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Assessment of antemortem tests used in the control of an outbreak of tuberculosis in llamas (Lama glama)
  1. D. F. Twomey, MVB, MRCVS1,
  2. T. R. Crawshaw, BVetMed, MSc, MRCVS1,
  3. J. E. Anscombe, BVetMed, MRCVS1,
  4. J. E. F. Barnett, BVSc, BSc, MRCVS2,
  5. L. Farrant, BVetMed, MRCVS3,
  6. L. J. Evans, BVetMed, MRCVS3,
  7. W. S. McElligott, MVB, MRCVS4,
  8. R. J. Higgins, BVM&S, MSc, MRCVS5,
  9. G. S. Dean, PhD6,
  10. H. M. Vordermeier, PhD6 and
  11. R. de la Rua-Domenech, DVM, PhD, DipECVPH, MRCVS7
  1. 1 Veterinary Laboratories Agency (VLA) — Starcross, Staplake Mount, Starcross, Exeter, Devon EX6 8PE
  2. 2 VLA — Truro, Polwhele, Truro, Cornwall TR4 9AD
  3. 3 Animal Health, Clyst House, Winslade Park, Clyst St Mary, Devon EX5 1DY
  4. 4 St Boniface Veterinary Clinic, 47 Mill Street, Crediton, Devon EX17 3AA
  5. 5 VLA — Lasswade, Pentlands Science Park, Bush Loan, Penicuik, Midlothian EH26 0PZ
  6. 6 VLA — Weybridge, New Haw, Addlestone, Surrey KT15 3NB
  7. 7 Bovine Tuberculosis Programme, Defra, Nobel House, 17 Smith Square, London SW1P 3JR
  1. E-mail for correspondence: f.twomey{at}vla.defra.gsi.gov.uk

Abstract

An outbreak of tuberculosis (TB) caused by Mycobacterium bovis in a llama herd is described. Over a 25-month period, a total of 70 llamas were selected for postmortem examination using four distinct criteria: clinical suspicion of disease (15 animals), positive tuberculin skin test result (three animals), antibody positive using a novel serological test (Rapid Test, 54 animals) and elective cull (five animals). Some animals qualified on more than one criterion. Gross lesions of TB were detected in 15 animals, with lung and lymph node lesions consistently observed. Samples were collected from 14 of 15 animals with visible lesions as well as those with no visible lesions, for histopathology and mycobacterial culture. All 14 llamas with visible lesions had caseonecrotic granulomatous lesions associated with acid-fast bacteria and variable mineralisation, and M bovis was isolated from 13. There were no histopathological lesions of TB in llamas with no grossly visible lesions, and M bovis was not isolated from any of these. The predictive value of suspicious gross lesions at postmortem examination was therefore high in the herd. Molecular typing results indicated that the outbreak was caused by a single strain likely to have originated from a local reservoir, probably cattle or wildlife. Antemortem indicators of infection assisted control of the outbreak, but no single test accurately identified all TB cases. Visible lesions were detected in nine of 15 llamas with clinical suspicion of disease, in two of three that had positive tuberculin skin test results and in 10 of 54 that were antibody positive; there was none (zero out of five) in llamas that were electively culled.

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TUBERCULOSIS (TB) is a chronic infectious disease caused by members of the Mycobacterium tuberculosis complex (MTBC), which includes Mycobacterium bovis. Although primarily a bovine pathogen, M bovis can infect a wide range of animal species and also accounts for a small number of human TB cases (O'Reilly and Daborn 1995). Disease associated with M bovis infection has been reported in South American camelids in many countries, including llamas (Lama glama) (Thoen and others 1977, Barlow and others 1999, Twomey and others 2007) and alpacas (Vicugna pacos) (Dinkla and others 1991, Ryan and others 2008, Twomey and others 2009). South American camelids are also susceptible to infection with Mycobacterium microti, another MTBC organism (Oevermann and others 2004, Lyashchenko and others 2007). However, their susceptibility to TB has traditionally been considered low (Fowler 1998).

