Article Text

Multispecies
Case of TB in a sheep caused by Mycobacterium bovis with transmission to another sheep and a steer in the same building
  1. Timm Konold1,
  2. James Dale2,
  3. John Spiropoulos1,
  4. Hugh Simmons1 and
  5. Ana Godinho3,4
  1. 1Pathology Department, Animal and Plant Health Agency, Addlestone, Surrey, UK
  2. 2Bacterial Characterisation Group (TB Genotyping), Animal and Plant Health Agency, Addlestone, Surrey, UK
  3. 3School of Medicine, University of Minho Life and Health Sciences Research Institute, Braga, Portugal
  4. 4ICVS/3B's-PT Government Associate Laboratory, Universidade do Minho Instituto de Investigacao em Ciencias da Vida e Saude, Guimarães, Portugal
  1. Correspondence to Dr Timm Konold; Timm.Konold{at}apha.gov.uk

Abstract

An adult sheep housed indoors developed chronic respiratory tract infection that was unresponsive to treatment and resulted in euthanasia. Granulomas were found in multiple organs and lymph nodes, which contained acid-fast bacilli identified in Ziehl-Neelsen stains, and Mycobacterium bovis was cultured from tissues, confirming tuberculosis. Tuberculin skin testing of other animals in the building subsequently identified another sheep as infected, which had been housed with the index case for 46 months, although no visible lesions were seen postmortem. A steer that shared common air space with the sheep in the same building was also identified as test reactor and M bovis was cultured from the retropharyngeal lymph node. Strain typing of the isolates from the index case and the steer revealed the same genotype, suggestive of transmission from sheep to steer. This is the first documented episode of M bovis transmission from sheep to cattle.

  • bovine tuberculosis
  • mycobacterium bovis
  • sheep
  • respiratory disease
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Background

Bovine tuberculosis (bTB), caused by Mycobacterium bovis, is a zoonotic disease of considerable economic importance in Great Britain. In 2018, more than 40 000 cattle were slaughtered as test reactors or direct contact animals.1 By contrast, sheep are rarely affected, and usually only single cases or cases from single flocks are reported.2–10 Sheep are considered spillover hosts, which become infected when the challenge level is relatively high but do not maintain the infection in the absence of continuing acquisition from maintenance hosts.11 The risk of TB spreading from sheep to cattle has been reported in the past12 and highlighted again more recently where sheep shared farms and pastures with cattle with bTB, resulting in outbreaks in several flocks in Spain, which—unless tested—may represent a potential risk to other susceptible species.6 However, to the authors’ knowledge, there have not been any documented reports of M bovis transmission from sheep back to cattle. Here we describe a clinical case of TB in a sheep that subsequently infected another sheep and a steer that was housed in the same building as part of a research project unrelated to bTB.

Case presentation

Animal movement and housing

Female cheviot sheep N376 (index case) generated by embryo transfer was born on 28 April 2006 in a classical scrapie-free closed flock maintained under strict biosecurity and tested regularly for specific sheep diseases.13 The flock was in a region of England not known to be endemic for bTB. The sheep was moved to a farm in Warwickshire (a county in the Edge Area of England with an intermediate incidence of the disease) on 10 September 2008 as part of an environmental study and was transported to APHA Weybridge on 21 May 2012. During the 44 months in Warwickshire, it was kept on pasture with up to 81 other sheep throughout the year and only housed when the ground conditions were poor. Sheep did not have contact with cattle. In July 2010, three cattle had bTB diagnosed (three skin test reactors, two with confirmed growth of M bovis) following a period of grazing on these pastures, which was only known retrospectively. At Weybridge, it was housed in a pen with another sheep from the same flock (N524) for a research project unrelated to bTB in buildings also occupied by other species (sheep, pigs and cattle) in different pens. These building companions had no history of clinical signs associated with bTB, did not originate from farms with a history of bTB, and the majority were on short-term experiments that were terminated more than 21 months before the index case was euthanased. Of particular interest is the last building that the animal occupied for 2 years before its death, which also housed cattle and sheep (see figure 1 for an overview of the building). This building was subject to specific biosecurity procedures: sheep pens were entered from side entrances with individual protective equipment (boots and overalls) for each pen, whereas the cattle pens were entered from the main entrance with boots and overalls dedicated to cattle pens. Foot dips for entering and exiting pens contained 2 per cent hypochlorite solution, which was renewed every 5 days or more frequently if too contaminated with organic matter. Each pen had its own tools for cleaning (shovels and scrapers), and manure was removed through a hatch at the rear of each pen, which was then taken to a dung heap by staff entering from a different entrance. Rodent pest control was carried out regularly.

