Detection of brucellosis in cattle and evaluation of risk factors associated with the disease in workers slaughterhouse

Brucellosis is anthropozoonosis caused by Brucella spp. Among the zoonotic species, B. abortus is the main species affecting cattle and can easily be transmitted to humans. The purpose of this study was to investigate, through epidemiological inquiry and serological analysis, animal and human health as related to smooth strain Brucella spp. in a slaughterhouse located in the southern region of the state of Bahia. For this purpose, blood samples were collected from workers and animals at a slaughterhouse together with the State Inspection Service. Then, the Buffered Acidified Plate Antigen test was performed for animals and humans, the Slow Agglutination Test was performed for humans only; and the Complement Fixation Test and the 2-Mercaptoethanol Brucella Agglutination test (2ME) were performed for animals only. In addition, an epidemiological inquiry was applied to workers in order to assess risk factors for the disease. After data analysis, it was concluded that infection by smooth strains of Brucella spp. was detected in 14.0% of the cattle. Additionally, one worker out 41 tested reactive to the disease.

Smooth strains of Brucella spp. can affect non-specific species. Therefore, the contaminated biological material in pastures and in animal facilities can act as transmission routes for other animals, such as equines, dogs, swine, sheep, goats, and including humans (Osterman & Moriyon, 2006;Emy, 2018;Petry, 2019;Kang, 2019).
In animals, the clinical signs are mainly related to the reproductive system, such as abortion, infertility, retained placenta and the birth of weak animals. These signs are explained because the bacteria have a preference for these tissues and because the presence of erythritol in testis and pregnant uterus increases bacterial multiplication (Antoniassi, 2017).
Transmission of brucellosis in humans occurs by oral route, through consumption of contaminated products of animal origin, such as milk, dairy products and raw or undercooked meat. Another important route is inhalation, through direct contact with infected animals or secretions and the products of abortion. Transmission can also occur by accidental inoculation of vaccines (Schneider, 2013). As regards occupational health, veterinarians, butchers and livestock professionals, who are in constant contact with potentially infected biological material, are at risk for brucellosis (Zigmunt, 2012;Skendros, 2013).
The incubation period for brucellosis in humans varies from 5 to 60 days, but it could even last for up to two years.
The clinical signs are nonspecific, they include fever, malaise, sweating (nightly and profusely), chills, lethargy, anorexia,   headache, muscle pain, abdominal pain, and, when chronic, the disease most frequently causes joint pain affecting the individual's quality of life (Corbel, 2006).
In Brazil, the serological diagnosis of brucellosis in cattle and buffalo herds is recommended by the National Program of Control and Eradication of Brucellosis and Tuberculosis (Programa Nacional de Controle e Erradicação da Brucelose e Tuberculose Animal -PNCEBT) (Brazil, 2017a). The official tests are the Buffered Acidified Antigen test (RBT), the 2-Mercaptoethanol Brucella Agglutination test simultaneously with the Slow Agglutination Test (2ME), the Fluorescence Polarization Assay (FPA), the Complement Fixation Test (CFT), and the Milk Ring Test (MRT), this last one for monitoring dairy herds (Brazil, 2017b).
The laboratorial diagnosis for humans includes the direct test (bacterial culture and PCR) and an indirect test, such as the 2ME, the SAT, the FPA and the Enzyme-Linked Immunosorbent Assay (ELISA) (Brazil,2019). Individuals exposed to the RB51 vaccine strain are not reagent in serological tests, and direct tests (Corbel, 2006;Brasil, 2019;Hyeda 2011) are recommended.
In view of its threat to public health and the economic loss in livestock, epidemiological research of this disease is essential. Therefore, the purpose of this study was to investigate, though epidemiological inquiry and serological analysis, animal and human health related to smooth strains of Brucella spp. in a slaughterhouse situated in the Southern region of the state of Bahia, Brazil.se the paragraph as a template. A total of 179 blood samples were collected, these were cooled and transported to the Microbiology Laboratory of UESC within 6 hours. Then, the samples were centrifuged and the serum was recovered and kept in microtubes at 0 °C. At the Instituto Biológico de São Paulo, RBT, 2ME and CFT tests were performed following instructions from the Ministry of Agriculture, Livestock and Supply (Ministério da Agricultura, Pecuária e Abastecimento -MAPA) (Brazil, 2017b).

