L.E. Sammartino^[6], A. Gil^[7] and P. Elzer^[8]

INTRODUCTION

Brucellosis is a worldwide zoonotic infectious disease caused by Gram-negative bacteria from the genus Brucella. Brucellosis, called undulant fever in man, remains an important disease that persists where the infection in animals has not been controlled and where the transmission of the disease to humans is still common. Ruminants, swine and wildlife contract this zoonotic agent. Ruminant brucellosis has been eliminated or greatly reduced in many developed countries; however, this is far from being a reality in non-developed countries.

1. THE AETIOLOGY

The genus Brucella is classified as an µProtobacteria and is composed of facultative intracellular bacteria that have a predilection for the reticuloendothelial system and the reproductive tract. The genus Brucella contains six well-defined species differing in their host preferences:

a) B. abortus, affecting cattle;
b) B. melitensis, causes disease in goats and sheep;
c) B. suis, affecting pigs;
d) B. ovis, causing epididymitis in rams;
e) B. canis, affecting dogs; and
f) B. neotomae affecting a desert wood rat found only in USA.

Recently, a new species was proposed called B. maris because it was isolated from marine mammals. Only B. ovis and B. neotomae are not known to cause infection in humans.

Briefly, Brucella has a similar structure to other Gram-negative bacteria. Brucella abortus, B. melitensis and B. suis exhibit a smooth (S) polysaccharide (LPS) that contains the O side chain, the immunodominant portion responsible for detecting the majority of antibodies resulting from a natural infection or for a response to vaccination. Strain 19 vaccine is used for preventing brucellosis in cattle and Rev 1 vaccine for goats and sheep. Both possess the O side chain. Brucella canis and B. ovis do not have the O side chain and are considered rough or "R" bacteria.

2. THE DISEASE IN MAN

2.1 Overview

Briefly, humans, in whom infection may cause a severe and a chronic form of the disease, are susceptible to B. abortus, B. suis, B. melitensis and B. canis. Human brucellosis is distributed worldwide, with regions of high prevalence such as the Mediterranean region, Middle East, Latin America and Asia. The true incidence of human brucellosis is unknown. This disease is well described by its original name "Undulant Fever", in that fever waxes and wanes like a wave. The disease does not have precise symptoms besides general malaise, making it difficult to diagnose clinically. Brucellosis is characterized by an intense fever, strong sweats, headaches, and symptoms that may be confused with the flu. The disease can have severe complications if not treated. The bacteria reach the lymph nodes, liver and spleen and may cause endocarditis, encephalitis and orchitis in man. However, there is no evidence of abortion in infected women.

Since the disease presents with a great variety of clinical manifestations the diagnosis must be confirmed by laboratory analyses. The diagnosis can be done directly by isolation of Brucellae from blood cultures, or indirectly by the detection of the immune response against the microbe's antigens. Asymptomatic infection is frequently characterized by antibody formation in people with no history of symptoms consistent with brucellosis. Hence, serological tests play a major role in diagnosis when the bacteria cannot be detected by culture. The treatment of brucellosis is based on antibiotic therapy with tetracyclines or doxycycline in combination with other drugs, such as rifampin or streptomycin, to aid in curing the disease.

2.2 Epidemiology

The main routes of Brucella transmission to man are ingestion, inhalation, or direct contact with infected animals or materials. Infection is also frequently a result of accidental inoculation. However, transmission between human beings does not usually occur; although a few instances have been described indicating sexual transmission, these all appear to be anecdotal. However, it should be considered a risk to transfuse potentially Brucella-infected blood.

Infection by ingestion is usually due to contaminated food. The consumption of untreated milk or cheeses in many places around the world is the cause of brucellosis outbreaks in man. Human infections may develop in people who are frequently in contact with infected herds of goats, goat manure, or who consume infected goat milk or its products. Therefore, the number of human brucellosis cases is expected to increase in the world as long as the disease persists among domestic animals. The habit of consuming soft cheese prepared from contaminated sheep and/or goat milk is a common reason for infection. This happens not only where the animals are breeding but also in towns and other places where those products are transported to be sold commercially. The importation of fresh cheese to brucellosis-free countries where migrant workers consume those products as they did in their original regions is also a source of infection. Beside, there are new trends in that local people are also developing a taste for this kind of food. Brucella-contaminated sheep milk and fresh cheeses are still a problem in Spain, Italy, Greece, the eastern Mediterranean countries and Asia.

Since Brucella-contaminated goat milk and cheese are very important in Latin America, Asia and Africa, unpasteurized cheeses from these areas may represent a particular risk for tourists. There are still regions where goat milk is given directly to the children because there is a local belief that this will be good for their health.

Infection by close contact may occur when humans assist animals during parturition or abortions, or handle stillbirths. It is also common for farmers to try to separate the placenta manually; in so doing they are being exposed to a probable source of brucellosis. Infection through the skin is not certain; however, rural workers or those persons working in abattoirs frequently have small lesions in their hands, which could facilitate the penetration of Brucella from contaminated tissues. At the slaughterhouse, Brucella contamination may take place in the processing of the carcasses, mainly through cutting infected lymph nodes and mammary glands. Brucella remains viable for a long time in refrigerated meat. Contamination of hands may occur from carrying objects such as cigarettes or dishes or glasses, which play a mechanical role in the transmission of Brucella to the mouth or conjunctiva of people. In some regions of the world there is a high risk of infections due to animal handling to aid in fostering. When the placenta from an aborted foetus is rubbed on to three- to five-day-old lambs, the fleece may still be severely contaminated with this bacterium.