Figure 1

Overview of the animal accommodation.

Clinical history

Loss of body condition was noticed in N376, 13 months after arrival at APHA Weybridge, which was attributed to teeth abnormalities of this sheep. However, it was treated 14 days later for suspected pneumonia (abdominal breathing, harsh lung sounds on auscultation, rectal temperature of 39.3°C and occasional coughing) with 5 mg/kg enrofloxacin (Baytril, Bayer) subcutaneously, 15 mg/kg long-acting amoxicillin (Amoxypen LA, MSD) intramuscularly and 0.5 mg/kg meloxicam (Metacam, Boehringer Ingelheim) subcutaneously. A 4-day course of 6.6 mg/kg gentamicin (Genta-Equine, Dechra) intramuscularly was then given instead of enrofloxacin, accompanied by a single injection of 0.06 mg/ml dexamethasone (Dexadreson, MSD) intramuscularly, because the sheep continued to display tachypnoea, and the daily rectal temperature ranged from 39.4°C to 40.1°C. Breathing and lung sounds improved eventually 15 days after the first treatment, and the sheep remained clinically unremarkable. Concerns were raised again 42 months after arrival at APHA Weybridge because of gradual loss of body condition and weight, evident tachypnoea (see online supplemental file 1) and frequent coughing. Rectal temperature was 39.5°C, and lung sounds were harsh with wheezes on auscultation. Treatment with 6 mg/kg gamithromycin (Zactran, Merial) subcutaneously and 0.5 mg/kg meloxicam subcutaneously was initiated, which resulted in an improvement, although the sheep continued to lose weight (see figure 2) so that additional food was provided.

Figure 2

Progressive weight loss of the index case compared with its companion. N376 was last weighed 1 day before euthanasia.

Due to deteriorating appetite a clinical examination was carried out at 46.5 months after arrival at APHA Weybridge. Findings were an abnormal posture, with the neck occasionally extended, further loss of body condition (score 1 on a scale of 1 of 5), rectal temperature of 39.3°C, reduced rumen contractions, very harsh lung sounds preventing auscultation of the heart, an absent menace response in the left eye and bilaterally weak pupillary light reflex. Examination of the eyes with a retinal lens revealed pale green spots in the tapetal fundus (single: left, multiple: right). Raising the hindquarters with the head held low (wheelbarrow test) did not elicit any nasal discharge. The superficial lymph nodes were normal to palpation.

Investigations

Results from haematology and blood biochemistry, carried out at APHA Shrewsbury, are shown in table 1. The key finding was hyperglobulinaemia, suggestive of chronic infection despite normal white blood cell count.

Table 1

Haematology and blood biochemistry values for the index case 2 weeks before cull

Differential diagnosis

The clinical signs were suggestive of a chronic condition affecting the respiratory system, such as pulmonary adenomatosis, chronic suppurative pneumonia, maedi or a malignant tumour with metastases in the lungs. Caseous lymphadenitis was considered as a cause of chronic weight loss and because of past occurrence in the sheep flock, but ELISA performed by SAC Consulting Veterinary Services, Penicuik, was negative for Corynebacterium pseudotuberculosis antibodies. No parasitological examination was undertaken because the sheep had been housed indoors for several years.

Treatment

Due to the poor prognosis, the sheep was euthanased with 40 mg/kg quinalbarbitone and 2.5 mg/kg cinchocaine (Somulose, Dechra) and a postmortem examination performed.

Outcome and follow-up

Main gross abnormalities were found in cardiopulmonary, digestive, lymphoreticular and urinary systems. Inadvertently, the eyes were not collected during the postmortem examination.