Infection with M bovis was first reported in British llamas by Barlow and others (1999) in a small herd kept in close proximity to infected cattle. Since then further cases of TB associated with M bovis and M microti have been diagnosed in South American camelids. British cases diagnosed under a Defra-funded VLA surveillance project (SB 4510) between 2003 and 2009 are summarised in Table 1. These include a herd, first disclosed in 2006, where M bovis infection was diagnosed in several llamas (Twomey and others 2007). Preliminary observations from the outbreak have been summarised elsewhere, but specific conclusions were not made at that time before the outbreak had concluded (Twomey and others 2007). The authors describe here a detailed investigation of the outbreak, including evaluation of antemortem skin and blood tests, together with clinical, pathological and bacteriological findings.

Materials and methods

Herd selection

The llama herd was selected for detailed investigation following the initial confirmation of TB caused by M bovis infection, as described by Twomey and others (2007). Following statutory movement restrictions, a herd intervention programme was put in place using immunological and clinical indicators of TB infection, with culling of suspect infected animals and confirmatory postmortem examination of all dead animals during the investigation period. All animals in the herd were included in these tests, regardless of their age. Restrictions were eventually lifted, two years later, following two consecutive herd tests in which no reactors were identified, using the single intradermal comparative tuberculin test (SICTT), an approach similar to the one adopted for infected cattle herds under European Union legislation.

Antemortem indicators of infection

SICTT

Llamas were screened for TB by SICTT at the postaxillary site, using Weybridge avian and bovine purified protein derivative tuberculins (25,000 iu/ml), except at the final herd test where Lelystad tuberculins were used (25,000 and 30,000 iu/ml for avian and bovine tuberculin, respectively). The initial thickness of a skin fold was measured with calipers immediately before injecting 0.1 ml avian and bovine tuberculin at separate sites. The response to each tuberculin injection was measured 72 (±4) hours later. Llamas were deemed reactors if they developed an increase in skin fold thickness over 2 mm, or had detectable oedema, at the bovine tuberculin injection site, and the increase in skin thickness exceeded that measured at the avian site. Whole-herd testing was carried out on five occasions: June 2006 (93 llamas tested), September 2006 (101 llamas), December 2006 to January 2007 (87 llamas), March to April 2007 (57 llamas) and February 2008 (56 llamas).

Serology – rapid test

Blood samples were collected for serology using the lateral-flow rapid test (RT) as described by Twomey and others (2007) and Dean and others (2009).

The first group, tested in August 2006, included 20 llamas that had been in closest contact with the first two confirmed TB cases (Table 2). RT was subsequently performed in tandem with SICTT in September 2006 (101 llamas), December 2006 to January 2007 (87 llamas) and March to April 2007 (57 llamas). In addition to blood samples collected on the day of tuberculin injection during the third and fourth SICTTs (December 2006 to January 2007 and March to April 2007), further blood samples were collected three weeks later to check for an anamnestic antibody response. Blood samples were also collected from llamas 2 and 11 at the time of euthanasia and were subsequently tested by RT.

Clinical disease

The owner was asked to notify of any llama in the herd that showed clinical disease, regardless of the clinical signs, which included animals found dead with no premonitory signs.

Postmortem examination

Llamas were selected for postmortem examination to confirm a diagnosis of TB using four distinct criteria: presence of clinical disease, positive SICTT result, positive RT result and elective cull, the latter not being chosen for the specific purpose of controlling the herd TB outbreak. The timescale over which these were examined is summarised in Fig 1. Each llama was weighed, and the presence of visible lesions suggestive of TB was recorded.

