Diagnosis of bovine tuberculosis: review of main techniques and applications
Diagnóstico de tuberculose bovina: revisão das principais técnicas e aplicações Daniela Fernandes Ramosa, Pedro Eduardo Almeida da Silvab, Odir Antônio
Dellagostina
ABSTRACT
Bovine tuberculosis (BTB) remains an important economic problem in Latin America with potential zoonotic consequences. Traditionally, the fight against bovine tuberculosis is initiated by the implementation of routine diagnostic tests for certification of free properties. The diagnosis of BTB can be made by direct and indirect methods, in which we can mention clinical, post mortem, histopathological, immunological, bacterial isolation and molecular diagnosis methods. Even with the need of futures studies, the renewal of scientific interest in tuberculosis in recent years, due to the need to develop more effective methods of diagnosis, prevention, control and eradication of bovine tuberculosis, has clarified significant gaps in understanding pathogenesis and epidemiology of the disease. The aim of this review is to present and discuss different diagnosis methods of M. bovis.
KEYWORDS: tuberculosis, bovine, diagnosis, Mycobacterium bovis
a
* Laboratório de Vacinologia, Núcleo de Biotecnologia, CDTec, Universidade Federal de Pelotas. CP 354, 96010-900, Pelotas, RS, Brazil, Phone: +55 53 32757350. E-mail: [email protected].
b
Laboratório de Micobactérias, Faculdade de Medicina, Universidade Federal do Rio Grande, Rio Grande, RS, Brazil.
RESUMO
A tuberculose bovina (BTB) continua sendo um importante problema econômico na América Latina, com potenciais consequências zoonóticas. Tradicionalmente, a luta contra a tuberculose bovina tem sido iniciada pela execução de testes de diagnóstico de rotina para a certificação de propriedades livres da doença. O diagnóstico de BTB pode ser feito através de métodos diretos e indiretos, nos quais podemos citar os métodos clínicos, post mortem, histopatológicos, imunológicos, isolamento bacteriano e diagnóstico molecular. Mesmo com a necessidade de futuros estudos, a renovação do interesse científico em tuberculose nos últimos anos, devido à necessidade de desenvolver métodos mais eficazes de diagnóstico, prevenção, controle e erradicação da tuberculose bovina, esclareceu lacunas significativas para compreender a patogênese e epidemiologia da doença. O objetivo deste artigo é analisar e discutir sobre os diferentes métodos de diagnóstico de M. bovis.
PALAVRAS-CHAVE: tuberculose, bovino, diagnóstico, Mycobacterium bovis
INTRODUCTION
Bovine tuberculosis (BTB) is a chronic bacterial disease caused mainly by Mycobacterium bovis, which can also infect and cause tuberculosis (TB) in badgers, deer goas, pigs, camelids (llamas and alpacas), dogs and cats, as well as man and other mammals (OIE, 2009). This disease is an important zoonosis that can spread to humans, typically by the inhalation of aerosols or the ingestion of unpasteurized milk. In developed regions, eradication programs have reduced or eliminated TB in cattle, and human disease caused by M. bovis is now rare.
This disease is still common in developing countries, and severe economic losses can occur from livestock deaths, chronic disease and trade restrictions. In some situations, this disease may also be a serious threat to endangered species (PHILLIPS et al., 2003). Consequently, about 70% of the cattle bred in Latin America are held in areas with high disease prevalence and nearly 17% in areas virtually free from tuberculosis (de KANTOR & RITACCO, 2006).
The disease is a major cause of economic losses, both in relation to individual herds, and for the economy of the countries where it still occurs (LILENBAUM et al., 1998). Brazil launched a nationwide program of BTB control in 2001, and added further regulations in 2004 (BRASIL, 2006). According to a tuberculin testing survey conducted in 1998, an average of 7.1% of herds was infected, ranging from 2.8% in the Central West to 58.3% in the Northern regions. M. bovis has been isolated from livestock pathological specimens (bovine, buffalo and swine lymph nodes), and from raw milk, confirming the risk for humans ingesting raw or un-pasteurized milk, as well as under-cooked animal products (LEITE et al., 2003).
In BTB, airborne infection is considered the most common route of transmission, and more than 19% of cattle with BTB shed the mycobacteria (de KANTOR & ROSWURM, 1978; VITALE et al, 1998), mainly early during infection (NEILL et al., 1989; NEILL et al., 1994) . The intermittent nature of bacilli shedding from infected animals, after a short initial post-infection period has been documented (NEILL et al., 1989; MENZIES & NEILL, 2000). Several studies indicate that between 8–26% of naturally infected animals shed mycobacteria, but the precise duration of the secretion period is unknown (VITALE et al., 1998; ROMERO et al., 1999; PALMER &
WATERS, 2006; ALVAREZ et al., 2009). In Brazil, the official rate is 1.3% of the national herd infected, which represent a large number in the order of 2.5 million animals. Recent research confirmed that the infection is more concentrated in dairy cattle, where infection rates can reach 15% of herds with at least one infected animal (BRASIL, 2006).