Methodology
The procedures involving humans were approved by the Research Ethics Committee of UESC under protocol 2.836.725. Approximately 20 days after the last collection of animal samples, all slaughterhouse workers were asked to take part in a serum-epidemiological inquiry for brucellosis. They were oriented as to all procedures that would be performed, enlightened about the risks and benefits of their participation in this research and informed about the occupational effects and the significance of brucellosis in public health. An Informed Consent Form was signed by 41 workers who agreed to take part in the survey. The blood collection on these workers was done by a nurse, following all ethical norms, and the epidemiological inquiry was carried out by a veterinarian and a veterinary student.
All human samples were identified and sent to the Special Public Health Service (Serviço Especial de Saúde Pública -SESP) of Itabuna, where the serum collection was performed in duplicate. One sample of serum (1 mL) from each participant was sent to a private clinical analysis laboratory (Hermes Pardini®) for performing standard tube (SAT), since the official Research, Society andDevelopment, v. 10, n. 3, e39710313248, 2021 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v10i3.13248 4 results needed to be laid out by a biomedical specialist. The remaining aliquots were transported to the UESC, frozen and subsequently tested through RBT, in accordance to MAPA instructions (Brazil, 2017b).
The epidemiological data and serological results from humans and cattle were initially stored in the Excel spreadsheet program. To perform epidemiological analysis both confirmatory tests (2ME and CFT) were considered gold standard. Then, both (the screening test and each confirmatory test individually) were compared to the gold standard. True and false positives and true and false negatives were calculated. Subsequently, the Open Epi version 3.01 was used to calculate sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV) and Cohen's Kappa coefficient, the latter to verify agreement between tests. The data related to risk factors for slaughterhouse workers was analyzed by Odds ratio and Fisher exact test with a 0.05 level of significance, both using the Open Epi program.
All samples were submitted to RBT and 2ME and CFT confirmatory tests. Of the tested animals, 39/179 (21.8%) were reagent in the screening test and 25/179 (14.0%) tested positive in both confirmatory tests ( Table 2). The South-Central mesoregion showed 10/67 (14.9%) animals testing positive, while in the South mesoregion the number of animals testing positive was 15/112 (13.4%) after confirmatory tests (Table 1).   (Table 2). Sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and Kappa coefficient of diagnostic tests of brucellosis calculated using OpenEpi software version 3.0. Frequency data based on sampling of 179 as 100%. Source: Authors.
Regarding the epidemiological survey and serological results of the 41 workers from the slaughterhouse, one of them was reagent in RBT and positive for brucellosis in SAT, and ELISA detected IgG. Based on answers obtained in the epidemiological inquiry, the Odds ratio was calculated, but it was not statistically significant in any category (p > 0.05). As regards the importance of answers referring to the disease transmission path, they have been described in Table 3. Table 3: Evaluation of risk factors related to brucellosis in workers of a slaughterhouse.

Category
Exposed Affected Frequency of exposed (%) Odds ratio P  During the epidemiological inquiry and before the blood sample collection, the individual found positive for brucellosis reported chronic joint pain, but dismissed fever episodes, sweating and reproductive dysfunction.

Discussion
The PNCEBT was created in 2001 and since then has gone adapting according to evolution of the disease in the country. In spatial and temporal studies of brucellosis carried out in Brazil between 2014 and 2018, 19,631 cattle tested positive, with incidence among 0.03 and 33.93/100,000 animals (Ribeiro, 2020). This research shows the occurrence of the disease in Brazil and indicates the need for improvement in controlling it.
In this study, 14.0% of sampled animals tested positive for brucellosis. These animals were selected from several different farms in the Southern Region of the state of Bahia and this number was found in only one slaughterhouse in the region. It is noteworthy that the products from this slaughterhouse qualify for the State Inspection Service (SIE) label, which allows the product to be marketed throughout the state, thus posing a threat to the population through contaminated animals.
The prevalence of brucellosis in the Southern region of Bahia was 0.86%, represented by 36 out of 3,565 females that tested positive (Alves, 2009). The higher proportion of animals testing positive during this study may be a consequence of the number of discarded animals in the slaughterhouse. It is widely known that reproductive problems are one of the main reasons for discarding dairy cattle (Ribeiro, 2003). Although the purpose of this study was not the identification of the prevalence of brucellosis, the high proportion of animals testing positive, especially in some cities, points to the need for further studies on this subject.

