CRL:CD[SD]) in this animal housing room had detected the presence of Pneumocystis carinii, but no other infectious agents (eg, Sendai virus, pneumonia virus of mice, sialodacryoadenitis virus, Kilham rat virus, Toolan H-1 virus, rat parvovirus 1, rat minute virus, nonstructural antigens-1 for parvovirus, reovirus, rat theilovirus, Mycoplasma pulmonis, or pinworms).
Clinical and Gross Findings
On examination, the rat had severe clinical signs; it had a body condition score of 1/5 and tachypnea with abdominal effort. Owing to the severity of the clinical signs, a decision to euthanize the animal immediately was made. At necropsy, the only gross abnormality was found in the lungs (Figure 1). Approximately 80% of the lung parenchyma contained 2- to 4-mm-diameter miliary to coalescing, raised, white nodules. Bilateral rib impressions were noted on the lung surfaces.
Photograph of the lungs of a 1-year-old athymic nude rat that was euthanized because of respiratory distress and emaciation. Notice the 2- to 4-mm-diameter miliary to coalescing, raised, white nodules throughout the lung parenchyma and the lung's dark red appearance and failure to deflate following removal. There are appreciable rib impressions on the lung lobes.
Citation: Journal of the American Veterinary Medical Association 256, 5; 10.2460/javma.256.5.563
Formulate differential diagnoses from the history, clinical findings, and Figure 1—then turn the page→
Histopathologic Findings
Sections of lung tissue were obtained aseptically for PCR assay and fungal and bacterial culture and submitted for analysis.d The lungs, spleen, reproductive tract (uterine body and ovaries), 1 liver lobe, kidneys, adrenal glands, and heart were fixed in neutral-buffered 10% formalin for 48 hours at room temperature (approx 21°C). Formalin-fixed organs were then trimmed and processed for paraffin embedding. Paraffin-embedded tissues were sectioned at a thickness of 4 μm, mounted on positively charged glass slides, and stained with H&E stain. Some lung tissue sections were stained with Grocott methenamine silver stain to identify intralesional organisms. Glass slides on which sections were mounted were examined with a light microscope. Histologic examination revealed that the alveolar septa were multifocally and moderately hypercellular because of infiltration by mononuclear cells (Figure 2). In addition, histiocytes, multi-nucleated giant cells, and eosinophilic, finely vacuolated material containing irregularly shaped (maximum dimension, 1 to 4 μm), uninucleate trophozoites and smaller numbers of 4- to 8-μm-diameter, thick-walled, multinucleate cysts with intracystic bodies filled the alveolar spaces and small airways within large confluent areas of the lung (Figure 3). The staining of the organisms with Grocott methenamine silver stain was indicative of Pneumocystis carinii (Figure 4). Lung tissue that underwent a PCR assay for P carinii yielded positive results, further confirming the identity of the intralesional organisms. Additional histologic lesions were found in the spleen, ovaries, and kidneys. The rat's spleen was devoid of mature lymphocytes and there was widespread extramedullary hematopoiesis. The lack of mature lymphocytes in the spleen was consistent with the strain of the rat (athymic nude). The ovaries contained several corpora hemorrhagica. The kidneys had multiple, small mineralized foci that were believed to be incidental lesions of unknown importance or cause and unrelated to the rat's clinical condition. No gross or histologic lesions were found in the liver, heart, or adrenal glands.
Photomicrographs of an affected area of lung tissue from the rat in Figure 1. A—The alveolar spaces are expanded by eosinophilic, finely vacuolated material (especially prominent in the center and the top of the image). Additionally, alveolar septa are moderately hypercellular (best seen in the center of the bottom left quadrant of the image). H&E stain; bar = 200 μm. B—At high magnification, the hypercellular area has alveolar spaces that are filled with large numbers of histiocytes (arrowheads) with finely vacuolated cytoplasm; some histiocytes have formed multinucleated giant cells (open arrows). There is also a mild lymphocytic and plasmacytic infiltrate (arrows) in the alveolar walls and type II pneumocyte hyperplasia (asterisks) in scattered alveoli. H&E stain; bar = 50 μm.