Inhalation of Brucella has been described from observations done in slaughterhouses, where the concentration of Brucella is very high, and also in laboratories (mainly producers of antigens or vaccines) where different kinds of accidents were described. These incidents, occurring in controlled environments are, in a way, experimental studies verifying this means of Brucella infection. Transmission of brucellosis by accidental inoculation due to mishandling of syringes and needles occurs in laboratories and, more often, in the field.

Special care should be taken by nomadic populations in some of the most Brucella-infected regions in the world where animals are moved from a place-to-place looking for better pastures. The risk of brucellosis increases during the parturition seasons. Typically, newborn animals are brought into the tents in order to avoid low temperatures, fed artificially with goat milk and frequently play with children. The temperatures in those regions are extreme and parturition seasons coinciding with cold weather increase still more the possibility of spread of the disease. It is impossible to clean the place where the animals are located owing to lack of water. Most of the time nomad populations save water for drinking and cooking and barely enough is reserved for cleaning. These are general considerations for nomadic populations, thus it should be considered that the specific cultural habits of raising animals and/or preparation of food (cooking, boiling or eating raw meat) are important factors related to the epidemiology of the disease in this kind of population.

Estimation of the economic impact of human brucellosis is calculated using as indicators the cost for each new patient based on days or months of leave, medical and laboratory examinations and treatments. The duration of the human illness and convalescence indicate that brucellosis is not only a medical problem but also an economic problem because of time lost from normal activities. Although antibiotics reduce the time that a patient could be incapacitated, still exists many regions where the medicine are not available and the programmes for the detection and prevention of the infection in man and animals are not adequately carried out. In these areas, the animal disease remains an important threat to human welfare.

2.3. Level of Risk in the human population

The level of risk is directly dependent on the amount of contact that a person has with the potential source of the infection. Dairy farmers who still milk animals with their hands have a greater chance of being infected than those farmers who use mechanical machines, although both are in contact with animals shedding Brucella. Slaughterhouse personnel who are in charge of opening the carcass, cutting nodes or udders, or opening the uterus are at higher risk of infection than those who work just processing the meat. There is also a risk from inhalation of aerosols when tables and floors are cleaned with hoses. Lately, there is a major commercial interest in collecting foetal blood to be used for tissue culture. Collecting this material from an infected uterus may be a major factor in brucellosis contracted inside the abattoir. Meat inspectors are not directly exposed to the bacteria but are at risk if they do not take care performing their duties. Other workers to be considered are those who perform artificial insemination. Although generally the semen used in this cases is brucellosis free, if inseminations of brucellosis-infected heifers or goats takes place then the worker could contract the disease if they do not take the necessary precautions. Laboratory workers making vaccines or reactive diagnostics (antigens) for brucellosis are directly exposed to the organism. Those who are seeding or harvesting the bacteria and those using centrifuges are at greater risk of becoming infected. In order to produce antigens or vaccines, live bacteria are being handled most of the time. Every single worker in a laboratory, whether for industrial, commercial or research purposes, is being constantly exposed to the organism if adequate safety measures are not taken.

3. THE DISEASE IN ANIMALS

3.1 Overview

The virulence of these organisms varies considerably according to the species, biovars and the numbers of the infecting inoculums. Host susceptibility is also variable and is directly associated with the reproductive status. Thus, in the field, all intermediate stages between typical acute infection and complete resistance may be observed. In addition, vaccinal immunity may modify the parasite-host relationship. The major routes of infection are the mucous membranes, mouth and upper respiratory tract or the conjunctiva. Goat and sheep can get the infection through the mucous membranes of the male or female genital tract. This route is not typically seen in cattle. After gaining entrance to the body, the organisms encounter the cellular defences of the host but generally succeed in arriving via the lymph channels to the nearest lymph node. The fate of invading bacteria is mainly determined by the cellular defences of the host, chiefly macrophages and T lymphocytes, although specific antibody undoubtedly plays a part. The outcome depends on the host species infected, age, immune and pregnancy status, and the virulence and number of the invading Brucellae. When the bacteria prevail over the immune system, a bacteraemia is generally established.

If the animal is pregnant, bacteraemia often leads to the invasion of the uterus. At the same time infection becomes established in various lymph nodes and organs, often in the udder and sometimes in the spleen. During this first stage of infection the main clinical manifestation of brucellosis in all female ruminants is reproductive failure, abortion or birth of weak offspring. Brucellae may localize in other tissues causing signs such as orchitis, epididymitis, hygroma, arthritis, metritis or subclinical mastitis.

There are some important facts of pathogenesis that should be known for the correct management of infected herds or flocks. For example, numerous animals develop self-limiting infections or they become asymptomatic latent carriers and potential shedders of the bacteria. Abortion generally does not occur if the female becomes infected at the later phases of pregnancy. The disease is characterized by either elimination of the organism or, more frequently, by a persistent infection of mammary glands and supramammary and genital lymph nodes with constant or intermittent shedding of the organisms in the milk and genital secretions. Animals generally abort once during mid-gestation, but reinvasion of the uterus occurs in subsequent pregnancies with shedding in fluids and membranes. The pregnancy can also continue to full term. The proportion of newly infected females that abort varies with the circumstances. The percentage of infected females lambing/kidding in a flock may reach 40 percent. Females that are born into an infected environment and subsequently infected, generally abort less than others. This explains the high level of abortions in newly infected flocks and their relatively low frequency in flocks where infection is enzootic. The mammary gland is a very important site for Brucella because of its predilection for supramammary lymph nodes.

