In cooperation with
Pathology in Practice
Figure 1—Photomicrographs of blood films from a 9-week-old male Australian Shepherd mix with a chronic cough. A—Blood film stained with a modified Wright stain; bar = 20 µm. B—Blood film stained with an aqueous Romanowsky-type stain; bar = 20 µm.
History A 9-week-old 5.6-kg (12.3-lb) male Australian Shepherd mix was evaluated at the Kansas State University Veterinary Medical Teaching Hospital because of a chronic cough. The cough had been present since the puppy was obtained as a stray 4 weeks prior. The cough was unresponsive to amoxicillin and doxycycline (given in combination, dose unknown), trimethoprim-sulfamethoxazole (dose unknown), and enrofloxacin (dose unknown), which were administered in succession. The puppy was found with 5 other littermates and was the runt of the litter. Clinical and Clinicopathologic Findings On physical examination, the puppy had a constant dry, nonproductive cough that was present without and This report was submitted by Marlene Y. Townsell, DVM; Lisa M. Pohlman, DVM, MS; and Kenneth R. Harkin, DVM; from the Department of Clinical Sciences, College of Veterinary Medicine, Kansas State University, Manhattan, KS 66506. Dr. Townsell’s present address is Small Animal Teaching Hospital, College of Veterinary Medicine, Auburn University, Auburn, AL 36832. Address correspondence to Dr. Townsell ([email protected]. edu). JAVMA, Vol 246, No. 6, March 15, 2015
elicited by tracheal palpation. Mucoid nasal discharge was present bilaterally. Crackles were auscultated in all lung fields. Rectal temperature (38.4°C [101.2°F]), pulse rate (120 beats/min), and respiratory rate (40 breaths/min) were within reference intervals. Thoracic radiography revealed a diffuse bronchiolar pulmonary pattern with a pulmonary fissure line between the right cranial and middle lung lobes. The dog underwent venipuncture to provide blood samples for serum biochemical analysis and a CBC. No major abnormalities were detected via serum biochemical analysis. The CBC revealed mild nonregenerative anemia (Hct, 33% [reference interval, 37% to 55%]; reticulocyte count, 34,000 cells/µL [reference interval, > 60,000 cells/µL indicative of regenerative anemia]) and lymphopenia (lymphocyte count, 300 cells/µL [reference interval, 1,500 to 5,000 cells/µL]). A modified Wright-stained blood film preparation and an aqueous Romanowsky-type–staineda blood film preparation were examined microscopically (Figure 1). The aqueous Romanowsky-type stain was part of a commercially available kit composed of methanol, eosin, and thiazin. Formulate differential diagnoses from the history, clinical findings, and Figure 1—then turn the page → Vet Med Today: Pathology in Practice
613
Microscopic Findings On the modified Wright-stained blood film (Figure 2), several erythrocytes contained single round light blue-gray inclusions that were 2 to 4 µm in diameter. Rare neutrophils contained similar inclusions, albeit inclusions were generally more oval and pale in color. When stained with the aqueous Romanowsky-type staina (Figure 3), the leukocyte and erythrocyte inclusions appeared intensely eosinophilic.
Cytologic Interpretation and Case Summary Interpretation: the appearance and distribution of the intracytoplasmic inclusions in the leukocytes and erythrocytes were consistent with canine distemper viral inclusions. Case summary: canine distemper virus disease in a dog. Comments
Figure 2—Same image of the modified Wright-stained blood film in panel A of Figure 1. Several erythrocytes contain round, light blue inclusions that are 2 to 4 µm in diameter (arrowheads). The neutrophil contains pale blue-gray inclusions (arrows). Modified Wright stain; bar = 20 µm.