The infection leads to a decrease from 10 to 20% of milk production, loss of weight and a reduction of fertility. In addition, there is condemnation of carcasses of infected animals, and restrictions to export meat to countries where BTB is controlled (BRASIL, 2006). As the cattle industry is one of the highlights of Brazilian agribusiness, with the second largest herd in the world, with about 200 million head of cattle, BTB has been the subject of intense eradication campaigns where several programs have been established.
It is believed to occur under-reporting of reactive cases that, combined with fewer tests than necessary, contribute to the inaccuracy of official data (BRASIL, 2006). In recent years, more diagnostic methods has helped more effective programs of prevention, control and eradication of disease. The implementation of control programs and effective eradication is extremely important in order to reduce the prevalence and incidence of the disease in countries where TB is widespread, and also to reduce the risk of transmission from animals to humans.
The diagnosis of BTB can be made by direct and indirect methods. The direct detection involves the identification of the etiologic agent in biological material. The indirect method a host immune response to the etiologic agent, which can be humoral (production of circulating antibodies) or cellular (mediated by lymphocytes and
macrophages) (RUA-DOMENECH et al., 2006)[8]. So, this work describes and discusses the diagnostic methods of M. bovis infection.
CLINICAL DIAGNOSIS
Tuberculosis is usually a chronic debilitating disease in cattle, but it can occasionally be acute and rapidly progressive. In countries with eradication programs, most infected cattle are identified early and symptomatic infections are uncommon. In the late stages, common symptoms include progressive emaciation, a low–grade fluctuating fever, weakness and inappetence. Animals with pulmonary involvement usually have a moist cough that is worse in the morning, during cold weather or exercise, and may have dyspnea or tachypnea. In the terminal stages, animals may become extremely emaciated and develop acute respiratory distress (UNE & MORI, 2007).
In some animals, the retropharyngeal or other lymph nodes enlarge and may rupture and drain. Greatly enlarged lymph nodes can also obstruct blood vessels, airways, or the digestive tract. If the digestive tract is involved, intermittent diarrhea and constipation may be seen. In cervids, bovine tuberculosis may be a subacute or chronic disease, and the rate of progression is variable. In some animals, the only symptom may be abscesses of unknown origin in isolated lymph nodes, and symptoms may not develop for several years. In other cases, the disease may be disseminated, with a rapid, fulminating course (UNE & MORI, 2007).
The diagnostic is of little clinical relevance at the initial stage of the disease, presumably due to the presentation of the healthy animal that can only be infected and lesions. However, in animals with early evolution of the disease, detailed clinical
examination and careful epidemiological research are of great value to the formulation of definitive diagnosis. This diagnosis includes auscultation, percussion, palpation of thermometry and superficial lymph nodes and mammary glands. In the latter case, it is mainly evaluated the exacerbation of pain sensitivity and the presence of lymph node enlargement. They manifestation of intense fatigue, the occurrence of non-productive cough, nasal eliminating secretion and the manifestation of dyspnea are also clinical signs that should be considered (ROXO, 1997). So, even TB is difficult to diagnose by clinical signs only, in developed countries, with few infections become symptomatic; most are diagnosed by routine testing or found at the slaughterhouse.
POST MORTEM DIAGNOSIS
The pathologic diagnosis, or post mortem, the BTB, while performing autopsies or sanitary inspection of carcasses in slaughterhouses refrigerated presents considerable difficulty, since many granulomatous inflammation caused by pathogens such as Actynomices bovis, Corynobacterium pyogenes and others, have morphologic characteristics similar to TB. Despite the difficulties, anatomo-pathological is crucial for the diagnosis of BTB, based on the results achieved by the control programs, where the conventional post-mortem examination detects approximately 47% of presumptive BTB lesions in carcasses of cattle slaughtered (BRIET et al., 2005).
In developed countries, the inspection of carcasses for evidence of BTB has come increasingly to be regarded as an extension of the national animal health programme rather than solely as a control point in the prevention of human cases of tuberculosis caused by M. bovis. Now, however, it is important to focus also on the contribution which efficient meat inspection, coupled with real-time data retrieval, and
supported by advanced diagnostic bacteriology including DNA-based strain typing, can make to the eradication of this zoonotic disease (BERENDS et al., 1993).
