Int.J.Curr.Microbiol.App.Sci (2018) 7(1): 1848-1877

International Journal of Current Microbiology and Applied Sciences ISSN: 2319-7706 Volume 7 Number 01 (2018) Journal homepage: http://www.ijcmas.com

Review Article https://doi.org/10.20546/ijcmas.2018.701.225

Brucellosis in Sheep and Goats and its Serodiagnosis and Epidemiology

Neharika Saxena1*, Balbir Bagicha Singh1 and Hari Mohan Saxena2

1School of Public Health and Zoonoses, Guru Angad Dev Veterinary and Animal Sciences University, Ludhiana, Punjab, India 2Department of Veterinary Microbiology, Guru Angad Dev Veterinary and Animal Sciences University, Ludhiana, Punjab, India

*Corresponding author

ABSTRACT

Brucellosis is an important zoonotic disease globally that causes huge economic losses to the livestock owners and is of public health significance. Brucellosis in animals is endemic K e yw or ds in India. In sheep and goats, Brucellosis is mainly caused by Brucella melitensis whereas Brucella ovis causes the disease in sheep. The symptoms in infected sheep and goats are Ovine Brucellosis, abortions, stillbirths and the birth of weak offsprings. Animals that abort may retain the Caprine Brucellosis, Serodiagnosis , placenta. Sheep and goats usually abort only once, but reinvasion of the uterus and Seroepidemiology shedding of organisms can occur during subsequent pregnancies. Several studies have been carried out on seroepidemiology of caprine and ovine Brucellosis in India. The tests Article Info commonly used for diagnosis of Brucellosis are the milk ring test, Rose Bengal Plate Test (RBPT), Standard Tube Agglutination Test (STAT), Microtiter Plate Agglutination Test Accepted: (MAT) and ELISA. The RBPT is a rapid screening test for the diagnosis of Brucellosis. 14 December 2017 Available Online: The sensitivity of RBPT is very high (>99%) but the specificity can be low and it could sometimes give a false positive result. Its positive predictive value is low and a positive 10 January 2018 test result requires confirmation by a more specific test. Isolation and culture of Brucella organisms is the gold standard test for the diagnosis of Brucellosis. Brucellosis consequences of the infection. Brucellosis is the second most important zoonotic disease in Brucellosis is an important zoonotic disease the world after rabies. The disease in animals that causes huge economic losses to the is also called as Bang’s disease, Enzootic livestock owners and is of great public health Abortion, Epizootic Abortion, Slinking of significance. It is a chronic infectious disease Calves, Ram Epididymitis and Contagious of livestock, rodents, marine animals and Abortion and in humans it is named as Malta human beings and is caused by facultative fever, Undulant Fever, Mediterranean fever, intracellular coccobacilli of genus Brucella. It Rock Fever of Gibraltar, Gastric Fever etc. is an important cause of reproductive losses in animals. Abortions, placentitis, epididymitis Abortion is the most obvious manifestation. and orchitis are the most common Infections may also cause stillborn or weak

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Int.J.Curr.Microbiol.App.Sci (2018) 7(1): 1848-1877 newborn, retained placentas, and reduced milk their host specificity; B. melitensis affects yield. Usually, general health is not impaired sheep and goats, and B. ovis infects sheep. B. in uncomplicated abortions. Seminal vesicles, melitensis, B. suis, and B. abortus are the most ampullae, testicles, and epididymes may be virulent and cause human disease in the infected in males and organisms are present in majority of cases. In sheep and goats, the semen. Agglutinins may be demonstrated Brucellosis is mainly caused by Brucella in seminal plasma. Testicular abscesses may melitensis, a Gram–negative coccobacillus or occur (Franco et al., 2007). short rod. This organism is a facultative intracellular pathogen. B. melitensis contains Brucellosis is important from public health three biovars (biovars 1, 2 and 3). All three point of view. Humans are often infected due biovars cause disease in small ruminants, but to direct animal contact or ingestion of their geographic distribution varies. Brucella contaminated dairy products, causing acute abortus and Brucella suis infections also occur febrile illness – undulant fever – which may occasionally in small ruminants, but clinical progress to a more chronic form and can disease seems to be rare. Most breeds of goats produce serious complications affecting the are readily infected, but sheep breeds vary musculo-skeletal, cardiovascular and central greatly in susceptibility. B. melitensis nervous systems (Mantur et al., 2007). It is a infections have also been reported well characterized occupational disease in occasionally in cattle, camels and dogs, and shepherds, abattoir workers, veterinarians, rarely in horses and pigs. Infections in sheep dairy industry professionals and laboratory and goats can spill over into wild ruminants. personnel (Agasthya et al., 2007). However, there is no evidence that these animals serve as reservoir hosts for The predominant symptoms in naturally domesticated sheep and goats. B. melitensis is infected sheep and goats are abortions, very contagious to humans. stillbirths and the birth of weak offspring. Animals that abort may retain the placenta. The main characteristic of organisms of the Sheep and goats usually abort only once, but genus Brucella is their ability to survive reinvasion of the uterus and shedding of within phagocytic and non-phagocytic cells organisms can occur during subsequent (Celli and Gorvel, 2004). pregnancies. Some infected animals carry the pregnancy to term, but shed the organism. Three species of Brucella affect humans: B. Milk yield is significantly reduced in animals melitensis, B. abortus and B. suis (other that abort, as well as in animals whose udder species can cause infection in humans, but becomes infected after a normal birth. only rarely). Of these three species, infections However, clinical signs of mastitis are by B. melitensis are the most common in uncommon. Acute orchitis and epididymitis humans and are also the most serious (Pappas can occur in males, and may result in et al., 2005). infertility. Arthritis is seen occasionally in both sexes. Many non-pregnant sheep and The organisms of genus Brucella are goats remain asymptomatic. facultatively intracellular bacteria that cause disease in a broad range of animal hosts. In Etiology of brucellosis in small ruminants vivo they often occur within the cytoplasm of the cells in closely packed clusters (Corbel The genus Brucella is currently known to and Brinley-Morgan, 1984). They are partially contain nine species classified on the basis of acid fast as they are not decolorized by 0.5%

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Int.J.Curr.Microbiol.App.Sci (2018) 7(1): 1848-1877 acetic acid in the modified Ziehl-Neelsen was found to be 11.80% in cattle, 10.67% in (MZN) staining (Alton et al., 1988). Flagella, buffaloes and 3.60% in goats. Analysis of data endospore and capsule are absent although revealed rise in prevalence of Brucellosis in capsule like structures have been reported in dairy animals during 1990-99 (Gill et al., preparations treated with antiserum. Most of 2000). Grewal and Kaur (2000) reported the strains require anaerobic conditions, incidence of Brucellosis to be 7% in sheep and optimal temperature of 370C and 5% in goats, respectively in Sangrur district of supplemented CO2 for growth. All strains lose Punjab, India. viability at 560C. Optimal pH conditions are from 6.6 to 7.4. Brucella strains are fairly Prevalence of brucellosis in sheep and goats resistant to drying and can survive in in India biological material for long periods, especially at low temperature. They are sensitive to a B. melintensis is the major cause of sheep and wide variety of disinfectants including goat Brucellosis in many countries including formaldehyde, hypochlorite, iodophors and India. The infection is wide spread in India phenols provided that excess organic matter is (Kapur and Grewal, 1974). In a national not present. The organisms are killed by heat survey of sheep and goat Brucellosis, Isloor et under pasteurization conditions. al., (1998) tested serum samples originating from 10 states, which included 6305 from Nearly all strains of Brucella are susceptible sheep and 3849 from goats. to gentamicin, tetracycline and rifampicin. Additionally, many strains are also susceptible The cumulative incidence was 7.9% in sheep to ampicillin, chloramphenicol, erythromycin, whereas in goats it was 2.2%. This survey kanamycin, streptomycin and trimethoprim. indicated widespread prevalence of Susceptibility to antibiotics can differ among Brucellosis in small ruminants in the country. species, biovars and even strains (Vemulapalli, 2000). Brucellosis is endemic in India and is prevalent in all parts of the country. Recent Epidemiology of ruminant brucellosis serological surveys of small ruminant Brucellosis indicated varying levels of Brucellosis is worldwide in distribution and is infection in different states - 4.9% of sheep more common in countries with poor animal and 7.6% of goats in Karnataka (Desai et al., and public health programs (Capasso, 2002). 1995), 11% sheep and 18% of goats in Though it has been eradicated from many northern state of Delhi, 50% sheep and 16% developed countries like Australia, Canada, goats in Punjab and 33% sheep and 30% goats Israel, Japan, New Zealand and Europe in the western state of Rajasthan (Kumar et (Geering et al., 1995), it remains an al., 1997), 55% of goats in Andhra Pradesh uncontrolled problem in regions of high (Mrunalini et al., 2000) and 24% of goats and endemicity such as Africa, the Mediterranean, 4.7% of sheep in Uttar Pradesh (Singh et al., Middle East, and parts of Asia and Latin 2000), respectively. It was observed that America (Refai, 2002). flocks that had a history of abortion had high incidence of Brucellosis (Mrunalini et al., Brucellosis is endemic in India and is 2000). The serological evidence of B.ovis prevalent in all parts of the country. In a study infection in 6 out of 102 rams has been conducted in various districts of Punjab in reported in the northern state of Himachal 2000, the overall prevalence of Brucellosis Pradesh (Katoch et al., 1996).

