Occurrence of BTB-suggestive lesions in lymph nodes in the front portion of carcasses during post mortem inspection in 41,193 cattle in slaughterhouses from different municipalities in the state of Mato Grosso, between May and October 2009.
Bovine tuberculosis (BTB) is an infectious disease of chronic evolution and debilitating effects. The etiologic agent of this disease is
Cattle are the primary hosts for
An important feature of mycobacterial infections is the cell-mediated immune response developed by the infected host, due to the intracellular location of mycobacteria. This leads to the development of granulomatous inflammations in the host, resulting in tuberous lesions [26, 27]. These lesions often occur in organs rich in reticuloendothelial tissue, especially in the head, neck, mediastinal and mesenteric lymph nodes, but also in the lungs, intestines, liver, spleen, pleura and peritoneum [28, 29]. Although tuberous lesions are not considered pathognomonic for BTB in cattle, their presence is closely linked to the appearance of clinical signs of BTB in animals [4, 5].
In developed countries, where TB control programs have been established longer and executed with rigor, BTB control is accomplished through mandatory procedures such as pasteurization of cow milk and its derivatives and sanitary inspection of cattle during slaughter, thereby drastically reducing cases of the disease in humans and animals . Although a tuberculosis control and eradication plan exists in Brazil, illegal sales of meat, milk and dairy products not inspected by sanitary control agents still occur and constitute a risk to public health .
The detection of the pathogen responsible for BTB is crucial for the control and eradication of the disease and should be performed as recommended by the OIE , by late hypersensitivity reactions in cattle (intradermal tuberculin tests), sanitary inspection in slaughterhouses, tracing the origin of diseased animals and disease sanitation .
With the aim of reducing the prevalence and incidence of new BTB outbreaks, the Brazilian national program for control and eradication of brucellosis and tuberculosis (PNCEBT) was instituted in 2001. This program is based on the performance of intradermal tuberculin tests and the slaughter of reactive animals (test and slaughter), associated with the health inspections carried out in slaughterhouses . Although the intradermal tuberculin test is widely used worldwide for BTB diagnosis, this test presents sensitivity and specificity problems, generating false-positive or false-negative results. These flaws are important, since the reference microbiological methods for BTB diagnosis also exhibit low sensitivity and are effective for pathogen detection only when the number of viable bacilli is higher than 100 bacilli/mL. In addition, microbiological testing procedures are laborious and time consuming, taking from 1 to 3 months for bacilli isolation and a further two or three weeks for the biochemical identification of the isolates .
Despite the occurrence of BTB, there is no official data on the current prevalence of the disease in Brazil. Data from official reports from 1989-1998 indicate that the national average prevalence was of 1.3% of infected cattle . Since the beginning of the PNCEBT program, however, few studies have been conducted to determine the prevalence of the disease, and estimates vary from 0.7% to 3.3% [33, 34-35, 36]. According to the data obtained by Roxo and Kantor [37, 38], the estimated national prevalence was of 0.83% and the region with the lowest prevalence of BTB was the Brazilian Midwest (0.37 %), where beef cattle in its majority is raised. In studies conducted by Salazar and Furlanetto [6, 39], in slaughterhouses in the state of Mato Grosso, located in the Midwest region of the country, a very low BTB prevalence was detected, of only 0.007%.
One of the main economic activities in the state of Mato Grosso is cattle production. This state is prominently the largest producer of beef cattle, with around 28 million cattle heads, and the second largest beef exporter the country , increasing beef exports to EU countries each year. However, countries that buy Brazilian beef are increasing pressure to implant effective, quick and definitive BTB diagnosis methods to identify tuberculosis-suspected lesions. In 2012, the Ministry of Agriculture, Livestock and Supply (MAPA), determined that farms in which suspected cases of BTB had been detected could no longer export beef to the Customs Union of Belarus, Kazakhstan and Russia, and that all lots of animals of the property must be sequestered during slaughter, until confirmation of the diagnosis of the BTB-suspected lesions by official MAPA laboratories [41, 42].
