Joint Pathology Center

Veterinary Pathology Services

Wednesday Slide Conference

2019-2020

Conference 17

5 February, 2020

 

 

CASE I: 1235813-006 (JPC 4117518).

 

Signalment: Three year old, male cynomolgus macaque (Macaca fascicularis) from China

 

History: This animal was found dead on day 109 of a 6 month routine general toxicity study. There were no reported clinical signs. Routine infectious agent screens and three consecutive tuberculosis skin tests were negative with the exception of measles which was positive after a live vaccine.

 

Gross Pathology There were moderate, focal to multifocal, tan foci on the liver and lung with moderate swelling of the mediastinum. Skin discolorations with abrasions / scabs were present on the ventral abdomen and right forelimb.

 

Laboratory results: The tan foci were PCR positive for Mycobacterium tuberculosis group and culture yielded slow growing acid fast bacilli identified as Mycobacterium tuberculosis.

 

Microscopic Description: Throughout approximately 90% of examined lung there was multifocal to coalescing granulomatous inflammation often forming discrete granulomas with central eosinophilic flocculent debris (caseating necrosis) with or without basophilic granular mineralization. This central area was surrounded by epithelioid macrophages, multinucleated giant cells, a rim of lymphocytes and variable fibroblasts/fibrosis. Similar solitary discrete granulomas were seen in the spleen, liver, and mediastinum (mediastinum not submitted). There was an increase in number and prominence of germinal centers in the spleen (follicular hyperplasia) as well as frequent multifocal type 2 pneumocyte hyperplasia in the lung.

 

Ziehl-Nielsen acid fast staining of the lung revealed rare acid fast bacilli within the cytoplasm of macrophages or multinucleated giant cells, and/or free within the necrotic/mineralized areas.

 

Contributor Morphologic Diagnosis:

Granulomatous pneumonia, severe, diffuse with focal hepatic and splenic granulomas and scant acid-fast bacilli.

 

Contributor Comment: Mycobacterium tuberculosis (M.tb) is a cosmopolitan organism that infects approximately 1/3 of the world?s human population and is a leading cause of death due to infectious disease. It is also an increasing public health concern due to 1) emerging multidrug resistant and extensively drug resistant M.tb and 2) the increasing risk of death in HIV infected people.²⁰ Tuberculosis was considered the cause of 40% of all HIV-related deaths in 2016.²⁰ Roughly 10% of people infected with M.tb go on to develop active contagious disease with the remaining individuals either clearing the bacteria (approximately 10%) or harboring latent disease. These latent carriers create a reservoir of tuberculosis which can reactivate during times of stress or immune suppression and disseminate M.tb to naïve populations of humans²⁰ or spread the disease to susceptible animals (reverse zoonosis).¹³

 

In an NHP necropsy, M.tb often presents as tan or creamy white nodules (granulomas), predominantly within the lung, but also with systemic involvement. Other common causes of tan nodules or granulomas within the lung includes foreign body (kaolin), protozoan (Toxoplasma gondii), bacterial (Nocardia sp), mycotic (Coccidioides immitis, Coccidioidoes posadasii, Histoplasma capsulatum, Blastomyces dermatitidis, and Cryptococcus neoformans) and parasitic organisms (Pneumonyssus simicola (acariasis), Taenia cysts, Echinococcus cysts, Mesocestoides tetrathyridia, Spirometra spargana, and Filaroides and Filariopsis nematodiasis), Histology can distinguish between these diagnoses based on the appearance of the organisms.¹¹

 

M.tb and mycobacteria in general are unusual among bacteria due to the high lipid content and relative thickness of their cell wall compared to gram-positive and gram-negative bacteria. The thick mycobacterial cell wall is relatively impermeable, does not take up crystal violet (Gram stain variable), and is responsible for the acid fast staining properties of mycobacteria. Immunologically, the lipid component of the mycobacterial cell wall is particularly effective at recruiting an immune response and has been commonly used as an adjuvant in vaccines to recruit a desired robust immune response to antigens of interest. The protein (antigen) and lipid components together during mycobacterial infection result in sensitization of the animal to tuberculosis proteins. Sensitized animals then react to purified mycobacterial protein derivative (PPD) in a skin test with a Type IV hypersensitivity reaction resulting in a cell mediated immune reaction after 2-3 days. Non-responders, or animals that fail to demonstrate a positive response to the PPD skin test in the face of mycobacterial infection (as seen in this monkey) are thought to be due to immunologic anergy.³ Anergy is a failure of the animal to produce a Type IV delayed type hypersensitivity reaction upon PPD exposure. The exact cause of anergy in M.tb infection is unknown; although, in general, it is a lack of response of cell mediated immunity due to tyrosine kinase blockage and reduced IL-2 receptor signaling, among other mechanisms.¹⁷ In M.tb infection a weak cell-mediated immune response (high IL-4) is thought to be associated with anergy.⁷

