Systemic mycobacteriosis caused by Mycobacterium avium subspecies hominissuis in a sheep

Abstract

Mycobacterium avium subsp. hominissuis (MAH) causes pulmonary and disseminated infections in immunocompromised humans. In pigs, MAH commonly causes subclinical infections that lead to granulomatous lesions in the mesenteric lymph nodes, liver, spleen, and jejunum. MAH infections have been reported less commonly in other animal species and in birds. Here, we report a case of systemic mycobacteriosis caused by MAH in a sheep. The affected animal had a history of hypoproteinemia, weight loss, and fever. Gross pathology findings included serous fluid accumulation in the peritoneal and pleural cavities, patchy dull granular light-red mucosa of the small intestine, diffuse orange-brown discoloration of the liver, and noncollapsing lungs that were mottled pink to dark-red. Histologically, granulomas were present in the intestines, mesenteric lymph nodes, liver, spleen, kidneys, lungs, and brain. MAH was isolated from the liver, spleen, and feces. Whole-genome sequencing revealed a mobile genetic element—an ISMAP02-like sequence—in the MAH genome. An ISMAP02-like sequence in mycobacteria other than Mycobacterium avium subsp. paratuberculosis (MAP) may interfere with the detection of MAP by PCR assays targeting ISMAP02.

Keywords

mycobacteriosis ovine sheep

The Mycobacterium avium complex (MAC) includes slow-growing, non-motile, non–spore-forming, gram-positive, acid-fast bacteria that persist in the environment and cause disease in both humans and animals.¹⁷ The MAC is a heterogeneous group of several species of mycobacteria that includes M. avium, M. intracellulare, and other recently reclassified bacteria such as M. arosiense, M. bouchedurhonense, M. chimaera, M. colombiense, M. lepraemurium, M. marseillense, M. paraintracellulare, M. timonense, M. vulneris, and M. yongonense.³⁰ MAC organisms account for ~80% of lung diseases caused by nontuberculous mycobacteria in humans.²¹ M. avium comprises 4 subspecies: M. avium subsp. avium (MAA), M. avium subsp. hominissuis (MAH), M. avium subsp. paratuberculosis (MAP), and M. avium subsp. silvaticum (MAS).²² Among members of the MAC, MAH is the most common cause of pulmonary and disseminated infections in immunocompromised humans.¹⁸ MAH also commonly causes subclinical infection in pigs, which is associated with the development of granulomatous lesions in the mesenteric lymph nodes, liver, spleen, and jejunum, and is often detected during postmortem inspection at slaughter plants.⁸ Sporadic cases of MAH infection have been reported in cattle,^13,23 cervids,^10,33 horses,¹⁹ dogs,¹¹ cats,¹⁶ domestic rabbits,¹² and less commonly in birds.^6,25 Here, we report clinical and gross and microscopic pathology findings in an adult sheep infected with MAH.

A 2-y-old, castrated male sheep was referred to the University of Pennsylvania’s New Bolton Center Hospital (Kennett Square, PA, USA) in March 2024 for evaluation of hypoproteinemia, weight loss, and fever. On presentation, the animal was recumbent, tachycardic (144 bpm; RI: 70–80 bpm), tachypneic (88 breaths/min; RI: 20–30 breaths/min), and febrile (40.5°C [104.9°F]; RI: 38.5–40°C [101.3–104°F]). The patient initially appeared mentally appropriate and later was paddling. Laboratory findings included a low packed cell volume of 0.18 L/L (RI: 0.26–0.41 L/L), low total solids of 44 g/L (RI: 50–70 g/L), and increased lactate (4.7 mmol/L; RI:1.0–1.4 mmol/L) and ammonia (265 µmol/L; RI: 15–64 µmol/L) concentrations. Point-of-care ultrasound revealed a small amount of free abdominal fluid. The patient died while abdominal radiographs were being obtained.

Postmortem examination revealed satisfactory body condition and excellent postmortem condition. The mucous membranes were pale pink. The peritoneal and pleural cavities contained ~700 mL and 400 mL of serous fluid, respectively. The liver was diffusely orange-brown and mildly firm with an enhanced lobular pattern. The spleen was mildly enlarged with a meaty consistency. The mucosal surface of the abomasum had rare 2-cm long filamentous nematodes with a red-and-white spiral appearance (Haemonchus sp.). The small intestinal mucosa was multifocally dull, granular, and light-red. The kidneys had diffuse cortical and medullary pallor. The lungs were mottled pink to dark-red, diffusely heavy, and noncollapsing with faint rib impressions on the pleural surface. Serous fluid oozed from the lung parenchyma when incised. No gross abnormalities were observed in the appendicular joints, brain, or remainder of the viscera.

