Wernery U

Review Article

J Bacteriol Mycol. 2016; 3(1): 1019.

Camelid Brucellosis: A Review

Wernery U**

Central Veterinary Research Laboratory, Dubai, UAE

*Corresponding author: U Wernery, Central Veterinary Research Laboratory, Dubai, UAE

Received: December 16, 2015; Accepted: March 03, 2016; Published: March 05, 2016

Abstract

Camel brucellosis has been diagnosed in all camel-rearing countries except Australia. In many countries the infection is on the rise in Old World camels (OWCs) due to the uncontrolled trade of live animals. Knowledge of camelid brucellosis has increased over the last decade through field investigations, experimental infection trials and comprehensive laboratory testing. Infection with Brucella melitensis is frequent in OWCs and rare with B. abortus. New World Camels (NWCs) rarely contract brucellosis. In East African countries the seroprevalence of brucellosis can reach 40% (herd level) and depends on the management system. The highest incidence is found when camels are kept together with infected small ruminants. Only a combination of serological methods can detect all serological reactors. However, many brucellosis antibody ELISAs for serum or milk are not suitable for diagnosis. Culturing the pathogen is still the preferred test method, although several assays based on polymerase chain reaction have been developed. through milk. Also, the blood of dromedary calves was negative in culture and Polymerase Chain Reaction (PCR). Interestingly, camel calves of serologically positive dams were all serologically negative, using RBT and cELISA techniques, at the age of six months. The calves therefore do not appear to be at risk for an acute brucellosis infection even after the disappearance of maternal antibodies. However, for confirmation of these findings, further investigations need to be performed [9]. Ostrovidov [37], and Solonitsyn and Pal’gov [38], proposed separating calves from their dams at the age of seven to eight months, when their maternal antibodies have disappeared. If this does not occur, they may contract infection from infected dams at the next parturition. The Brucella-negativity of female camel calves from chronically infected dams is controversially discussed among Dubai-based veterinarians and some researchers believe that confirmation of the Brucella-negativity can only be confirmed when camel calves remain serologically negative after parturition. In males, it is an even more complicated unsolved issue.

Keywords: Brucellosis; Camelid; Diagnosis; Epidemiology; Treatment

Introduction

Many countries, such as the United Kingdom, Australia and Japan, as well as parts of the United States of America (USA) and some countries in North Europe have succeeded in eradicating brucellosis through intensive health control measures, but elsewhere the disease remains widespread in domesticated and wild animal populations and presents a great economic problem for tropical animal husbandry [1]. Brucellosis is also one of the most important zoonoses in developing countries. Old World camels (OWCs) are frequently infected with brucellosis, particularly when they are in contact with infected ruminants [2-6]. The disease is rare in New World Camels (NWCs) but outbreaks with classical signs of brucellosis have been described [7].

Aetiology

Brucellosis is a contagious disease caused by bacteria of the genus Brucella. Taxonomically, the genus Brucella. is divided into ten classified species and subdivided into biovars. The subdivision is based on biochemical reactions and agglutination with mono-specific sera. Recently, Brucella strains have been isolated from numerous marine mammal species; molecular typing methods have not been able to classify these isolates within the described species, and therefore they have received their own names: Brucella cetacea (dolphins) and B. pinnipeda (seals, fur seals, walruses). Brucella bacteria are Gram-negative coccobacillae that are non-motile and non-sporeforming. They grow an aerobically and certain strains need a 5% to 10% carbon dioxide atmosphere. Brucella organisms grow slowly, but can be enhanced by using enriched media, such as Farrell’s media supplemented with 5% horse serum and six added antibiotics.

The growth of B. ovis and B. abortus, biotype 2, always requires media enriched with serum or blood incubated in an atmosphere of 5% to 10% carbon dioxide.

