Bovine Tuberculosis - Analysis of 10-year cases and impact of visual inspection in the surveillance at the slaughterhouse in Portugal

Bovine Tuberculosis (bTB) is a One Health issue that interconnects human health with animal and environmental health. Due to its importance, cattle (the main host) it is under a national eradication programme in Portugal. Within it, meat inspection is considered a cost-effective procedure in bTB surveillance. The Commission's Implementing Regulation (EU) 2019/627 changed the post-mortem meat inspection requirements applied to cattle, essentially reducing handling and incisions in young cattle (<8 months). To study the impact of these changes on the surveillance of bovine Tuberculosis, data (age, sex, fitness, breed, lesions, laboratory results and post-mortem decisions) on suspected and confirmed cases of bTB detected at routine slaughter of the period from October 2010 to January 2020 were analysed. From a total of 3,279,548 slaughtered animals, meat inspection detected 808 suspected cases. Of these, 381 cases were laboratory confirmed. The estimated global prevalence was 11.6 cases/100,000, increasing with age: 3.5, 5.9 and 27.3 cases/100,000 in, respectively, the groups <8 months, 8 to 19 months, and ≥ 20 months. The prevalence was higher in females (16.3 vs 7.3 cases/100,000 in males) and in beef cattle (16.3 vs 1.4 cases/100,000 in dairy cattle). Among the confirmed cases, mediastinal and bronchial lymph nodes were the most reported locations. Based on the results, there is no evidence of a negative impact on the application of Regulation (EU) 2019/627 on bTB surveillance in animals ≥8 months. For the <8 months group the change to a visual inspection protocol could lead to a failure in the detection of bTB cases. Considering the epidemiological situation, the incision of the mediastinal, bronchial and retropharyngeal lymph nodes in cattle <8 months should be maintained.


Introduction
Bovine Tuberculosis (bTB) is a chronic zoonotic disease caused by bacteria from Mycobacterium tuberculosis Complex, mainly by Mycobacterium bovis [1]. Regarding public health concerns and the economic impact on animal production, in Portugal there is an eradication programme (PETB) since 1991 aimed to control the infection in susceptible animal hosts, thus reducing the risk for human infection [2][3][4].
Under the PETB, at the farm level, the screening for bTB is based on the single intradermal comparative tuberculin skin test (CITT), which is considered the most widely used test [5][6][7][8]. After a positive test, the animal is slaughtered in the abattoir under special conditions, a sanitary slaughter. A detailed post-mortem inspection is performed to identify granulomatous lesions compatible with bTB infection [3] as a supplementary means of monitoring the effectiveness of surveillance screening tests [9,10]. The gamma-interferon test can also be used as a complementary diagnostic test, when there is a significant number of doubtful results in CITT and as an alternative to compulsory sanitary slaughter on herds with chronic positivity on CITT [3,6].
Meat inspection is essential to verify compliance with requirements for human and animal health and animal welfare and it is also a valuable tool in animal health surveillance and monitoring [11][12][13]. With this perspective, meat inspection plays a primary role in detecting animals that despite being CITT negative, do present macroscopic lesions at postmortem examination, being considered a cost-effective procedure in bTB surveillance [7,8,10,[14][15][16].
Practical arrangements for post-mortem inspection are defined in the Commission Implementing Regulation (EU) 2019/627, which introduced changes in official controls moving forward to risk-based meat inspection [17]. Particularly in young cattle (<8 months), if there are no indications of possible risk for public health, animal health or welfare, palpations and incisions are reduced to minimize cross contaminations. The main procedures that were suppressed included the incisions of the retropharyngeal, bronchial and mediastinal lymph nodes, and may have repercussions on the detection of granulomatous lesions of bTB, since they occur frequently in those locations [18]. A change in these procedures under a routine slaughter may be associated with a reduction in the likelihood of detecting lesions consistent with bTB and, consequently, may have a negative impact on disease control programmes and public health [19,20]. To ensure the surveillance of their tuberculosisfree status Member States may decide to carry out further investigations [11] Therefore, each EU member state should assess the possible consequences of the implementation of this new inspection protocol on bTB surveillance.
The objectives of this study were to characterize the cases of bTB detected through post-mortem inspection at the abattoir over a 10-year period and to investigate the possible impact of the change in the meat inspection protocol on the surveillance of the disease.