Figure 3—Same image of the aqueous Romanowsky-type–stained blood film in panel B of Figure 1. Several erythrocytes contain round, pink inclusions that are 2 to 4 µm in diameter (arrowheads). The neutrophil contains an irregularly shaped, pink, cytoplasmic inclusion (arrow). Aqueous Romanowsky-type stain; bar = 20 µm. 614
Vet Med Today: Pathology in Practice
Canine distemper virus is a paramyxovirus within the genus Morbillivirus.1,2 The widespread use of vaccinations has markedly decreased the incidence of canine distemper virus disease among dogs, but outbreaks among unvaccinated and, occasionally, vaccinated dogs still occur. Most canine distemper virus infections in dogs are believed to be subclinical or only cause mild upper respiratory tract signs. Most infections are thought to resolve without treatment.3 Severity of clinical signs depends on the ability of the infected dog to mount an immune response against the virus. If the dog has a strong immune response, the virus is cleared from the tissues prior to development of clinical illness and the dog makes a full recovery. If the virus is not cleared from the tissues within approximately 3 to 6 days after infection, the dog typically becomes pyrexic and anorexic as a result of widespread virus proliferation. This initial phase of disease is typically followed by coughing and labored breathing, ocular and nasal discharge, and then vomiting and diarrhea. Dogs that mount a weak immune response may develop variable degrees of illness and ultimately recover, but the virus persists in the skin, uvea, CNS, and urothelium. In these dogs, hyperkeratosis (of the footpads or nasal planum) and delayed CNS signs may develop. When a dog has minimal immune response to the virus, the acute form of disease progresses to involve the CNS, resulting in ataxia, hyperesthesia, myoclonus, and seizures. Dogs that develop neurologic signs usually die within 2 to 4 weeks after infection.1,2 Canine distemper virus is surrounded by a lipoprotein envelope and has a single-stranded negative-sense RNA genome.1 The primary mode of transmission is inhalation of airborne virus, with infected dogs shedding viral particles in almost all bodily secretions. After the virus becomes trapped in the upper respiratory tract mucosa, hemagglutinin facilitates attachment of the virus to host JAVMA, Vol 246, No. 6, March 15, 2015
cell receptors,4 enabling the union of the host cell membranes with the viral envelope1 and facilitating prompt replication in macrophages and lymphoid tissue.1,2 Viral replication within B and T lymphocytes results in cellular damage and peripheral lymphopenia (as seen in the case described in the present report).1 On microscopic examination of a peripheral blood smear, viral antigen can appear as inclusion bodies in lymphocytes and, less commonly, in neutrophils and macrophages.2 Inclusion bodies in Wright-stained WBCs appear as large (approx 3 µm), oval, gray structures. With this staining technique, it is less common to see viral inclusion bodies in RBCs, but when detected, those inclusion bodies appear round, eccentrically placed, and blue.2,5,6 The appearance of these inclusion bodies is different with the use of an aqueous Romanowsky-type staina; with this technique, inclusion bodies in both WBCs and RBCs appear pink.5 Romanowsky-type stains use a combination of basic dyes that bind to DNA, RNA, and acidic proteins and acidic dyes that bind to hemoglobin and basic proteins. Originally, these dyes were aqueous in nature and were later modified to improve stability. Such aqueous dyes still have widespread use in general practice because they are easy to use and provide results rapidly. Most clinical pathology laboratories use the modified form of these staining techniques known as methanolic Wright or Wright-Giemsa staining. Although the differences between the 2 are subtle, it has been long established that canine distemper viral inclusions are better visualized during microscopic examination of blood film preparations stained by aqueous staining methods.5,6 Characteristic cytoplasmic inclusions in RBCs and WBCs are not detected in blood film preparations from all cases of canine distemper viral infection, which may just reflect an early phase of disease.2 Therefore, the absence of detectable inclusion bodies in a blood film preparation obtained from a sick dog should not be used to rule out the diagnosis of canine distemper virus infection if that is suspected clinically. If inclusions are found, this can be a simple, cost-effective way to make a diagnosis
JAVMA, Vol 246, No. 6, March 15, 2015
of canine distemper virus infection without more expensive or time-consuming diagnostic techniques. More conventional and reliable diagnostic techniques for the diagnosis of canine distemper virus infection include immunofluorescent antibody testing and PCR assay. Immunofluorescent antibody testing performed on scrapings or smears from conjunctival, tonsillar, nasal, or vaginal epithelium can be used to diagnose canine distemper virus infection within the first 3 weeks.1 Reverse transcriptase PCR assay and real-time reverse transcriptase PCR assay can be used to detect canine distemper virus in buffy coat preparations and samples of whole blood, CSF, and other affected tissues and do not require that the infection be in the acute phase.1 Later in the course of disease, viral inclusions may be detected in cells from the lower respiratory tract.3 In the case described in the present report, the client was informed of the guarded prognosis and elected to provide supportive care. The dog was sent home, and treatment included clarithromycin, interferon, hydrocodone, and vitamin C. The outcome for this dog remains unknown. a.