FIG 1

Chronology of postmortem examinations of 70 llamas over the course of the TB outbreak

All visible lesions were sampled for histopathological examination, excluding llama 11, which was only examined grossly on farm. An extensive standardised sampling protocol was implemented for llamas 3 to 56 (excluding llama 11), irrespective of individual postmortem findings, consisting of brain, liver, small intestine, large intestine, left and right kidney, spleen, lung, tonsil and lymph nodes. Lymph node groups were chosen to represent specific anatomic drainage areas as follows: head and neck (mandibular, parotid, retropharyngeal, cervical), thoracic (sternal, tracheobronchial, mediastinal), abdominal and pelvic (gastric, mesenterics, anorectal, iliacs, hepatic, renal) and superficial (prescapular, axillary, superficial inguinal, prefemoral, popliteal). In some cases, stomach (sections of all three stomach compartments from 26 animals), mammary gland (16 animals) and trachea (12 animals) were additionally sampled. Fixed tissues were not collected for histopathology from llamas 57 to 70 following a revision of the original protocol.

All visible lesions were sampled for mycobacterial culture, except for llama 11. Lymph node pools were cultured from the animals with no visible lesions, excluding llamas 69 and 70, following a final revision of the sampling protocol.

Histology

After primary fixation in 10 per cent neutral buffered formalin, trimmed tissue slices were processed to paraffin wax blocks. Sections cut at 4 μm were stained with haematoxylin and eosin and a cold Ziehl-Neelsen method for acid-fast bacilli detection (Swisher 2002).

Mycobacterial culture and spoligotyping

Tissues were processed for mycobacterial culture with spoligotyping of positive isolates using methods described by Daniel and others (2009).

Results

Findings from 70 llamas subjected to postmortem examination are summarised in Tables 2 and Table 3.

Clinical disease

A total of 15 llamas displayed clinical disease. Visible lesions were detected in nine of these. Clinical signs in confirmed TB cases included weight loss (six animals), respiratory distress (three), discharging skin lesion with acid-fast bacilli in exudate (one), anorexia (one), lethargy (one) and agitation (one); one case was found dead with no premonitory signs.

SICTT

Three llamas were positive by SICTT at the third herd test. TB wassubsequently confirmed at postmortem examination in two of these.

RT serology

A total of 54 llamas gave a positive RT result, following herd group tests: six of 20 (30 per cent) tested positive in August 2006; 10 of 101 (10 per cent) tested positive in September 2006; 11 of 87 (13 per cent) tested positive at the third SICTT between December 2006 and January 2007, with 26 of 87 (30 per cent) testing positive at the anamnestic test three weeks later; and five of 57 (9 per cent) tested positive at the fourth SICTT between March and April 2007, with 12 of 57 (21 per cent) testing positive at the anamnestic test. Of these 54 seropositive llamas, TB was subsequently confirmed postmortem in 10 of them. Llamas 2 and 11, blood sampled at euthanasia, were seronegative and seropositive, respectively.

Elective cull

Five electively culled llamas showed no evidence of TB at postmortem examination.

Postmortem findings

A total of 70 llamas were selected for postmortem examination between February 2006 and February 2008. Visible lesions were identified in 15 llamas during the first year of the investigation, and a diagnosis of TB was subsequently confirmed by histopathology and/or positive culture in all animals, except llama 11, from which confirmatory samples were not available. Thirteen animals with visible lesions were female adults. The other two were juveniles (llamas 26 and 27) whose dams had visible lesions (llamas 11 and 15, respectively). The average bodyweight of adult female llamas with confirmed TB was 115 kg (n=11) compared with 134 kg (n=42) for uninfected adult females. The difference between these groups was not statistically significant.

The lungs were the most frequently and severely affected organs with extensive, coalescing, caseonecrotic lesions and consolidation. In most cases, the pleura was also affected. Gross cavitation was observed in llamas 2 and 40. In llama 2, multifocal caseonecrotic ulcerated tracheal lesions of approximately 15 mm × 10 mm in size were also seen.

