History
A 2-year-old vaccinated neutered male Golden Retriever was imported from Turkey, at which time it weighed 28.3 kg (62.3 lb). It had a high titer of circulating antibodies against Ehrlichia canis and hyperglobulinemia. Initial treatment for ehrlichiosis included doxycycline. One month later, the dog weighed 28.7 kg (63.1 lb) and it had hyperglobulinemia with hypoalbuminemia. Ultrasonographic findings included enlarged hepatic lymph nodes, mesenteric mineralization, and diffusely hyperechoic hepatic parenchyma with ill-defined, hypoechoic nodules. Over 2 months, multiple acquired portosystemic shunts, portal hypertension, and mineralized hepatic lymph nodes, mild hypoglycemia, high serum total protein concentration with hyperglobulinemia, hypoalbuminemia, and markedly high serum alkaline phosphatase activity were identified, and the dog's weight decreased to 24 kg (52.8 lb). Despite palliative treatment, the dog's condition worsened during the following week, and it developed lethargy, inappetence, diarrhea, and urinary incontinence. The owners opted for euthanasia of the dog by injection of pentobarbital sodium and submittal of the carcass for necropsy.
Gross Necropsy Findings
At necropsy, the dog had a body condition score of 3/9 and weighed 21.5 kg (47.3 lb). The oral and conjunctival mucous membranes and the skin were diffusely yellow. A prescapular lymph node was diffusely red on cut section. Scant frothy fluid was in the bifurcation of the main stem bronchi, and small amounts of serous fluid exuded from cut sections of the lungs. The liver was diffusely enlarged and friable with randomly distributed, coalescing, soft, tan foci that ranged from 0.2 to 1 cm in diameter and encompassed 80% of all lobes (Figure 1). The spleen was diffusely enlarged and mildly firm. Both kidneys had smaller, multifocal tan nodules, mostly localized to the cortex.
Formulate differential diagnoses from the history, clinical findings, and Figure 1—then turn the page→
Histopathologic and Microbiological Findings
Samples of a hepatic lymph node, a prescapular lymph node, liver, lungs, kidneys, spleen, brain, heart, adrenal glands, thyroid gland, and gastrointestinal tract were fixed in neutral-buffered 10% formalin and processed routinely for histologic examination. Fresh samples of a hepatic lymph node and the liver were frozen and submitted for microbial culture and other ancillary testing. Histologically, the liver was most severely affected and effaced by multifocal to coalescing, necrotizing granulomas. The granulomas were composed of central caseous necrosis encircled by epithelioid macrophages and few multi-nucleated giant cells and surrounded externally by lymphoplasmacytic inflammation and scant fibroplasia. Similarly, both kidneys had multifocal to coalescing granulomas (Figure 2). The hepatic lymph node was diffusely necrotic and infiltrated with numerous neutrophils. The prescapular lymph node had few, multifocal granulomas. The leptomeninges were infiltrated by small numbers of lymphocytes and plasma cells, and a few small granulomas composed of epithelioid macrophages were evident (Figure 3). The adrenal glands, lungs, heart, and mesentery of the small intestine also had variably sized but subjectively smaller granulomas. Ziehl-Neelsen staining of tissue sections revealed myriad acid-fast, elongated bacilli in the cytoplasm of epithelioid macrophages of granulomas of the liver, kidneys, leptomeninges, and the hepatic lymph node.
A sample of liver tissue underwent a PCR assay targeting the groEL gene, which generated a 439–base pair product, consistent with Mycobacterium spp. A partial region of the 16S rRNA gene was sequenced and compared with entries in a bioinformatic search tool,a which indicated a 100% similarity match with Mycobacterium bovis and Mycobacterium tuberculosis. Liver tissue specimens then underwent another PCR assay (targeting the hsp65 gene of Mycobacterium spp), and sequencing of these results was indicative of M bovis. A section of frozen liver was sent to the National Jewish Health Mycobacteriology Laboratory in Denver for use in a line probe assay to confirm M tuberculosis complex and to test for the presence of Mycobacterium-associated resistance mutations for rifampin and isoniazid. The presence of M tuberculosis complex and M bovis was confirmed. No katG, inhA, or rpoB mutations were detected, which could have conferred resistance to isoniazid and rifampin.
