1. Introduction
Mycobacterium kansasii is a zoonotic pathogen capable of causing chronic, debilitating disease that resembles pulmonary tuberculosis.
M. kansasii belongs to the phylum
Actinomycetota and the family
Mycobacteriaceae [
1,
2,
3].
M.
kansasii is an aerobic, Gram-positive, slow-growing, and photochromogenic species with a genome size of 6.4 Mb [
4].
M. kansasii is classified as a non-tuberculous mycobacterium (NTM), distinguishing this species from the
Mycobacterium tuberculosis complex (MTBC) [
4].
M. kansasii is ubiquitous in the environment worldwide, particularly in soil and tap water [
3,
5].
M. kansasii was first described as an opportunistic pathogen in human patients with pulmonary disease resembling tuberculosis in Kansas, USA in the 1950s [
6]. Clinical signs of
M. kansasii in humans include anorexia, progressive weight loss, weakness, low-grade intermittent fever, and respiratory signs [
7].
M. kansasii is more common in immunocompromised patients [
8,
9,
10,
11] and patients with existing pulmonary conditions, such as chronic obstructive pulmonary disease [
12,
13]. Human-to-human transmission of
M. kansasii has not been confirmed [
4,
14].
M. kansasii is ubiquitous in the environment, yet is a rare pathogen in livestock and wildlife [
4]. The clinical presentation of
M. kansasii infection in animal hosts is highly variable, ranging from asymptomatic infection to overt clinical disease that may feature chronic weight loss, granulomatous pneumonia, bronchial lymphadenopathy, or cutaneous lesions [
15,
16,
17,
18,
19,
20,
21,
22,
23,
24,
25,
26,
27,
28]. Disease associated with
M. kansasii infection has been reported in wild boar (
Sus scrofa) [
27], free-ranging black-tailed deer (
Odocoileus hemionus columbianus) [
17] and white-tailed deer (
Odocoileus virginianus) [
25], bonteboks (
Damaliscus pygargus dorcas) [
20], laboratory rhesus monkeys (
Macaca mulatta) [
22], captive Florida manatees (
Trichechus manatus) [
16], domestic goats (
Capra hircus) [
15], cattle (
Bos taurus) [
19,
23,
24,
28], domestic cats (
Felis catus) [
26], Sichuan takins (
Budorcas taxicolortibetana) [
21], siamangs (
Hylobates syndactylus) [
21], and alpacas (
Vicugna pacos) [
18]. Herein, we report the first known case of
M. kansasii infection in a farmed white-tailed deer.
2. Materials and Methods
In March 2018, an approximately 7-year-old farmed white-tailed deer doe was transported to a private property in Levy County, Florida. The owner of the doe reported progressive weight loss and pelvic limb weakness but did not initiate treatment as the doe was believed to be pregnant. The doe eventually delivered premature twin fawns, both of which died after birth. The owner administered a corticosteroid (intramuscular (IM), dexamethasone) at dosages varying between 4 and 12 mg daily for 28 days, a macrolide antibiotic (1.5 cc, IM, tylosin, Tylan 200 Injection, Elanco US, Inc., Greenfield, IN, USA), a gastric cytoprotectant (1 scoop, orally, sucralfate, Carafate©, Aspen Pharmacare, Durban, South Africa), supplemented B vitamin complex, vitamin B12, and thiamine at unknown dosages. Due to the lack of response to treatment, the doe was euthanized by the owner in August 2018. The University of Florida’s Cervidae Health Research Initiative (CHeRI) was contacted to request a post mortem examination.
At necropsy, the doe presented in poor nutritional condition with a dull, rough-hair coat. The lungs were pale, with multifocal, 0.2–0.5 cm yellow-white-to-gray, granular, nodular lesions distributed across the pleural surface and throughout the parenchyma (
Figure 1). The greatest density of nodular lesions was observed in the cranial lobes bilaterally. Paired lung, heart, liver, kidney, and spleen samples were chilled at 4 °C and fixed in 10% neutral buffered formalin. Fresh and fixed lung tissues were submitted to the University of Florida College of Veterinary Medicine Diagnostic Laboratories for histopathological and microbiological characterization.