 

Another feature of the mycobacterial cell wall is its role in allowing the bacteria to evade the host immune system. In part, the success of M.tb is due to the highly infectious nature of the bacteria. It is thought that as few as 10 bacteria can cause disease.¹⁸ Once within the host, mycobacteria are engulfed by macrophages into phagosomes. Under normal circumstances within a macrophage, phagosomes mature and fuse with lysosomes exposing the bacteria to destructive chemicals and lytic enzymes leading to bacterial death. Mycobacteria have evolved mechanisms to avoid phagosome-lysosome fusion and survive within macrophages. For example, components of the mycobacterial cell wall interrupt the normal traffic of the phagosome to the lysosome by preventing recruitment of key molecules in phagosomal maturation such as the Rab5 to Rab7 conversion.¹ Phagosome maturation arrest in M.tb is thought to involve specific cell surface receptors (i.e. mannose receptor) and mycobacterial cell wall components (i.e. lipoarabinomannan and SapM enzyme)⁵. Ultimately, whether the mycobacteria are destroyed or remain active or latent within the host depends upon the infectious dose, the host immune response, and the environment of the infection.³ A host response characterized by a Th2 cytokine response (IL-4) is associated with mycobacteria infection and Th1 cytokine response (IL-12, TNF-α, and IFN-γ) is associated with bacterial clearance.¹

 

There are many mycobacteria that cause significant human and animal diseases. The histologic features of these diseases range from structured granulomas (often associated with few bacteria/paucibacillary infection) to more diffuse macrophage infiltration (often associated with numerous bacteria/multibacillary infection). Granulomas, or tubercles, have four histologic features: 1) central caseous necrosis with or without mineralization, 2) surrounded by epithelioid macrophages and Langhans multinucleated giant cells, 3) surrounded by lymphocytes with or without neutrophils and peripheral fibrosis, 4) tubercles contain rare acid fast bacilli which are found within macrophages, multinucleated giant cells, and/or the caseous core. In paucibacillary mycobacterial diseases, gross observations are crucial to identifying areas to look for these rare bacilli as bacteria are not typically found outside of the structured granulomas.³. Multibacillary infection is more variable with few to sheets of infiltrating macrophages containing few to numerous acid fast bacilli. Leprosy (Mycobacterium leprae) is centered on nerves and can be paucibacillary and granulomatous or multibacillary and diffuse depending upon the strength of the cell mediated immune response with a stronger cell mediated response associated with the paucibacillary infection.⁷ Common mycobacterial species involved in disease are listed in Table A.

 