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Int.J.Curr.Microbiol.App.Sci (2018) 7(1): 1848-1877

In a study conducted by Avinash Reddy et al., In yet another study undertaken in North (2014), a total of 252 serum samples were Gujarat and Kutchh regions of Gujarat, a total collected from goats of Karnataka and of 434 serum samples, collected from 14 subjected to 5 different serological tests, i.e., different locations, were subjected to RBPT, Rose Bengal Plate Test (RBPT), Standard STAT and ELISA. The seropositivity was the Tube Agglutination Test (STAT), 2- highest by i-ELISA (18.20%), intermediate by mercaptoethanol test (2-MET), Indirect RBPT (11.98%) and lowest by STAT ELISA (I-ELISA) and Dot-ELISA to detect (9.44%). In comparison to males, higher the anti-Brucella antibodies. The seroprevalence was recorded in female sheep. seroprevalence in goats was 5.15% by RBPT, The highest seroprevalence was recorded in 6.34% by STAT, 1.98% by 2-MET, 9.52% by Marwari breed followed by Patanwadi and I-ELISA and 7.14% by Dot-ELISA. Taking I- Magara while none was found positive in ELISA as reference, the tests revealed the Chokhla breed. The highest seroprevalence relative sensitivity values in the order: Dot- was recorded in Kutchh district followed by ELISA>STAT>RBPT>2-MET. Banaskantha, Patan, Sabarkantha and Mehsana districts (Kotadiya et al., 2015). In a study conducted in various districts of Punjab in 2000, the overall prevalence of In a study conducted on Caprine Brucellosis Brucellosis was found to be 11.80% in cattle, by Priya et al., (2010) in Wayanad district of 10.67% in buffaloes and 3.60% in goats. Kerala, a total of 24 sera were positive by the Analysis of data revealed rise in prevalence of RBPT with a seroprevalence of 5.7%. Upon Brucellosis in dairy animals during 1990-99 STAT testing of all the 420 sera, 18 out of 24 (Gill et al., 2000). Grewal and Kaur (2000) samples (4.3%) showed presence of Brucella reported 7 % incidence of Brucellosis in sheep antibodies. Of the 420 sera examined, 24 and 5% incidence of Brucellosis in goats, (5.7%) were seropositive to RBPT, out of respectively in Sangrur district of Punjab, which only 18 (4.3%) reacted positively to India. STAT. The prevalence was higher in female goats (6.1 % by RBPT and 4.7 % by STAT) Lone et al., (2013) conducted a study on than in males (3.9 % by RBPT and 2.6 % by seroprevalence of Brucellosis in sheep from STAT). both organized and unorganized sectors of the Kashmir valley in which sera of 6615 sheep Several serological surveys of small ruminant were analyzed and the prevalence of Brucellosis indicated varying levels of Brucellosis was recorded by RBPT and STAT. infection in different states. Incidences of 4.9% in sheep and 7.6% in goats in Karnataka The overall prevalence of Brucellosis recorded (Desai et.al., 1995) 11% sheep and 18% in in sheep was 6.50%. In this study, the goats in Delhi, 50% sheep and 16% goats in prevalence of Brucellosis was recorded higher Punjab and 33% sheep and 30% goats in the in unorganized sector (14.14%) as compared Rajasthan (Kumar et.al, 1997), 55% goats in to organized sector (3.23%). In a study Andhra Pradesh (Mrunalini et al., 2000) and conducted in North Gujarat on a total of 1000 24% goats and 4.7% sheep in Uttar Pradesh serum samples comprising 485 from sheep (Singh et al., 2000) have been recorded. In a and 515 from goat, the seroprevalence of national survey of sheep and goat Brucellosis, Brucellosis in small ruminants was 11.30%, Isloor et al., (1998) tested serum samples, 11.10%, and 8.80% by RBPT, STAT, and I- which included 6305 from sheep and 3849 ELISA, respectively (Sadhu et al., 2015). from goats originating from 10 states. The

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Int.J.Curr.Microbiol.App.Sci (2018) 7(1): 1848-1877 cumulative incidence was 7.9% in sheep nations, but the cost of surveillance to remain whereas in goats it was 2.2%. This survey B. melitensis - free is significant. B. melitensis indicated widespread prevalence of brucellosis is very contagious to humans (Nicoletti, in small ruminants in the country. The 2013). In humans, Brucellosis is a serious, serological evidence of B. ovis infection in 6 debilitating and sometimes chronic disease out of 102 rams has been reported in the state that can affect a variety of organs. Brucella of Himachal Pradesh (Katoch et al., 1996). melitensis is a Gram negative coccobacillus or short rod. This organism is a facultative In another study (Suryavanshi et al., 2014) a intracellular pathogen. B. melitensis contains total of 181 and 164 serum samples of sheep three biovars (biovars 1, 2 and 3) and goats were tested for brucellosis by RBPT (Behroozikhah et al., 2012). All three biovars in which an overall prevalence of 7.32% cause disease in small ruminants, but their and17.68% was reported in goats and sheep, geographic distribution varies. Brucella respectively. abortus and Brucella suis infections also occur occasionally in small ruminants, but clinical Ovine and caprine brucellosis disease seems to be rare. Genetic and immunological evidence suggests that all Caprine and ovine Brucellosis, caused by members of the genus Brucella are closely Brucella melitensis, is an economically related, and some microbiologists have important cause of abortion in small proposed that this genus be reclassified into a ruminants. It is also called as Undulant Fever, single species (B. melitensis), which contains Malta Fever, Mediterranean Fever, and many biovars. However, both taxonomic Contagious Abortion. This infection causes systems are currently in use. significant losses from decreased productivity and lost trade in much of the developing B. melitensis is particularly common in the world. Most species of Brucella are primarily Mediterranean. It also occurs in the Middle associated with certain hosts; however, East, Central Asia, around the Persian Gulf infections can also occur in other species, (also known as the Arabian Gulf), and in some particularly when they are kept in close countries of Central America (Garner et al., contact. Brucella melitensis mainly infects 2003). This organism has been reported from sheep and goats. Most breeds of goats are Africa and India, but it does not seem to be readily infected, but sheep breeds vary greatly endemic in Northern Europe, North America in susceptibility. B. melitensis infections have (except Mexico), Southeast Asia, Australia, or also been reported occasionally in cattle, New Zealand. Biovar 3 is the predominant camels and dogs, and rarely in horses and pig biovar in the Mediterranean countries and the (Tibary et al., 2006). Infections in sheep and Middle East, and biovar 1 predominates in goats can spill over into wild ruminants; Central America. Sporadic cases or incursions however, there is no evidence that these are occasionally reported in B. melitensis - animals serve as reservoir hosts for free countries. In the U.S., cases have mainly domesticated sheep and goats. been reported in imported goats and rarely in cattle (USDA APHIS CEI, 1999). Brucella melitensis Epidemiology of B. melitensis B. melitensis is considered to be a re-emerging pathogen in the Middle East (Tibary et al., Brucella melitensis is the most virulent species 2006). It has been eradicated from some of the Brucella genus and has three biovars,