Diff-Quick, Medix Corp, Los Angeles, Calif.
References 1. 2. 3. 4. 5. 6.
Martella V, Elia G. Canine distemper virus. Vet Clin North Am Small Anim Pract 2008;38:787–797. Greene CE, Appel MJ. Canine distemper. In: Greene CE, ed. Infectious diseases of the dog and cat. 3rd ed. St Louis: Saunders Elsevier, 2006;25–41. Taylor MS. Encephalitis, myelitis, and meningitis. In: Nelson RW, Couto CG, eds. Small animal internal medicine. 4th ed. St Louis: Mosby, 2009;1059–1060. Zachary JF. Nervous system. In: McGavin MD, Zachary JF, eds. Pathologic basis of veterinary disease. 4th ed. St Louis: Mosby Elsevier, 2007;934–935. Harvey JW. Hematology tip—stains for distemper inclusions. Vet Clin Pathol 1982;11:12. Allison RW, Velguth KE. Appearance of granulated cells in blood films stained by automated aqueous versus methanolic Romanowsky methods. Vet Clin Pathol 2010;39:99–104.
Vet Med Today: Pathology in Practice
615
Pathology in Practice
Figure 1—Photomicrographs of blood films from a 9-week-old male Australian Shepherd mix with a chronic cough. A—Blood film stained with a modified Wright stain; bar = 20 µm. B—Blood film stained with an aqueous Romanowsky-type stain; bar = 20 µm.
History A 9-week-old 5.6-kg (12.3-lb) male Australian Shepherd mix was evaluated at the Kansas State University Veterinary Medical Teaching Hospital because of a chronic cough. The cough had been present since the puppy was obtained as a stray 4 weeks prior. The cough was unresponsive to amoxicillin and doxycycline (given in combination, dose unknown), trimethoprim-sulfamethoxazole (dose unknown), and enrofloxacin (dose unknown), which were administered in succession. The puppy was found with 5 other littermates and was the runt of the litter. Clinical and Clinicopathologic Findings On physical examination, the puppy had a constant dry, nonproductive cough that was present without and This report was submitted by Marlene Y. Townsell, DVM; Lisa M. Pohlman, DVM, MS; and Kenneth R. Harkin, DVM; from the Department of Clinical Sciences, College of Veterinary Medicine, Kansas State University, Manhattan, KS 66506. Dr. Townsell’s present address is Small Animal Teaching Hospital, College of Veterinary Medicine, Auburn University, Auburn, AL 36832. Address correspondence to Dr. Townsell ([email protected]. edu). JAVMA, Vol 246, No. 6, March 15, 2015
elicited by tracheal palpation. Mucoid nasal discharge was present bilaterally. Crackles were auscultated in all lung fields. Rectal temperature (38.4°C [101.2°F]), pulse rate (120 beats/min), and respiratory rate (40 breaths/min) were within reference intervals. Thoracic radiography revealed a diffuse bronchiolar pulmonary pattern with a pulmonary fissure line between the right cranial and middle lung lobes. The dog underwent venipuncture to provide blood samples for serum biochemical analysis and a CBC. No major abnormalities were detected via serum biochemical analysis. The CBC revealed mild nonregenerative anemia (Hct, 33% [reference interval, 37% to 55%]; reticulocyte count, 34,000 cells/µL [reference interval, > 60,000 cells/µL indicative of regenerative anemia]) and lymphopenia (lymphocyte count, 300 cells/µL [reference interval, 1,500 to 5,000 cells/µL]). A modified Wright-stained blood film preparation and an aqueous Romanowsky-type–staineda blood film preparation were examined microscopically (Figure 1). The aqueous Romanowsky-type stain was part of a commercially available kit composed of methanol, eosin, and thiazin. Formulate differential diagnoses from the history, clinical findings, and Figure 1—then turn the page → Vet Med Today: Pathology in Practice
613
Microscopic Findings On the modified Wright-stained blood film (Figure 2), several erythrocytes contained single round light blue-gray inclusions that were 2 to 4 µm in diameter. Rare neutrophils contained similar inclusions, albeit inclusions were generally more oval and pale in color. When stained with the aqueous Romanowsky-type staina (Figure 3), the leukocyte and erythrocyte inclusions appeared intensely eosinophilic.