Data collection and case definition
The study population, the total of cattle slaughtered in Portugal from October 2010 to January 2020, was obtained from the Animal Identification and Registration National Database (SNIRA). The National Competent Veterinary Authority Official Database for meat inspection (SIPACE) was consulted to collect data on the suspected and confirmed cases of bTB detected during routine slaughter from October 2010 to January 2020 in all Portuguese bovine slaughterhouses. These cases correspond to animals that had tested negative for CITT but presented detectable macroscopic lesions on post-mortem examination at slaughter. A suspect case was defined as an animal slaughtered for human consumption that in the post-mortem examination performed by the official veterinarian (OV), presented one or more macroscopic lesions compatible with bTB. According to the national guidelines, all suspected lesions of bTB detected in animals originating from officially bTB-free herds, and those not officially free but without isolation of bacteria of the Mycobacterium tuberculosis complex, are collected by the OV and examined for histopathologic and/or bacteriological confirmation at the national Reference Laboratory [3]. The confirmed cases of bTB are the result of the bacteriological isolation of any bacteria of the Mycobacterium tuberculosis complex and/or compatible histological examination, as defined in the PETB [3,4].
The data collection of suspected and confirmed cases included animal age, sex, breed, type of production, lesions distribution, laboratory results and post-mortem decision. When some information was missing, other official databases were consulted, such as the national animal identification and registry database SNIRA. Despite these efforts, in some cases data were missing, namely the location of lesions.
The carcasses were declared fit or unfit for human consumption by the Official Veterinarian, according to EU law [11].
Following the age categorization of the Commission Implementing Regulation (EU) 2019/627, of 15 March 2019, animals were divided into three classes: <8 months, 8 to 19 months, and ≥ 20 months. The type of production was categorized as beef cattle and dairy cattle and subdivided into two other groups, autochthonous (Portuguese) and exotic breeds.
The location of lesions included lungs, pleura, and lymph nodes (retropharyngeal, parotid, mandibular, mediastinal, bronchial, gastric, mesenteric, hepatic, renal and retro mammary), spleen, liver, kidneys, peritoneum, genitals, mammary gland. The assessment of the location of lesions was carried out in the slaughterhouse and registered in accordance with the official guidelines [3] In this study, lesions were categorized as single, if only one organ or lymph node was affected, and multiple if else.

Data analysis
The prevalence of abattoir cases of bTB was calculated by dividing the number of confirmed cases by the population and multiplying by 100,000. The prevalence was reported as cases per 100,000. The confirmation rate was calculated by dividing the number of confirmed positive cases by the number of suspected cases of bTB and was reported as a percentage. Absolute frequencies and percentages were used to describe the distribution of suspected and confirmed cases of bTB. The distribution of lesions in bovines suspected and infected was assessed. Age in months was described as median and the interquartile range (IQR).
The data were analysed using Excel and SPSS version 26.