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Int.J.Curr.Microbiol.App.Sci (2018) 7(1): 1848-1877 with biovars 1 and 3 being the ones isolated infected animals are silent carriers of the most frequently in small ruminants in the disease. It is therefore recommended that Mediterranean, the Middle East and Latin infected females and their offspring be culled America. Brucellosis is a barrier to trade in as part of an eradication programme in animals and animal products and causes infected herds (Banai, 2007). The exact significant losses from abortion, as well as mechanism enabling latent Brucella infection from being a serious zoonosis. Goats are the to develop is unknown (Blasco and Molina- classic and natural hosts of B. melitensis and, Flores, 2011). Some female hoggets testing together with sheep, are its preferred host. In seropositive to Brucellosis have been found to pathological and epidemiological terms, B. shed B. melitensis in milk postpartum, melitensis infection in small ruminants is whereas others do not shed Brucellae despite similar to B. abortus infection in cattle: the being infected. While lambs sampled for main clinical manifestations of brucellosis in seven months showed seropositivity, some ruminants are abortions and stillbirths, which tested seronegative for Brucellosis in routine usually occur in the last third of the pregnancy tests even though a post-mortem study later following infection and usually only once in revealed them to have been infected with B. the animal’s lifetime (Blasco and Molina- melitensis. This was also observed in lambs Flores, 2011). from the same herd that had been born from mothers seronegative for Brucellosis Healthy animals can be exposed to Brucella (Godfroid et al., 2011). B. melitensis was infection in many ways, as a large number of successfully isolated from the vaginal bacteria are shed in the birth fluids or fetus, discharge of a goat that had aborted but tested placenta and abortion secretions of infected seronegative for Brucellosis, making the females. The bacteria have the ability to animal a potential risk for spread undetectable survive several months outdoors, especially in by serological diagnosis (Herrera et al., 2011). cold, wet conditions, where they remain While orchitis and epididymitis are infectious to other animals, mainly through uncommon in rams and billy goats, they do ingestion. Brucellae also colonise the udder occur (Chand et al., 2002). Brucella melitensis and contaminate milk (Banai, 2007). Although can infect not only cattle but also calves, females calve apparently normally in through the ingestion of infected milk (Verger pregnancies following the first abortion, they et al., 1989). continue to shed large numbers of bacteria into the environment. As with B. abortus The isolation of B. melitensis in dogs has been infection in cows, B. melitensis can be demonstrated and this has been observed to transmitted congenitally in utero but only a favour incidence of the disease, as dogs can small proportion of lambs and kids are drag placentas or aborted fetuses to uninfected infected in this way and most latent infections areas (Hinić et al., 2012). B. melitensis biovars of B. melitensis are probably acquired by 1 and 3 are the most frequently isolated in ingesting colostrum or milk (Grillo et al., Mediterranean countries. In most 1997). circumstances, the primary excretion sources are foetal fluids and vaginal discharges after Despite the low transmission rate, the abortion or full-term parturition. Then, existence of such latent infections makes it excretion of B. melitensis is important in milk even more difficult to eradicate the disease and is also common in semen. As it happens in because, as the bacteria persist without cattle, B. melitensis can be transmitted inducing detectable immune response, congenitally from ewes to lambs, the animals

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Int.J.Curr.Microbiol.App.Sci (2018) 7(1): 1848-1877 remain infected but seronegative. However, a low, particularly when it is below freezing. majority of latent infections are probably Cattle and other species can be infected with acquired through colostrum or milk (Grillo et B. melitensis after contact with infected sheep al., 1997). and goats (Tibary et al., 2006). It has not been established whether cattle can maintain this Transmission of B. melitensis species indefinitely in the absence of contact with small ruminants. Cattle with infected In animals, B. melitensis is usually transmitted udders can shed B. melitensis in the milk for by contact with the placenta, fetus, fetal fluids months or years. Camels also shed this and vaginal discharges from infected animals. organism in milk. Humans usually become Small ruminants are infectious after either infected by ingesting organisms (including abortion or full-term parturition. Goats usually contaminated, unpasteurized dairy products) shed B. melitensis in vaginal discharges for at or by the contamination of mucous least 2 to 3 months, but shedding usually ends membranes and abraded skin (Sauret and within three weeks in sheep. This organism Vilissova, 2002). Incubation period i.e., the can also be found in the milk and semen; period between infection and abortion or other shedding in milk and semen can be prolonged reproductive signs is variable. or lifelong, particularly in goats. Kids and lambs that nurse from infected dams may shed B. melitensis is highly pathogenic for humans; B. melitensis in the feces. this organism is considered to be the most severe human pathogen in the genus. Most animals become infected by ingestion or Occupational exposure is seen in laboratory through the mucous membranes of the workers, farmers, veterinarians and others oropharynx, upper respiratory tract and who contact infected animals or tissues conjunctiva, but Brucella can also be (Yongqun, 2012). Brucellosis is one of the transmitted through broken skin. Although the most easily acquired laboratory infections. mammary gland is usually colonized during People who do not work with animals or the course of an infection, it can also be tissues usually become infected by ingesting infected by direct contact, with subsequent unpasteurized dairy products. The Rev-1 B. shedding of the organisms in the milk. In melitensis vaccine is also pathogenic for utero infections also occur. Transmission humans and must be handled with caution to during breeding is possible, but seems to be avoid accidental injection or contamination of uncommon during natural mating. B. mucous membranes or abraded skin. melitensis can be spread on fomites, and could Asymptomatic infections can occur in be disseminated mechanically by carnivores humans. In symptomatic cases, the disease is that carry away infected material (Moreno et extremely variable and the clinical signs may al., 2002). appear insidiously or abruptly.

In conditions of high humidity, low Clinical signs in affected sheep and goats temperatures and no sunlight, Brucella can remain viable for several months in water, The predominant symptoms in naturally aborted fetuses, manure, wool, hay, equipment infected sheep and goats are abortions, and clothes. Brucella species can withstand stillbirths and the birth of weak offspring. drying, particularly when organic material is Animals that abort may retain the placenta. present, and can survive in dust and soil. Sheep and goats usually abort only once, but Survival is longer when the temperature is reinvasion of the uterus and shedding of

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Int.J.Curr.Microbiol.App.Sci (2018) 7(1): 1848-1877 organisms can occur during subsequent minimum of 10% (Tibary et al., 2006). pregnancies (Ashraf et al., 2015). Some Fertility in males can be permanently infected animals carry the pregnancy to term, impaired. Deaths are rare except in the fetus. but shed the organism. Milk yield is significantly reduced in animals that abort, as Post mortem lesions well as in animals whose udder becomes infected after a normal birth. However, At necropsy, granulomatous inflammatory clinical signs of mastitis are uncommon. lesions may be present in the reproductive Acute orchitis and epididymitis can occur in tract, udder, supramammary lymph nodes, males, and may result in infertility. Arthritis is other lymphoid tissues, and sometimes in the seen occasionally in both sexes (Nicoletti, joints and synovial membranes. Necrotizing 2013). Many non-pregnant sheep and goats orchitis, epididymitis, seminal vesiculitis and remain asymptomatic. B. melitensis has also prostatitis have been reported. The fetus may been associated with abortions in cattle, and be autolyzed, normal or have an excess of abortions, orchitis and epididymitis in camels. blood stained fluid in the body cavities and an In dogs, infection with B. melitensis is often enlarged spleen and liver. Placentitis, with asymptomatic, and rapid elimination of this edema and / or necrosis of the cotyledons and organism has been reported. However, a thickened and leathery intercotyledonary abortion, orchitis and epididymitis, and other region can be seen. These lesions are not symptoms of canine Brucellosis can also occur pathognomonic for Brucellosis (Xavier et al., (Dénes and Glávits, 1994). 2009).