Histologically, granulomas were present in the small and large intestines, mesenteric lymphatic vessels and lymph nodes, liver, spleen, kidneys, lungs, brain, and femoral bone marrow ( Fig. 1A–E , and data not shown). Intrahistiocytic acid-fast bacilli were found in Ziehl–Neelsen acid-fast–stained granulomas in the intestines, mesenteric lymph nodes, liver, spleen, kidneys, and lungs ( Fig. 1F , and data not shown). Additional histologic findings included foci of necrosis and mineralization within the inflamed mesenteric lymph nodes, hepatocellular degeneration and canalicular cholestasis at the periphery of the granulomas in the liver, and minimal myocardial fibrosis and histiocytic infiltrates in the heart. The brain had mild cerebrocortical Alzheimer type II astrocytosis and cerebellar white matter spongiosis, compatible with hyperammonemic encephalopathy.

Figure 1.

Systemic mycobacteriosis caused by Mycobacterium avium subsp. hominissuis in a sheep. A–E. Granulomatous inflammatory infiltrates (arrows) of epithelioid macrophages, multinucleate giant cells, lymphocytes, and plasma cells within the A. small intestine, B. liver, C. lung, D. kidney, and E. brain. H&E. F. Epithelioid macrophages and multinucleate giant cells in the kidney contain numerous intracytoplasmic acid-fast bacilli (arrow). Ziehl–Neelsen acid-fast stain.

Based on gross pathology findings, infection with MAP was included in the rule-outs, with PCR testing of feces and organ tissues. DNA was extracted from a fecal sample obtained from the colon (MagMAX core nucleic acid purification kit with mechanical lysis module; ThermoFisher) as described previously.²⁸ A positive extraction control (PEC; a fecal sample from a high-MAP-shedding animal obtained from the National Veterinary Services Laboratories [NVSL, Ames, IA, USA; reagent code: MAP-POS, sample 554]) also was included. DNA from liver and spleen were extracted (Wizard genomic DNA purification kit; Promega), following the manufacturer’s instructions.

Extracted DNA samples were tested using 3 PCR assays (in-house insertion sequence 900 [IS900] PCR, VetAlert Johne disease real-time PCR [TC-9828-100; Tetracore], and VetMAX-Gold MAP detection kit [A29809; Applied Biosystems]). The in-house IS900 used the primers described previously (forward primer, 5′-CCGCTAATTGAGAGATGCGATTGG-3′ and the reverse primer, 5′-AATCAACTCCAGCAGCGCGGCCTCG-3′) to amplify a 229-bp MAP-specific sequence.^27,31 Briefly, each 25-μL reaction contained 1× iQ SYBR Green Supermix (Bio-Rad), a 0.5 μM concentration of each primer, and 2 μL of DNA template. PCR amplifications were performed (CFX96 touch real-time PCR detection system; Bio-Rad) with the following conditions: 1 cycle of initial denaturation at 95°C for 10 min, 45 cycles of denaturation at 95°C for 30 s, annealing/extension at 63°C for 45 s, with fluorescence acquisition at the end of the annealing/extension step. The conditions for melting curve analysis of the amplified products included denaturation at 95°C for 10 min, renaturation at 55°C for 15 min followed by gradual increase of temperature from 55°C to 95°C by 0.5°C increments, each lasting 10 s. Melting temperature (Tm) of the PCR amplification products was determined by analyzing the graph of the negative first derivative of the change in fluorescence plotted as a function of temperature (−dF/dT). MAP-specific IS900 amplicons had a Tm of 88.5°C. A MAP DNA positive amplification control, a PEC, and a no-template control were included with each PCR run. In addition, the presence or absence of the expected 229-bp amplicon was verified by capillary electrophoresis (QIAxcel automated capillary electrophoresis system; Qiagen). The VetAlert Johne disease kit targets the hspX gene; the VetMAX-Gold MAP kit has been reported to target the MAP genetic element, insertion sequence MAP02 (ISMAP02).⁴ Both the VetAlert Johne disease and VetMAX-Gold MAP assays were performed following the manufacturers’ instructions. Both the IS900 PCR and the hspX gene PCR assays repeatedly failed to produce any amplicons; the VetMAX-Gold kit produced positive results with low Ct values (14–18) for all 3 specimens tested.