Due to the demands imposed by countries that import Brazilian beef and the difficulties in achieving quick and specific BTB diagnoses, molecular tests based on PCR assays and its variations (
In the present study, different BTB diagnosis tests, used singly or in combination with each other, were evaluated. Different methods, i.e. macroscopic analyses, histopathological examinations and
2. Geographic region and study conditions
The study of the prevalence of BTB in animals slaughtered in the state of Mato Grosso, Brazil, was carried out by monitoring cattle slaughter and by
Seven slaughterhouses inspected by the SIF were monitored, located in six different cities in the state of Mato Grosso (Figure 1). As mentioned above, this region is considered the largest producer and second largest beef exporter in the country . For the sampling to be considered representative with regard to cattle herds in this geographical area, slaughterhouses in four areas of Mato Grosso which have significant cattle herd production were selected: Southeast, south central, southwest and north. No sampling was conducted in the northeast area due to the unavailability of establishments with SIF inspection. However, animals from that region were slaughtered at the Paranatinga municipality, an area fortunately covered by this study. The selected sampling sites covered the four Mato Grosso cattle-producing circuits, divided according to Negreiros , in: Pantanal-represented by the Cáceres (16º 04' 14" S, 57º 40' 44" W) and Várzea Grande (15º 38' 48" S, 56º 07' 57" W) municipalities; Milk-represented by the Rondonópolis municipality (16º 28' 15" S, 54º 38' 08" W); Fattening-represented by the Paranatinga municipality (14º 25' 54" S, 54º 03' 04" W); and Reproduction-represented by the Juara (11º 15' 18" S, 57º 31' 11" W) and Tangará da Serra (14º 37' 10" S, 57º 29' 09" W) municipalities. Cattle slaughter at Juara was only monitored in two slaughterhouses.
During carcass inspections, all fragments of lesions classified by SIF as lymphadenitis or tuberculosis lesions located in the head, neck, chest cavity or cervical area lymph nodes (areas frequently affected by BTB) were sampled, according to official standards . Once identified, the lesions were photographed, divided into samples and properly packaged. Information on body condition score, age and sex, origin (municipality and property where the cattle were raised) and health status of animals (participation or non-participation in the PNCEBT program), were obtained and recorded during sampling by means of the Animal Traffic Guide (GTA) of each lot.
2.1. Prevalence of bovine tuberculosis in herds slaughtered in 2009 in the state of Mato Grosso, Brazil, determined using conventional tests 
The inspected carcasses 41.193 carcasses belonged to 492 herds, from 85 (60%) municipalities in the state of Mato Grosso (Figure 2). From the 41.193 carcasses assessed during the
The HE histopathological examination performed on 198 samples of BTB-suspected lesions, indicated that 83.8% of the lesions were granulomatous, 8.1% were pyogranulomatous, 6.1% were suppurative, and 2.0% were lesions characteristic of interstitial pneumonia. The ZN histopathological examination indicated no AARB in the samples. The absence of AARB in BTB-suspected lesions has been reported by Salazar , and may occur due to the low bacilli concentrations in the examined lesions (paucibacillary lesions) .
Although granulomas are a classic BTB lesion, they cannot be considered pathognomonic of the disease [20, 32 – 59]. This statement was confirmed in the present study, where 91.9% (182/198) of the samples were granulomatous or piogranulomatose lesions (classic BTB lesions) and only 1.64% (3/182) of the lesions were affected by
Samples stored at-20°C were processed for bacteriological analyses within three months after their collection. Approximately 3g of each sample were macerated with ground glass, and subjected to the hexadecylpyridinium chloride (HPC) 0.75% decontamination method and an adapted Petroff method (4% NaOH). The 0.75% HPC decontamination method was performed as described by Ambrosio , and the Petroff method  was adapted for the simultaneous processing of up to five samples, respecting the collection order and slaughterhouse of origin. When colony growths were observed, samples were reprocessed individually to identify the infected sample. After decontamination, the samples were plated in duplicate in Stonebrink and Lowenstein-Jensen (LJ) culture media, and incubated at 37°C. The samples were observed weekly during the first month and subsequently every two weeks until 90 days of culture. After isolate growth, the samples were stained by ZN to indicate the presence of AARB , as recommended in the National Manual of tuberculosis and other mycobacteria laboratory surveillance, by the Ministry of Health .