Impact on Human Health

In humans, the disease, which is often referred to as ‘undulant fever’ or ‘Malta fever’ is a serious public health problem. Human brucellosis remains one of the most common zoonotic diseases worldwide, with more than 500,000 new cases annually (World Health Organization (WHO) and Food and Agriculture Organization of the United Nations (FAO), [8]. Infection prevalence in the animal reservoirs determines the incidence of human cases [9]. Brucella spp. are also potential agents of bioterrorism and are classified in group B (second-highest priority agent) of the Centres’ for Disease Control and Prevention (CDC) in the USA. Brucella melitensis and B. abortus are the two species most commonly found in human cases, and B. melitensis is responsible for the most serious infections. Human brucellosis is mainly an occupational disease, and the main modes of transmission are contact through skin with animal tissues, blood, urine, vaginal discharge, aborted foetuses and, especially, placentas, and consumption of raw milk and other unheated dairy products. Airborne infections occur in animal pens, stables, laboratories [10] and abattoirs. Some cases have also occurred from accidental selfinoculation with live vaccines [11] World Organisation for Animal Health (OIE), [12]. Moreover, it was also shown by Bradenstein et al. [13], that Rev 1 vaccine strain can cause human infections. In their study humans became infected after consuming milk from vaccinated adult pregnant animals which excreted the vaccine strain in milk for a long period of time. The high and increasing herd and animal prevalence of camel brucellosis in many countries is of grave concern [14] therefore, veterinary authorities, consumers, camel owners and camel keepers, as well as responsible persons in the Ministry of Health and Agriculture of each country, should make every effort to address this issue.

During investigations conducted by Radwan et al. [15], it was found that brucellosis was diagnosed in 30% of the camel handlers and milkers and the same B. melitensis biovars were cultured from aborted sheep and goats sharing the same premises.

In humans, the incubation period lasts from five to 60 days, but can also be longer. Clinical signs are not specific and can be acute or chronic (Table 1) [16].

Table 1: Signs of brucellosis in humans.




  
    Clinical signs 
    Patients affected in % 
  
  
    Fever
    90–95
  
  
    Malaise
    80–95
  
  
    Body ache
    40–70
  
  
    Sweats
    40–90
  
  
    Arthralgia
    20–40
  
  
    Splenomegaly
    10–30
  
  
    Hepatomegaly
    10–70



Table 1:  Signs of brucellosis in humans.

Brucella infections in pregnant women in early pregnancy may lead to high rates of fetal loss (up to 40%) and infection in men can lead to orchitis and epididymitis. Brucella melitensis DNA persists in human blood for many years after infection despite appropriate treatment and apparent recovery [17]. Humans are at risk through consumption of unheated milk or through handling Brucella-positive animals [8,15,18-19]. Shimol et al. [20] described a brucellosis outbreak that affected 15 people who consumed unpasteurized camel milk. Affected people suffered mainly from arthralgia and fever and 50% had positive blood culture for B. melitensis, whereas 60% had serum agglutination titres of 1:60 or higher.

During a B. melitensis outbreak which occurred in a herd of alpacas in Peru, over 25% of the alpaca handlers were seropositive to brucellosis and some developed clinical signs [21].

Extreme care must be exercised when working with Brucella organisms in laboratories. It is estimated that up to 2% of all diagnosed brucellosis cases are laboratory-acquired infections, mainly through inhalation when handling diagnostic specimens [22].

Incidence of Camelid Brucellosis

Camelid brucellosis caused by B. melitensis and B. abortus has been reported in all camel-rearing countries except Australia and the incidence appears to be closely related to breeding and husbandry practices [23], which Omer et al. [24] were able to prove in Saudi Arabia. They compared the brucellosis seroprevalence of a female dromedary herd which was in close contact with small ruminants (n = 165) with a closed female dromedary herd (n = 95). The brucellosis prevalence in the open camel herd was 8.5%, whereas only one animal (1%) was diagnosed in the closed herd. The diagnostic tests used were the Rose Bengal test (RBT), serum agglutination test (SAT) and competitive enzyme-linked immunosorbent assay (cELISA). High animal and herd prevalences have been reported from many countries, which not only pose a severe risk to humans but also to other livestock. The infection rate in some regions of the former Union of Soviet Socialist Republics (USSR), where Bactrian camels were kept on large farms, was 15% [25], whereas in countries with more extensive forms of husbandry, such as Chad or Ethiopia, the brucellosis seroprevalence was 3.8% [26] and 5.5% [23], respectively. Similar differences in seroprevalence have been reported from Saudi Arabia by Radwan et al. [27] and Ghoneim and Amjad [28]. They reported a higher incidence of camel brucellosis in intensively farmed camels than in free-grazing desert camels. In Sudan, prevalence varies according to the system of camel husbandry: agro pastoralists reported a higher prevalence of brucellosis (31.5%) than nomads (21.4%) [29-31]. A seroprevalence of dromedary brucellosis of 40% has been reported from Sudan [32], and the United Arab Emirates (UAE) has experienced a drastic increase of brucellosis in camel populations due to the uncontrolled import of dromedaries from East African countries. Also, introduction of camels into cattle, sheep and goat areas in the Darfur region of Sudan led to high incidence levels, as shown by Musa and Shigidi [33]. In another study in Sudan, conducted by the same authors, in 3,413 dromedaries that were intermingled with cattle and small ruminants, the herd infection rate was 45.5%, with prevalence rates of between 1.4% and 90%.