Table A3
Mycobacteria	Species Affected	Systems involved and/or Gross appearance	Typical histologic appearance
M. tuberculosis	humans, NHPs, elephants, psittacine birds (zoonosis and reverse zoonosis)   exotic ungulates, marine mammals, carnivores14, dogs, cats, pigs, cattle   Potential reservoir species: humans	Granulomas ? lung and systemic (i.e. liver, spleen, etc.)	granulomas
M bovis	cattle, dogs/cats, humans, cervids, swine, horses   deer, elk, bison, buffalo, goats, camels, llamas, swine, elephants, rhinoceroses, dogs, foxes, cats, mink, badgers, humans, NHPs, pigs, goats, warthogs, cats, mustelids, humans   Potential reservoir species: badgers, brush tailed possums, swamp buffalo, buffalo, bison, various species of deer	cattle: granulomas ? lung and systemic (lymph nodes, gastrointestinal tract, etc)   dogs and cats: skin nodules or thickening/inflammation   humans: pulmonary granulomas or gastrointestinal inflammation   cervids: pulmonary granulomas, superficial lymphadenitis with draining tracts (DDx: Fusobacterium necrophorum   horses: Gastrointestinal thickening/ granulomas   swine: systemic granulomas	granulomas   dogs and cats: unstructured pyogranulomatous inflammation of the skin dissecting along facial planes. (Note: central necrosis and paucibacillary help to distinguish from feline leprosy); often more granulation tissue with few scattered macrophages.
M avium subspecies avium and hominissuis6	birds > swine, cattle, horses, sheep, NHPs, humans	birds: thin body condition with intestinal and hepatic > systemic granulomas (lungs, air sacs, spleen, bone marrow and skin)   humans: lymphadenitis, granulomas ? lung and/or systemic	granulomas
M. avium paratuberculosis	cattle, goats, sheep	muscle atrophy/wasting; thickened mucosa of the ileum, large intestine, enlarged draining nodes; intimal plaques/mineral in thoracic aorta   small ruminants: granulomas (enteric or vascular/lymphatic)	multibacillary ? unstructured granulomatous enteritis with villus atrophy   sheep: lepromatous neuritis reported
M.microti18	voles, humans	voles: gastrointestinal granulomas   humans: pulmonary granulomas	granulomas
M.africanum4	humans	Pulmonary granulomas	granulomas
M.avium intracellularae complex (MAC)	dogs, cats	atypical mycobacterial skin infection - singe to multiple cutaneous and subcutaneous nodules, plaques, macules or diffuse swelling In cats: thickened, hardened tissue in the inguinal fat	multibacillary unstructured pyogranulomatous dermatitis and panniculitis. Can resemble feline leprosy. Occasionally fibroplasia may be marked and the lesion may appear as a spindle cell neoplasm
M. marinum10	fish   humans	fish: systemic disease (potential route of infection skin trauma or gastrointestinal tract)   humans: skin granulomas > pulmonary granulomas	granulomas
M. kansasii	humans	cervical lymphadenitis; pulmonary granulomas	granulomas
M.scrofulaceum15	humans	cervical lymphadenitis ?scrofula?	granulomas
saprophytic Mycobacteria (M. smegmatis, M. fortuitum, M. chelonae8, M. phlei, M. thermoresistible, M. xenopi)	dogs/cats	Focal thickened skin / inflammation	atypical mycobacterial skin infection - Similar to MAC although paucibacillary and clear vacuoles surrounded by neutrophils and peripheral epithelioid macrophages. Organisms can be found within the clear vacuoles
M. leprae9	armadillos, sooty mangabeys, chimpanzees, cynomolgus macaques, red squirrels	Multifocal to coalescing granulomatous dermatitis with loss of sensation/body parts on face, hands, and feet.	granulomas (tuberculoid) to unstructured (lepromatoid) granulomatous inflammation centered on nerves
M lepraemurium	rodents, cats	often head and limb cutaneous or subcutaneous nodules with or without alopecia or ulceration, often 1-3 year old cats	granulomatous dermatitis and/or panniculitis ? large, pale, foamy macrophages; tuberculoid (organized granulomas) to lepromatous (sheets of cells); nerve involvement is only sporadic (distinct from human leprosy
Mycobacterium?	Cats	Feline tuberculosis syndrome in adult cats with firm skin nodules with draining tracts	pyogranulomatous dermatitis and panniculitis
Mycobacterium?	Dogs	canine leproid granuloma syndrome ? firm painless nodules mainly on the head and dorsal pinnae of short coated breeds	acid fast bacteria identified, but not cultured; histo description unavailable

 

 

 

Contributing Institution:

MPI Research; a Charles River company

https:///www.crl.com

https://www.mpiresearch.com

 

JPC Diagnosis:

1. Lung: Pneumonia, bronchointerstitial, necrogranulomatous, multifocal to coalescing, severe, with Langhans multinucleated giant cells, mineralization, and edema.

2. Spleen: Splenitis, necrogranulomatous, multifocal to coalescing, severe, with mineralization.

 

JPC Comment: The contributor has provided an excellent review of infection by M tuberculosis, (which has also been a popular submission in non-human primates as well as various models over the years).

The very early history of Mtb is still conjecture, but early strains may have infected hominids up to three million years ao with modern strains originating some 15,000-20,000 years ago. Egyptian mummies have demonstrated skeletal deformitis reminiscent of "Pott&146;s disease", and are even present in Egyptian art. The first written documents about TB appeared in India and Chinica dating back to 3000 to 2000 years ago.²

TB was well known and referred to as phthisis in ancient Greece. Isocrates first suggested the contagious nature of "the king?s evil". The Romans recognized the fist symptomatology of TB and recommended fresh air, milk, and sea voyages as treatment².