Infection in lactating non-pregnant goats is likely to lead to colonization of the udder with excretion of Brucella in the milk. In goats, about two thirds of acute infections acquired naturally during pregnancy lead to infection of the udder and excretion of the bacteria in the milk during the subsequent lactation. In some goats excretion may cease during this lactation, but in many it persists and often continues during the next pregnancy and lactation. Greatly reduced milk yield follows abortion, and infection of the udder following a normal birth also leads to a considerable reduction in yield. In spite of this, clinical signs of mastitis are seldom detectable in naturally infected cattle and goats. Sheep that abort often excrete the bacteria in the milk, but generally for not more than two months. However, in cattle, excretion may continue for 60 days by vaginal discharges, and organisms may be shed intermittently in the milk in succeeding lactations. Vertical transmission is also described and Brucella can be transmitted from infected cattle or dams to calf, lambs or kids. A small proportion of lambs or kids may be infected with Brucella but the majority of infections are probably acquired by consumption of contaminated colostrums or milk.

The joint FAO/WHO Expert Committee on Brucellosis summarized the pathogenesis of animal brucellosis indicating that "Infection with Brucella usually results in the induction of both humoral and cell-mediated immune responses, but the magnitude and duration of these responses is affected by various factors including the virulence of the infecting strain, the size of infecting inoculums, pregnancy, sexual and immune status of the host".

3.2 Epidemiology

Brucellosis can be found in both domestic and wild animals. In cattle, the disease is caused by B. abortus, and in goats and sheep by B. melitensis, although in special circumstances B. melitensis can infect cattle. Sheep also can get B. ovis, causing epididymitis of the ram, but this species does not affect man. Both B. abortus and melitensis can been found in other species, including water buffalo, bison, deer, elk, camels, horses, pigs, dogs and in several wild animals. Dogs have been shown to be mechanical and biological vectors of brucellosis. The spread of the disease via waterways is rare and can only be effective over short distances. Exposure of animals to Brucella depends on the region where they live. For instance, camels and dromedaries are exposed to B. abortus and/or B. melitensis in the Middle East while bison or elks are exposed to B. abortus in North America.

Of the three different biovars of B. melitensis, biovar 3 predominates almost exclusively in Mediterranean countries and the Middle East, while biovar 1 predominates in Latin America. Biovars 1 and 2 have also been reported in some southern European countries. In bovines, B. abortus biovar 1 predominates in Latin America, however biovar 2 has been described in the Argentine Republic. Brucella abortus biovar 3 has been reported frequently in Africa.

Susceptibility to brucellosis is associated with two main factors. First, brucellosis primarily affects sexually mature animals. Second, susceptibility increases dramatically with pregnancy. The incubation period is shorter in pregnant animals and abortions take place frequently. Understanding the transmission of brucellosis is necessary because it plays a key role in the epidemiology of the disease. In most cases, transmission of Brucella through uterine fluids and the placenta expelled by infected animals, either when they abort or have full term parturition, is the main cause of disease transmission. The probability of the surrounding animals being infected will depend on their own susceptibility, the numbers of bacteria to which they are exposed, and also infected animals shedding Brucella.

Major risk factors for animal infection will directly depend on the husbandry practices, local habits, and management of the herd/flock. First, the factors contributing to intraherd transmission should be addressed. In a herd/flock infected with brucellosis all animals in the farm must be tested as a starting point. A plan must be formulated to control the disease, but it should be complemented with other very important measures. For example, the sources of purchase must be chosen very carefully. Worldwide, most infections or reinfections in disease-free herds originate from buying infected animals. New animals should be introduced into the herd/flock after being tested negative for brucellosis; vaccination reports for these animals must also be required. Another major risk factor is the proximity of infected herds/flocks. The disease may be eliminated from a farm but if the neighbours have infected animals, despite all efforts made, sooner or later the disease will come back. Community pastures should be treated as one herd/flock and control measures must be applied to all animals. Other factors to be considered include the ability of Brucella to persist outside the mammalian hosts under suitable conditions. For example, when environmental conditions are favourable, such as high humidity, low temperature and absence of direct sunlight, Brucella may retain infectivity for several months in water, aborted foetuses, placental membranes, liquid manure, hay, buildings, equipment and clothes.

Another factor to be considered is that dogs are present with herds or flocks worldwide. Both B. abortus and B. melitensis infections in farm dogs have been reported. Although clinical signs are uncommon, abortion, epididymitis and arthritis may occur. Dogs may be infected through ingestion of infected bovine placental tissue. If a pregnant dog is infected with B. abortus it may abort and the tissue and vaginal discharges have a great potential for transmitting Brucella to susceptible cattle.

Vaccinated animals will have a better chance of avoiding the disease. The size of the herd, the housing methods and the population density are factors that may be considered in the progress of the disease. For instance, dairy animals have a much greater chance not only of contracting brucellosis but also of spreading it faster than beef animals. The reason is far from being a genetic or physiological factor, but instead is due to husbandry. Those animals that live concentrated in smaller areas come into close contact when they are grazing and when they are milked.

4. PREVENTION OF BRUCELLOSIS

4.1 Prevention of human brucellosis

Similar to other major zoonosis, prevention of brucellosis mainly involves education and control of food and personal hygiene. Although, according to the literature, vaccination for humans has been employed in some countries, such methodology is not routinely available. The risk of getting this disease is recognized in industrial countries more than in the developing countries. Cultural ways of raising domestic animals tends to involve closer contact in developing countries, with either nomadic populations or shepherds looking for better pastures and moving animals from one place to another.