Moustafa et al. [34] reported on a serological survey in dromedaries and a brucellosis eradication campaign in the eastern regions of the UAE during a five-year period. The highest prevalence was in 1991, with a reactor rate of 5.8%, whereas the lowest was in 1996, with a rate of 0.01%. Since no camels had been culled due to brucellosis, it is believed that the reduction in camel brucellosis was caused by the reduction in brucellosis in sheep and goats.

Epidemiology

The disease has a worldwide distribution and affects cattle, pigs, sheep, goats, camelids, dogs and, occasionally, horses. Brucella infections have also been documented worldwide in a great variety of wildlife species and, more recently, in marine mammals. A spill over of infection from domestic animals to bison, elks or African buffalos may also be possible [35].

The infection occurs via the mucous membranes, including oralnasopharyngeal, conjunctival and genital mucosa, and also through cutaneous abrasions. Animals become infected through feed, water, colostrum, contaminated milk and, especially, by licking or sniffing at placentas and aborted foetuses. The spread of brucellosis during sexual activity plays a subordinate role. The primary shedding routes of Brucella organisms remain uterine fluids (lochia) and placenta expelled from infected animals. In cattle it is known that abortion is associated with the shedding of 1012 to 1013 Brucella bacteria. Survival of the organisms in the environment is enhanced by cool temperatures and humidity; however, it was proven that two dromedaries in a Brucella-negative dromedary herd were infected with B. melitensis through contaminated dust particles from aborted camel foetuses 500 m apart, indicating that organisms can also survive in a hot desert environment. Many placental mammals, including herbivores, participate in placentophagy, with camelids as a noted exception, which may contribute to the spread of Brucella bacteria through wind. In bovines, shedding of up to 103 B. abortus bacteria/ml through milk following abortion may last for a period of up to three months, which is considered an important fact from an epidemiological point of view. The situation in camelids is unknown. Excretion of the pathogen through milk is intermittent [36]. However, in chronically infected (serologically positive) dromedaries from the UAE which gave birth to healthy off springs, no Brucella organisms were isolated from expelled placentas, and no shedding occurred

In general, abortions occur mainly during the first pregnancy and infected camelids are clinically well. The pathogen is found intracellular in mononuclear phagocytes, in which it also multiplies. In pregnant camels, the bacteria localize in the placenta and are most abundant in abortion material (up to 1013 bacteria) including the fetal stomach, vaginal discharge and colostrums [39]. Brucella melitensis and/or B. abortus organisms have been isolated from camel milk, aborted foetuses, the placenta, fetal stomach fluid, lymph nodes, vaginal swabs, testes and hygromas (Table 2).

Table 2: Brucella species isolated from camelids in different countries.




  
    Country 
    Author
    Year
    Species
    Organs 
  
  
    Jordan 
    Al-Majali
    2006
    B. melitensis biovar 3
    Aborted    foetuses vaginal swab
  
  
    Russia 
    Solonitsyn
    1949
    B. abortus
    n/a
  
  
    Pal’gov
    1950
    B. abortus
    Bactrian,    foetuses
  
  
    Iran 
    Zowghi and Ebadi
    1988
    B. melitensis biovar 1
    Lymph    nodes
  
  
    Kuwait 
    Zowghi and Ebadi
    1988
    B. melitensis biovar 3
    Lymph    nodes
  
  
    Libya 
    Al-Khalaf and El-Khaladi
    1989
    B. abortus biovar 1
    Foetal    stomach
  
  
    Gameel et al.
    1993
    B. melitensis biovar 1
    Milk
  
  
    Saudia Arabia 
    Gameel et al.
    1993
    B. melitensis biovar 1
    Milk,    vaginal swab, aborted foetus
  
  
    Radwan et al.
    1992
    B. melitensis biovar 1 and 2
    Milk
  
  
    Radwan et al.
    1995
    B. melitensis biovars 1, 2, 3
    Milk
  
  
    Ramadan et al.
    1998
    B.    melitensis
    Carpal    hygroma
  
  
    Al Dubaib
    2007
    B.    melitensis
    n/a
  
  
    Sudan 
    Agab et al.
      Musa et al.
    1996
      2008
    B. abortus biovar 3
      B. abortus biovar 6
      B. melitensis biovar 3
    Teats,    lymph nodes, vaginal swab, testis
      Lymph    nodes
  