|Tangará da Serra||9,689||20||0.21|
The bacteriology analyses isolated
Because of the characteristics of the present study, in which the purpose was to estimate the prevalence of BTB through animals destined for slaughter without possessing knowledge of the number of cattle specimens from each sampled property, it was not possible to calculate the actual prevalence of BTB in the state. Therefore, we calculated only the simple apparent prevalence of BTB in cattle and herds slaughtered in the state of Mato Grosso under SIF supervision, according to the method described by Martin . BTB prevalence was calculated as 0.007% [CI 95%=-0.001%; 0.016%] for cattle and 0.61% [CI 95%=-0.08%; 1.30%] for herds. These results were similar to those found by Salazar , 0.007%, when surveying 57.641 cattle slaughtered in the state of Mato Grosso under supervision of the state sanitary inspection (SSI) service, from November 2004 to August 2005, during the PNCEBT program deployment by bacteriological analyses . Of the total inspected cattle, 0.05% (27/57.641) showed BTB-suggestive lesions according to SISE, with four of these animals (14.8%) confirmed as BTB-positive by the bacteriological analyses. Similar results observed in the present study, four years after the start of the PNCEBT program, indicate a slow progress of the BTB eradication program in Mato Grosso, with the need for greater involvement of all the public and private links involved in this process.
Between 1993 and 1997, the prevalence of BTB in cattle slaughtered at ten slaughterhouses in the state of Minas Gerais (southeastern region) under SIF supervision, was of 0.08%, considering only official macroscopic findings . Meanwhile, estimates on the apparent prevalence of infected animals in the same state, considering the results of the comparative cervical tuberculin test (CTT) (official
According to Kantor , estimates lower than 0.1% suggest areas considered low-prevalence or virtually tuberculosis-free. Therefore, the results of the present study may be underestimating up to 14 times the total number of infected animals and yet, even with this underestimation, Mato Grosso would still be considered a low prevalence or virtually tuberculosis-free area. To provide confirmatory estimates of the disease in Mato Grosso, it would be necessary to conduct a representative BTB sampling survey of the main cattle raising properties in the region .
The low prevalence status found in Mato Grosso was expected, since the area presents certain characteristics that hinder the spread of BTB, including a tropical climate, cattle raised predominantly by the extensive system, aimed at beef exports, low pasture stocking, and early slaughter of the animals. Because of this, the animals end up having less contact with each other and, consequently, shorter exposure to possibly infected animals .
As a result of the low prevalence status observed in this study, the state of Mato Grosso may advance to the stage of BTB eradication, using strategies such as the implementation of an efficient monitoring system, performed alongside inspection officers and the health defense service, so that, together, they are able to detect remaining BTB foci in the region, the application of
2.2. Use of complementary tests in the
post-mortem inspection of suspected bovine tuberculosis infections 
The association of molecular tests and conventional tests was evaluated to contribute to the choice of additional tests in order to reach the BTB-eradication stage in Mato Grosso, identifying the limitations and benefits of each approach regarding their use in
DNA extraction was performed using the commercial Qiagen extraction and purification kit (DNeasy® Blood & Tissue kit), with modifications in the protocol as described by Figueiredo . Five microliters of template DNA-about 100 ng-were used for the m-PCR test based on the method described by Figueiredo  using a reaction mixture of 5 μL reaction buffer (Invitrogen, USA), 0.2 mM dNTPs (Fermentas, USA), 1.5 U of recombinant Taq polymerase (Platinum® Taq – Invitrogen, USA), 5 mM MgCl2 (Invitrogen, USA) and 20 pmols of each primer (Invitrogen, USA) for the amplification of IS6110 genomic sequences (245 bp) Ixlink: (5’-CGTGAGGGCATCGAGGTGGC-3’) and INS2: (5’-GCGTAGGCGTCGGTGACAAA-3’)  present only in MBC members, and
On the revaluation of the macroscopic analyses of the carcasses inspected in the present study, a high incidence of lesions in the pre-scapular and pre-pectoral lymph nodes, of approximately 73.2% (145/198) was observed (Table 2). Despite
The affected retrofaringeal lymph nodes showed increases in size and number of lesions (Figure 5-A, B and C). However, the lesions were localized (restricted to the retropharyngeal node) with no BTB-suggestive lesions in other areas of the carcasses.