As described previously, a major risk of getting brucellosis for man is directly related with occupational labour. In some parts of the world, entire villages are dedicated to raising animals, with families taking on tasks according to capabilities. Those populations whose cultural habits include consumption of milk and the use of its products raw or poorly cooked will be at higher risk than those populations where it is the habit to heat the milk before consumption. Pasteurization can be conducted by heating products to 63 °C for 30 minutes or 72 °C for 15 seconds. Pasteurization has been shown to effectively reduce or eliminate a number of pathogenic organisms, including Brucella.

We should also consider husbandry practices. In most industrialised countries, brucellosis is not a disease with an important impact in public health. However, in developing countries where dairy production has been moved to suburban or completely urban places, brucellosis in man is not only still present but has increased. The possibility of maintaining the disease increases if abortions take place and they are not removed, contributing to the transmission of the disease on the farm due to lack of hygienic conditions.

4.2 Prevention of Animal Brucellosis

Although the word "prevention" is strongly associated with vaccination, in animal brucellosis, other aspects must be taken into consideration to avoid the disease. Basic practical management has the same importance as vaccination. For example:

• All contact between infected animals and those that are Brucella free should be avoided.

• Abortions should be removed as soon as possible and aborting females isolated until a serological diagnosis is confirmed.

• Replacement animals must be brucellosis free and should be kept in quarantine no less the one month during which a serological test should be carried out.

In other words, both, vaccination and herd management should be considered to have the same level of importance in the control of the disease.

Vaccination has been practiced for many years with different kinds of vaccines being developed. However, nowadays live vaccines are the only ones that give adequate levels of protective immunity. The most commonly used vaccine worldwide is B. abortus strain 19 (S19), a smooth live vaccine developed in the 1930s to protect cattle against brucellosis. The characteristics of S19 are stability, low pathogenicity, and it is naturally attenuated. Protection from the infection and abortion is around 60 to 70 percent depending on the status of the infection. The major inconvenience of this vaccine is the induction of antibodies indistinguishable from antibodies originating from field strains. Recently, a new vaccine has been developed for cattle called B. abortus RB51, which is a rough vaccine, rifampin resistant, and has similar attributes to S19. However, RB51 does not induce antibody formation that can be detected by the conventional diagnostic tests used in brucellosis; this is a major advantage for the control programmes.

Vaccination against brucellosis has usually been recommended in early calfhood, so, serological test are negative by breeding age. The age when most vaccines are applied is between four and eight months, therefore, by 18 months of age the animal should be negative on conventional serological tests. However, in practice some S19 vaccinated animals, still have serological titres present at a low percentage at breeding age and also during pregnancy. Some countries that are not able to control the disease implement S19 adult vaccination using a reduced dose of the vaccine. This practice has the major benefit of improved immunity of the herd; however, permanent serological titres are increased and abortion may occur if the vaccine is given during pregnancy. The results of different experiments either under controlled conditions or in "real life" situations are influenced by many factors including the virulence of the field strain.

Brucella melitensis Rev 1 live vaccine is the most widely used vaccine in control programmes against brucellosis in small ruminants. When properly used, Rev 1 vaccine confers a long-lasting protection against field infections in a high proportion of animals. This vaccine, however, shows a considerable degree of virulence and induces abortions if the first vaccine dose is administered during pregnancy. The antibody response to vaccination cannot be differentiated from the one observed after field infection, and this therefore impedes control programmes. Attempts have been made to develop new vaccines based on genetically modified strains of the Brucella species; those vaccines await further evaluation in field experiments.

5. SURVEILLANCE OF BRUCELLOSIS

5.1 Rationale for surveillance

As described above, brucellosis is one of the most widespread zoonotic diseases with an important global impact on human health and the animal industry. The main measures to control the disease are based on prevention. Surveillance is a key factor for management of prevention, control and eradication programmes. In order to be useful a brucellosis control programme should have a very well implemented surveillance system, where a precise data collection methodology from the field, must be established. The main purpose of the surveillance system would be to detect infected animals that are the cause of the transmission and to determine the prevalence of the disease, so that appropriate measures for its control can be taken.

5.2 Recommended types of surveillance

There is not a single surveillance system for all situations. As brucellosis typically cannot be diagnosed clinically, laboratory diagnostic technology is required. The isolation of Brucella is the "gold standard" for diagnosis. It is an excellent tool but technically very difficult to perform owing to many factors such as timing, high costs and low chance of isolation of the organism. Thus, most diagnosis occurs only through epidemiological investigations. Most methods rely on the correct application of serological tests to indicate brucellosis infection. Different factors could affect such methodology and should be analysed. The main characteristic of the serological tests used for control and eradication must be considered carefully. Sensitivity and specificity of the tests must be considered and should be used according to the objectives of the programme. If a test with high sensitivity but low specificity is used, the number of false positives will be increased and the positive predictive value will be low. For example, the very well known agglutination tests, the Rose Bengal/card test and the buffer plate antigen agglutination test, are very sensitive, inexpensive, easy to use and can be done everywhere; however, these tests lack specificity, mainly for those regions using S19 or Rev 1 vaccination in cattle or goats. These vaccines induce antibodies that are indistinguishable from those antibodies originating from field strains of Brucella. Nevertheless, plate agglutination tests are very often the only means to do diagnostic testing in rural areas. Other conventional tests, such as rivanol or 2-mercaptoethanol, have greater specificity, giving more precise results and a better panorama of the brucellosis situation. Although these tests do not take into account vaccinal antibody levels, they are extensively used in several countries. The complement fixation test (CFT) is highly specific but it is laborious and requires highly trained personnel as well as suitable laboratory facilities. In spite of this inconvenience, CFT has been used as the confirmatory test in many control and eradication programmes. It does, however, make this test less suitable for use in developing countries. In dairy cattle herds, the milk ring test is used worldwide for surveillance.

