Systemic Mycobacterium avium Infection in a Dog Diagnosed by Polymerase Chain Reaction Analysis of Buffy Coat

Clinical Course

A 4-year-old, spayed female Great Pyrenees was presented for evaluation of weight loss, anemia, diarrhea, and intermittent right front limb lameness of 2 years’ duration. The owner reported that the lameness occurred sporadically and had responded to treatment with corticosteroids or carprofen. Occasional small bowel diarrhea was noted for 1 year and had been partially responsive to various antibiotics and dietary trials. In the 6 months prior to presentation, the dog had intermittent lethargy, anorexia, dyspnea, and nonspecific pain. In the month prior to presentation, the dog was given oral prednisone (0.9 mg/kg per os [PO] q 12 hours for 1 week, then gradually reduced over several weeks to 0.3 mg/kg PO q 12 hours), which seemed to alleviate some of the clinical signs.

On physical examination, the dog was normothermic, tachycardic (140 beats per minute), and tachypneic (120 breaths per minute). The dog was tetraparetic, moderately dehydrated, and depressed. She weighed 33 kg, with a body condition score of 1.5/5. Initial diagnostic tests included a complete blood count (CBC), serum biochemical panel, urinalysis, and thoracic and abdominal radiographs.

Abnormal results on the CBC included a moderate leukocytosis (22,900/μL; reference range 5800/μL to 11,700/μL), consisting of a mature neutrophilia (18,778/μL; reference range 3000/μL to 7100/μL), monocytosis (1145 μL; reference range 0 to 800/μL), and eosinophilia (916/μL; reference range 0 to 100/μL); a nonregenerative anemia (packed cell volume [PCV]=23%; reference range 38% to 59%); and thrombocytopenia (112,000/μL; reference range 157,000/μL to 394,000/μL). Abnormal results on the biochemical panel included an elevated alanine transaminase (ALT) (8100 U/L; reference range 21 U/L to 76 U/L) and alkaline phosphatase (ALP) (757 U/L; reference range 14 U/L to 72 U/L), and slight hypoalbuminemia (2.3 g/dL; reference range 2.5 g/dL to 3.8 g/dL). On urinalysis, the urine specific gravity was >1.035, and proteinuria (2+), bilirubinuria (2+), and hemoglobinuria (3+) were noted. Urine sediment was unremarkable.

Abdominal radiographs were unremarkable except for gas-filled loops of small intestine. Thoracic radiographs revealed a diffuse, patchy interstitial pattern throughout the thorax and mild pleural effusion. No evidence of intrathoracic lymphadenopathy was seen.

The dog was admitted to the hospital, and intravenous (IV) fluid therapy was started. Prednisone^a was continued (0.3 mg/kg PO q 12 hours). The dog became pyrexic (103.6°F) and progressively more lethargic throughout the day. Considering the possibility of sepsis or disseminated intravascular coagulation, blood was drawn for bacterial culture and activated clotting time measurement. The clotting time was normal. After blood cultures were obtained, IV antibiotic therapy with ticarcillin/clavulanic acid^b (50 mg/kg IV q 8 hours) was initiated.

The following day, abdominal ultrasonography revealed a hyperechoic mesentery, suggestive of infiltrative disease, with a hypervascular congested mesenteric vessel. A hypo-echoic structure within the mesentery, consistent with a granuloma, was also imaged. The duodenum was diffusely thickened, with an approximate wall thickness of 1 cm. Fine-needle aspirates of the liver were obtained and showed occasional macrophages containing large, nonstaining rods suggestive of a Mycobacterium species.

A second CBC demonstrated an increase in the leukocytosis (37,200/μL), with a mature neutrophilia (31,992/μL), monocytosis (1860/μL), and eosinophilia (1488/μL); persistent anemia (PCV 27%); and a further decrease in platelet count (59,000/μL). Close scrutiny of a blood smear showed a neutrophil with a nonstained intracytoplasmic rod. The dog was moved to an isolation ward, and amikacin^c (30 mg/kg IV once daily) was initiated to specifically address a suspected mycobacterial infection. Because of continued clinical deterioration, the grave prognosis associated with mycobacterial infections in dogs, and the possible zoonotic threat posed by some mycobacterial species, the dog was euthanized.