Morbidity and mortality Control of B. melitensis

B. melitensis is a significant problem in small B. melitensis is most likely to be introduced ruminants, particularly in developing nations into a herd by an infected animal. Semen where infections can be widespread. The could also be a source of infection. This relative importance of B. melitensis for sheep organism can be eradicated from a herd by test and goats varies with the geographic region, and slaughter procedures, or by depopulation. and can be influenced by husbandry practices In areas where B. melitensis is not endemic, and the susceptibility of sheep breeds in the infected herds are usually quarantined and the region. Management practices and animals are euthanized. Because dogs can be environmental conditions significantly infected, some countries require that shepherd influence the spread of infection. Lambing or dogs also be euthanized, or treated with kidding in dark, crowded enclosures favors the antibiotics and castrated, when flocks are spread of the organism, while open air depopulated (Sauret and Vilissova, 2002). parturition in a dry environment results in Any area exposed to infected animals and decreased transmission (Yilma et al., 2016). their discharges should be thoroughly cleaned The abortion rate is high when B. melitensis and disinfected. Infections in other species are enters a previously unexposed and generally prevented by controlling B. unvaccinated flock or herd, but much lower in melitensis in sheep and goats. flocks where this disease is enzootic. Ruminants usually abort only during the The B. melitensis Rev1 vaccine is used to gestation when they are first infected. control this disease in infected areas. Rev 1 Inflammatory changes in infected mammary can cause abortions in pregnant animals glands usually reduce milk yield by a (Blasco, 1997). This vaccine also interferes

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Int.J.Curr.Microbiol.App.Sci (2018) 7(1): 1848-1877 with serological tests, particularly when it is mortality in lambs. Ovine epididymitis is injected subcutaneously, but conjunctival caused by Brucella ovis, a Gram-negative administration to lambs and kids between the coccobacillus or short rod. This organism is a ages of 3 and 6 months minimizes this facultative intracellular pathogen. Species problem. affected with B. ovis infects sheep, as well as farmed red deer (Odocoileus virginianus) in Brucella species are readily killed by most New Zealand (Nicoletti, 2013). Experimental commonly available disinfectants including infections have been reported in goats and hypochlorite solutions, 70% ethanol, cattle, but there is no evidence that these isopropanol, iodophores, phenolic species are infected in nature. disinfectants, formaldehyde, glutaraldehyde and xylene; however, organic matter and low Global distribution and transmission of B. temperatures decrease the efficacy of ovis disinfectants. Brucella on contaminated surfaces gets destroyed by disinfectants that B. ovis has been reported from Australia, New include the following components like 2.5% Zealand, North and South America, South sodium hypochlorite, 2-3% caustic soda, 20% Africa, and many countries in Europe. It freshly slaked lime suspension, or 2% probably occurs in most sheep-raising regions formaldehyde solution (all tested for one of the world (Alton and Forsyth, 1996). B. hour). Ethanol, isopropanol, iodophores, ovis is often transmitted from ram to ram by substituted phenols or diluted hypochlorite passive venereal transmission via ewes. Ewes solutions can be used on contaminated skin. can carry this organism in the vagina for at Alkyl quaternary ammonium compounds are least two months and act as mechanical not recommended for this purpose (Adel et al., vectors. Some ewes become infected, and shed 2015). Autoclaving [moist heat of 121°C for at B. ovis in vaginal discharges and milk. Rams least 15 minutes] can be used to destroy often become persistently infected, and many Brucella species on contaminated equipment. of these animals shed B. ovis intermittently in These organisms can also be inactivated by the semen for 2 to 4 years or longer. B. ovis dry heat [160-170°C for at least 1 hour]. can also be transmitted by direct non-venereal Boiling for 10 minutes is usually effective for contact between rams (Cutler et al., 2005). liquids. Xylene (1ml/liter) and Calcium cyanamide (20 kg/m3) are reported to Ram-to-ram transmission is poorly understood decontaminate liquid manure after 2 to 4 and may occur by a variety of routes, weeks. Brucella species can also be including oral transmission. Shedding has inactivated by gamma irradiation (e.g. in been demonstrated in the urine as well as in colostrum) and pasteurization (Yantorno et al., semen and genital secretions. Red deer can be 1978). infected by venereal transmission, direct contact between infected stags, and Brucella ovis experimentally by the intravenous, conjunctival, nasal and rectal routes (Ridler et Brucella ovis is an economically important al., 2002). Similarly to rams, infected stags cause of epididymitis, orchitis and impaired shed B. ovis in semen; however, most stags fertility in rams. Similar symptoms have been eliminate the infection within a year and do reported in male red deer in New Zealand. B. not seem to transmit the organism long term ovis is occasionally associated with abortion in (Cerri et al., 2002). B. ovis has been found in ewes, and can cause increased perinatal the urinary bladder and kidneys of infected

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Int.J.Curr.Microbiol.App.Sci (2018) 7(1): 1848-1877 stags. Contamination of pastures does not vary from a slight enlargement of the seem to be an important method of epididymis to large indurations. Epididymal transmission for B. ovis. enlargement can be unilateral or bilateral, and the tail is affected more often than the head or Pathogenesis of B. ovis body. Spermatoceles containing partially inspissated spermatic fluid may be found in In experimentally infected rams, clinically the epididymis. Fibrous atrophy can occur in detectable lesions become apparent from 3 the testis. The tunica vaginalis is often weeks to 8 weeks after inoculation. B. ovis can thickened and fibrous, and can have extensive cause epididymitis, orchitis and impaired adhesions. Placentitis may be observed in fertility in rams. Initially, only poor quality ewes (Poester et al., 2013). semen may be seen; sperm motility and concentration may be decreased, and Control of B. ovis individual sperms are often abnormal. Later, palpable lesions may occur in the epididymis B. ovis is generally introduced into a flock by and scrotum. Epididymitis may be unilateral infected animals or semen (Ridler et al., or, occasionally, bilateral. The testes may 2002). The prevalence of infection can be atrophy. Palpable lesions are often permanent, reduced by examining rams before the although they are transient in a few cases. breeding season and culling rams with Some rams shed B. ovis for long periods palpable abnormalities. A commercial killed without clinically apparent lesions (Poester et B. ovis vaccine is used in New Zealand. In al., 2013). B. ovis can also cause abortions and other countries, weaner rams may be placentitis in ewes, but this appears to be vaccinated with the B. melitensis Rev-1 uncommon. Infected ewes may give birth to vaccine (Adosinda et al., 2015). weak lambs that die soon after birth. Systemic signs are rare in adult ewes and rams (Xavier Vaccination is not practiced in the U.S. et al., 2010). Antibiotic treatment has been used successfully in some valuable rams, but it is Morbidity and Mortality usually not economically feasible for most animals. Fertility may remain low even if the Approximately 30-50% of all infected rams organism is eliminated. Infections in ewes are have palpable lesions of the epididymis. B. generally prevented by controlling infections ovis has little effect on sperm quality in some in rams. B. ovis has been eradicated from individual animals, but causes severe sheep flocks in the Falkland Islands, as well as decreases in sperm motility, concentration and some individual flocks in New Zealand by test morphology in others. Estimates of the and removal methods directed at rams (Lim abortion rate in ewes and perinatal mortality and Rickman 2004). vary. Some sources report rates of 1% to 2%, while others suggest that these outcomes are Antigenic components of Brucella rare. Limited experimental studies have reported abortion rates from 0% to 8%. Even though a number of antigenic components of Brucella have been Post mortem lesions characterized, the antigen that dominates the antibody response is the lipopolysaccharide Lesions are mainly found in the epididymis, (LPS). The difference in linkage influences tunica vaginalis and testis in rams. The lesions the shape of the LPS epitopes. The specificity