According to the Regulation of Industrial and Sanitary Inspection of Products of Animal Origin (Regulamento da
Inspeção Industrial e Sanitária de Produtos de Origem Animal -RIISPOA) (Brazil, 2017c) animals bred for milk and meat production must be monitored for brucellosis and, in the case of animals testing positive, they must be euthanized or slaughtered in an authorized slaughterhouse. However, it can be assumed that diagnosis frequency is insufficient, since the animals that tested positive were routed and received by the slaughterhouse without any indication of positive reaction to brucellosis.
The RIISPOA provides for the release of carcasses for consumption of animals that are positive for brucellosis in the absence of indicative lesions. The regulation also provides, that if this is the case, the udder, genital tract and blood should be condemned (Brazil, 2017c). However, a previous study performed by De Macedo ( 2019) demonstrated Brucella spp. in bursitis lesions in some animals. It should be noted that bursitis is not a typical sign of brucellosis and is not included in the RIISPOA. Therefore, these animals are not condemned in the slaughter line and may pose a risk of contamination to the population.
The first paragraph of the aforementioned RIISPOA (Brazil, 2017c) article states that animals with brucellosis must be slaughtered separately. Reagent and non-reagent animals investigated in this study passed through the slaughter line in a concomitant period. This is a non-conformity to regulation, which probably occurred due to lack of awareness of the serological condition for brucellosis in these animals.
According to the PNCEBT, all cows should be vaccinated against brucellosis (Brazil, 2017a)  The 2ME and CFT confirmatory tests showed maximum agreement in this study, detecting the same animals as positive. In a previous study, Meirelles-Bartoli & Mathias (2010) found a Kappa coefficient of 0,86 for these tests. The efficacy of both confirmatory tests has already been proven in vaccinated and unvaccinated animals (Paulin, 2002). Beyond that, the reliability of results is higher when serology is performed by two confirmatory tests concomitantly (Meirelles-Bartoli & Mathias, 2010). On the other hand, RBT showed 100% sensitivity and negative predictive value in this study, which are desirable characteristics for a screening test.
Regarding risk factors, 48.8% of workers claim to have consumed raw milk, 75.6% have already consumed products made from unpasteurized milk and 73.2% have already eaten undercooked meat. This behavior is worrying because it increases the risk of transmission of brucellosis and other zoonoses. In a previous study, DNA from Brucella spp. was identified by PCR in 10 samples of unpasteurized milk out of 80 tested, confirming the risk factor (Paula, 2015).
As to contact with infectious sources, 85.4% are in direct contact with bovine blood, while 43.9% have already handled birth or abortion remains of domestic animals. Fetal remains, placenta and aborted fetuses of infected animals can contain the bacteria and be an important reservoir (Antoniassi 2017). As regards to inhalation as a route of transmission (Corbel, 2006) individuals exposed to this factor are more likely to be infected (Skendros, 2013), especially in slaughterhouses which are closed locations with reduced air circulation.
In addition, it was found that 63.4% of workers act professionally only at slaughterhouses, while 19.5% also perform some livestock activity and 17.1% have already vaccinated animals against brucellosis. According to MAPA standards, vaccination against brucellosis is to be performed by veterinarians holding Official Veterinary Service authorization and assistant vaccinators are to be previously trained and registered (Brazil, 2017d). In this study, the workers acting as vaccinators did not mention any training. Performing vaccination against brucellosis without procedural knowledge increases the accident risk factor. It is a well-documented fact that these accidents can occur even with authorized professional workers and may result in infection (Brazil, 2019).
Despite the importance of risk factors, no association was found between exposure and the occurrence of human brucellosis, and the Odds ratio was also not significant, p > 0,05. It is worth mentioning that the number of volunteers in this study was not high, which may explain the absence of association. However, the biological character of the data reinforces the relevance of discussing the values found. Note that one volunteer (2.4%) submitted to at least four risk factors tested positive for brucellosis in this study. Of these risk factors, some, such as direct contact with animals and consumption of raw milk and its derivatives, are often related in infected individuals (Soares, 2015). This data stresses the importance of educational measures for the population at risk.

Conclusion
Infection from smooth strain Brucella spp. was found in cattle slaughtered in the slaughterhouse studied. The seropositivity of one individual for brucellosis, associated to the epidemiological data suggests that the disease is present and may be explained through exposure of workers to Brucella spp. in slaughterhouses, of occupational origin, and because of educational and cultural habits, due to lack of information or consumption of potentially contaminated food. Therefore, the data alert as to the occurrence of brucellosis among both animals and humans, reinforces the need for measures to be taken towards prevention, education, and control in order for the country to achieve eradication and promote public health development as proposed by the PNCBET.