Speciation Tests

A blood sample was submitted for acid-fast staining and PCR analysis of buffy-coat preparations, and the body was submitted for necropsy. Deoxyribonucleic acid (DNA) was extracted from nucleated buffy-coat cells from the dog, using the QIAamp DNA Mini Kit,^d following the manufacturer’s instructions. Extracted DNA was used as a template in a specific PCR assay for M. tuberculosis complex, using primers amplifying a 123 base pair fragment of insertion sequence IS6110, as previously described.³ The sample from this dog did not produce an amplicon. Infection with M. tuberculosis complex in this dog was considered unlikely, since IS6110-negative strains of M. tuberculosis are extremely rare in the United States.⁴ Polymerase chain reaction assay specific for the IS901 gene of M. avium was performed as described and was negative.⁵ The negative result of this PCR did not eliminate M. avium, however, since not all strains of M. avium have this insertion sequence.

Mycobacterium avium was confirmed by sequencing following PCR amplification of a 447 base pair portion of the 65 kDa heat shock protein gene (hsp65) as previously described.⁶ Successful PCR amplification was determined by ethidium bromide staining following the resolution of PCR products by 1.5% agarose gel electrophoresis. Samples were viewed under ultraviolet illumination in a gel docking system.^e The amplicons from the sample were cut from the agarose gel using a sterile scalpel blade, and DNA was purified using a Quantum Prep Freeze ‘N Squeeze DNA Gel Extraction Spin Column,^f following the manufacturer’s instructions. To obtain the most rapid diagnosis, direct nucleotide sequencing of the purified amplicons was performed by Amplicon Express, Pullman, Washington, using the Applied Biosystems Big Dye Terminator Kit,^g and it was analyzed on an ABI 377 DNA Sequencer.^h The sequence of a 393 base pair fragment was obtained with nine nucleotides that could not be resolved. Base reading errors are common technical artifacts of direct sequencing, but they often provide sufficient information for a presumptive identification. This sequence was analyzed with a GenBank search for similarities using BLAST (Basic Local Alignment Search Tool) and FASTA programs online.^i,7 The sequence from this dog’s sample had 97% identity with the hsp65 gene of M. avium and M. avium subspecies paratuberculosis, and it had 92% identity with M. tuberculosis and M. bovis [see Table]. Based on this information, a presumptive diagnosis of M. avium infection was made.

The identity of the sequence was then confirmed by sequencing the amplified fragment from a plasmid vector, which resolved the base reading errors in the direct sequence and also allowed analysis of a larger number of nucleotides. A GenBank search for similarities was performed on a 473 base pair fragment using BLAST and FASTA programs and resulted in 100% identity with the hsp65 gene of M. avium, 99% identity with M. avium subspecies paratuberculosis, and 92% to 93% identity with next-closest matches, which were M. marinum, M. ulcerans, M. tuberculosis, and M. bovis. Subsequent to the diagnosis by sequencing the hsp65 gene, the laboratory incorporated a technique targeting 16S ribosomal rDNA specific for M. avium, following a described protocol.⁸ This protocol gave a positive result for the dog.

Necropsy Findings

Because of the zoonotic potential associated with some mycobacterial species, necropsy was delayed for 2 days pending PCR speciation, as directed by the public health veterinarian. During this time, the body underwent considerable autolysis. At necropsy, the spleen was diffusely enlarged, with rounded edges. The peripancreatic and duodenal mesentery were severely thickened with numerous 0.1- to 0.5-cm, white to yellow nodules adhered to the surface. The omentum contained a small number of similar nodules. Mesenteric lymph nodes and Peyers’ patches were mildly enlarged. The liver was mildly enlarged. The lungs were firm with multifocal, pinpoint white nodules scattered throughout the lobes.

Histopathological examination of the spleen, small intestines, liver, adrenal cortices, mesenteric lymph nodes, bone marrow, and lungs showed a large number of macrophages with intracytoplasmic acid-fast bacilli, consistent with a disseminated M. avium infection. Additionally, lung sections contained evidence of lungworm infestation, and myocardial and brain sections revealed protozoal cysts identified by PCR as Neospora caninum (N. caninum).

The immunological status of this dog was not determined, but the possibility of immune deficiency was considered likely because of the severity of the mycobacterial lesions and presence of protozoal and lungworm infestations. Treatment with glucocorticoids may also have contributed to immunocompromise in this animal. A source for the intermittent lameness could not be found grossly. Bacteriological culture of various tissues isolated a large number of acid-fast bacilli with the phenotypic and growth rate characteristics of M. avium. Based on molecular biology and cytological, histopathological, and bacteriological culture findings, a diagnosis of disseminated M. avium infection was made.