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Int.J.Curr.Microbiol.App.Sci (2018) 7(1): 1848-1877 of the R-LPS is, therefore largely determined fixation test (CFT) are the most widely used by the core polysaccharide. Numerous outer tests for the serological diagnosis of sheep and and inner membrane, cytoplasmic and goats Brucellosis (Farina, 1985; MacMillan, periplasmic protein antigens have also been 1990); they are also the official tests for characterized. Some are recognized by the international trade (European Commission, immune system during infection and are 2001). The antigenic suspensions (whole cells) potentially useful in diagnostic tests. Recently, used in both tests are made with an A- ribosomal proteins have re-emerged as dominant B. abortus biovar 1 (Alton et al., immunologically important components 1988) and, theoretically, infections due to M- (Corbel, 1976). L7/L12 ribosomal proteins are dominant strains (i.e. B. melitensis biovar 1) important in stimulating cell mediated could be misdiagnosed (Alton et al., 1988; responses (Oliveira and Splitter, 1994). MacMillan, 1990). However, existence of a Brucella outer membrane protein was also common (C) epitope in the immunodominant found to induce lymphocyte proliferation and S-LPS can account for the high sensitivity of strong delayed type hypersensitivity reaction the B. abortus biovar 1 antigens to detect B. in infected cattle (Winter, 1987). melitensis biovar 1 infections and vice-versa (MacMillan, 1990; Dıaz-Aparicio et al., Antigens of diagnostic significance 1993). In fact, no significant differences have been found in the sensitivity of the classical B. Due to the high cost and low individual abortus 1 RBT antigen (AC) between ovine sensitivity of culture and PCR techniques, the populations infected either with B. melitensis indirect diagnosis of disease is recommended biovar 1 (MC) or 3 (AMC) (Blasco et al., for large-scale surveillance and/or eradication 1994a). Moreover, the indirect ELISA purposes. Detection of antibodies (and at a (iELISA) sensitivity in sheep, goats and cattle lesser degree the measure of the cell mediated is not affected by the epitopic composition immunity) against relevant Brucella epitopes (AC or MC) of the antigens used (Alonso- is the more practical approach. However, Urmeneta et al., 1998). precise antigens and adequate tests have to be used for a proper efficacy and reliability. There is limited information on the value of Particularly relevant is the problem of the outer membrane (OMP) and inner cytoplasmic specificity of serological tests since antibodies proteins for diagnosis of Brucellosis in sheep against Brucella epitopes may be present in and goats. The immunoelectrophoretical the animal population due to vaccination and / pattern of cytoplasmic proteins, considered or of contacts with other Gram-negative specific for the genus Brucella (Dıaz and bacteria (mainly, Yersinia enterocolitica O:9) Bosseray, 1974) shows little differences sharing cross-reactive epitopes with Brucella. between the several Brucella species when assayed with polyclonal sera (Dıaz et al., There is no agreement on what should be the 1967; Dıaz et al., 1968). The cytoplasmic nature and characteristics of a universal antigens have been reported to be sensitive antigen for diagnosing Brucellosis. One of the and specific enough when used in most critical and controversial points precipitation tests (Dıaz-Aparicio et al., 1994). concerning serological diagnosis of B. In contrast, when used in iELISA, there was melitensis infection in small ruminants is high background IgG reactivity in sera from related to which Brucella species and biovars Brucella free animals (D´ıaz-Aparicio et al., are used in production of antigens. The Rose 1994; Debbarh et al., 1996a). An important Bengal Test (RBT) and the complement drawback of tests using uncharacterised

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Int.J.Curr.Microbiol.App.Sci (2018) 7(1): 1848-1877 cytosolic proteins is the lack of specificity antimicrobial peptides, and inflammatory when testing Rev-1 vaccinated animals, cytokines orchestrated and regulated by host although a partially purified soluble protein of genome (Adams and Schutta 2010). Brucellae 26 kDa (CP26) from the cytosoluble protein usually enter the body via the oral route and extract (CPE) of B. elitensis has been reported lodge in the mucosa, where the bacteria are as specific when used in an iELISA. However, ingested by professional phagocytes beneath this test is significantly less sensitive than both the sub-mucosa. Once internalized, Brucella is RBT and CFT tests to diagnose infected ewes localised in a vacuole that matures from an (Debbarh et al., 1996a). A competitive ELISA early to a late endosome and, unless (cELISA) using CPE extracts and some of the destroyed, goes on to multiply in the above monoclonal antibodies improved endoplasmic reticulum of macrophages. sensitivity in infected sheep, with no antibody However, not all Brucellae survive: where responses being detected in Rev-1 vaccinated bacteria are not sufficiently numerous and the animals (Debbarh et al., 1996b). Several animal has a competent immune system, they authors have attempted to identify the main are directed towards the lysosomes where they specificities of the antibody response to OMP are destroyed and the major histocompatibility extracts of B. melitensis by using either complex on the cell surface presents the immunoblotting or cELISAs with specific peptides to Th1 and Th2 lymphocytes to elicit monoclonal antibodies (Zygmunt et al., 1994a, an immune response. b; Hemmen et al., 1995; Debbarh et al., 1996b). However, while OMPs of 10, 16, 19, The B-lymphocytes govern the humoral arm 25–27 and 31–34 kDa were found suitable as of adaptive immunity characterized by potential antigens by immunoblotting or production of antigen specific antibodies. The ELISA, the antibody responses detected in role of humoral immunity against intracellular infected sheep were scanty and heterogeneous bacterial infections is limited and not (Zygmunt et al., 1994). protective. Antibody mediated opsonization by immunoglobulins (IgM, IgG1, IgG2a and Immune response to Brucella IgG3) enhances phagocytic uptake of bacteria, limiting the level of initial infection with Immune response of host to Brucella infection Brucella, but has little effect on the is mediated through both humoral and cell intracellular course of Brucella infection mediated immunity. It is mentioned that (Bellaire et al., 2005; Baldwin and Goenka, immune response mechanisms may diverge 2006). From a clinical perspective, detection and they are dependent on the host and the of antibodies against Brucella LPS is species or strain of Brucella. Adaptive commonly used for the diagnosis of immunity expands after the activation of Brucellosis (using seroagglutination tests) in innate immunity in order to mount and sustain livestock and humans. Absence of B cells is an antigen-specific response aimed at associated with marked antibody independent eradicating bacteria and protecting the host. resistance to Brucella (Al Dahouk, 2003). Brucella antigens induce the production of T Natural or innate resistance mechanisms helper type 1 (Th1) cytokines and an adequate include the complex of host cell surface Th1 immune response is critical for the receptors for Brucella pathogen associated clearance of Brucella infection. Skendros and molecular patterns, factors mediating effective Boura (2013) clearly demonstrated the Th1 macrophage and dendritic cell maturation and nature of adaptive immunity in Brucellosis. activation, carbohydrate binding proteins, Studies on experimental and human