  
    Omer et al.
    2010a
    B. abortus biovar 6
    Lymph    nodes, testis
  
  
    Peru 
    Acosta et al. (alpacas)
    1972
    B.    melitensis
    Organs
  
  
    UAE
    Wernery et al. (camels from Sudan)
    2007b
    B. melitensis biovars 1 and 3
    Milk,    lymphnodes, placenta
  
  
    Moustafa et al.
    1998
    B. melitensis
    Milk
  
  
    Senegal 
    Verger et al.
    1979
    Brucella    abortus biovars 1 and 3
    n/a
  
  
    Egypt 
    El-Seedy et al.
    2000
    B. abortus biovars 1 and 7
      B. melitensis biovar    3
    Organs



Table 2:  Brucella species isolated from camelids in different countries.

It was also shown by Von Hieber [9] that, during a period of two years, 5% (n = 118) of the dams had fluctuating titres from positive to negative to positive and 20% of the serologically positive dams turned negative with RBT and cELISA (latent infection?). This indicates that the pathogens can conceal themselves, most probably in lymph nodes, and do not produce detectable antibodies in those intracellular hiding places. However, evidence of spontaneous recovery from brucellosis had also been described by Gatt Rutter and Mack [44] and Ostroividov [37], with no further explanation. Further research by Wernery et al. [45], who investigated the question of where Brucella organisms were concealed in serologically positive lactating dromedaries which gave birth to healthy calves, revealed that they were in internal lymph nodes. They were mainly isolated in lung lymph nodes, indicating an inhalation infection route. These investigations in camelids clearly show that there are important epidemiological differences in dromedaries which abort (acute brucellosis) and chronically infected animals which do not abort. A chronic infection is certainly the most common occurrence, and in bovines it is known that 75% to 90% of cows abort once only [46].

Theoretically, the three Brucella species known to cause brucellosis in camels (B. abortus, B. melitensis, B. ovis) can cause infection anywhere [47]. However, it is surmised that B. melitensis is widespread in Africa and the Middle East and B. abortus is widespread in the former USSR. Solonitsyn [48] reported mixed infections with various Brucella species in Bactrian camels in Russia. (Table 2) demonstrates which Brucella species have been isolated from which organs in which country.

Although camels appear to be very susceptible to Brucella infection, isolation of Brucella organisms from camel samples is rare. But attempts to isolate Brucella from milk have been successful. Brucella abortus biovars 1 and 3 were isolated from camels in Senegal [49]. Radwan et al. [3] were able to isolate B. melitensis biovars 1 and 2 26 times from a total of 100 milk samples from seropositive Saudi Arabian dromedaries. Gameel et al. [50] were also able to isolate B. melitensis biovar 1 five times from the milk of Libyan dromedaries and four times from aborted foetuses and vaginal swabs from a herd of 124 Libyan dromedaries. The authors did not mention from how many affected dromedaries the samples were taken. Zaki [51] inoculated guinea pigs with milk samples from seropositive dromedaries and cultured the milk samples in vitro. Both tests (SAT and culture) were negative. Al-Khalaf and El-Khaladi [52] examined cultures of 209 milk samples from Kuwaiti dromedaries. The samples were obtained from herds with an increased incidence of abortion. The results were culture-negative. However, the authors were successful in isolating B. abortus from the gastric fluids of five aborted foetuses. Pal’gov [53] was able to isolate B. abortus from Bactrian camels in Russia. In the herds examined, 2% of all animals aborted in the first half of the pregnancy. Fifteen percent of the herds were seropositive to brucellosis using the complement fixation test (CFT). Zowghi and Ebadi [54] cultured 3,500 lymph nodes from 300 slaughtered dromedaries from Iran for Brucella organisms. Brucellosis melitensis biovars 1 and 3 were isolated from these lymph nodes in 1% (3/300) of the camels. The authors are of the opinion that the B. melitensis infections in the dromedaries originated from neighbouring sheep and goat herds.