Actually, more accurate tests are available, like the enzyme immunoassay (ELISA) and the fluorescence polarization assay (FPA), which are fast, have excellent sensitivity and specificity values, and are not subjective. Indirect ELISA is a precise and very sensitive test that can be used to detect bovine and caprine brucellosis and it is also indicated for testing milk in bovines too. The disadvantage of this test is its inability to differentiate vaccinal antibody. Competitive ELISA however, beside its high sensitivity and specificity, overcomes this problem. The FPA is a new test that can be performed either in the laboratory, using a more sophisticated fluorescent polarization analyser, or in the field by using a portable analyser. This test is simple and very fast to do, and results can be obtained rapidly. The FPA is able to differentiate S19 vaccinal antibodies and can be used with whole blood in cattle. The diagnostic performances of both tests are comparable to, or are better than, CFT. Brucellosis is primarily diagnosed by serological methods. As new technology has developed, rapid and more accurate tests have been created. The polymerase chain reaction (PCR) has been developed and evaluated during recent years. This test can be used for identification of Brucella species or biovars. The test should be very useful for epidemiological trace-back or species identification in any brucellosis programme. PCR technology should be incorporated into those programmes that have the laboratory capability.

Independent of the tests applied, critical aspects of the success of a programme are the cutoffs established, which play a key factor in the control and eradication programme. When the levels of titres for each test are established they will determine positive and negative animals. Thus, this stage of the programme should be considered before implementation of the techniques to be applied. For instance, if the control programme is in an early stage tests should be more sensitive; however, if the programme is nearing completion, highly specific and sensitive tests are recommended to decrease the number of false positives identified while maintaining a good detection level. Another major issue to be considered when a diagnostic method is selected is the predictive value of the test. For a positive test the predictive value can be defined as the proportion of positive animals that are really infected, whereas, the predictive value of a negative test is the proportion of negative animals that are truly free of the infection. The predictive values are also influenced by the prevalence of the disease in the population; for example, in a low prevalence situation, most of the cattle tested are negatives, and the number of false positives may be higher than the number of true positives (infected animals) decreasing the positive predictive values.

If the prevalence in a zone is unknown, randomly selected herd testing can be used as well other methodology for monitoring the brucellosis situation at the early stage of the programmes. This method would supply important information to determine the basic logistics for the programme. The data collected could be useful for the selection of the sampling method and the sample herds to be tested. When brucellosis infection is detected in a farm, the plan should include testing the adjacent herds. This is a basic and very important epidemiological method for controlling the disease. Some programmes select a determined geographical area for testing in which every single herd in the area should be analysed. Monitoring randomly will not indicate all infected herds. Testing in specific areas is applied by departments, counties or sectors where brucellosis in several herds has been detected and should be complemented with other strict measures (like movement control with prior testing).

6. STRATEGIES FOR CONTROL AND ERADICATION OF BRUCELLOSIS IN MAN AND ANIMALS

6.1 Good practices to follow when handling infected animals or contaminated materials

People working on a farm that is infected with brucellosis must follow basic rules to avoid getting the disease regardless whether the farm is for raising cattle, sheep or goats. Protective clothes must be used (overalls, rubber gloves, rubber boots, glasses) which should be disinfected after use. Disinfection may be done by soaking the clothes in a solution of 2 percent chloramines for 30 to 40 minutes. Where chemical products are unavailable clothes can be boiled and scalded. Realistically, many farmers will not use gloves when handling animals or cleaning installations, etc. They should wash their hands with a solution of quaternary ammonium 1 percent which is very effective for killing Brucella and then use alcohol and wash their hands and arms with soap. Any entrance where the animals are located must have a container laid in the floor filled with disinfectant. Treatment with solutions such as 2.5 percent sodium hypochlorite, 2-3 percent caustic soda, 20 percent freshly slaked lime suspension, or 2 percent formaldehyde solution for approximately one hour is enough to destroy Brucella on contaminated surfaces.

If an abortion is found, ideally it must be burned and covered with cal, but if that is not possible, it should be covered with slaked lime and absolution of chlorine should be poured over the area. Leaving the foetus without treatment is the best way to disseminate the disease. Another practice to be avoided is that of burying the foetus because wild animals may dig, take the foetus out and disseminate the disease.

Laboratory personnel working with Brucella could be constantly exposed to the organism. Basic safety measures must be taken in such laboratories. The World Health Organization (WHO) laboratory biosafety manual classifies Brucella in risk group III. All workers involved with Brucella must be completely protected, and manipulation and handling of these bacteria must be done under biohazard safety cabinets level II or above. Biohazard hoods should be located in a separate room, away from desks, office, or other non-laboratory accommodation. The requirements of biosafety must be very strict in those places where antigen or vaccine production takes place. The centrifugation of Brucella-containing media must be done under stringent conditions. Aerosols produced during centrifugation can easily infect operators. Management of cultures for bacteriology diagnosis or research can be done on open benches if workers wear proper protective equipment (glasses, gloves etc.). All samples (tissues, milk etc.) or specimens studied must be placed in metal trays, covered and put in an autoclave as soon it is possible. The autoclave must be programmed to be ready for using after work with Brucella-suspected tissues or research plates.