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Brucellosis indicate that interferon-γ (IFNγ) is response of the animal to other the principal cytokine active against Brucella microorganisms which share epitopes with infection. Mackaness (1964) confirmed cell Brucella species OPS. False negative reactions mediated immunity in Brucellosis suggesting occur in acidified antigens, especially in the important role of interaction between T RBPT. The classes of antibodies involved in lymphocytes and macrophages in defense the two tests RBPT and ELISA are different. against intracellular pathogens. Brucella has Even differences in sensitivities of C-ELISA developed sophisticated mechanisms in order and I-ELISA have been reported (Mythili et to escape from cellular immunity and to al., 2011). Furthermore, the prevalence rate of achieve the intracellular persistence. Brucella the disease would vary among various reports can even cause asymptomatic latent-disease because variations occur in different and late reactivation (Ogredici et al., 2010). populations on account of differences in breeds, animal husbandry and management Cross-reactions and false positive test results practices and preventive and control measures can occur in Brucella antibody tests. The adopted. primary immunodeterminant and virulence factor for Brucella species is the cell wall Common serodiagnostic tests for brucellosis surface lipopolysaccharide, which is antigenically similar to the lipopolysaccharide The most incontrovertible diagnosis of of other gram-negative rods. False positive Brucellosis is made by bacteriological Brucella antibody test results can be caused by isolation which has draw-backs like low cross-reactivity of antibodies to Escherichia sensitivity, time consuming and cumbersome. coli O157, Francisella tularensis, Moraxella At times, isolation is not possible even from phenylpyruvica, Yersinia enterocolitica, and known positive cases (Seleem et al., 2010) certain Salmonella species (Corbel, 1997). because of many factors like slow growth and Most cross-reacting antibodies are IgM poor sensitivity. The low sensitivity for (Corbel, 1985), making interpretation of any isolation is attributed to many factors like the IgM assay difficult because of false positivity. individual laboratory practices, quantity of pathogen in clinical samples, stage of Although the International Office of infection, use of antibiotics before diagnoses Epizootics has recommended the RBPT as one and the methods used for culturing and the of the tests for the diagnosis of bovine cultured strain (B. melitensis is more readily brucellosis, some authors (Saravi et al., 1990) cultured from clinical sample than B. abortus). have reported unacceptable rate of false As a result, recourse is taken in serological negatives with the RBPT. The sensitivity of tests. A large number of serological tests are RBPT is low, so chances of getting a false available for diagnosis of Brucellosis of which positive outcome are more when compared to RBPT, STAT and CFT have been used ELISA. False positive results are a major extensively to diagnose Brucellosis in animals. problem which makes serological diagnosis of brucellosis difficult in some cases. Virtually The conventional serological tests for all serological tests for antibody to smooth diagnosis of Brucellosis have their limitations. Brucella species use LPS, part of LPS or It has been shown that culture positive animals whole cells as antigens. The immunodominant were negative in STAT, RBPT and CFT epitope on the surface of the smooth cell is (Sutherland et al., 1986). On the other hand, OPS the outer most portion of LPS. Most, but culture positive animals negative by RBPT not all, of the problems arise from an immune and CFT have been found positive by I-

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ELISA (Araj, 1989). I-ELISA has been test in the diagnosis of brucellosis (Oomen reported to be highly sensitive and specific and Waghela, 1974). The sensitivity of RBPT and it can be used for the determination of is reported to be very high (>99%) but the specific IgG, IgM and IgA Brucella antibodies specificity can be disappointingly low in blood, serum and CSF (Nielsen et al., (Barroso et al., 2002). As a result, the positive 1996). Further, I-ELISA has been considered predictive value of the test is low and a as gold standard test by many workers to positive test result thus requires confirmation compare the results of other tests in case of by a more specific test (Smits and Kadri, Brucellosis (Isloor et al., 1998). The speed and 2005). Gram negative bacteria such as ease with which the I-ELISA can be Yersinia enterocolitica, Vibrio cholera, performed, the high sensitivity and specificity Campylobacter fetus, Bordetella values and the use of an easily obtainable bronchiseptica and Salmonella spp. may cross antigen make the indirect I-ELISA an react with smooth Brucella spp. (Corbel and excellent test for the diagnosis of Brucellosis. Brinley-Morgan, 1984). STAT measures the total amount of agglutinating antibodies (IgM and IgG). IgG Despite the scanty and sometimes conflicting antibodies are considered better indicators of available information (Fensterbank and an active infection than IgM. Maquere, 1978; Farina, 1985; Alton, 1990b; MacMillan, 1990; Blasco et al., 1994a, b), this Serodiagnosis of brucellosis in sheep and test is internationally acknowledged as the test goats of choice for the screening of Brucellosis in small ruminants (Garin-Bastuji and Blasco, It is widely assumed that serological tests used 2004). for B. abortus infection in cattle are also adequate for diagnosis of B. melitensis However, standardization conditions suitable infection in small ruminants. Accordingly, for diagnosing cattle infection (MacMillan, RBT and CFT are the most widely used tests 1990; European Commission, 2001; Garin- for the serological diagnosis of Brucellosis in Bastuji and Blasco, 2004) are not adequate in ruminants (Farina, 1985; Alton, 1990a; sheep and goats (Blasco et al., 1994a, b) and MacMillan, 1990). Although the Rev-1 account for the low sensitivity of RBT vaccine is an essential tool to control small antigens in small ruminants (Blasco et al., ruminants Brucellosis, when applied under 1994a; Falade, 1978); along with the fact that standard conditions (i.e. full dose via the a high proportion of animals in infected areas subcutaneous route), it induces long lasting give results negative in RBT, but positive in serological responses that interfere with CFT, question the efficacy of the present RBT subsequent serological screening (Alton, as an individual test (Blasco et al., 1994a). 1990a; MacMillan, 1990). This problem impedes combined use of vaccination and test Standard tube agglutination test and slaughter programmes for eradicating Brucellosis. The Standard tube agglutination test (STAT) (Fig. 2) detects antibodies to the S-LPS. Rose Bengal Plate Test (RBPT) Antibodies reacting against S-LPS can also be detected by other tests, such as ELISA The Rose Bengal Plate Test (RBPT) (Fig. 1) is (enzyme-linked immunosorbent assay) and the often used as a rapid screening test (Ruiz- Coombs test. Mesa et al., 2005) and considered as a reliable

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Brucella microagglutination test association of the conjunctival vaccination procedure and the presence of a moderate CDC utilizes a test called the Brucella interval after vaccination minimize or microagglutination test (BMAT) (Fig. 3), a abrogate the specificity problems. A similar modified version of the serum (tube) indirect technique has been also proposed for agglutination test (SAT), that can detect diagnosing sheep Brucellosis in individual or antibodies to Brucella species - pooled milk samples (Alonso-Urmeneta et al., abortus, melitensis or suis. 1998), but the test lacks sensitivity when compared with serological tests. Enzyme linked immunosorbent assays (ELISA) A highly immunogenic periplasmic protein from B. abortus (Rossetti et al., 1996) or B. Good diagnostic results have been obtained in melitensis (Cloeckaert et al., 1996a) has been sheep and goats with iELISA or, at a lesser applied to Brucellosis diagnosis in different degree, cELISA using various antigens, but host species. Indirect and competitive ELISA generally those with a high content of smooth with this antigen could be sensitive and lipopolysaccharide (LPS) are the most reliable specific tests for diagnosing B. melitensis (Fig. 4). These ELISA provide similar or infection in sheep and have been reported to better sensitivity than both RBT and CFT, but be useful in differentiating Rev-1 vaccinated like classical tests, ELISA are unable to from infected animals (Debbarh et al., 1995, differentiate infected animals from animals 1996b; Cloeckaert et al., 1996b). All these recently vaccinated with the Rev-1 vaccine ELISA have potential advantages in (Jimenez´ de Bagu¨es´ et al., 1992; Blasco et sensitivity and specificity with respect to both al., 1994b; D´ıaz-Aparicio et al., 1994; RBT and CFT, but their diagnostic efficacy at Delgado et al., 1995; Ficapal et al., 1995; large scale is unknown and a great deal of Ferreira et al., 2003) or animals infected with standardisation work is still required (Garin- cross-reacting bacteria. However, the Bastuji and Blasco, 2004).

Fig.1 RBPT testing of sera from animals suspected of Brucellosis

Left: Brucellosis positive serum; Right: Brucellosis negative serum

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Fig.2 Standard Tube Agglutination Test

Left: Tube with matt formation (positive); Right: button formation (negative)

Fig.3 Microtiter agglutination test showing negative (button) and positive (matt) wells