The thoracic surface of the diaphragm was lined by multiple white nodules from 1 to 10 mm in diameter. There were large fibrous adhesions between the left cranial lung lobe and the parietal pleura, with mineralisation of the connective tissue. The left cranial lung lobe presented a cystic structure of approximately 10 cm in diameter containing a greenish pasty substance. Multiple white nodules similar to those described previously, with hard consistency and gritty to the cut, were identified on the pulmonary pleura (see figure 3) and throughout the pulmonary parenchyma. Areas of atelectasis and emphysema, with mild to moderate congestion and oedema, were observed microscopically. Multifocally, there were several granulomas, from areas with a few small size granulomas, capsulated, with several giant cells, epithelioid cell, lymphocytes and macrophages, to big areas of coalescent granulomas that also had necrotic centres and calcification.

Figure 3

Costal pleura of index case N376. Multiple granulomas from 0.3 to 1.0 cm in diameter were observed, with a pearl to white appearance, surrounded by fibrotic tissue. At the cut surface, they were yellow with gritty consistency.

Some areas in the left ventricle wall of the heart were discoloured and the thickness of the ventricular wall was 1.0:1.5. The surface of the heart valves was slightly rough. Histopathologically, there was mild pericardial oedema with mild lymphocytic inflammatory infiltrate. In the myocardium, there were three small granulomas, composed mainly by macrophages, giant cells and lymphocytes, with a few neutrophils, surrounded by a capsule of connective tissue, and, diffusely, mild lymphocytic inflammatory infiltrate was observed.

Multiple round to oval, white, nodular structures of approximately 5 mm in diameter were found on the serosa of the ileum, which were well demarcated and of hard consistency and gritty to the section, containing mineralised/calcified material. The liver was enlarged, with round edges, rough surface and a slightly yellow in colour. There were numerous nodular structures similar to the ones described earlier, with a multifocal distribution throughout the organs, and in sizes ranging from 2 mm in diameter to several centimeters and with the presence of coalescent lesions. On histopathology, there were distension of the hepatic sinusoids by a mild inflammatory infiltrate of neutrophils and lymphocytes, vacuolar degeneration and necrosis of hepatocytes and one non-capsulated granuloma, composed by lymphocytes, and a few neutrophils, surrounding foamy macrophages and epithelioid cells.

Most of the lymph nodes examined, including those of the head and the thoracic and abdominal cavity, were enlarged and firm, with cartilage-like or bone-like texture. The cut surface showed the presence of gritty sandy material and, in a few cases in the thoracic cavity, a creamy yellow to green material. A single hard white nodule of around 1–2 mm in diameter was found in one kidney. These changes were identified as granulomas similar to the ones found in the lungs. The findings of granulomatous inflammation in most of the organs (granulomatous pleuropneumonia, lymphadenitis, focal myocarditis and mild hepatitis with leucocytosis) were suspicious of tuberculosis (TB). Acid-fast bacilli were identified by Ziehl-Neelsen stains of sections of the lung, kidney, parietal pleura, medial retropharyngeal and mesenteric lymph nodes with and without formic acid treatment (figure 4). A mix of liver, kidney and mesenteric lymph node was prepared for culture and molecular typing. Approximately 20 g of tissue was ground in a stomacher, decontaminated with oxalic acid, centrifuged and the pellet resuspended in sterile phosphate-buffered saline (PBS) and centrifuged again. The homogenates were then resuspended in PBS and cultured at APHA Sutton-Bonington in Mycobacteria Growth Indicator Tubes (BBL MGIT, Becton-Dickinson) used with automated mycobacterial detection system (BACTEC MGIT, Becton-Dickinson),14 which gave a positive reaction after 6 weeks of incubation. Molecular typing of the positive, heat-killed culture using spoligotyping and variable number tandem repeat (VNTR) typing to characterise the M bovis strain15 16 identified the isolate as genotype 17:a (spoligotype 17 (SB0263), VNTR type 7-5-5-5*−3–3.1)).

Figure 4

Ziehl‐Neelsen staining of a granuloma from index case N376. Staining of a granuloma composed by a central necrotic area, surrounded by cell debris, macrophages, multinucleated giant cells, few neutrophils and lymphocytes, as well as fibroblasts in the external layer. Two acid fast-resistant bacilli are observed (black arrows). © Crown copyright 2020.

