Naturally acquired Mycobacterium tuberculosis complex in laboratory pig-tailed macaques

Figures & data

Table 1 History and pathology characteristics of seven pig-tailed macaques on a SHIV study where MTBC went undetected

	Animal A	Animal B	Animal C	Animal D	Animal E	Animal F	Animal G
Date challenged with SHIV	27 Mar 2009	26 Mar 2009	27 Mar 2009	27 Mar 2009	26 Mar 2009	26 Mar 2009	26 Mar 2009
Date of death	8 Jun 2010	29 Nov 2010	9 Dec 2010	16 Dec 2010	20 Dec 2010	1 Apr 2010	17 May 2010
%Weight loss	14%	10%	No	No	No	22%	13%
Diarrhea	Yes	No	No	No	No	Yes	Yes
TST	NEG 08/2008	NEG 08/2008	NEG 08/2008	NEG 08/2008	NEG 08/2008	NEG 08/2008	NEG 08/2008
Findings on gross pathology	Emaciation, extensive, chronic, multifocal mesenteric lymph node granulomas	Emaciation; mild, multifocal atelectasis; lymphadenopathy	No significant disease	No significant disease	No significant disease	Emaciation	Emaciation
Histological findings	Histiocytic infiltration of the jejunum, ileum, colon and cecum; mesenteric granulomas; hepatic microgranulomas with giant cells	Lymphohistiocytic, plasmacytic, eosinophilic gastro-entero-colitis; enteric villar blunting and fusion	Lymphohistiocytic and eosinophilic gastro-entero-colitis;enteric villar blunting and fusion	Lymphohistiocytic, plasmacytic, eosinophilic gastro-entero colitis; enteric villar blunting and fusion; colonic spirochetosis	Lymphohistiocytic, plasmacytic, eosinophilic gastro-entero colitis; enteric villar blunting and fusion; colonic spirochetosis	Plasmacytic and eosinophilic gastro-entero-colitis; lymphoid depletion; hemosiderin deposition in lamina propria of small intestine	Lymphohistiocytic, plasmacytic and eosinophilic gastro-entero-colitis; enterocolonic amyloidosis
AFB detected	+ intestinal, mesenteric, inguinal and axillary lymph nodes	Not performed	Not performed	Not performed	Not performed	Not performed	Not performed
Cause of death on pathology report	SAIDS; opportunistic atypical mycobacteriosis	End of Study	End of study	End of Study	End of Study	SAIDS; IBD, food allergy, hypersensitivity, dietary intolerance	IBD food allergy, hypersensitivity, dietary intolerance; secondary amyloidosis from GI tract inflammation

Abbreviations: AFB, acid fast bacteria; IBD, inflammatory bowel disease; SAIDS, simian autoimmune deficiency syndrome; SHIV, simian-human immunodeficiency virus; TST, tuberculin skin test results prior to inoculation with SHIV.

short-legendFigure 1

Table 1 IS6110 amplification of swabs^a collected at live and/or necropsy time points from SHIV-infected pigtailed macaques

Animal ID	A		B		C		D		E		F		G
	mtDNA^b	IS6110	mtDNA	IS6110	mtDNA	IS6110	mtDNA	IS6110	mtDNA	IS6110	mtDNA	IS6110	mtDNA	IS6110
Necropsy oral swab	+	+	+	+	+	+	+	+	+	+	+	+	+	+
Necropsy liver swab	+	+	−	−	+	+	+	+	+	+	+	+	+	+
Necropsy lung swab	+	+	−	−	+	+	+	+	+	−	+	+	+	+
Necropsy granuloma swab^c	+	+	NA	NA	NA	NA	NA	NA	NA	NA	NA	NA	NA	NA
Oral swab collected while living^d	NC	NC	+	+	+	+	+	+	+	+	NC	NC	NC	NC

Abbreviations: NA, not applicable (no granulomas detected); NC, not collected.

^aThe specificity of PCR using IS6110 primers to detect MTBC infection and disease has been measured in several human populations to be in the range of 98% and higher.¹⁶ Measurement of PCR test specificity in human populations is facilitated by the availability and routine use of multiple diagnostic modalities, including history and physical exam, radiography, PPD and sputum culture, which constitute a ‘gold standard’ against which new tests can be compared. Similar population data for primates are unavailable, in part because diagnostic tests for MTBC in primates are unwieldy to apply, inaccurate and lack a reliable gold standard for detecting MTBC.⁵ However, indirect evidence for high specificity of IS6110 PCR in primates comes from studies in free-ranging macaques showing that IS6110 is not detected (0/39) in macaques living alongside human populations with very low levels of MTBC infection, while IS6110 in macaques living alongside human populations with high MTBC prevalence, such as Indonesia, are much higher (68%).²

Swabs of buccal mucosa and necropsy tissues were individually placed in sterile lysis buffer and DNA was subsequently extracted following a standard phenol-chloroform procedure. To avoid cross-contamination of samples, or contamination from positive controls, all pre-PCR work was performed in a dedicated laboratory with dedicated instruments, consumables, and reagents. Work flow was unidirectional from pre-PCR to post-PCR laboratories. Extraction and PCR-setup hoods were UV irradiated for at least 20 min between each extraction and PCR preparation. Hoods were cleaned with 10% commercial bleach solution and RNase between work sessions. Alongside each DNA extraction, we ran at least one negative extraction blank. All PCR amplification procedures were performed in a separate post-PCR laboratory, and multiple PCR blanks were included in each PCR run. Additionally, tissue was obtained as a negative control from an infant pigtailed macaque that was never in contact with any of the animals reported in this study. DNA extracted from this negative control was consistently negative for IS6110.

^bFollowing DNA extraction, we assessed recoverability of DNA from samples by amplifying a 101 base-pair fragment of macaque mitochondrial DNA using the primers MT12Sa′ 5′CTG GGA TTA GAT ACC CAC TAT3′ and MT12So 5′GTC GAT TAT AGG ACA GGT TCC3′.¹⁷ In samples that contained DNA, a 123 base-pair fragment of the MTBC-specific repetitive element, IS6110³ was targeted using the primers TB130-F and TB130-R.¹⁸ Real-time PCR was performed with the SsoFast EvaGreen supermix (Bio-Rad Laboratories, Inc.), in a 20 μl reaction containing each primer at a final concentration of 0.2 μM and 2 μl DNA template for 40 cycles. Positive controls for M. tuberculosis H37Rv and for M. bovis BCG were added immediately prior to thermal cycling and without opening the tubes containing swab sample DNA or blanks. Product high resolution melt peaks were assessed relative to the positive and negative controls. Samples with positive melt curves matching IS6110 were then directly sequenced to ensure that the amplicon was indeed from IS6110.

^cGross pathological examination identified granulomas only in animal A.

^dBuccal swabs of the oral mucosa were collected from live animals B–D 3 days after the death of animal A.

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