Fig.4 ELISA plate showing positive (colorless) and negative (yellow) wells

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Sensitivity and specificity of various hand, MacMillan (1990) reported that TAT diagnostic tests failed to show significant titres in recent and chronic Brucella infection. While Corbel Different diagnostic tests have been validated (1972) reported that TAT gives false positive for diagnosing Brucellosis in small ruminants, reaction as a results of cross reaction between but only the Rose Bengal test (RBT) and the the antigen of Brucella and other organisms complement fixation test (CFT) are approved or due to the presence of non-specific for diagnosis of small ruminant Brucellosis in agglutinins in bovine sera. The presence of the European Union (EU) legislation on intra- some samples collected from non-reactors Community trade (Council Directive (Brucella free cows) reacted positively with 91/68/EEC). However, there is evidence that RBPT, BAPT and TAT. This may be both tests are less sensitive and specific for attributed to the presence of some bacteria as the diagnosis of Brucellosis in sheep and Escherichia coli, Salmonella Dublin, Yesinia goats than in cattle (Blasco et al., 1994b; enterocolitica 0:9 and Pasteurella tularensis Garin-Bastuji et al., 1998). in the body fluids and secretions which react positively with the tests used in diagnosis of According to Biancifiori et al., (2000), the Brucellosis causing faults or error in the cELISA has a diagnostic sensitivity (99.4%) interpretation of the results. and specificity (98.9%) in sheep and goats comparable to that of many standard indirect The FPA has been reported to be the test with ELISA methods. In addition, the test proved the smallest sensitivity and iELISA that with able to distinguish between vaccinated and the smallest specificity. iELISA was found to infected animals with an accuracy of up to be the most sensitive test, whilst cELISA the 90% and result reproducibility of 93%. They most specific. The cELISA had the best and concluded that cELISA could be useful for FPA the second best positive predictive value. differentiation of Rev.1 vaccinated and cELISA had the best negative predictive naturally infected sheep and goats. value, which is expected due to the use of monoclonal antibodies. This test may be The serological tests used in previous studies ultimately used for confirming negative were the Rose Bengal Test (RBT), Standard samples, as it is not influenced by non- Tube Agglutination Test, ELISA or Plate specific reactions (Nielsen et al., 1996). Agglutination Test. None of the iELISA would be valuable for use at the early aforementioned tests are perfect. So, the stage of a control program, whilst cELISA at prevalence reported using these tests are not its final stage. Although the FPA did not true prevalence due to misclassification of appear to be as accurate, when compared to some of the tested animals. Moreover, the the other two methods when testing performance of these tests has not been vaccinated animals, it was the easier, cheaper validated in naturally infected small (use of reagents is minimal) and quicker to ruminants. Tests are normally validated by perform; further investigation should be comparing with the gold standard or perfect carried to establish its value in large scale test. However, the gold standard for the surveys, where a great number of samples diagnosis of Brucellosis is isolation and need to be tested. Furthermore, Nielsen and identification of the organism (Alton et al., Gall (2001) have reported its use in testing 1988; OIE, 2008). The TAT has a high stored whole blood samples with an almost sensitivity in respect of IgM rather than IgG 100% sensitivity and specificity; this can be as reported by Alton (1977). On the other an added advantage. The test may also be

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Int.J.Curr.Microbiol.App.Sci (2018) 7(1): 1848-1877 used in samples from all animal species, as al., 1973; Jones and Marly, 1975; Fensterbank well as in poor quality samples. et al., 1985). However, since mixed DTH- antibody mediated intradermal reactions are Cell-mediated immunity (CMI) based occasionally observed, a reading time of 72 h diagnosis seems advisable for a better assessment of true DTH reactions (Blasco et al., 1994a, b). An alternative diagnostic test is the brucellin Anergy induced by repeated skin testing, well skin test, which can be used for screening known in bovine tuberculosis (Radunz and unvaccinated flocks, provided that a purified Lepper, 1985), is not absolute in Brucellosis, (S-LPS free) and standardized antigen but the responses lessen within the 24 days preparation is used (Bhonghibhat et al., 1970; that follow a positive skin test (Blasco et al., Jones et al., 1973; Jones and Marly, 1975; 1994a, b). No information has been published Fensterbank, 1985; Blasco et al., 1994a, b). S- on the diagnostic value of in vitro CMI tests LPS (Blasco et al., 1994a, b) does not take (e.g. IFN detection) for Brucellosis of small part in DTH reactions (Jones et al., 1973; ruminants. Jones, 1974) and may provoke antibody- mediated inflammatory reactions or induce Diagnosis of B. melitensis infection in sheep antibodies that interfere with subsequent and goats serological screening. One such commercially available preparation is brucellin INRA, Clinical Brucellosis should be considered in prepared from a rough strain of B. melitensis flocks and herds when abortions and (Blasco et al., 1994 a, b). The brucellin skin stillbirths occur without concurrent illness. test has a high sensitivity and, in the absence For differential diagnosis, other diseases of vaccination, is considered one of the most causing abortion in small ruminants, specific diagnostic tests (Alton et al., 1988). particularly chlamydiosis and coxiellosis, This test is of particular value for the should be considered. B. ovis can also cause interpretation of FPSR due to infection with epididymitis and orchitis in rams. cross-reacting bacteria (FPSR animals are Microscopic examination of smears stained negative in the skin test), especially in with the Stamp's modification of the Ziehl- Brucellosis-free areas. Despite its high Neelsen method can be useful for a sensitivity, not all infected animals show presumptive diagnosis, particularly if the positive skin test responses and, moreover, direct examination is supported by serology Rev-1 vaccinated animals can react in this test (CDC, 2005). Other organisms that cause for years (Fensterbank et al., 1982; Garin- abortions such as Chlamydophila abortus and Bastuji et al., 1998; Pardon et al., 1989). Coxiella burnetii can resemble Brucella ovis, Therefore, this test cannot be recommended which causes epididymitis and orchitis in either as the sole diagnostic test or for the rams, can also be confused with B. melitensis purposes of international trade. The site and (Nicoletti, 1989; Garner et al., 2003). route of allergen inoculation are not important factors affecting its sensitivity (Fensterbank, The clinical diagnosis is complicated by 1985; Alton, 1990; Blasco et al., 1994a, b). variable incubation period and absence of apparent clinical signs, except abortion. While The method considered more efficient and isolation and culture of Brucella organisms is practical for sheep and goats is the regarded as the gold standard test for subcutaneous inoculation in the lower eyelid laboratory diagnosis of Brucellosis, its with reading 48 h after inoculation (Jones et sensitivity is low because the Brucellae are

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Int.J.Curr.Microbiol.App.Sci (2018) 7(1): 1848-1877 fastidious micro-organisms that can easily be al., 2003). A definitive diagnosis can be made overgrown by other contaminating bacteria. if B. melitensis is cultured from an animal. More importantly, the procedure is associated with high risk of infection to laboratory The vaccine strain (B. melitensis strain Rev.1) personnel (Alton et al., 1988). Therefore, can be distinguished from field strains by its serological tests are often relied upon for the growth characteristics and sensitivity to diagnosis of Brucellosis. antibiotics and other additives. Animal inoculation is uncommonly used for isolation, The tests commonly used for diagnosis of but occasionally necessary when other Brucellosis are the milk ring test (MRT), techniques fail. Guinea pigs or mice can be serum agglutination test (SAT), Rose Bengal used for this purpose. Polymerase chain Plate Test (RBPT), anti-globulin (Coombs’) reaction (PCR) techniques, PCR restriction test, 2 – mercaptoethanol test, rivanol, and the fragment length polymorphism and Southern enzyme- linked immunosorbent assay blotting are available in some laboratories (ELISA) (Morgan, 1982). The RBPT is a very (CDC, 2005). sensitive test. However, it could sometimes give a false positive result because of S19 Milk samples and vaginal swabs are vaccination or of false positive serological particularly useful for diagnosis in live sheep reactions (OIE, 2009a). Also, Gram negative and goats. B. melitensis can also be cultured bacteria such as Yersinia enterocolitica, from aborted fetuses (stomach contents, Vibrio cholera, Campylobacter fetus, spleen and lung) or the placenta (Alton et al., Bordetella bronchiseptica and Salmonella 1988). The spleen, mammary and genital spp. may cross react with smooth Brucella lymph nodes, udder and late pregnant or early spp. (Corbel and Brinley-Morgan, 1984). postparturient uterus are the most reliable samples to collect at necropsy. This organism Immunostaining is sometimes used to identify can also be cultured from semen, the testis or Brucella in smears. Serology can be used for epididymis, and arthritis or hygroma fluids. a presumptive diagnosis of brucellosis, or to screen flocks. The most commonly used Diagnosis of ovine Brucellosis serological tests in small ruminants are the buffered Brucella antigen tests (the card and B. ovis infections should be considered when Rose Bengal plate agglutination tests) and the rams develop epididymitis and testicular complement fixation test. Indirect or atrophy, or poor semen quality is seen (Ridler competitive enzyme-linked immunosorbent et al., 2002). Some but not all rams have assays (ELISAs) are also used. In vaccinated palpable lesions. For differential diagnosis, sheep and goats, the native hapten-based gel other bacteria that cause epididymitis and precipitation tests (gel diffusion or radial orchitis should be considered. Commonly immuno-diffusion tests) are sometimes used isolated organisms include Actinobacillus to distinguish vaccination from infection seminis, A. actinomycetemcomitans, (Kahn and Line, 2003). Other serological tests Histophilus ovis, Haemophilus spp., are in development or in use in research and Corynebacterium pseudotuberculosis ovis, other special situations. A brucellin allergic Chlamydophila abortus and B. melitensis, but skin test is sometimes used to test many other organisms can also cause these unvaccinated sheep and goats for B. conditions. Sterile, trauma-induced spermatic melitensis. This test is performed by injecting granulomas should also be ruled out (Webb et the allergen into the lower eyelid (Garner et al., 1980).