Personnel working in slaughterhouses have the potential of being exposed to animals infected with brucellosis. Most regulations in different countries indicate that positive animals must be sent to slaughter, including in industrialized countries. Meat processing facilities may differ widely throughout the world. They can go from fully mechanized buildings to rudimentary places where animals are killed in open facilities. The latter can be frequently observed in developing countries. The risk of handling infected animals in abattoirs is always high, but the level of risk varies according to the facilities of the building. If animals are processed right after delivery or abortion caused by brucellosis, then the bacteria are present in the lymph nodes and udder. If an infected animal is killed when pregnant, the bacteria are in nodes, udder and mainly in the placenta and foetuses. The concentration of Brucella in infected placentas or foetuses is very high with counts of up to 10 to 14 million/gram. In order to prevent infection several measures must be taken in these kinds of facilities. First, all reactor animals should be slaughtered in a special sanitary room or after brucellosis-free animals have been slaughtered. The place must be fully equipped with disinfectants for the floor and building (chloramines), and for personal use (quaternary ammonium), and with rooms for storing sanitary clothing and disposable uniforms. Food and smoking must be forbidden in the whole facilities.

Special precautions should be taken by those people involved in the collection of foetal serum. These people should be trained to avoid contamination with Brucella or other pathogens. Protection provided should include disposable clothes if available, or sanitary clothing, gloves and rubber boots that are easy to disinfect or that can be autoclaved. All clothes can be disinfected in a 2 percent solution of chloramines and hands must be disinfected wit 1 percent solution of chloramines or quaternary ammonium. Finally, hands should be washed with soap and water.

Processing of foodstuffs and raw materials originating from Brucella-infected animals requires very special handling. Although in a perfect world such material should be discarded, in the real world this does not happen. Different levels of risk can be observed according to the kind of food processed. Generally speaking, meat does not have a high risk of contagion for brucellosis, but lymph nodes, udder and uteri do. Occasionally small numbers of bacteria may be present and humans may contract brucellosis if consuming infected raw meat. Published literature does not record cases of brucellosis from consuming cooked meat but it should be remembered that the conservation of meat in some parts of the world by either salting or freezing is not enough to kill Brucella. Working with milk and its products required special considerations. It is clear that people milking infected domestic animals by hand are directly exposed to the disease. There are also places where there is are insufficient water supplies or the facilities to keep such supplies free of contaminants; thus maintaining correct hygiene for the milking process is very difficult. It is well known that boiling or pasteurizing milk will prevent the risk of Brucella infection. However, there are many places where, from ignorance, negligence, or cultural reasons, these rules are not followed.

Cheese production, either from cattle, sheep or goat milk, is an important vehicle of transmission. The ripening of cheese is crucial in order to have safe food. Brucella does not persist for a long time in ripened fermented cheese. The optimal fermentation time to ensure safety is not known, but is estimated at three months. In normally acidified soft cheese, the strictly lactic and short-time fermentation and drying increase the survival time of Brucella. Previous pasteurization of milk or cream is the only means to ensure safety for those manufacturing such products. However, production and handling of fresh cheeses are important risks for the employer manufacturing such products. In contrast to dairy products, the survival time of Brucella in meat seems extremely short, except in frozen carcasses where the organism can survive for years. The number of organisms per gram of muscle is small and rapidly decreases with the fall in pH of the meat in the maturation process.

6.2 Brucellosis control methods

Control of brucellosis should have different steps. First, it must be attractive for farmers and other people involved in animal production. Farmers are working with a disease without treatment and infected animals must be eliminated. Frequent blood sampling and vaccination make it very difficult for the farmer to perceive immediate benefits. Explanations that in the long term great advantages, including economic profits, will be achieved are not easily accepted. Thus, it is very important to explain all the rationale and advantages of the control programmes. The economic impact of the disease must be explained and it should be indicated that controlling the disease will eliminate a major risk of contagion for all personnel working in the farm.

Education is essential for the success of a brucellosis programme. Education has several levels according to who will receive the information. Here, there are some basic points to be considered:

• The programme, including all the phases, must be discussed and planned first by the brucellosis task force.

• Governmental authorities and politicians involved in agriculture and public health sectors must be informed and their support in running the programme is essential.

• The leaders of the animal production and food industries must be motivated to participate in the programme.

• During all stages of the programme all participants must be involved regardless of their role. Thus if the disease is controlled, they must remain alert and follow all planned measures to avoid reinfection.

7. ELABORATION OF THE PROGRAMME

7.1 Education and extension programme

Brucellosis is a disease present in non-developed countries and in areas where ancient ways of raising animals (mainly for milk and its derivatives) and the use of their products for consumption are still practiced. Thus, in order to be successful a programme must be realistic. There are programmes established in industrialized countries where the measures implemented can be followed step by step, however, these programmes will not work for most of the regions of the world that have brucellosis problems. The first bottleneck for any brucellosis programme is the elimination of infected animals, which requires more effort than education. Monetary compensation is necessary to replace infected animals, but this is not available everywhere. The government involved in the programme must help those producers who have brucellosis in their animals. "Indemnity" is a magic word for the successful programme. The authorities may not be able to subsidize replacement of infected animals but perhaps could reduce taxes to compensate for such important losses.

Of all participants in the brucellosis control programme, the animal caretakers, milk handlers, shepherds and farmers are most exposed to brucellosis. Educational materials that are easy to read and interpret should be provided, as necessary. This material must be adapted to the local or regional conditions, including geography and cultural habits. Education must be continued after brucellosis is controlled, and emphasis on surveillance should be stressed in order to avoid possible reinfection.