Results
A total of 3,279,548 animals were slaughtered from October 2010 to January 2020, 808 animals were classified as suspected cases, and of these 381 (47.2%) were confirmed by laboratory analysis (Table 1). Mycobacterium bovis was isolated in 80.3% (n = 306) of the positive cases; Mycobacterium caprae in 1,8% (n = 7), Mycobacterium tuberculosis in 0,3% (n = 1) and Mycobacterium tuberculosis complex in 1,3% (n = 5). The remaining 13,6% were divided between compatible with bTB by histopathological examination (6%) and positive results registered in the database without any information about laboratory results. The confirmation rate and the number of confirmed cases reached the highest values in 2018 and 2019, respectively 79.1% (n = 53) and 71.1% (n = 59). The estimated prevalence of bTB over the ten-year period showed an increasing trend from 2015 to 2020. The estimated global 10year prevalence was 11.6 cases/100,000, increasing with age: 3.5, 5.9 and 27.3 cases/100,000, respectively, in the groups <8 months, 8 to 19 months, and ≥ 20 months ( Table 2). The prevalence was higher in females (16.3 vs 7.3 cases/100,000 in males) and in beef cattle (16.4 vs 1.4 cases/100,000 in dairy cattle). All dairy animals were of exotic breeds, while beef cattle included both exotic and autochthonous breeds. Among beef cattle, autochthonous breeds account for only 14% of slaughtered animals, but the estimated prevalence of bTB in these was higher than in exotic breeds (32.2 vs 13.9 cases/100,000).
The median age of the confirmed cases was 56 months (interquartile range: 16 to 137 months), higher in females (median = 110 and interquartile range: 34 to 161 months) than in males (median = 17 and interquartile range: 12 to 38 months), and higher in beef cattle (median = 61 and interquartile range: 16 to 140 months) than in dairy cattle (median = 25 and interquartile range: 12 to 50.5 months).
Regarding the distribution of lesions, of a total of 799 suspected cases with information, 432 (54.1%) had multiple lesions and the rest had single lesions. Detailed information on the location of lesions (Table 3) was only available in 767 records. The most reported location in suspected cases was the mediastinal lymph nodes (46.3%), followed by the lungs (46%) and bronchial lymph nodes (37.5%). In the confirmed cases of bTB, the locations of the lesions were the mediastinal (62.5%) and bronchial lymph nodes (53.9%), the lungs (49.2%), and retropharyngeal lymph nodes (33.1%). Considering the three age groups in the confirmed cases, mediastinal and bronchial lymph nodes were also the most reported locations. However, retropharyngeal lymph nodes were the third most reported location in the two youngest groups, versus lungs in the oldest group. Considering the anatomical regions, in the confirmed cases the highest proportion of lesions was in the thoracic region (83.9%) and the head (34.7%).
Analysing the occurrence of single lesions in confirmed cases (n = 109), only 107 had information on the anatomical location (Table 4). In these cases, retropharyngeal lymph nodes (44.9%), followed by mediastinal lymph nodes (27.1%) were the most common locations of lesions. The anatomical distribution of single lesions across the three age groups was very similar and in accordance with the distribution of the total number of cases. In the confirmed cases, the total condemnation rate of carcasses was higher (57.6%) than in suspected cases (45.2%).