Movement restrictions were imposed and single intradermal comparative cervical tuberculin (SICCT) testing17 was carried out in all animals housed in the same building 5 months after euthanasia of the index case. The TB incident occurred in pen 6, which housed the other sheep N524 and another sheep introduced as companion replacement for TB case N376. Pen 7, adjacent to pen 6, housed two sheep and pens 3, 2 and 1 on the other side housed four, four and three steers, respectively (see figure 1). Sheep N524, which had been housed with the index case for 46 months, was identified as test reactor based on the skin test technique described previously,7 with a skin thickness difference of 8.5 cm between the reactions to avian and bovine tuberculin measured 72 hours after injection. The sheep was euthanased (40 mg/kg quinalbarbitone, 2.5 mg/kg cinchocaine, Somulose), together with the companion sheep, which was test-negative. No lesions suggestive of TB were detected on gross examination. One steer (7201) in pen 2 had a skin thickness difference of 2 mm between the avian and bovine tuberculin injection site, which was classified as an inconclusive skin test result. It was subsequently quarantined in another building that was otherwise empty, with a steer (7197) of the same pen as companion. A SICCT retest 77 days later identified this steer as reactor with a skin thickness difference of 6 mm, and it was euthanased with 140 mg/kg pentobarbital (Pentobarbital, Ayrton Saunders). Visible lesions of TB were observed in the left medial retropharyngeal (see figure 5) and left bronchial lymph nodes, from which M bovis of a genotype identical to the sheep (genotype 17:a) was cultured. There was no record of TB on the source farm of this steer around the time when this animal was moved to Weybridge.

Figure 5

Ziehl-Neelsen staining of the retropharyngeal lymph node of the bovine tuberculosis reactor. medial retropharyngeal lymph node showing (A) grade IV granulomas displaying central necrosis and mineralisation (H&E) and (B) acid fast bacilli in the necrotic milieu of a granuloma (ZN).

While the companion steer was culled due to an unrelated disease before further TB tests could be carried out (no visible lesions observed, negative culture on medial retropharyngeal lymph node), all sheep and cattle sharing the accommodation were subjected to a further SICCT test with negative results. In addition, blood samples from the remaining nine cattle in the building tested negative by the gamma interferon test (BOVIGAM TB Kit, Thermo Fisher Scientific according to the manufacturer’s instructions)18 so that the restrictions were eventually lifted. All cattle in the pen had been subject to weekly passive behavioural observations19 for 15 minutes late morning as part of a research project where any incidence of coughing was recorded. Retrospective analysis of the coughing frequency in all cattle (7195, 7197 and 7203), which were in contact with the bTB reactor 7191 in this pen during a period of 31 months when the sheep index case moved in the building and the bTB reactor was culled, revealed that coughing was observed quite regularly from 8 months before cull in this steer but not in the companions (see table 2). A timeline of events is displayed in figure 6.

Figure 6

Timeline of events. d, days; m, months; TB, tuberculosis.

Table 2

Coughing frequency of the bTB reactor and its pen companions

Discussion

bTB caused by M bovis is an endemic disease of cattle in England and Wales, which continues to spread despite extensive efforts to control or eradicate it. Although all terrestrial mammals, including humans, are susceptible to infection, cattle and other bovine species are considered the primary reservoirs for maintaining the infection (maintenance hosts), whereas other species are considered spill-over hosts, where the infection occurs within the species only as long as there is input from a maintenance host. Sheep were long considered dead-end hosts that did not play any significant role in the transmission to the same or other species. Indeed, sheep had been considered fairly resistant to infection, although there have been various reports of outbreaks in sheep caused by M bovis.2–5 7–10 20 21 A study in Spain,6 which demonstrated bTB in the majority of sheep cohabitating with bTB-infected cattle herds, led the authors to conclude that sheep may pose a risk of disease transmission to other susceptible species, although, to our knowledge, there has not been any report confirming transmission from sheep to other species. TB in sheep is most often diagnosed at slaughter of supposedly healthy animals5 21 or diagnosed retrospectively when carcases are submitted for further examination.7 20

The current report describes an unexpected clinical case of TB in a sheep that was permanently housed after arrival at APHA Weybridge. Biosecurity measures, such as pest control, personal protective equipment and use of 2 per cent sodium hypochlorite solution as disinfectant, which has been shown to be effective against Mycobacterium tuberculosis at this concentration,22 made the introduction of M bovis into the building via fomites or wildlife vectors very unlikely.