Morphologic Diagnosis and Case Summary
Morphologic diagnosis: severe, chronic, granulomatous hepatitis with myriad acid-fast bacilli consistent with Mycobacterium spp; severe, chronic, granulomatous nephritis with acid-fast bacilli; severe, necrotizing, neutrophilic lymphadenitis (hepatic lymph node) with acid-fast bacilli; and mild, multifocal granulomatous meningitis with acid-fast bacilli.
Case summary: disseminated tuberculosis attributable to M bovis infection in a Golden Retriever.
Comments
The dog of the present report had severe, disseminated tuberculosis, which had resulted in multifocal granulomas and granulomatous inflammation with characteristic acid-fast bacilli throughout many tissues, particularly the liver, kidneys, and hepatic lymph nodes. The diagnosis of tuberculosis associated with M bovis infection was confirmed with PCR assays and sequencing. Although M bovis infection is rare in dogs, the most common portal of entry is via the gastrointestinal tract through ingestion of contaminated milk or meat,1 which was the proposed route of infection for this dog. Mycobacterium bovis is part of the M tuberculosis complex, a group of highly pathogenic, facultative intracellular bacteria of the macrophage-monocyte system.1–3 Infection causes chronic, progressive granulomatous disease in the lungs, lymph nodes, and other organs.2,3 Approximately one-third of the world's human population has tuberculosis; M bovis is the second most common causative agent after M tuberculosis.4 To reduce confusion, M bovis is generally referred to as bovine tuberculosis or zoonotic tuberculosis to distinguish it from tuberculosis attributable to M tuberculosis. Cattle serve as the primary reservoir for M bovis; however, the organism has a wide host range and can infect various domestic and wildlife species as well as humans.1,5 In industrialized countries, control measures such as milk pasteurization and test-and-slaughter practices have largely eliminated M bovis as a cause of tuberculosis. However, in developing countries, M bovis persists in cattle populations because of insufficient or absent control measures and continues to be an important cause of tuberculosis.6
Mycobacterium bovis can be transmitted to humans via respiratory secretions, especially in crowded cattle production settings, and via human consumption of unpasteurized milk or meat. Mycobacterium spp can also be shed in feces and remain viable for months within the fecal organic matter.1 Cutaneous infection can also develop in humans after puncture or bite wounds inflicted by affected animals.1,5 Once any animal is infected, the bacteria can be disseminated via erosion into the vascular system by the growing granuloma or via the lymphatic vessels, thereby resulting in disseminated granulomatous disease. Gross lesions consist of focal areas of caseous necrosis in lymph nodes or organs. Histologically, focal granulomas composed of epithelioid macrophages with or without lymphocytes and plasma cells and with variable degrees of central necrosis and mineralization are typically seen.2,3
Clinical signs in dogs with tuberculosis reflect the site of granuloma formation and are related to the affected organ systems. Respiratory tract infection is associated with mild to severe respiratory disease and can progress to pneumonia, whereas gastrointestinal tract disease results in nonspecific clinical signs such as weight loss, anorexia, and lymphadenopathy.2 In dogs, the liver and spleen are also commonly affected either through direct extension from gastrointestinal tract infections or in association with disseminated disease.7 Oropharyngeal infection occasionally occurs and results in local lymphadenopathy and lesions that cause coughing, ptyalism, or dysphagia.2,7 Clinical laboratory findings similarly reflect the organ system affected. In addition, radiographic and ultrasonographic signs include visible mass effects within various affected organs and lymphadenopathy.1,2
In the case described in the present report, the dog was presented to the veterinary medical specialist with a history of progressive weight loss, icterus, and seropositivity for E canis. However, the dog did not respond to treatment for ehrlichiosis, and further diagnostic testing identified end-stage hepatic nodular disease as a probable cause of its declining health. Additionally, at postmortem examination, the hepatic nodules were soft and the examined hepatic lymph node was necrotic and neutrophilic, in contrast to inspissated nodules (tubercles) that are often associated with tuberculosis. Such uncharacteristic findings can lower clinical suspicion of mycobacterial disease and highlight the need to obtain a detailed patient history to accurately diagnose the underlying condition and institute appropriate personal protective measures during clinical examination and at the time of necropsy.