This disease is characterized by the formation of granulomas (tubercles) where bacteria are located. These granulomas are usually yellowish and either caseous, caseo- calcareous or calcified. They are often encapsulated. In some species such as deer, the lesions tend to resemble abscesses rather than typical tubercles. Some tubercles are small enough to be missed by the naked eye, unless the tissue is sectioned. In cattle, tubercles are found in the lymph nodes, particularly those of the head and thorax. They are also common in the lung, spleen, liver and the surfaces of body cavities. In disseminated cases, multiple small granulomas may be found in numerous organs (de KANTOR & RITACCO, 1994).
Lesions are sometimes found on the female genitalia, but are rare on the male genitalia. In countries with good control programs, infected cattle typically have few lesions at necropsy. Most of these lesions are found in the lymph nodes associated with the respiratory system. However, small lesions can often be discovered in the lungs of these animals if the tissues are sectioned.
As evidenced by findings at post-mortem examination, the tuberculosis in cattle is primarily a respiratory disease. The origin of the infected aerosol exhaled by the tuberculous animal bovine or otherwise, and its role in the dissemination of M. bovis among animals and in the environment is all too often overlooked in the investigation of breakdowns. This is more likely to be the case when the data relating to the tuberculin test results, rather than the natural history of the disease, are the main focus of attention. Again, the fundamentals of the control of an infectious disease require to be kept in
mind at all stages of the investigation and management of the outbreak; otherwise, wrong conclusions and decisions may result, with untoward consequences both for the herdowner and the programme (COLLINS, 2006).
An adequate system of disease control and epidemiological surveillance relies on slaughterhouse inspection. This implies a sound infrastructure, highly trained staff and a reliable register system for tracingback to the herd of origin. Official inspection is currently in vigour in 22 countries. Nevertheless, the scarcity of qualified veterinary inspectors and trained personnel limits the efficiency of post-mortem examination in several countries (de KANTOR & RITACCO, 2006). Therefore, the abattoir surveillance has been important to lesion detection during commercial slaughter that is used as cost-efficient method for passive surveillance of BTB. The success of such investigations is highly variable. Generally speaking, lesion detection exhibits a major lack in sensitivity.
Histopathological Diagnosis
A presumptive diagnosis can also be made by histopathology and/or the microscopic demonstration of acid-fast bacilli, as a complementary form of post mortem lesions diagnostic presumptive BTB. More direct methods for tuberculosis diagnosis are based on the isolation or detection of the bacterium in sputum samples or biopsies (mostly in humans) or at post mortem, from tuberculous organ lesions (generally in animals). The presence of mycobacteria in a given sample can be assessed by Ziehl- Neelsen staining followed by light microscopy or auramine O staining and fluorescence microscopy (MARAIS et al., 2008; MICHEL et al., 2010). These techniques are based on the tinctorial properties common in mycobacteria and microorganisms of the genus
Nocardia, Rhodococcus and Corynebacterium, known as acid resistant bacilli. That is named because they can retain the fuchsin heated material after treatment for alcohol- acid. In this type of coloring, alcohol acid resistant microorganisms can be observed under the microscope (MARAIS et al., 2008; MEDEIROS et al., 2010; MICHEL et al., 2010).
The presumptive diagnosis of mycobacteriosis can be made if the tissue has characteristic histological lesions such as caseous necrosis, mineralisation, epithelioid cells, multinucleated giant cells and macrophages. As lesions are often paucibacillary, the presence of acid-fast organisms in histological sections may not be detected, although M. bovis can be isolated in culture. However, large numbers of acid-fast organisms are seen in lesions in primates, felids, mustelids (badgers) and marsupials (brush-tailed possums (SCHILLER et al., 2010; CORNER et al., 2011).
Recent work with M. tuberculosis suggests that the auramine O staining technique may be more sensitive and specific than Ziehl-Neelsen staining (MARAIS et al., 2008). However, microscopic detection of mycobacteria shows a generally low sensitivity (from 50 to 70%) for human sputum samples. In addition many features, including the dyeing property, overlap in the genus Mycobacterium and Nocardia, making it difficult in some cases, the differentiation between both. This is mainly due to the requirement of a high bacterial load for microscopy. A much higher sensitivity can be achieved by prior culture of the bacteria. Culture is still regarded as the gold standard for tuberculosis diagnosis despite certain deficiencies (MICHEL et al., 2010).
Immunological Diagnosis
The immunological diagnosis of BTB is based on delayed-type hypersensitivity (DTH) reaction in vivo, represented by the tuberculin test (SCHILLER et al., 2010). This evidence is in an indirect method of diagnosis of tuberculosis and can reveal incipient infections, with three to eight weeks after contact with the M. bovis, since techniques are employed using standard reagents and equipment. It is a test widely used since it was recommended by Robert Koch in 1890 (MONAGHAN et al., 1994).
