Companion animal tuberculosis: clinical presentations, outbreak investigations, improved diagnostics and the early macrophage response
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Date
04/07/2020Item status
Restricted AccessEmbargo end date
04/07/2021Author
O'Halloran, Conor John
Metadata
Abstract
Tuberculosis caused by the Mycobacterium (M.) tuberculosis-complex (MTBC) of organisms
remains one of the most prevalent and deadly infectious diseases of man and other animals.
The mycobacteria responsible are a highly conserved group of pleomorphic acid-fast bacilli
which cause chronic granulomatous infections. Tuberculous infections in humans and cattle
often remain latent for prolonged periods of time before progressing to disease that has
severe, negative consequences for the health and welfare of the infected host. Some of the
organisms within the MTBC are highly specialised and limited to just a single or small number
of host species whereas others, such as M. bovis, can infect a broad range of mammals
including humans. Companion animals are susceptible to MTBC infections and understanding
of the significance and frequency of these infections has grown in recent years. Cats and dogs
share unrivalled proximity to their owners and therefore pose a small but real risk for the
zoonotic transmission of tuberculous infections. Despite the high frequency of mycobacterial
infections observed in companion animals, diagnostic tests to identify the commonly
encountered mycobacterial species are lacking.
The first aim of this work was therefore to improve on the currently available diagnostic test
methodologies for companion animals. A diagnostic PCR assay was developed and applied
to 380 histologically confirmed feline and eight canine mycobacteriosis samples. This novel
assay specifically targeted the mycobacterial species most frequently identified by
mycobacterial culture (M. bovis and M. microti) and was optimised for use with formalin-fixed
tissue; a prerequisite for the safe handling of tuberculous tissue from companion animals by
UK laboratories. The assay was suitable for both feline and canine tissue with a significantly
quicker turnaround time, higher rate of test positive results and a significant increase in the
proportion of M. microti diagnoses compared to culture results. Since evaluation of cytokines has shown diagnostic potential in other species, this project
explored the potential of cytokine profiling in cats for the rapid and sensitive detection of
mycobacteriosis. By evaluating serum/plasma from 116 naturally infected cats, this study
demonstrated a consistent elevation in the cytokines associated with macrophage activation
and antigenic stimulation compared to control cats. Sub-group analysis showed that elevations
in PDGF-BB were specifically associated with M. microti infections whereas elevated TNF-α
sensitively identified cats infected with M. bovis.
Investigation of an unprecedented outbreak of M. bovis associated with the ingestion of a
putatively contaminated raw food product led to the use of the interferon-γ release assay
(IGRA) as a screening test for clinically healthy cats, a purpose for which it had not previously
been evaluated. Nearly a third of clinically normal IGRA test-positive cats were subsequently
found to have structural disease detected by diagnostic imaging. Though this raises questions
regarding the specificity of the IGRA in clinically normal cats, it was an invaluable diagnostic
tool to evaluate individual cats involved in the outbreak.
The work on feline TB was complemented by similar investigations of canine TB. When an
outbreak of M. bovis tuberculosis occurred in a kennel of 164 working Foxhounds, a testing
strategy to successfully bring the outbreak under control and investigate the cause was
developed. Collaborative work undertaken to screen at risk humans exposed to the hounds
identified a latently infected person, highlighting the zoonotic risk posed by M. bovis infections
in companion animals. Eight novel and existing diagnostic testing methodologies were
evaluated for use in dogs of which a cell-based IGRA, and three serological tests comprising
a novel comparative peptide ELISA, the Chembio DPP VetTB assay and the Idexx M. bovis
Ab ELISA showed diagnostic potential for canine TB. Additional analysis employing a
Bayesian latent class modelling approach revealed that the IGRA developed herein was as
sensitive and specific as comparable tests in other species. The serological assays were
shown to have markedly lower sensitivity than the IGRA but had higher specificities. All four
tests had positive and negative predictive value estimates which indicate that these tests can
be informative to clinicians who suspect cases of canine TB. A review of 1012 cases of canine TB highlighted an apparently lower incidence of infections
in dogs compared to cats, but an increased severity of clinical signs when disease occurred.
To investigate this discrepancy a protocol to derive macrophages from canine and feline bone
marrow was developed. These cells acquired cell surface molecules indicative of a
macrophage phenotype during ten days of culture with recombinant CSF-1. The response of
primary macrophages and the DH82 canine histiocytic cell line was assessed following
stimulation with LPS, infection with M. bovis Bacille Calmette Guerin, M. bovis AF2122/97 (the
reference strain) or a clinical isolate of M. bovis. These investigations consistently revealed
that DH82 cells do not accurately represent primary canine macrophage biology which was
associated with altered morphology, lack of nitrite production and significantly reduced
secretion of the pro-inflammatory cytokines IL-6 and TNF-α. Overall, the work presented in
this thesis demonstrates novel advancement of the diagnostic methodologies for identifying
cases of companion animal mycobacteriosis and in particular cases of TB. It further begins to
explore the immunological basis for the clinical differences seen between species that may
further contribute to novel testing and treatment strategies in the future.