Radwan et al. [15] examined a large camel herd with 2,536 dromedaries in Saudi Arabia from which a 12% abortion rate had been reported. A Brucella seroprevalence of 8% was found with RBT and the standard buffered plate agglutination test (BPAT) of the United States Department of Agriculture (USDA). The authors also isolated B. melitensis biovars 1, 2 and 3 from aborted camel foetuses. Brucella abortus biovar 3 was recovered from an inguinal lymph node, three vaginal swabs and one supramammary lymph node obtained from free-ranging camels in eastern Sudan which had histories of abortion, presence of hygromas or testicular lesions [55]. It is worth mentioning that both isolates of B. abortus biovar 3 from Senegal and Sudan are the only oxidase-negative biovars reported in the literature. Ramadan et al. [56] have recovered B. melitensis from a hygroma of an Indian camel. Brucella melitensis was isolated twice from two-quarters of milk samples from three seropositive camels in the UAE [36].

Brucellosis is not a major disease in NWCs, but severe outbreaks, such as the outbreak in Peru referred to earlier, have occurred from time to time. It was thought that sheep were the source of infection in this alpaca herd [21]. In an experimental infection trial in llamas in the USA, it was found that llamas are susceptible to B. abortus and that they develop positive serological titres. The authors used five conventional serological tests (CFT, standard tube test, standard plate agglutination test, RBT and BPAT) in addition to an ELISA developed at Iowa State University. The llamas also developed histological lesions similar to those found in cattle, sheep and goats [57].

Three llamas died at London zoo after they came into contact with camels which were newly imported from Moscow [58]. The authors claimed that the high serological titre (type of test not given) for B. melitensis was indicative of an acute infection.

Clinical Signs

Brucellosis is characterized by abortion and to a lesser extent by orchitis and infection of the accessory sex glands in males. According to various researchers, the clinical signs of brucellosis in breeding camelids are the same as those in bovines and small ruminants, although infection in breeding camelids causes fewer abortions than it does in bovines and small ruminants [8,15,21,55,59]. Infections may cause stillborn calves, retained placenta, foetal death, and mummification and reduced milk yield. Also, delayed service age and fertility have been reported [33]. A retained placenta is rare in Camelidae. This may be a result of the difference in the placental attachment [58]. Camelids possess a placenta diffusa like the horse and not a cotyledonary placenta.

Non-pregnant dromedaries (n = 6) artificially infected subcutaneously in the right lower back of the neck with two strains of B. abortus (four with S19, two with field bovine strain, × 106 bacteria,) developed only mild clinical signs. Reduced appetite, slight lameness and bilateral lacrimation were observed. On necropsy the pathogen was re-isolated 45 to 65 days later from the cranial and genital lymph nodes. No clinical signs were observed in the four camels inoculated with S19, whereas slight non-specific signs were found in the dromedaries infected with the bovine B. abortus field strain. On necropsy no gross lesions were detected, but histological results revealed focal granulomas in the liver and a generalised lymphadenitis (supramammary lymph node). The pathogen was re-isolated from the lymph nodes of the genital tract and head [60].

Pathology

Little is known about the pathological changes caused by Brucella organisms in camelids. These bacteria have a predilection for the pregnant uterus, udder, testicles, accessory male sex glands, lymph nodes, joint capsules and bursae. Lesions may be found in these tissues. Nada and Ahmed [61] described lesions in non-pregnant dromedaries. They found inflammation of the uterus lining with reddening, oedema and necrotic foci in the uterus epithelium, as well as fibrosis of the endometrium and atrophy of the uterine glands. The

authors also observed an increased number of ovariobursal adhesions and hydrobursae. The adhesions occurred between the bursa ovarica and the ovary and in several cases also between the bursa ovarica and the salpinges, causing a severe induration of the latter. Hydrobursitis was often observed in brucellosis-positive dromedaries causing an enlargement of the bursa, which was then filled with a clear ambercoloured fluid. No lesions have been described so far in aborted camelids and in brucellosis-positive camelid males except orchitis and epididymitis. The testes and epididymis of 360 dromedaries were examined for gross and histopathological lesions. Around 12% of the tested organs originated from seropositive camel bulls. From the investigations it is not clear if the epididymitis, orchitis or testicular degeneration was caused by Brucella infection or was a normal pathological feature [62]. A pregnant llama was experimentally infected by inoculating viable B. abortus bacteria into the conjunctival sac. Forty-three days post inoculation; the llama aborted an eightmonth- old fetus. Brucella abortus was isolated from the placenta and all fetal specimens, including the brain, small and large intestines, spleen, kidney, liver, stomach fluid, heart blood and lung. Bacteria were also isolated from numerous mammary gland lymph nodes in the dams. Histologically there was a moderate, multifocal, lymphocytic and histiocytic, subacute placentitis, with a marked loss of trophoblastic epithelial cells. The chorioallantoic stroma contained abundant necrotic and mineralised debris and the swollen capillaries were expanded by large numbers of Brucella organisms [57,63].