The tuberculin by Koch's discovery, after some modifications, is currently called purified protein derivative (PPD), widely used for the indirect diagnosis of bovine tuberculosis in vivo. Currently is available in most of the world, including Brazil, presentation corresponding to bovine PPD (antigen extract purified protein) derived from the sample AN5 M. bovis and avian PPD which is synthesized from sample D4 M. avium (MONAGHAN et al., 1994). Advantages the use of PPD and reasons for its wide use are low costs, high availability, long history of use and, for a long time, the lack of alternative methods to detect BTB. Still, this test has many known limitations including difficulties in administration and interpretation of results, need for a second-step visit, low degree of standardization, and imperfect test accuracy (RUA-DOMENECH et al., 2006; SCHILLER et al., 2010).
However, test specificity is not only influenced by the purity, potency, and dosage of the tuberculin and strictness of interpretation of the response in the animal, but it is also influenced by sensitization of the animal. While the single most important cause of sensitization is that the tuberculin skin test using PPD does not distinguish between infection with M. tuberculosis/ bovis and BCG vaccination, or exposure to
environmental mycobacteria. These cross-reactions are assigned, usually in the presence of PPD antigens common to different species of mycobacteria (YOUNG, 1992). However, this test is the only validated and used routinely and widely for more than 85 years (HUEBNER et al., 1993).
The predominant immunological response in M. bovis-infected cattle is affected by T lymphocytes [8]. Ante mortem tests of cellular immunity are very important for the control of bovine tuberculosis since they can identify M. bovis-infected animals very early. The tuberculin skin test and the interferon-gamma test (MENON et al., 2002) test are both based on the detection of the early cell-mediated immune response in tuberculosis infection. However, at late disease stages, the cell-mediated immune response can wane as opposed to a generally increasing humoral immune response and these tests can therefore give false negative results (RUA-DOMENECH et al., 2006). This is of importance for the diagnosis of bovine tuberculosis in settings where no or little disease control measures are applied and where the percentage of late stage diseased animals is believed to be high. Therefore, in developing countries, serological tests, which are based on the detection of the humoral immune response, may be of particular use. Unfortunately, to date, no satisfactory serological test is available. Some of the problems related to the development of serological tests for tuberculosis diagnosis include the observed highly variable antibody responses between individuals to mycobacterial antigens and antigenic variation between mycobacterial strains (PHEIFFER et al., 2005).
This is a enzyme-linked immunosorbent assay (ELISA) through which it is possible to verify the existence of cell-mediated immune response developed by the
body of the animal in response to mycobacterial infection. IFNγ produced by T lymphocytes of the infected animal is detected by capture ELISA, using monoclonal anti-IFNγ (MEDEIROS et al., 2010). The lack of detection of IFNγ characterizes the negativity of the animal to infection M. bovis since lymphocytes from uninfected cattle do not produce this chemokine in specific ways. As this is an in vitro test that has the advantage of not interfering with the immune status of the animal and may be repeated in the same animal is the need to respect the period of desensitization (WOOD & JONES, 2001; ).
Although serological assays cannot be considered first choice diagnostic methods, many researchers describe strategies for their use. The indirect ELISA technique measures the binding of specific antibodies to an antigen (RUA- DOMENECH et al., 2006). In order to diagnose cattle infected by M. bovis, antigens usually employed are the PPD and single or associated purified antigens from M. bovis such as antigens of the Ag85 that complex represents a major part of the secreted proteins, and mpb70 and its highly homologous protein MPB83, secreted mycobacterial proteins with limited species distribution. This antigens has achieved a sensitivity and especificity of around 90%. and their recommendations are based on the existence of anergic animals, as well as increased antibody titres in more advanced stages of the disease (McNAIR et al., 2001; POLLOCK & NEILL, 2002; WELSH et al., 2005; MEDEIROS et al., 2010).
Recently, the development of a lateral flow test that is based on the detection of more than one antigen has shown promising results for tuberculosis diagnosis in certain animal species (e.g. in elephant), although it may not be suitable for others, such as
buffaloes (LYASHCHENKO et al., 2008; GREENWALD et al., 2009; MICHEL & SIMOES, 2009). Another recently developed serological test for animals is based on antibody detection using fluorescence polarization but has shown variable effectiveness in different settings (JOLLEY et al., 2007; NGANDOLO et al., MEDEIROS et al., 2010).
Bacterial Isolation
The isolation of M. bovis is a direct method of diagnosis considered "gold standard". However, the long period required for the isolation and biochemical identification, is one of its critical points, and may require more than twelve weeks to complete the final diagnosis, and also low sensitivity (COLLINS et al., 1994). The major limitation for systematic cultivation of M. bovis of animals is to obtain samples,