Cytologic Interpretation and Case Summary Interpretation: the appearance and distribution of the intracytoplasmic inclusions in the leukocytes and erythrocytes were consistent with canine distemper viral inclusions. Case summary: canine distemper virus disease in a dog. Comments
Figure 2—Same image of the modified Wright-stained blood film in panel A of Figure 1. Several erythrocytes contain round, light blue inclusions that are 2 to 4 µm in diameter (arrowheads). The neutrophil contains pale blue-gray inclusions (arrows). Modified Wright stain; bar = 20 µm.
Figure 3—Same image of the aqueous Romanowsky-type–stained blood film in panel B of Figure 1. Several erythrocytes contain round, pink inclusions that are 2 to 4 µm in diameter (arrowheads). The neutrophil contains an irregularly shaped, pink, cytoplasmic inclusion (arrow). Aqueous Romanowsky-type stain; bar = 20 µm. 614
Vet Med Today: Pathology in Practice
Canine distemper virus is a paramyxovirus within the genus Morbillivirus.1,2 The widespread use of vaccinations has markedly decreased the incidence of canine distemper virus disease among dogs, but outbreaks among unvaccinated and, occasionally, vaccinated dogs still occur. Most canine distemper virus infections in dogs are believed to be subclinical or only cause mild upper respiratory tract signs. Most infections are thought to resolve without treatment.3 Severity of clinical signs depends on the ability of the infected dog to mount an immune response against the virus. If the dog has a strong immune response, the virus is cleared from the tissues prior to development of clinical illness and the dog makes a full recovery. If the virus is not cleared from the tissues within approximately 3 to 6 days after infection, the dog typically becomes pyrexic and anorexic as a result of widespread virus proliferation. This initial phase of disease is typically followed by coughing and labored breathing, ocular and nasal discharge, and then vomiting and diarrhea. Dogs that mount a weak immune response may develop variable degrees of illness and ultimately recover, but the virus persists in the skin, uvea, CNS, and urothelium. In these dogs, hyperkeratosis (of the footpads or nasal planum) and delayed CNS signs may develop. When a dog has minimal immune response to the virus, the acute form of disease progresses to involve the CNS, resulting in ataxia, hyperesthesia, myoclonus, and seizures. Dogs that develop neurologic signs usually die within 2 to 4 weeks after infection.1,2 Canine distemper virus is surrounded by a lipoprotein envelope and has a single-stranded negative-sense RNA genome.1 The primary mode of transmission is inhalation of airborne virus, with infected dogs shedding viral particles in almost all bodily secretions. After the virus becomes trapped in the upper respiratory tract mucosa, hemagglutinin facilitates attachment of the virus to host JAVMA, Vol 246, No. 6, March 15, 2015
cell receptors,4 enabling the union of the host cell membranes with the viral envelope1 and facilitating prompt replication in macrophages and lymphoid tissue.1,2 Viral replication within B and T lymphocytes results in cellular damage and peripheral lymphopenia (as seen in the case described in the present report).1 On microscopic examination of a peripheral blood smear, viral antigen can appear as inclusion bodies in lymphocytes and, less commonly, in neutrophils and macrophages.2 Inclusion bodies in Wright-stained WBCs appear as large (approx 3 µm), oval, gray structures. With this staining technique, it is less common to see viral inclusion bodies in RBCs, but when detected, those inclusion