Abu Damir et al. [64] as well as Wernery et al. [36] described only a few lesions in non-pregnant B. abortus-infected dromedaries and in lactating dromedaries that were seropositive for B. melitensis (B. melitensis was also isolated from milk samples). Cranial and genital lymph nodes from which the pathogen was isolated showed marked sinusoidal oedema and follicular hyperplasia of cortical and paracortical areas, with active germinal centres and histocytosis. There were no lesions in the reproductive tract.

In Saudi Arabia, pathological and histopathological studies of non-pregnant dromedaries naturally infected with B. melitensis biovar 3 [24] revealed the following alterations in the following organs:

lymph node (especially supramammary): oedema, enlargement, lymphoid hyperplasia, granulomatous reaction in the cortical area of the lymphoid follicle
spleen: enlargement with granular surface in some cases, depletion of some lymphoid follicles, proliferation of fibrous tissue, histiocytosis
mammary gland: granulomastitis in some cases, proliferation of interlobular fibrous connective tissue
uterus: moderate amount of mucous and ulceration of endometrial mucosa, endometrial stroma showed oedema and diffuse and heavy infiltration (mainly of macrophages and lymphocytes in the lamina propia), blood vessels were dilated and congested.

Diagnosis

The morphology of the Brucella bacterial colonies is associated with the presence of lipopolysaccharides (LPS) in the external membrane of the bacterium.

Smooth (S-LPS) and rough (R-LPS) phenotypes are differentiated. The S-LPS phenotype is found in most Brucella species, only B. canis and B. ovis possess the R-LPS. Some proteins of Brucella are responsible for serological cross-reactions between Brucella spp. and other bacterial species [65]. Cross-reactivity exists to:

Yersinia enterocolitica O: 9
Escherichia hermannii
E. coli O: 157
Francisella tularensis
Stenotrophomonas maltophilia
Vibrio cholera O: 1
Salmonella serotypes group N

Therefore, difficulties may arise in the diagnosis of brucellosis. Abortion and reduced fertility in the camel frequently have other causes, such as salmonellosis, trypanosomosis, or infections with Campylobacter or Tritrichomonas fetus [66-68], making laboratory testing essential. An incorrect diagnosis of brucellosis may occur when based on serology alone.

Culture

Brucellosis is usually diagnosed in the laboratory by culture of blood, milk or tissue or the detection of antibodies in sera. Brucella organisms can be recovered from the placenta, but, more conveniently, in pure culture from the stomach and lungs of aborted foetuses. It should be stressed that only fresh material is suitable for culture to avoid overgrowth by a number of opportunistic bacteria. Culture of Brucella spp. is still the gold standard but also time consuming, expensive, difficult and dangerous.

For isolation, the recommended medium is Farrell’s medium, which contains six antibiotics. But other selective Brucella media are also in use for the growth of this pathogen from fresh camel milk and camel tissue samples [15]. During intensive investigations using selective media it was found that on a camel farm in Saudi Arabia 34% of all Brucella seropositive milking dromedaries were Brucella shedders. The high number suggests that it is preferable to use selective media.

Tissue specimens from Brucella-positive dromedaries were examined by Omer et al. [24] with the immunoperoxidase test, with very good results. Brucella organisms were detected in the cytoplasm of macrophages (visible as brown granules), in the lymphocytes of the lymph nodes and spleen, within the epithelial lining of the endometrium and endothelium of blood vessels, and within mononuclear cells around blood vessels.

Polymerase Chain Reaction

The isolation of Brucella organisms is still the preferred method of diagnosis. This method also allows typing of the isolated strains. However, new PCR techniques are now being implemented for both identification and phenotypic bio typing [35]. These PCRs can discriminate between Brucella species, and between wild and vaccine strains, but do not discriminate between Brucella biovars. So far, only monoclonal antibodies against different epitopes of the Brucella LPS can be used for biovar differentiation.