Pulmonary TB was well known in Middle Ages Europe, with "scrofula", a form of TB affecting the cervical nodes, described as a new clinical form. Before the first surgical interventions in the removal of scrofula, the touch of English or French royalty was considered a cure (and continued in France until 1825).²

The true infectious nature of "phthisis" and "consumption" was expounded upon by English physician Benjamin Martin, and the disease was first called "tuberculosis" Johan Schonlein in the mid 19^th century. The industrial revolution and poorly ventilated and overcrowded workplaces and housing with poor sanitation resulting in a fluorishing of TB, with up to 15% of workers dying of tuberculosis in 1838-1839 in England alone. The anemic pllor of the afflicted resulted in another name ? "the white plague". The "white plague" reached a zenith in the mid-1800s, resulting in almost one in four deaths in Europe and North America².

The 1800s also brought a new and revised view of tuberculosis, with numerous renowned clinicians connecting the dots between miliary disseminated disease, tubercules, consolidation, pleurisy and pulmonary cavitation. In 1843, German physician Philipp Klencke was able to reproduce the human and bovine disease n rabbits. In 1850, the "sanatorium cure" was described for the first time by Hermann Berhmer, a botany student suffering from TB himself, and was used for the next 100 years as a mainstay of treatment for TB patients.²

In 1882, Robert Koch identified, isolated, and for the first time visualized the tubercle bacillus of Mycobacterium tuberculosis in a number of manifestations of tuberculosis, including pulmonary, scofulaceous, extra-pulmonary, and meningeal lesions. Following on his work, Paul Ehrlich developed alternate methods of staining, which in the hands of Franz Ziehl and Friedrich Neelson, became the first acid alcohol method for demnstrating the bacillus in tissue.¹²

In 1895, Wilhelm Konrad von Rontgen discovered the use of X-rays and became the first physician to visualize and detect TB lesions in the lungs; X-rays were an inexpensive method of diagnostic screening together with the tuberculin skin sensitivity test until the 1950s. In 1890 Koch himself developed a concentrated filtrate of cultured tubercle bacilli which he called "tuberculin" or "Koch?s lymph" which he believed could actually cure the disease. However, when injected, it cause violnt ractions, high fever, expansion of pre-existing lesions, and a severe reaction at the site, which he noted on self-inoculation. While it never did cure the disease, it was noted that infected individuals would develop "allergy" upon injection, resulting in an effected diagnostic method. The tuberculin and its method of use in the skin continued to be refined until the development of tuberculin purified protein defivative (PPD) by American biochemists Florence Seibert and Esmond Long in 1930, which was able to also detect latent subclinical infections.¹²
Vaccination for TB over the years has been problematic at worst, ineffective most of the time, and today is used only in aras with a high prevalence of childhood disease and in people in the military and healthcare with a high risk of exposure. The most successful advance in vaccination came in 1905 by two French bacteriologists, Albert Calmette and Camille Guerin at the Pasteur Institute, who developed a live attenuated vaccine from a non-virulent stratin. Over 100,000 children were vaccinated with this strain in the 1930s across Europe. Unfortunately 250 children were vaccinated with a BCG vaccine accidentally contaminated with a virulent strain in Lubeck Germany; 73 died and 135 developed clinical tuberculosis. Childhood vacination continued in post-WWII Europe and Asia to combat a resurgence of TB, but ultimately was discontined in the 1970s, when strict control programs were considered sufficient to control the spread.¹²

In 1993, the World Health organization declared TB a global emergency, and in 1995 defined the directly observed treatment, short-course strategy (DOTS) control policy, which has decreased the global incidence of TB by 1.5% each year, and overall mortality from TB has declined 22% from 200-2015. With new and effective anti-tuberculosis chemotherapeutic agens, the strategy of early diagnosis and targeted therapy is now considered to have the best cost/benefit ratio for treating this worldwide disease.¹²

The moderator and attendees reviewed a number of genpath concepts regarding mycobacterial infections, a number of of species and concepts regarding mycobacterial infections in a variety of species, and some diseases that might grossly cause nodules in the lungs of non-human primates. She mentioned the current theory of aortic mineralization in cattle with severe granulomatous disease as being the result of production of 1,25-cholecaliferol by the large numbers of macrophages in this disease and hypercalcemia.