Communication is the "key" for the educational part of the programme. This is not a problem for those countries or regions where the level of literacy is high and electronic communications, such as television, radio or the Internet, are available. Unfortunately, for a great number of the regions affected by brucellosis or other major zoonoses, the situation is completely different. Although radio is spread worldwide, and it is an excellent media for mass communication, it should not be the primary method for transmitting the basic aspects of the programme. The process of diffusion should make the programme available to everybody according to their particular needs and abilities to get such information. Beside, advertising by radio or television, notes in pamphlets or local newspapers will help spread information. However, there are places where few literate people are involved in raising infected animals, and where there is a major risk of infection. In such places local authorities should find a community leader to organize and explain basic aspects of the programmes and set up small discussion groups that can be educated. In those regions where distances are considerable, some members of the group should be selected to be in charge of communicating aspects of the plan to other farmers and animal caretakers. Thus, not only every single person will be informed but also leaders or subordinates will be motivated to follow the rules of the plan. If available, explanation should be in the form of very simple audiovisual information, such as posters, where the basic precautions to be taken to prevent infection are explained in a simple and effective way.

Educational material must be selected to be adequate and appropriate for the target populations. The message should be very simple, easy to follow and to understand. It must consider local habits even if, for scientific purposes, it would look "absurd". Those beliefs can be modified, but this should be done by personal talks and demonstrations. For example, some farmers may be scared of bleeding animals because of cultural belief. Educators should introduce the subject and explain that nothing will happen, not only using printed material but also, if possible, by practical demonstrations. Sometimes, farmers have logical reasons for not bleeding their animals. In summertime, many regions have an abundance of flies or other insects, and farmers know that animals with wounds will have problems and production will decrease. Thus, the educator must be aware of the local situation. Always, the economic impact must be pointed out.

These educational issues must be included in any strategy, no matter the region or the cultural level of the target population. A sensible idea may be to show the material first to leaders of the group to get them involved in the "whole idea". Thus, all the key players will interact with each other and deliver the information according to the needs of the receivers (politicians, breeders or householders).

7.2 Additional components of the Brucellosis Programmes

The programme to be implemented should always be feasible according to regional practices and should include all aspects described previously in this report. Major issues to be considered for the control and eradication programmes of brucellosis are:

• They should include different issues that demonstrate at the end a major contribution to improving public health, animal health and animal production.

• They must be precise, have clear and specific objectives, and a distinct duration should be defined. Timing should be analysed and possible problems identified according to regional peculiarities.

• The cost of a programme is a major issue. Although precise details will not be available for the overall programme, all expenditure should be sensibly considered. Non-developed countries can request monetary support from international organizations. Such requests should be reasonable and complement local programmes.

• Individuals responsible for the programmes must be "open minded". They should be capable of consulting external help and makes progress reports, and be open to external evaluation. External evaluation is of major importance and should never be interpreted as "critical control"; instead it should be considered as a "friendly advice".

• The information obtained must be issued not only to the decision-makers but also to all the members participating in the programme, so that they can be informed about the progress of their work.

• The programme must have political support and the correct government authorities should be fully involved.

• Collaboration between veterinary services and public health services is required.

Epidemiological surveillance is one of the major issues in any sanitary programme, but it is key for a brucellosis programme. It will include analysis and interpretation of the collected data. The surveillance system allows for the optimization of resources, for alternative actions to be designed, for the continuous justification of expenses, and for evaluation of activities performed and assessment of achievements. Once a precise surveillance system is in place and is supplied with valid data collected from the field, the progress, impact, adequacy, efficiency, and efficacy of a control programme can be continuously evaluated.

Zoonosis control programmes are distinctive, in that the advantages of such programmes are not as obvious as in other diseases where occurrence is characterized by heavy losses in an animal population. There are many cases where zoonosis control programmes have failed because the people participating lost motivation when they did not see any immediate or obvious benefit.

8. FINAL RECOMMENDATIONS

There is not a magical formula to control, eliminate or eradicate brucellosis. The strategies outlined below should be implemented for the control of the disease.

8.1 Selection of strategy

Decisions as to the appropriate strategy for the control and/or elimination of brucellosis are the responsibility of the country conducting the programme. In large countries where regional differences are appreciable, implementation of the strategy may be delegated to regions or provinces or made feasible for individual islands or communities. Factors to be considered are:

• the financial, technical and personnel resources available;

• laboratory capabilities;

• the prevalence of the disease;

• animal husbandry techniques;

• the geography of the country, region, or area; and

• acceptance of the strategy by the livestock owners.

Definitions and rules should be written in a document. It will outline the actions, authority, and responsibility of the chief veterinary officer of each country/region/state/territory, and act as guidelines when formulating detailed operating procedures in response to any suspected case of brucellosis.

8.2 Immunization of the susceptible animals

Control of brucellosis can be achieved by using vaccination to increase the population's resistance to the disease.

• Vaccination of young animals: calf vaccination with B. abortus strain 19 or RB51; goat and sheep vaccination with B. melitensis REV 1.

• Vaccination of whole herd or flock: adult cattle with B. abortus S19 or RB51 (full or reduced doses); adult goats and sheep with B. melitensis REV 1 reduced doses.

• Combination of both alternatives shown above.

• Massive vaccination is recommended where there is a high prevalence of brucellosis and serological diagnostic tests cannot be conducted.

8.3 Election and application of the serological diagnostic tests

There are many serological tests developed to diagnosis brucellosis. However, to ensure the best outcome for the brucellosis campaign it is advisable to use only a few tests that are most compatible with the laboratories where those tests will be done.