According to these results, it is advisable that
|Apical lymph node||4||2||0||0||1||0.5|
|Esophageal lymph node||7||3.5||0||0||0||0|
|Mediastinal lymph node||4||2||0||0||0||0|
|Tracheo-bronchial lymph node||6||3||0||0||2||1|
|Retropharyngeal lymph node||19||9.5||3||1.5||5||2.5|
|Parotid lymph node||1||0.5||0||0||0||0|
|Sublingual lymph node||2||1||0||0||0||0|
|Pre-pectoral lymph node||108||54.5||0||0||4||2|
|Pre-scapular lymph node||37||18.6||0||0||1||0.5|
|Ischiatic lymph node||2||1||0||0||0||0|
When comparing macroscopic analyses, bacteriological cultures and m-PCR, the results indicate that the macroscopic analyses correctly identified 93% (184/198) of the samples, considering these lesions as common lymphadenitis (non-tuberculosis) samples. However,
The state of Mato Grosso has emerged in the Brazilian national scene as the largest beef cattle producer and second largest beef exporter in the country , leading to annual increases in the amount of meat exported to EU countries. Consequently, the pressure on Brazil by countries that buy Brazilian products to implant an effective, rapid and definitive diagnosis of BTB in tuberculosis-suspected lesions has also increased.
In 2012, the Ministry of Agriculture, Livestock and Supply (Ministério da Agricultura, Pecuária e Abastecimento-MAPA) determined that farms where the detection of BTB cases took place can no longer export beef to the Customs Union of Belarus, Kazakhstan and Russia, recalling all lots from these animal farms until the diagnosis of suspicious lesions in samples collected after slaughter could be conducted at an official MAPA laboratory [41, 42].
In view of these commercial and sanitary restrictions, the difficulties in tuberculosis diagnosis must be overcome. Diagnosis alternatives for the quick and specific identification of BTB in clinical samples or isolated colonies have emerged, such as molecular methods based on PCR and its variants (simplex PCR,
In this context, the purpose of the present study was to evaluate the performance of diagnostic tests, such as m-PCR, culture and histopathology, on the detection of MTC species directly from suspected BTB lesions. The apparent prevalence of BTB among animals slaughtered in the state of Mato Grosso was also re-evaluated and discussed, due to the great importance of this geographic region in meat production and export to several consumer countries worldwide, including the European Community.
When comparing macroscopic analyses, bacteriological cultures and m-PCR, the results indicate that the macroscopic analyses correctly identified 93% (184/198) of the samples, categorizing these lesions as common lymphadenitis (non-tuberculosis) samples. However,
As a result of the low prevalence status established in this study, BTB in the state of Mato Grosso may advance to the stage of eradication, using strategies such as the implementation of an efficient monitoring system, performed alongside inspection officers and the health defense service. Alongside the application of
Currently, there is no diagnosis test (
The results of the present study indicate that mistakes can occur during rulings of suspected bovine tuberculosis lesions in cattle, particularly those presenting paucibacillary lesions. These mistakes cause a distortion in BTB estimates in slaughterhouses, with harmful consequences to the success of the Brazilian Tuberculosis Control Program (PNCEBT). The results point to the use of complementary molecular assays for rapid diagnoses of lesions situated in frequently BTB-affected carcass areas, thus minimizing mistakes in judging the disease in slaughterhouses. m-PCR was the most sensitive, rapid and specific method among the complementary methods tested in the present study when compared to conventional methods for BTB-diagnosis. It is, therefore, a promising alternative in disease surveillance to be used by the federal inspection service to contribute to the bovine tuberculosis control and eradication program, for disease surveillance in slaughterhouses and for tracking remaining BTB foci in the state of Mato Grosso, as well as in other regions of the country, contributing even further to the success of the PNCEBT program.
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