The historical records strongly suggest that the sheep (index case) became infected while on pasture on the farm in Warwickshire, although the original source of infection remains unknown. Cattle with confirmed M bovis infection had previously grazed the same fields as the sheep. The genotype of the sheep isolate (SB0263, 17:a) did not match the genotype isolated from the infected cattle that used the same pasture (SB0272, 10:a). Even so, results from other TB incidents in the county in 2012 showed that, while spoligotype 10 was the most common, spoligotype 17 was also found and predominant in the neighbouring counties of West Midlands and Worcestershire.23

The disease in this sheep was chronic and slowly progressive, and the main clinical signs were dyspnoea with coughing and weight loss, identical to those described for clinical cases of ovine TB in Spain,6 21 although ill-thrift was the only sign reported in an outbreak of bTB in Lleyn sheep in England.7 Clinical pathological findings were unspecific despite the widespread pathological changes in multiple organs. Marianelli and others reported a generalised infection in sheep, where no clinical signs were observed,5 but a systemic infection with disemination in other organs seems to be rarer in sheep than a respiratory infection affecting lungs and associated lymph nodes.6 24 Hyperglobulinaemia was suggestive of a chronic infectious disease, such as pulmonary adenomatosis,25 which was considered the most likely diagnosis, even though the wheelbarrow test used as clinical marker for this disease was negative. Serum haptoglobin, described as a useful marker for acute bacterial infections in sheep, although its usefulness in chronic infections was not known,26 was not increased. More advanced diagnostic techniques, such as radiography or ultrasonography, were not available to make a more definite clinical diagnosis.

M bovis infection was subsequently transmitted to the companion sheep of the index case based on its positive SICCT result. The absence of any gross pathological lesions of bTB in this sheep was relatively surprising, given that both were housed together for over 46 months (since the arrival of the second sheep). This supports the view that sheep are naturally less susceptible than cattle to M bovis infection, as mentioned before. However, a steer housed in the same building but not in a pen adjacent to the index case became infected and lesions suggestive of bTB were found in the medial retropharyngeal lymph node, which revealed M bovis in culture and the same genotype as the index case. As different protective clothing was worn for the sheep and cattle pens, different equipment was used and pens were accessed via different entrances, infection was likely acquired air-borne because both animals shared the same air space. The infected steer had a habit of standing with his head reaching over the door into the main corridor that connected all pen entrances, which may have increased the probability of infection. Indeed, a study in cattle to investigate cattle-to-cattle transmission of bTB in a building demonstrated that transmission can occur between cattle kept in different pens and without having nose-to-nose contact,27 either through transfer of contaminated material, which is unlikely in the current case, or through the shared air space.

This report confirmed for the first time that sheep can act as a source of TB in cattle, which has previously only been hypothesised.6 28 The housing of cattle and sheep for a prolonged period in the same building is unlikely to be representative of normal farming practice, but this case nevertheless demonstrates that excretion of viable bacilli and potential contamination of the environment by infected sheep is possible.

Learning points

  • Tuberculosis (TB) should be considered in sheep reared in areas of endemic bovine TB with chronic weight loss and respiratory signs that are unresponsive to treatment.

  • Knowledge of previous locations and movement records of this sheep would have helped to include TB in the list of differential diagnoses.

  • Transmission of TB between sheep and between sheep and a steer sharing the same air space was demonstrated.

Acknowledgments

The authors are grateful to Patrick Johnson at Drayton Animal Health and past and present members of staff at APHA Sutton-Bonington, the APH England Field Delivery, the Pathology Department, Animal Sciences and the TB units at APHA Weybridge for their help and support. The animals were part of a Defra-funded project (SE1959, SE1960) unrelated to the outbreak of TB. We thank Dr de la Rua-Domenech for proofreading the manuscript.

References

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Supplementary materials

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Footnotes

  • Contributors TK carried out the clinical examinations and investigations, supported by HS, and, together with AG, drafted the manuscript. JD was responsible for the characterisation of the isolates; JS and AG carried out the pathological examinations. All authors read, contributed to and approved the final manuscript.

  • Funding The authors have not declared a specific grant for this research from any funding agency in the public, commercial or not-for-profit sectors.

  • Competing interests None declared.

  • Provenance and peer review Not commissioned; externally peer reviewed.

  • Data availability statement All data relevant to the study are included in the article.

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