Molecular Examination
DNA from the lung and isolated bacteria was extracted using DNeasy Blood and Tissue Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. Polymerase chain reaction (PCR) for
Mycobacterium spp. was carried out using the primer pair KY18-KY75, which amplified a fragment (~500 bp) of the 16S rRNA [
25]. Total reaction volumes were 30 µL, which consisted of 0.15 µL of Platinum Taq DNA Polymerase (Invitrogen), 3.0 µL of 10× PCR Buffer, 1.2 µL of 50 mM MgCl2, 0.6 µL of 10 mM dNTPs, 1.5 µL of 20 µM of each forward and reverse primer, 17.6 µL of molecular-grade water, and 4.5 µL of DNA template (100 ng). An initial denaturation step of 95 °C for 5 min was followed by 40 cycles of a 95 °C denaturation step for 5 s, a 55 °C annealing step for 5 s, a 68 °C extension step for 25 s, and a final extension step at 71 °C for 1 min. PCR products were subjected to gel electrophoresis using a 1% agarose gel stained with ethidium bromide. Amplicons of the expected size were purified using a QIAquick PCR Purification Kit (Qiagen). The concentration of purified amplicons was determined fluorometrically using a Qubit 3.0 Fluorometer and dsDNA BR Assay Kit (Life Technologies, Carlsbad, CA, USA) before submitting to Functional Biosciences, Inc. (Madison, WI, USA) for Sanger sequencing. The sequences were visualized and assembled using BioEdit 7.1.3.0.
3. Results
3.1. Microscopic Observations
Microscopically, the lung architecture was effaced by multifocal small-to-large granulomas with central areas of necrosis varying from caseous to lytic, with mild to prominent mineralization within necrotic centers (
Figure 2). The necrotic centers were bordered by peripheral rims of epithelioid macrophages that often had finely vacuolated cytoplasm, low numbers of Langhans-type multinucleated giant cells, and scattered lymphocytes, plasma cells, and neutrophils. The granulomas were bordered by variable degrees of fibrosis, with more prominent fibrosis around granulomas with prominent central mineralization (likely indicating chronicity). The alveolar septa were mildly to moderately expanded by lymphocytes, plasma cells, macrophages, several neutrophils, small amounts of fibrin, and congested capillaries (interpreted as interstitial pneumonia). Ziehl–Neelsen staining revealed low numbers of approximately 5–15 μm long, occasionally beaded, slender, acid-fast rods that were extracellular and within macrophages and Langhans-type multinucleated giant cells. Significant lesions were not detected in the heart, spleen, liver, and kidney. The presence of acid-fast rods within granulomas with caseous necrosis and mineralization was highly suggestive of infection with tuberculoid
Mycobacterium spp. and prompted the molecular identification of the bacteria.
3.2. Molecular Examination
A
Mycobacterium-screening culture (Lowenstein–Jensen agar slants at 35–37 °C and 5% CO
2, and Middlebrook 7H11 agar at room temperature without added CO
2) isolated a rapid-growing
Mycobacterium sp. at around 7 days after culturing. A collected colony and a fragment of the lung were tested for
Mycobacterium using a specific PCR assay [
17] followed by Sanger sequencing. The obtained sequence was 375 bp. A standard nucleotide–nucleotide BLAST (BLASTN) search revealed 100% query coverage and 99.48% identity to 79 strains/isolates of
Mycobacterium kansasii within the National Center for Biotechnology Information GenBank database.