Based on the acid-fast bacilli in several organs and discrepant MAP PCR results, the fecal, liver, and spleen samples were sent to the NVSL for mycobacterial culture and identification. Mycobacteria were isolated from all 3 specimens. Partial sequences of the 16S ribosomal RNA gene and the RNA polymerase β-subunit (rpoB) gene were amplified from one of the cultured isolates using previously published primers,^1,2 and the PCR amplicons were subjected to Sanger sequencing. An 818-bp 16S rRNA gene sequence had >99.6% identity to M. avium species sequences (data not shown, Suppl. Fig. 1 , GenBank PZ099870.1). The rpoB gene, along with the heat-shock protein 65 (hsp65) gene, is commonly used for distinguishing M. avium at the subspecies level, given that some species or subspecies within the MAC share identical 16S rRNA sequences.^1,2 The 576-bp rpoB gene had 100% sequence identity to MAH sequences (data not shown), and the maximum-likelihood phylogenetic analysis distinguished it from other M. avium subspecies ( Suppl. Fig. 2 , GenBank PZ106367.1).

We hypothesized that the genome of the MAH isolate from the sheep carried the mobile genetic element, ISMAP02, which resulted in a positive MAP PCR. The genome was sequenced (MiSeq; Illumina) by the paired-end method (GenBank PRJNA1272106). The sequencing data from each of reads 1 and 2 contained >3.8 million reads. The average read length was ~147 bp, with 92.9% of reads having a Phred Q score of ≥30, indicating high-quality sequencing. The GC content of the genome was 68.5%.

The genome was assembled de novo (Unicycler v.0.5.1³²) for downstream classification. Kraken (v.1.1.1)¹⁵ profiling identified the genome as M. avium but could not resolve the subspecies given the database composition. We therefore applied FastANI (v.1.34, https://github.com/ParBLiSS/FastANI ), which had >98% average nucleotide identity, with >85% coverage to MAH.

To further verify the identity of the genome, we examined the MAC marker IS elements in the sequenced MAH genome. IS900 (GenBank X16293.1), which is considered specific for MAP, and IS901 (GenBank X59272.1), which is common in MAA and MAS, were absent in the MAH genome. IS1245 (GenBank L33879.1), which is commonly present in MAA, MAS, and MAH,²⁹ was present in the MAH genome from sheep with 100% identity. Notably, the ISMAP02 sequence was identified in the assembled MAH genome, with near-complete coverage and >99% identity to the 6 ISMAP02 loci reported in the MAP K-10 reference genome (GenBank AE016958; Table 1 ).¹⁴

Table 1.

BLAST identification of ISMAP02 and IS1245 within the assembled Mycobacterium avium subsp. hominissuis genome.

Insertion sequence	Fragments	Reference length	Coverage, bp	Coverage, %	Mean identity, %	Reference accession
ISMAP02	2	1,697	1,660	99	~99	AE016958
IS1245	5	1,414	1,085	77	100	L33879.1

Several PCR assays have targeted ISs, particularly IS900 and ISMAP02, given that the ISs are present in high copy numbers and considered specific to MAP for the diagnosis of paratuberculosis.^3,26 Genomes of C-type (type II) MAP strains contain 16 or 17 copies of IS900 compared with 19–22 copies in the S-type strains.^5,7 Six copies of ISMAP02 are known to be present in the MAP genome.²⁶ However, IS900 and ISMAP02 have been reported in mycobacteria other than MAP, which can potentially cause false-positive PCR test results.^9,20 One or 2 copies of ISMAP02 were reported in 14 MAH genomes of 190 MAH genomes analyzed.⁴ Furthermore, the utility of incorporating multiple targets—IS900, ISMAP02, and F57 gene—in a PCR assay was demonstrated for the sensitive and specific detection of MAP from bovine fecal and environmental samples.⁴