bodies appear round, eccentrically placed, and blue.2,5,6 The appearance of these inclusion bodies is different with the use of an aqueous Romanowsky-type staina; with this technique, inclusion bodies in both WBCs and RBCs appear pink.5 Romanowsky-type stains use a combination of basic dyes that bind to DNA, RNA, and acidic proteins and acidic dyes that bind to hemoglobin and basic proteins. Originally, these dyes were aqueous in nature and were later modified to improve stability. Such aqueous dyes still have widespread use in general practice because they are easy to use and provide results rapidly. Most clinical pathology laboratories use the modified form of these staining techniques known as methanolic Wright or Wright-Giemsa staining. Although the differences between the 2 are subtle, it has been long established that canine distemper viral inclusions are better visualized during microscopic examination of blood film preparations stained by aqueous staining methods.5,6 Characteristic cytoplasmic inclusions in RBCs and WBCs are not detected in blood film preparations from all cases of canine distemper viral infection, which may just reflect an early phase of disease.2 Therefore, the absence of detectable inclusion bodies in a blood film preparation obtained from a sick dog should not be used to rule out the diagnosis of canine distemper virus infection if that is suspected clinically. If inclusions are found, this can be a simple, cost-effective way to make a diagnosis
JAVMA, Vol 246, No. 6, March 15, 2015
of canine distemper virus infection without more expensive or time-consuming diagnostic techniques. More conventional and reliable diagnostic techniques for the diagnosis of canine distemper virus infection include immunofluorescent antibody testing and PCR assay. Immunofluorescent antibody testing performed on scrapings or smears from conjunctival, tonsillar, nasal, or vaginal epithelium can be used to diagnose canine distemper virus infection within the first 3 weeks.1 Reverse transcriptase PCR assay and real-time reverse transcriptase PCR assay can be used to detect canine distemper virus in buffy coat preparations and samples of whole blood, CSF, and other affected tissues and do not require that the infection be in the acute phase.1 Later in the course of disease, viral inclusions may be detected in cells from the lower respiratory tract.3 In the case described in the present report, the client was informed of the guarded prognosis and elected to provide supportive care. The dog was sent home, and treatment included clarithromycin, interferon, hydrocodone, and vitamin C. The outcome for this dog remains unknown. a.
Diff-Quick, Medix Corp, Los Angeles, Calif.
References 1. 2. 3. 4. 5. 6.
Martella V, Elia G. Canine distemper virus. Vet Clin North Am Small Anim Pract 2008;38:787–797. Greene CE, Appel MJ. Canine distemper. In: Greene CE, ed. Infectious diseases of the dog and cat. 3rd ed. St Louis: Saunders Elsevier, 2006;25–41. Taylor MS. Encephalitis, myelitis, and meningitis. In: Nelson RW, Couto CG, eds. Small animal internal medicine. 4th ed. St Louis: Mosby, 2009;1059–1060. Zachary JF. Nervous system. In: McGavin MD, Zachary JF, eds. Pathologic basis of veterinary disease. 4th ed. St Louis: Mosby Elsevier, 2007;934–935. Harvey JW. Hematology tip—stains for distemper inclusions. Vet Clin Pathol 1982;11:12. Allison RW, Velguth KE. Appearance of granulated cells in blood films stained by automated aqueous versus methanolic Romanowsky methods. Vet Clin Pathol 2010;39:99–104.
Vet Med Today: Pathology in Practice
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