Tubercular lymphadenitis involved thoracic lymph nodes (13 animals), hepatic lymph nodes (10), gastric/mesenteric lymph nodes (six), cervical lymph nodes (three), retropharyngeal lymph nodes (two), axillary lymph nodes (two), iliac lymph nodes (one), superficial inguinal lymph nodes (one) and prescapular lymph nodes (one). TB-affected lymph nodes were easy to locate due to their marked enlargement and often extensive caseous tubercular lesions distorting or effacing their architecture. This was in contrast with small, unaffected lymph nodes, which were often difficult to locate, a previously recorded feature of South American camelids (Fowler 1998).

Visible lesions in other organs that were subsequently confirmed as TB by histopathological examination included liver (nine animals), spleen (three), skin (two), pericardium (one) and intestine/mesentery (one). A discharging skin lesion was detected antemortem in llama 31, and a similar lesion was detected postmortem in the inguinal skin of llama 27; both lesions, subsequently confirmed by histopathology as granulomatous cellulitis, were associated with tubercular lymphadenitis in their respective drainage lymph nodes (retropharyngeal and superficial inguinal) and are assumed to have been sequelae to the lymph node pathology.

Histopathological findings

Histopathological lesions consistent with mycobacterial infection were identified in all 14 llamas with visible lesions from which fixed tissues had been collected. Their distribution is summarised in Table 3. There was strong correlation between gross and histopathological findings. Small lesions that had not been detected at postmortem examination were subsequently found in a further eight tissue samples (seven lymph nodes and one spleen), all from llamas with visible lesions in other tissues. There was no histopathological evidence of TB in the wide range of tissues examined from cases with no grossly visible lesions.

Tubercles were consistent with those described in a preliminary report of some animals from this herd (Twomey and others 2007). These typically showed central caseous necrosis surrounded by predominantly macrophage and lymphocytic infiltrations within variably developed outer fibroplastic stroma. Classical Langhans' giant cells were never seen. A significant neutrophil component at the interface was often a feature of small active lesions. Acid-fast bacilli were detected in Ziehl-Neelsen-stained sections from all 14 llamas with visible lesions examined, with the majority showing only sparse numbers, usually concentrated at the reactive viable interface. In contrast, two llamas had large numbers of acid-fast bacilli present in tuberculous organs. Multifocal central calcification of necrotic tissue within tubercles was consistently observed in only four llamas, and overall, mineralisation was noted in less than 10 per cent of all tissues that had grossly visible lesions. A noticeable feature of many well-established tubercles was acute vascular damage with thrombosis and haemorrhage, which were often associated with localised coagulative necrosis.

Mycobacterial culture and spoligotyping

Mycobacteria were isolated from 13 of the 14 llamas with visible lesions that were subjected to culture, and these were identified as M bovis spoligotype SB0274 (VLA type 11). M bovis was never isolated from llamas with no visible lesions. Non-MTBC organisms were isolated from three llamas with no visible lesions: Mycobacterium smegmatis (llama 54), Mycobacterium intracellulare (llama 57) and Mycobacterium celatum (llama 68).

Discussion

The primary purpose of this report was to compare the antemortem indicators of infection within the herd. Although clinical disease, tuberculin testing and serological testing contributed to the control of the outbreak, none of them was completely reliable in accurately identifying individual TB cases. The sensitivities and specificities of these methods could not be determined, as the investigation did not represent a structured epidemiological survey performed with these outputs in mind, particularly because no animals from confirmed TB-free holdings were available for testing. Detailed studies using a sufficient number of animals of known infection status are required to further investigate these parameters.

Clinical identification of disease was the initial criterion for identifying llamas with visible lesions in this outbreak and was subsequently the most consistent indicator of visible lesion status. Weight loss was the most consistently observed sign, and the mean bodyweight of adult females with TB was at the lower end of the reference range (113 to 250 kg) (Fowler 1998). This clinical feature has previously been reported with M bovis and M microti infections in South American camelids (Barlow and others 1999, Oevermann and others 2004, Ryan and others 2008). It was surprising that overt respiratory disease was only observed in three llamas in the herd that had been confirmed as positive for TB, despite the often severe chronic lung pathology. However, in experimental M bovis infection of llamas via the tracheal route, respiratory signs were usually either mild and transient, associated with stress during handling, or terminal, despite significant lung pathology being present in all cases (Stevens and others 1998). Although TB is typically considered a chronic progressive condition, one llama was found dead without premonitory signs; therefore, TB needs to be considered in the differential diagnosis of sudden death as well (Dinkla and others 1991, Barlow and others 1999, Lyashchenko and others 2007, Ryan and others 2008).