Infection via microbial aerosolization poses a risk to human health that is highest among farmers, veterinary staff, and rural slaughterhouse workers.8 A necropsy room is a unique environment wherein microorganisms in animal tissues are easily transferred or even aerosolized by the use of electronic oscillating saws and other equipment. Transmission of M tuberculosis to veterinary staff after the use of an electronic saw for brain removal from a dog during necropsy has been reported.9 Once the diagnosis of zoonotic tuberculosis was confirmed, tuberculin skin testing (purified protein derivative test) was recommended for the faculty, staff, and students who assisted with the necropsy, and further treatment of infected persons was performed on the basis of those test results. More recently, a similar case of tuberculosis as a result of infection with M bovis in a dog from Brazil was reported.10 In that case, the dog had nonspecific clinical signs, and chronic canine ehrlichiosis was initially suspected as the cause for its declining health and severe weight loss. However, M bovis infection was confirmed at necropsy with the presence of acid-fast staining bacteria in the liver, kidneys, and mesentery.10
In some large agricultural states, such as Michigan, M bovis is endemic, and infections in beef and dairy cattle have also been detected in 21 states, including Iowa, Kansas, Minnesota, and Ohio. In Michigan in particular, bovine tuberculosis continues to pose a threat to wild white-tailed deer, and surveillance and eradication efforts have been in place for > 20 years.11
For the dog of the present report, ingestion was the suspected route of infection; however, given the limited history for the dog prior to its entry into the United States, the exact route of infection was not confirmed. Additionally, no underlying factors that could have predisposed the dog to immunosuppression were identified.
Footnotes
BLASTn, National Center for Biotechnology Information, National Library of Medicine, Bethesda, Md. Available at: blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=blastn&PAGE_TYPE=BlastSearch&LINK_LOC=blasthome. Accessed Jul 14, 2016.
References
1. Greene CE, Gunn-Moore DA. Mycobacterial infections. In: Green CE, ed. Infectious diseases of the dog and cat. 4th ed. St Louis: Elsevier Saunders, 2012;495–510.
2. Lopez A, Martinson SA. Respiratory system, mediastinum and pleurae. In: Zachary JF, ed. Pathologic basis of veterinary disease. 6th ed. St Louis: Elsevier, 2017;471–560.
3. Caswell JL, Williams KJ. Respiratory system. In: Maxie MG, ed. Jubb, Kennedy, and Palmer's pathology of domestic animals. Vol 2. 6th ed. St Louis: Elsevier, 2016;547–551.
4. CDC. Data and statistics: tuberculosis. Available at: www.cdc.gov/tb/statistics/default.htm. Accessed Aug 24, 2016.
5. Grange JM, Yates MD. Zoonotic aspects of Mycobacterium bovis infection. Vet Microbiol 1994;40:137–151.
6. Cosivi O, Grange JM, Daborn CJ, et al. Zoonotic tuberculosis due to Mycobacterium bovis in developing countries. Emerg Infect Dis 1998;4:59–70.
7. Brown DL, Van Wettere AJ, Cullen JM. Hepatobiliary system and exocrine pancreas. In: Zachary JF, ed. Pathologic basis of veterinary disease. 6th ed. St Louis: Elsevier, 2017;412–470.
8. Michel AL, Müller B, van Helden PD. Mycobacterium bovis at the animal-human interface: a problem, or not? Vet Microbiol 2010;140:371–381.
9. Posthaus H, Bodmer T, Alves L, et al. Accidental infection of veterinary personnel with Mycobacterium tuberculosis at necropsy: a case study. Vet Microbiol 2011;149:374–380.
10. Rocha VC, Figueiredo SC, Rosales CA, et al. Infection by Mycobacterium bovis in a dog from Brazil. Braz J Microbiol 2017;48:109–112.
11. Orloski K, Robbe-Austerman S, Stuber T, et al. Whole genome sequencing of Mycobacterium bovis isolated from livestock in the United States, 1989–2018. Front Vet Sci 2018;5:253.