Citation: Journal of the American Veterinary Medical Association 256, 5; 10.2460/javma.256.5.563
High-magnification photomicrograph of a section of affected lung tissue. The alveolar spaces are filled with acellular, eosinophilic, and finely vacuolated material. Within this material there are innumerable basophilic organisms (arrows). H&E stain; bar = 20 μm.
Citation: Journal of the American Veterinary Medical Association 256, 5; 10.2460/javma.256.5.563
High-magnification photomicrograph of a section of affected lung tissue following staining with Grocott methenamine silver stain. There are 4- to 8-μm-diameter, thick-walled, multinucleated cysts with intracystic bodies in the alveolar spaces and small airways within large confluent areas of the lung tissue. The staining of the cysts (arrows) is indicative of Pneumocystis carinii. Grocott methenamine silver stain; bar = 20 μm.
Citation: Journal of the American Veterinary Medical Association 256, 5; 10.2460/javma.256.5.563
Morphologic Diagnosis and Case Summary
Morphologic diagnosis and case summary: severe, chronic, interstitial pneumonia with intralesional organisms (P carinii) in a rat.
Comments
Pneumocystis carinii is an opportunistic fungal pathogen of the family Pneumocystidaceae. Pneumocystis infection commonly develops in immunodeficient animals of all species, with P carinii being host specific to rats.1 Immunocompetent animals can become infected with the fungus, and infection is characterized by lymphohistiocytic interstitial pneumonia with dense perivascular lymphoid cuffs; typically, immunocompetent animals clear the infection and do not develop clinical signs. In the past, a mild form of similar interstitial pneumonia in immunocompetent rats was incorrectly attributed to infection with rat respiratory virus,2 but has more recently been attributed to infection with P carinii.3,4 However, immunodeficient animals that are unable to clear the infection develop chronic progressive interstitial pneumonia that is characterized by alveoli filled with foamy, eosinophilic material.1,5
Pneumocystis carinii is generally spread through aerosol transmission6 although fomites, most notably soiled bedding, are another source of transmission among rats.2 Immunodeficient rats with Pneumocystis infection typically develop cachexia, which progresses to dyspnea, cyanosis, and death.7,8 Clinical diagnosis of infection is somewhat difficult because this organism cannot be grown in in vitro culture.9 In humans, a diagnosis of Pneumocystis infection is usually made on the basis of the results of Giemsa or direct immunofluorescence staining of bronchoalveolar lavage fluid samples (which are not easy to obtain from rats) or PCR assay of respiratory tract samples.9 The latter procedure is typically performed on samples from nasal swabs or, more preferably, of lung tissue1,10 to provide the diagnosis of Pneumocystis infection in rodents. This disease can also be tentatively identified by its typical gross and microscopic findings. Characteristically, the lungs of an affected animal are rubbery and enlarged with pale, gray, or red areas of consolidation; a pathognomonic feature of Pneumocystis infection is the failure of the lungs to deflate on postmortem examination. Microscopically, lymphohistiocytic interstitial pneumonia is present with alveoli filled with foamy, eosinophilic exudate and large numbers of Pneumocystis organisms.2,5,11 Methenamine silver or periodic acid-Schiff staining of lung tissue sections aids in highlighting the organisms within the alveoli.
The athymic nude rat of the present report had typical clinical signs of Pneumocystis infection, namely severe cachexia and respiratory distress. Cachexia is a classic sequela of Pneumocystis infection, and Pneumocystis infection should be high on the differential diagnosis list for any cachexic immunodeficient-strain rodent, especially if respiratory signs are evident. Routine testing of animal housing facilities for Pneumocystis organisms is recommended to identify contaminated rooms. Pneumocystis infections among laboratory rodents can confound research results, especially those of pulmonary studies; therefore, infected animals are poor research subjects.12 Rederivation of infected animals, which is the process of performing embryo transfers from rodents with Pneumocystis infection to noninfected rodents to breed disease-free animals is ideal. However, limiting contact of disease-free rodents with actively infected rodents may aid in conversion of a contaminated room to a clean room over time.
Footnotes
Thoren Caging Systems Inc, Hazleton, Pa.
ALPHA-dri, Shepherd Specialty Papers, Lawrenceville, Ga.
Purina LabDiet 5010, St Louis, Mo.
College of Veterinary Medicine, University of Georgia, Athens, Ga.
References
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