Discussion
A post-mortem examination at the slaughterhouse constitutes an additional surveillance method for detecting cases that may have been missed in ante-mortem testing programmes [10,15,[21][22][23]. In our study, nearly half (47.2%) of the suspected cases detected in the slaughter line were confirmed as positive cases, a lower proportion than reported by other authors [24,25]. Mycobacterium bovis was isolated in 80.3% (n = 306) of the positive cases, in accordance with the sensitivity of the bacteriology testing previously described [26]. Surprisingly, since the PETB is in force for a period of 31 years, it was observed an increasing trend in the detection of cases since 2015 reaching the highest value in 2019 ( Table 1). As meat inspection procedures were maintained in the reporting period, this may reflect the increased sensitivity of the OV to detect lesions through post-mortem examination, encouraged by the Competent Authority under the PETB. During the last four years of the period under analysis, the confirmation rate has also increased. This can indicate an increase in the submission to the laboratory of granulomatous lesions with a macroscopic appearance clearly indicative of bTB, improving the accuracy in the laboratory results.
The prevalence was higher in beef cattle and within these, in autochthonous breeds. These animals are raised in a more traditional way, preferably outdoors with access to pasture that can potentially lead to direct or indirect pathogen transmission at the wildlife-livestock interface in a multi-host system [27][28][29][30]. In our study, older animals (≥ 20 months) and females (median age 110 months) had the highest proportion of confirmed cases. Our results are in accordance with previous studies, regarding the fact that for sake of production and economic interests, cows usually reach an older age than males because of their role in calving and the increase in incidence with age reflects an intrinsic property of bTB transmission [29,31,32].
In confirmed cases, the most recorded locations of lesions (Table 3) support the prevalence of respiratory infection and are in agreement with previous studies [18,[33][34][35] Interestingly, 71.2% of the confirmed cases (279 out of 378) presented multiple lesions in opposition to other studies, that reported above 70% of single lesions [36,37]. Whenever a granulomatous lesion is detected, the OV performs a more detailed examination with additional palpations and incisions, increasing the probability of finding multiple lesions [11].
Several authors and studies confirm that post-mortem inspection including palpation and incision is more effective to detect bTB lesions when compared with the visual inspection method [13,15,24,25,38,39] can be hypothesized that the use of a simplified inspection method decreases the chance to find gross lesions and can delay the success of an eradication program [10,25]. However, there is a trend in the modernization of meat inspection in the EU food law moving forward to risk-based meat inspection, avoiding unnecessary manipulation and consequent cross-contamination [12,17].
A qualitative risk assessment of the visual-only meat inspection concluded that Mycobacterium bovis was the only risk confirmed as nonnegligible for animal health and welfare, since the omission of palpation and incision of lymph nodes and organs is likely to reduce the sensitivity of detection of bTB-associated lesions on post-mortem examination to almost zero [38]. The risk to animal health was considered higher in regions or countries where bTB was officially eradicated, given the Table 1 Number and distribution of suspected cases (N = 808) and confirmed cases (N = 381) of bovine tuberculosis per year in the period 2010-2020 and estimated annual prevalences.  longer time interval between CITT controls [38]. The main changes introduced by the Regulation (EU) 2019/627 regarding post-mortem inspection for the detection of bTB are, in bovine ≥8 months, the suppression of visual inspection and of incision of the sub-maxillary and parotid lymph nodes and the lungs. In our study, out of 344 animals ≥8 months, only one case with a single lesion in the submaxillary lymph node and six cases with lesions in the lungs were identified. In these latter six cases, either a complete description of the location of the lesions was missing in the data base or the respective lymph nodes were not carefully examined, since additional lesions should be expected. This can be explained by the pathogenesis of tuberculosis. A primary complex is complete if lesions in the primary focus of infection and respective draining lymph nodes are both present, and incomplete, i.e., with single lesions in the lymph nodes, when the lesion at the portal of entry has already disappeared [14].
In young bovine animals <8 months, the meat inspection protocol was deeply altered by Regulation (EU) 2019/627. Post-mortem inspection became mostly visual, maintaining only the palpation of lungs, retropharyngeal, mediastinal and bronchial lymph nodes and suppressing all incisions. In this age group, except for one case with generalized lesions, we found that all cases presented lesions of early stages of infection -primary complexes that could be unnoticed with a visual-only post-mortem examination. Single lesions were found in 7 out of 18 confirmed cases and these were located in the retropharyngeal or mediastinal lymph nodes. In the retropharyngeal lymph nodes, 80% of the confirmed cases had single lesions and, in the mediastinal lymph nodes, the opposite was found, revealing multiple lesions in 75% of the cases. However, most of these multiple lesions (78%) were associated to primary complexes. Despite the estimated prevalence of bTB being low in this group (3.5 cases/100.000), the importance of early detection of bTB outbreaks should not be neglected, avoiding the dissemination and increase of the infected herds. In a future study, it would be useful to compare the results of meat inspection on the detection of new bTB outbreaks with the testing in live animals through CITT. This practice would be a good tool to evaluate the contribution of post-mortem inspection to the detection of bTB cases.

Conclusion
To our knowledge, this is the first study with a detailed analysis of the cases of bTB detected in slaughtered cattle in Portugal. This zoonotic disease is a global veterinary public health issue, which clearly justifies the One Health approach. In Portugal, due to its importance, it is under an eradication program (PETB) in cattle, the main reservoir. The results from this study highlighted that post-mortem inspection of routinely slaughtered cattle have proven to be a valuable tool for screening bTBinfected animals that for some reason were not detected at the farm level under the PETB. The variety in the typology and topography of lesions found in bTB-confirmed animals, allowed us to conclude that there was no evidence of a negative impact on the surveillance in cattle ≥8 months old by changing post-mortem inspection protocol to implementing Regulation (EU) 2019/627. On the contrary, for the group <8 months of age, despite the limited number of cases confirmed (n = 18, 3.5 cases /  100,000) our data suggest that changing the procedures to a visual-only inspection protocol, could lead to a reduced detection of bTB cases, resulting in a detrimental effect in public and animal health. Based on this Regulation, incisions may be maintained considering the epidemiological situation of the Member State. The results of our study can provide scientific support to the national Competent Veterinary Authority's decision to maintain the incision of mediastinal, bronchial and retropharyngeal lymph nodes in bovine animals <8 months.

Funding information
This work was financed by National Funds (FCT/MCTES, Fundação para a Ciência e a Tecnologia and Ministério da Ciência, Tecnologia e Ensino Superior) under the project UIDB/00211/2020.
The participation of Madalena Vieira-Pinto was supported by the projects UIDB/CVT/00772/2020 and LA/P/0059/2020 funded by the Portuguese Foundation for Science and Technology (FCT).