M. kansasii is considered a slow-growing
Mycobacterium species; however, PCR and Sanger sequencing conclusively confirmed the presence of this bacterium. Aerobic culture (general blood agar, chocolate agar, Columbia CNA agar, and MacConkey agar at 35–37 °C and 5% CO
2) of lung tissue found no growth at 24 h and very scant growth of mixed bacterial and fungal flora, without any clear predominance of the four morphotypes observed after an extended culture period.
4. Discussion
We report the first known isolation of
M. kansasii from a farmed white-tailed deer. The histopathological changes observed in this case are consistent with lesions described in
M. kansasii infections reported in a hunter-harvested, free-ranging black-tailed deer in Washington [
17] and a free-ranging white-tailed deer in Louisiana [
25].
M. kansasii more commonly infects immunocompromised individuals. Corticosteroids have known immunosuppressive activity, and oral dexamethasone is associated with the reactivation of latent
Mycobacterium spp. infections in HIV patients. In this case, the history of extended corticosteroid therapy and transportation to a new facility may have produced an immunocompromised state that promoted the reactivation of a latent
M. kansasii infection. No comorbidities were detected via post mortem examination or ancillary diagnostics, suggesting that
M. kansasii was the primary cause of death. Though aerobic culture found bacterial and fungal flora in cultured lung tissue, microbial growth was scant, and no known pathogens were isolated.
5. Conclusions
This case highlights the importance of positively identifying mycobacterium species associated with tuberculous disease in captive or managed wildlife and underscores the risk of introducing disease agents when translocating livestock and captive wildlife. Mycobacteriosis associated with M. kansasii is not a reportable disease; however, bovine tuberculosis (bTB) caused by Mycobacterium bovis is a reportable zoonotic agent, and the diagnosis of bTB in captive and free-ranging wildlife has significant regulatory and public health implications for both livestock producers and wildlife managers. Identifying the Mycobacteria species associated with tuberculosis-like disease in captive wildlife is critical in mitigating risks to domestic livestock and free-ranging wildlife populations.
Despite M. kansasii not being a reportable disease, the pathogen still poses a risk as an opportunistic pathogen in immunocompromised humans. Deer farming brings humans into close contact with captive cervids, and this increased interaction between farmed cervids and human caretakers elevates the risk of transmission of potential zoonotic pathogens, including M. kansasii. It is crucial to maintain basic biosecurity and hygiene practices to reduce the risk of zoonotic disease transmission.
Author Contributions
Conceptualization, J.M.C.K. and S.L.C.; methodology, S.L.C., A.-C.C., P.H.d.O.V., K.S., W.F.C., M.E.I., J.M.C.K. and S.M.W.; software, P.H.d.O.V., A.-C.C. and K.S.; validation, S.L.C., J.M.C.K. and S.M.W.; formal analysis, S.L.C., P.H.d.O.V., K.S. and A.-C.C.; investigation, J.M.C.K., S.L.C., W.F.C., M.E.I. and K.S.; resources, J.M.C.K. and S.M.W.; data curation, S.L.C. and J.M.C.K.; writing—original draft preparation, S.L.C. and J.M.C.K.; writing—review and editing, S.L.C., J.M.C.K., W.F.C., M.E.I., K.S., P.H.d.O.V., A.-C.C. and S.M.W.; visualization, S.L.C. and J.M.C.K.; supervision, J.M.C.K. and S.M.W.; project administration, J.M.C.K.; funding acquisition, J.M.C.K. and S.M.W. All authors have read and agreed to the published version of the manuscript.
Funding
This research was funded by the University of Florida, Institute of Food and Agricultural Sciences, CHeRI, with funds provided by the Florida legislature #6000CHERI.
Institutional Review Board Statement
Not applicable.
Informed Consent Statement
Not applicable.
Data Availability Statement
Data are contained within the article.
Acknowledgments
We are grateful to all CHeRI technicians for their assistance. This study was made possible through the co-operation of farmed-deer producers throughout Florida; we are grateful for their participation.
Conflicts of Interest
The authors declare no conflicts of interest.
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