The limited sensitivity of SICTT has been discussed in another report (Dean and others 2009), although discussion of the timescale over which tests were performed in relation to postmortem examination was outside the scope of that study; however, it is important when considering field evaluation of diagnostic tests. SICTT was performed in the three months preceding postmortem examination of 14 of the llamas with visible lesions, with a positive result in only two animals, demonstrating a sensitivity of only 14 per cent in that particular group. However, because the incubation period following experimental infection with M bovis can be as short as 62 days (Stevens and others 1998), five llamas (llamas 2, 5, 7, 8 and 11) should be excluded from this sensitivity calculation because they may have become infected in the period between SICTT in June 2006 and subsequent postmortem examination in August 2006. This argument cannot, however, be applied to seven animals with visible lesions, which were SICTT negative within one month (llamas 14, 15, 28, 31 and 40) or two months (llamas 25 and 26) of confirmatory postmortem examination, because some of them were likely to have already been infected with M bovis at the time of SICTT. This low sensitivity might possibly have been influenced by the SICTT technique used, particularly tuberculin dosage and the time interval chosen to read the test, neither of which might be optimal for South American camelids. Anergy to tuberculin when an infected animal fails to give a measurable delayed hypersensitivity cutaneous response is another hypothetical cause of a false-negative result (Ryan and others 2008), but this phenomenon has not been proven in South American camelids. The identification of a positive SICTT result in llama 29, which had no detectable pathological or bacteriological evidence of mycobacterial infection, concurred with earlier reports (Hesketh and others 1994, Connolly and others 2008), but the reason for the lack of correlation is not clear.

The RT results from llamas with visible lesions in this herd have already been reported in a study comparing diagnostic methods used to identify llamas with visible lesions (Dean and others 2009). In the present report, the usefulness of RT as an ancillary diagnostic field test for M bovis infection to enhance the sensitivity of antemortem screening was considered, which requires consideration of the results from all animals tested, including llamas with no visible lesions. Although 10 llamas with visible lesions were identified from 54 RT-positive animals during group blood sampling, the other 44 animals had no demonstrable evidence of disease by pathology or culture. The identification of only two animals with visible lesions from 22 that were RT positive only at the post-SICTT 'anamnestic' sampling suggests that trying to detect a genuine anamnestic response was of little additional benefit in this herd, especially as both had already been selected on clinical disease status and positive SICTT. It is not clear why so many animals that were RT positive had no postmortem evidence of M bovis infection, as the same test has been used in other animal species with good accuracy of detection (Chambers 2009). In considering these results, the possibility of latent infection undetected by the methods used in this study cannot be discounted. They could also be associated with cross-reactivity to other mycobacterial antigens such as non-MTBC mycobacteria isolated from three RT-positive llamas that had no visible lesions. The only other report of RT in South American camelids is that of Lyashchenko and others (2007), who used it in M microti-infected animals, but that study was unable to accurately determine sensitivity or specificity.

Although South American camelids are not considered to be particularly susceptible to TB (Fowler 1998), incidents of high morbidity have been reported in some countries, similar to that observed in the herd described here (Dinkla and others 1991, Bleem and others 1993, Ryan and others 2008). The apparently low incidence reported by Fowler (1998) may reflect a lack of exposure rather than a natural species resistance. Outbreaks are more likely in South American camelid herds kept in bovine TB endemic areas, probably associated with spillover from other species, which might explain the rising number of cases in parts of Great Britain. This hypothesis is supported by findings in the present study, in which the molecular type of M bovis was the same as that found in the endemically infected local cattle and wildlife population (Twomey and others 2007). Similar circumstantial evidence was also noted in other British and Irish South American camelid cases (Barlow and others 1999, Connolly and others 2008).