PCR-based assays have been developed for brucellosis diagnosis and are based on the detection of specific sequences of the pathogen, such as genes of the locus 16S – 23S, the IS711 insertion sequence or the bcsp 31 gene encoding for a protein of 31kDa. Von Hieber [9], who used a PCR assay designed with hybridisation probes and primers targeting the insertion sequence of IS711 of the BMEI 1162 gene, has shown reliable results in the amplification of pure target DNA in bacterial dilutions, but the assay was less sensitive when tissue samples were tested. The reasons for this may be explained by the extraction method used, the intracellular presence of the pathogen and the distribution pattern of Brucella organisms [69].

Serology

The majority of studies on camelid brucellosis use serological methods for diagnosis, but none of the serological brucellosis tests are validated for use in camels yet, as acknowledged by the World Organisation of Animal Health (OIE). Similarly, none of the tests have been validated for the diagnosis of human brucellosis [70]. However, it was found that a combination of different serological tests can increase diagnostic efficacy in camels, although none of the serological tests can differentiate between a B. abortus or B. melitensis or B. ovis infection. Sunaga et al. [71] reported that five dromedaries imported into Japan were positive in the CFT and SAT. The animals were immediately slaughtered. No Brucella organisms were isolated; however, Yersinia enterocolitica serotype 0:9 was identified. It is known that false-positive (unspecific) reactions with various other bacterial species can occur [72-73].

Many authors regard the CFT as being the most sensitive and specific test for brucellosis because CFT antibodies remain in the serum for longer than SAT antibodies [25,44,74-75]. Shumilov [76] determined that the CFT was four times more sensitive than the SAT. He tested Bactrian’s in Mongolia, where brucellosis is widespread among camels. He examined two herds with the following results:

Herd 1: 3751 camels: CFT 4.3% and SAT 0.6%
Herd 2: 54,673 camels: CFT 3.7% and SAT 1.0%.

In the SAT an end titre of 1:20 (40 IU) was regarded as suspicious by different researchers [53,77-80], Fayed et al. [80] Salem et al. [81], and El-Sawally et al. [82] believe that the SAT or tube agglutination test (TAT) detect a higher percentage of reactors to brucellosis than other assays due to their greater sensitivity to immunoglobulin M (IgM) than immunoglobulin G (IgG). In order to eliminate unspecific reactions in the SAT, Wernery and Wernery [83] utilised a 5% solution of phenol sodium chloride, which increases the specificity of the test and reduces the cross-reactivity. The specificity is also increased by adding mercaptoethanol, dithiotreitol or a chelating agent such as ethylenediaminetetraacetic acid (EDTA) to the antigen.

In addition to cross-reactivity with other bacteria that makes the serological diagnosis of brucellosis more difficult, Zhulobovski and Pal’gov [84] observed prozones in some sera of Bactrian camels in Russia, as did Nada [85] in dromedaries from Egypt. The absence of a visual positive reaction in low dilutions has also been observed in 1.5% of all positive dromedary sera in the UAE [86]. Nearly 30% of the 1,449 alpacas tested in Peru had a positive plate agglutination titre [21].

Other researchers have used ELISA for the detection of Brucella antibodies, not only in camel sera [2,87], but also in camel milk [88]. The camel milk ELISA seems to be an important alternative to the conventional serodiagnosis of camelid brucellosis. It must be noted that none of the commercially available brucellosis ELISAs (direct, indirect or competitive) for serum or milk has been evaluated for the diagnosis of camelid brucellosis. Our unpublished newest research clearly indicates that none of the tested ELISAs is suitable due to many false positive results and it is therefore highly recommended to establish a suitable camelid brucellosis antibody ELISA for milk and serum. False positive results may have their reasons in a poor cut-off level and /or the use of an anti-ruminant conjugate instead of a homologous system. It has been shown that dromedary IgG has 74.3% sequence identity to porcine and 73.1% to both equine and bovine, whereas anti-goat IgG has a much lower sequence identity of only 61.6% [89].

Several researchers have evaluated the different serological tests for the diagnosis of camel brucellosis [2,23,90-92]. It was concluded that the elimination of non-specific reactions to Brucella in camelid sera is essential for the correct diagnosis. It is also important to apply more than one test, one of which must be the TAT using 5% NaCl phenolised solution. Atwa [93] and Abou-Zaid [2] found a good agreement between five different serological tests (SAT using 5% NaCl-phenol lysed solution, SAT with 11.4% phenol-NaCl, BPAT, RBT, mercapto-ethanol test, and ELISA), ranging between 80.6% and 95.6%.