MAH has been reported to infect a wide range of hosts, with inhalation or ingestion as the principal mode of transmission.¹⁸ MAH primarily infects immunocompromised humans, resulting in chronic granulomatous lung disease with involvement of cervical lymph nodes.¹⁸ In pigs and other animals, MAH predominantly infects the gastrointestinal tract, causing granulomatous inflammation in the intestine, mesenteric lymph nodes, and abdominal organs, such as liver and spleen.⁸ We observed involvement of both abdominal organs and the lungs in the infected sheep. The gross histologic findings observed in our case were similar to those reported in other ruminants. MAH infection in a 5-y-old, intact female reindeer (Rangifer tarandus) resulted in miliary nodules in the small intestine, liver, spleen, lungs, and pleura, with enlarged mesenteric lymph nodes and dilated mesenteric and serosal lymphatic vessels.³³ Severe necrotizing, granulomatous inflammation with acid-fast bacilli was observed in the lungs, the proximal jejunum, liver, and spleen.³³ MAH infection in a 14-mo-old Japanese Black beef steer resulted in systemic infection with thickening of the intestinal mucosa, hemorrhage in the intestines, and enlarged mesenteric lymph nodes with caseous necrosis.¹³ In addition, discoloration of the liver, splenomegaly, and pulmonary edema were observed. Histologically, granulomas with numerous acid-fast bacilli were observed in the jejunum, ileum, cecum, liver, spleen, and mesenteric, hepatic, and pulmonary lymph nodes. Fewer granulomas were observed in the lung and abomasum of the infected steer.¹³ In contrast, MAP infection of a wild yearling male mule deer (Odocoileus hemionus) primarily affected the lungs, resulting in granulomas in the right caudal lung lobe and pyogranulomatous lymphadenitis in the tracheobronchial lymph node.¹⁰ The differences observed in the disease presentations and pathology findings most likely reflect variations in the genotypes, in addition to the dose and route of transmission.

Our searches of PubMed and Google Scholar using the search terms “sheep/Ovis aries”, “goats/Capra hircus”, and “Mycobacterium avium subsp. hominissuis” returned no cases of natural MAH infection in sheep and goats. After oral inoculation of 10–21-d-old goat kids with a high dose of MAH (2.13 × 10¹⁰ cfu/goat),²⁴ nearly half of the infected kids developed severe disease; the remaining kids developed mild disease reflecting host genetic heterogeneity. The animals with severe disease had fever, anorexia, depression, soft feces, and loss of body condition. These animals either succumbed to infection or were euthanized because of animal welfare concerns. Gross pathology findings included nodular thickening and ulceration of the intestinal mucosa. Histologically, diffuse granulomatous inflammation along with necrosis and mineralization were observed in the intestinal mucosa and gut-associated lymphoid tissue. Numerous MAH bacilli were observed in the lesions of the intestine and associated lymphoid tissues, but no granulomas were present. In contrast, the surviving animals developed subclinical infection with transient fever and mild depression before becoming clinically healthy. At the end of the 13-mo experimental period, autopsy revealed good nutritional condition and numerous small nodules in the intestinal mucosa. Histologically, well-formed granulomas, along with fewer MAH bacilli, were observed in the intestinal mucosa and gut-associated lymphoid tissue.²⁴

The source of MAH infection and the immune status of the sheep in our case were unknown. MAH is a ubiquitous environmental bacterium that is common in natural sources, such as water, soil, plants, and bedding material.¹⁸ Because MAH is a zoonotic agent, the absence of antemortem tests and the chronic nature of MAH infection in animals pose a significant risk to animal handlers and veterinarians.

Supplemental Material

sj-pdf-1-vdi-10.1177_10406387261464590 – Supplemental material for Systemic mycobacteriosis caused by Mycobacterium avium subspecies hominissuis in a sheep

Supplemental material, sj-pdf-1-vdi-10.1177_10406387261464590 for Systemic mycobacteriosis caused by Mycobacterium avium subspecies hominissuis in a sheep by Nagaraja Thirumalapura, Susan Bender, Manoj K. Sekhwal, Patrick Camp, David Farrell, Doris Bravo, Julia Livengood, Linh Nguyen, Kimberly Lehman and Deepanker Tewari in Journal of Veterinary Diagnostic Investigation

Footnotes

Acknowledgements

We thank Adam DiRocco for assistance with Mycobacterium avium subsp. paratuberculosis PCR assays.

Declaration of conflicting interests

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The authors received no financial support for the research, authorship, and/or publication of this article.

ORCID iDs

Nagaraja Thirumalapura

Kimberly Lehman

Deepanker Tewari

Supplemental material

Supplemental material for this article is available online.

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