This investigation was a useful opportunity to evaluate the pathology of M bovis infection in llamas. The extensive pathological damage observed in the respiratory system and associated thoracic lymph nodes is similar to previous reports of TB in South American camelids (Dinkla and others 1991, Stevens and others 1998) as well as other British cases (VLA, unpublished data) and suggests that respiratory aerosols are the most likely mechanism for dissemination of infection between llamas. Two llamas showed cavitating pulmonary lesions similar to those described in alpacas infected with M bovis (Dinkla and others 1991, Twomey and others 2009). Cavitation is recognised as a significant feature perpetuating TB in human beings and may also enhance host infectivity and disease transmission in animals (Cassidy 2006). Liver and associated hepatic lymph nodes were also commonly affected tissues in this herd, similar to observations in the study by Dinkla and others (1991). The pathogenesis of intestinal and mesenteric lymph node lesions present in several animals could not be determined, but possibilities include primary oral infection or secondary spread following bacteraemia or swallowing of infected sputum coughed up from the lung. The morphological characteristics of tubercles for the whole herd study were similar to those in the preliminary report of earlier cases from the same outbreak and in reports by other authors, although few histopathological studies have been carried out on South American camelids (Stevens and others 1998, Twomey and others 2007, Ryan and others 2008). Of particular note was the consistent absence of Langhans' giant cells, which are a characteristic feature of M bovis infection in cattle (Thoen and others 2009). The reason for this species variation is unclear.

In addition to lateral transmission via respiratory aerosols, vertical transmission should also be considered, as the two juveniles with visible lesions observed during the study were the progeny of adults that also had visible lesions. However, they were examined postmortem three months after their infected dams had died; thus, the possibility of lateral spread could not be ruled out. The absence of tubercular lesions in the mammary glands suggests that offspring were not infected by consumption of infected colostrum or milk. Other possible routes of infection in this herd included discharging skin lesions and faeces associated with intestinal infection.

The strong correlation between visible lesion/no visible lesion status and subsequent histopathological and bacteriological findings in this herd differs from observations in cattle where a small percentage of animals with no visible lesions are culture positive or have histopathological lesions consistent with TB (Liebana and others 2008). The reason for this difference between species is unclear and may simply reflect the number of animals examined in this single herd. Unlike with cattle, it is difficult to carry out structured pathological surveys in South American camelids due to their high financial value and comparatively low population, and further documentation of individual herd studies is recommended to advance knowledge on the pathology and other aspects of TB in these species.

The British South American camelid population has steadily been increasing (Davis and others 1998, D'Alterio and others 2006), but there is little information on the zoonotic risk associated with MTBC infections in these species. A single case of cutaneous TB caused by M bovis has been diagnosed in a British veterinary surgeon who had contact with tuberculous tissues from an alpaca (Twomey and others 2010). Human infection without clinical disease has also been suspected; in this case the infected person was in close contact with a heavily infected Dutch alpaca herd and had a strongly positive Mantoux reaction. The patient subsequently received anti-TB therapy as a precautionary measure (Dinkla and others 1991). TB should, therefore, be considered not only as a cause of clinical disease in South American camelids but also in humans with a history of camelid contact. Despite the severity of infection in this herd, there was no evidence of transmission to human contacts. Nevertheless, care should be taken when handling potentially infected animals.

No further cases of TB were diagnosed in this herd after restrictions were lifted. The herd has subsequently been dispersed, and TB has not been identified in other herds that purchased sale animals from this herd.

Acknowledgements

The authors are grateful to Keith Jahans (who is now retired from VLA – Weybridge) for mycobacterial culture and spoligotyping. Antemortem screening and postmortem confirmatory tests were funded by Defra.

References

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Footnotes

  • Provenance: not commissioned; externally peer reviewed

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