Mohammed [94] evaluated the RBT, the TAT, and the CFT for the diagnosis of brucellosis in camels. He found that the RBT and the CFT demonstrated equal ability in detecting positive and negative sera as well as prozone reactions. However, for optimal sensitivity, the RBT has to be used with serum-antigen at a 3:1 dilution. When using the CFT, the 1:10 diluted sera have to be inactivated at 54ºC for 30 min and the cold fixation technique has to be applied. Using the TAT, the classical neutral pH antigen has to be replaced by a buffered (pH 3.5) antigen to achieve optimal results. As mentioned earlier, none of these tests have been validated for use in camel brucellosis and the results are therefore difficult to compare.

Radwan et al. [15] examined a large camel farm comprising 2,536 dromedaries in Saudi Arabia for Brucella antibodies. The authors used a combination of two tests to identify seropositive dromedaries – the RBT and the standard USDA BPAT. With these two methods, the authors successfully eradicated the disease from the farm, where it had caused abortion in 12% of female camels. The authors adopted these tests due to their sensitivity, simplicity and applicability in the field.

The use of serological tests is the core of the control or eradication of brucellosis. Many such tests are available but, they must be used in accordance with strict standardisation rules and meet the requirements laid down by the OIE. For bovine brucellosis the OIE recommends the RBT, the BPAT the CFT, the ELISA and the Fluorescence Polarisation Assay (FPA). The activity of immunoglobulins during infection in the different serological tests allows the distinction between acute and chronic infection. Hence, the presence of both IgM and IgG indicates an acute brucellosis, whereas chronic brucellosis is characterised by the presence of IgG alone. Details of the sensitivity and specificity of the various serological tests are summarised in (Table 3).

Clinical signs	Patients affected in %
Fever	90–95
Malaise	80–95
Body ache	40–70
Sweats	40–90
Arthralgia	20–40
Splenomegaly	10–30
Hepatomegaly	10–70

Country	Author	Year	Species	Organs
Jordan	Al-Majali	2006	B. melitensis biovar 3	Aborted foetuses vaginal swab
Russia	Solonitsyn	1949	B. abortus	n/a
Russia	Pal’gov	1950	B. abortus	Bactrian, foetuses
Iran	Zowghi and Ebadi	1988	B. melitensis biovar 1	Lymph nodes
Kuwait	Zowghi and Ebadi	1988	B. melitensis biovar 3	Lymph nodes
Libya	Al-Khalaf and El-Khaladi	1989	B. abortus biovar 1	Foetal stomach
Libya	Gameel et al.	1993	B. melitensis biovar 1	Milk
Saudia Arabia	Gameel et al.	1993	B. melitensis biovar 1	Milk, vaginal swab, aborted foetus
	Radwan et al.	1992	B. melitensis biovar 1 and 2	Milk
	Radwan et al.	1995	B. melitensis biovars 1, 2, 3	Milk
	Ramadan et al.	1998	B. melitensis	Carpal hygroma
	Al Dubaib	2007	B. melitensis	n/a
Sudan	Agab et al. Musa et al.	1996 2008	B. abortus biovar 3 B. abortus biovar 6 B. melitensis biovar 3	Teats, lymph nodes, vaginal swab, testis Lymph nodes
Sudan	Omer et al.	2010a	B. abortus biovar 6	Lymph nodes, testis
Peru	Acosta et al. (alpacas)	1972	B. melitensis	Organs
UAE	Wernery et al. (camels from Sudan)	2007b	B. melitensis biovars 1 and 3	Milk, lymphnodes, placenta
UAE	Moustafa et al.	1998	B. melitensis	Milk
Senegal	Verger et al.	1979	Brucella abortus biovars 1 and 3	n/a
Egypt	El-Seedy et al.	2000	B. abortus biovars 1 and 7 B. melitensis biovar 3	Organs

Serological test	Sensitivity in %	Specificity in %
SAT	81.5	98.9
CFT	90–91.8	99.7–99.9
RBT	87	97.8
cELISA	95.2	99.7
iELISA	97.2	97.1–99.8
FPA	96.6	99.1
MRT	88.5	77.4

Tests	Positive	Negative
cELISA	20	3
CFT	16	7
RBT	14	9
SAT	7	16

Camelid Brucellosis: A Review

Abstract

Introduction

Aetiology

Impact on Human Health

Incidence of Camelid Brucellosis

Epidemiology

Clinical Signs

Pathology

Diagnosis

Culture

Polymerase Chain Reaction

Serology

Skin Test

Control and Treatment

Antibiotics

Vaccination

Conclusion

References