References:

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2.     Barberis I, Bragazzi NL, Galluzo L, Martini M. The history of tuberculosis: from the first historical records to the isolation of Koch?s bacillus. J Prev Med Hyg 2017; 58:E9-12.

3.     Caswell JL, Williams KJ. Respiratory System. In: Jubb, Kennedy, and Palmer?s Pathology of Domestic Animals. Vol 2. 5^th ed. Editor: Maxie MG. Philadelphia, PA: Elsevier; 2007: 606-610

4.     de Jong BC, Antonio M, Gagneux S. Mycobacterium marinum ? Review of an Important Cause of Human Tuberculosis in West Africa. 2010. PLOS Negl Trop Dis. 4(9):1-9

5.     Deretic V, Singh S, Master S, Harris J, Roberts E, Kyel G, Lee HH, Vergne I. Mycobacterium tuberculosis inhibition of phagolysosome biogenesis and autophagy as a host defence mechanism. 2006. Cell Microbio 8(5):719-727

6.     Dhama K, Mahendran M, Tiwari R, Singh SD, Kumar D, Singh S, Sawant PM. Tuberculosis in Birds: Insights into the Mycobacterium avium Infections. 2011. Vet Med Intern. 1-14

7.     Ginn PE, Mansell JEKL, Rakich PM. Skin and appendages. In: Jubb, Kennedy, and Palmer?s Pathology of Domestic Animals. Vol 1. 5^th ed. Editor: Maxie MG. Philadelphia, PA: Elsevier; 2007: 688-689

8.     Greer LL, Strandberg JD, Whitaker BR. Mycobacterium chelonae Osteoarthritis in a Kemp?s Ridley Sea Turtle (Lepidochelys kempii). 2003. J Wild Dis 39(3): 736-741

9.     Honap TP, Pfister LA, Housman G, Mills S, Tarara RP, Suzuki K, Cuozzo FP, Sauther ML, Rosenberg MS, Stone AC. Mycobacterium leprae Genomes from Naturally Infected Nonhuman Primates. 2018. PLOS Negl Trop Dis. 12(1):1-17

10.  Lescenko P, Matlova L, Dvorska L, Bartos M, Vavra O, Navratil S, Novotny L, Pavlik I. Mycobacterial Infection in Aquarium Fish. 2003. Vet Med ?Czech (3):71-78

11.  Lowenstine LJ, Osborn KG. Respiratory System Diseases of Nonhuman Primates. In: Nonhuman Primates in Biomedical Research: Diseases. Vol 2. Editors: Abee CR, Mansfield K, Tardif S, Morris T. London, UK: Elsevier; 2012: 413-48.

12.  Martini M, Besozzi G, Barberis I. The never-ending story of the fight against tuberculosis: from Koch?s bacillus to global control programs. J Prev Med Hyg 2018:E241-247.

13.  Messenger AM, Barnes AN, Gray GC. Reverse Zoonotic Disease Transmission (Zooanthroponosis): A Systematic Review of Seldom-Documented Human Biological Threats to Animals. 2014. PLOS One 9(2):1-9

14.  Montali RJ, Mikota SK, Cheng LI. Mycobacterium tuberculosis in Zoo and Wildlife Species. 2001. Rev Sci Tech 20(1):291-303

15.  Moulis G, Martin-Blondel G. Scrofula, the King?s Evil. 2012. CMAJ 184(9).

16.  Pieters J. Mycobacterium tuberculosis and the macrophage: Maintaining a Balance. 2008. Cell Host & Microbe 3:399-407

17.  Schwartz RH. T cell Anergy. 2003. Annu Rev Immunol 21:305-334

18.  Smith NH, Crawshaw T, Parry J, Birtles RJ. Mycobacterium microti: More Diverse Than Previously Thought. 2009. J Clin Microbiol. 47(8):2551-2559

19.  Yan L, Cui H, Xiao H, Zhang Q. Anergic Pulmonary Tuberculosis is Associated with Contraction of the Vd2+T cell Population, Apoptosis, and Enhanced Inhibitory Cytokine Production. 2013. PLOS One 8(8):1-8

20.  http://www.who.int/news-room/fact-sheets/detail/tuberculosis