• Screening tests: Rose Bengal, buffered plate antigen test. These tests can be done in the field, if necessary, or in the laboratory. (Modern technology such as FPA, which can also be done in the field, may be used for screening). Agglutination plate tests can be performed anywhere with minimum requirement and must be used as screening if other superior technology is not available.

• Confirmatory tests: conventional tests like rivanol, 2-mercaptoethanol or CFT. Complement fixation is the best conventional confirmatory test to use but it is cumbersome to perform. Rivanol and 2-mercaptoethanol are the second choice tests if CFT cannot be performed. These tests can be done only in laboratories. The incorporation of modern technology such as ELISA and FPA tests into all brucellosis programmes is strongly recommended.

• Surveillance test: the milk ring test can be used for dairy cattle. It should be used at least twice in those regions with low prevalence of brucellosis, and four times in those regions with medium or high prevalence of the disease.

• Market cattle testing: this is an excellent strategy for the control of brucellosis. It will provide data for surveillance and also for disease control. It is recommended that all programmes intending to eliminate brucellosis adopt this approach. It will include testing at slaughterhouses, livestock markets, or any livestock buying station. The most appropriate place to implement this method can be decided for a particular programme. It is crucial that animals can be identified and that records are kept for tracing purposes. Although it is well known that in many regions animals lack of identification due to high costs, field tests "in situ" are currently available which can minimize this inconvenience.

• In all cases, training of laboratory and field personnel in test interpretation and animal classification must be done in order to obtain consistent results.

• It is also recommended that in any area where signs of brucellosis are detected serological tests should be performed at least once a year to every herd/flock. According to the results obtained, and considering the local factors, an appropriate management plan can be developed including strategic diagnostic tests.

• It is strongly recommended that PCR technology be evaluated in a coordinated study by appropriate laboratories to find a more standardized test so that results can be compared worldwide and applied to the diagnosis of brucellosis.

8.4 Removal of infected animals - test and slaughter policy

According to previous experiences, a test and slaughter policy is justified by economic grounds only when the prevalence of infected animals in an area is about 2 percent or less.

• Voluntary or compulsive programme - it must be decided which one of these options will be chosen, according to local conditions.

• Animals must be individually identified and an efficient and well-organized veterinary service for surveillance and laboratory testing must be in place.

• Infected herds must be quarantined.

• Full cooperation of farmers is a critical issue to keep in mind if this policy is adopted.

• Economic compensation or replacement of animals should be considered.

In 1998, a document issued by the WHO indicated that "provided that the prevalence of disease is moderate, financial resources are available, and a well-functioning surveillance by the veterinary service is in place, vaccination of young animals can be combined with a test and slaughter policy in a long-term action to control brucellosis in small ruminants".

8.5 Monitoring brucellosis free herds/flocks and regions

The risk of acquiring the disease by means of animal movement must be evaluated.

• Movement of potentially infected animals into such areas must be prohibited.

• Importation of animals should be permitted only from certified brucellosis-free farms or areas. Control of movements of animals and animal products from regions of higher risk must be strict.

• Serologically negative animals should be accompanied by original certification, which must be checked when they arrive at their final destination.

8.6 Collaboration between veterinary services and public health services

To solve a major problem like brucellosis, both animal and public health services must work together. Having veterinarians and medical doctors with similar training will aid brucellosis control and eradication programmes and there is a greater chance of success than if the two professions work independently. Periodically holding joint meetings will be mutually beneficial for both services. For example, if cases of human brucellosis are reported veterinarians working in the region can be alerted to detect the focus and eliminate it, or if brucellosis is reported by veterinarians, physicians can be made aware of the possibility of a human brucellosis outbreak. The spread and the rate of infection in an animal population is an excellent indicator for a potential outbreak in the human population. An abortion "storm" could be a signal indicating to both professions that brucellosis is present in the area. It is recommended that similar tests for human and animal populations be employed in non-developed countries where the use of modern and expensive techniques is not possible. Laboratory assistance, techniques, diagnostic facilities, and the equipment are generally the same regardless of the species to be tested. In those countries where economic resources are limited, sharing of laboratory facilities between these two professions is strongly recommended, to be more cost effective.

Most countries have legislation for livestock production and include surveillance data generated that should be useful for analysis and employed for the public health services. Thus, these services can intensify preventive measures, through information to the general public, including schools. In countries where the veterinary service has an important role visiting farms, either for general disease control or specifically for bleeding animals for brucellosis control, they may frequently be questioned about symptoms of brucellosis. Here, the veterinarians play a very important role in notifying physicians and in advising the farmworkers how to protect themselves against the infection.

For controlling this disease, the interchange of information and surveillance data should include geographical areas where the outbreaks occur, species and number of animals affected, and human cases. The concept of "territoriality" between medical doctors and veterinarians must be avoided; these attitudes will not help the people who really need both professions working together. Working separately brings duplication of effort and tremendous expenditure that can be easily avoided simply with communication.

CONCLUSIONS

Brucellosis is still a major disease of worldwide distribution. There are many factors involved in both human and animal brucellosis that make the control and eradication of this disease an important challenge. Today, we have very powerful tools to fulfil the requirements, excellent serological methods, very effective immunogens and an overall knowledge of the pathogenesis of this disease. Efforts should be made by responsible authorities to make practical plans. The responsibility for the plan should belong to all participants together and there must be support for the programme, not only a strong economic structure. The most important aspect to be considered is education, which reaches all susceptible populations and is adjusted for each region and its culture.

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