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Laboratory tests like microscopic examination can be made by means of the microscopic of semen or smears stained with the Stamp's examination of stained smears from vaginal modification of the Ziehl-Neelsen method can swabs, placentas or aborted foetuses (Stamp’s be useful for a presumptive diagnosis. Other method). However, morphologically related organisms such as Chlamydophila abortus organisms such as Brucella ovis, and Coxiella burnetii can resemble. A Chlamydophila abortus or Coxiella burnetii definitive diagnosis can be made if B. ovis is can cause misleading diagnoses. Therefore, cultured from an animal. Brucella spp. can be isolation of B.melitensis on appropriate isolated on a variety of plain media, or culture media is recommended for accurate selective media such as Farrell's medium or diagnosis. Thayer-Martin’s modified medium. Enrichment techniques can also be used. B. Vaginal excretion of B. melitensis is usually ovis colonies usually become visible after copious and persists several weeks after three to four days (Nicoletti, 2013). The abortion (Alton, 1990a). Thus, taking vaginal colonies are round, shiny and convex, and swabs and milk samples is the best way to approximately 0.5-2.5 mm in diameter. B. isolate B. melitensis from sheep and goats. B. ovis is a rough (R) form of Brucella; this can melitensis does not require serum or CO2 for be observed by examining the colony by growth and can be isolated on ordinary solid oblique illumination (OIE, 2004). media under aerobic conditions at 370C. Nevertheless, due to the overgrowing B. ovis can often be identified to the species contaminants usually present in field samples, level by its cultural, biochemical and selective media are needed for isolation serological characteristics, although phage purposes. The Farrell selective medium, typing can be used for definitive developed for isolation of B. abortus (Farrell, identification. Pulse-field gel electrophoresis 1974), is also recommended for B. melitensis or specific polymerase chain reaction (Alton et al., 1988). restriction fragment length polymorphism (PCR RFLP) can also distinguish B. ovis from PCR assay has been shown to be a valuable other Brucella species. Serological tests used method for detecting DNA from different to detect B. ovis include enzyme-linked microorganisms and provides a promising immunosorbent assay (ELISA), agar gel option for diagnosis of Brucellosis. Several immunodiffusion (AGID) and complement authors reported good sensitivity of PCR, fixation (Webb et al., 1980). Other tests based on different molecular markers (16S including hemagglutination inhibition and rRNA, bscp31, IS 6501/711) (Romero and indirect agglutination have also been Lopez–Goni, 1999) for detecting of Brucella described, but are less commonly used. DNA with pure cultures (Farrell, 1974). Dichelobacter nodosus, which causes foot rot, However, few studies have been performed is reported to cross-react with B. ovis in with clinical or field samples and even fewer serological assays, but the practical have evaluated the PCR as a diagnostic tool significance is unknown. B. ovis can also be (Fekete et al., 1992). The possibility of using detected by PCR. the PCR technique to detect the DNA of dead bacteria or in paucibacillary samples and even The most reliable and the only unequivocal in samples highly contaminated with other method for diagnosing animal Brucellosis is microorganisms, could increase the rate of isolation of Brucella spp. (Alton et al., 1988). detecting infected animals. However, up to The bacteriological diagnosis of B. melitensis now, no technique appears sensitive enough

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Int.J.Curr.Microbiol.App.Sci (2018) 7(1): 1848-1877 to replace classical bacteriology on all kinds mainly detect antibodies against of biological samples. Several methods, lipopolysaccharide. However, antibodies mainly PCR-RFLP and Southern-blot analysis against lipopolysaccharide are also induced in have been employed to find DNA animals vaccinated with Brucella sp. polymorphism to differentiate some Brucella attenuated strains. Therefore, an important species and biovars. Specific molecular goal in Brucellosis research is the markers have been developed for identification of protein antigens that induce distinguishing the Rev-1 strain from B. an intense antibody response during infection melitensis wild strains (Cloeckaert et al., and that are not essential for the induced 2002). Recently, a new method has been protective immunity or for survival of the described for fingerprinting Brucella isolates bacterium. Vaccination with a mutant of the based on multi-locus characterization of a vaccine strain lacking the gene coding for a variable number, 8-base pair, tandem repeat. protein of interest, in association with a The technique is highly discriminatory among serological test based on the purified protein, Brucella species or strains (Bricker et al., should allow the differentiation between 2003). vaccinated and infected animals.The most encouraging results until present have been Biomarkers of Brucella and brucellosis obtained with the Brucella sp. BP26 protein, which has been simultaneously identified by In the absence of a single accurate three nonrelated research groups as an serodiagnostic test for Brucellosis based on immunodominant antigen in infected cattle, antibody detection, efforts are on for sheep, goats, and humans (Cloeckaert et al., identifying a specific biomarker of 1996; Rossetti et al., 1996). Accordingly, the Brucellosis for correct diagnosis. The region of BP26 between amino acids 55 and biomarker is a characteristic that is 152 might provide better specificity results objectively measured and evaluated as an than the entire recombinant BP26, avoiding indicator of normal biological processes, false-positive reactions with sera pathogenic processes, or pharmacologic from Brucella-free sheep, for the serological responses to a therapeutic intervention or diagnosis by indirect ELISA of sheep other health care intervention. The biomarker Brucellosis caused by B. melitensis or B. ovis. is either produced by the diseased organ or by This region of BP26 might be either obtained the body in response to disease. Biomarkers as a synthetic peptide or purified from are potentially useful along the whole recombinant E. coli/pCP28124 as fusion spectrum of the disease process. Before protein with LacZ. diagnosis, markers could be used for screening and risk assessment. During Goldbaum et al., (1993) obtained some anti- diagnosis, markers can determine staging, cytoplasmic protein monoclonal antibodies grading, and selection of initial therapy. (MAbs) from mice immunized by infection Genetics, genomics, proteomics and modern with Brucella ovis cells. One of these MAbs, imaging techniques and other high-throughput BI24, was used to purify by immunoaffinity a technologies allow us to measure markers. protein with a pI of 5.6 and a molecular mass However, there are few studies on biomarkers of 18 kDa. This protein was present in all of in the diagnosis of Brucellosis. the rough and smooth Brucella species studied, but it could not be detected in Diagnosis of Brucellosis is currently Yersinia enterocolitica 09. Three internal performed with serological techniques that peptides of this protein were partially

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Int.J.Curr.Microbiol.App.Sci (2018) 7(1): 1848-1877 sequenced; no homology with other bacterial to purify Omp31 on a larger scale by using proteins was found. The immunogenicity of procedures such as ion exchange-, reversed the 18-kDa protein was studied with both phase-, affinity- and gel filtration human and bovine sera by a capture enzyme- chromatography suggested that the outer linked immunosorbent assay system with membrane proteins were aggregated with MAb BI24. rough lipopolysaccharide. Only denaturing SDS-gel filtration chromatography was able In another study by Manat et al., (2016) a to separate proteins of about 29 kDa from purified recombinant outer membrane protein rough lipopoly- saccharide but did not 28 (rOMP28) of Brucella species produced in separate Omp31 from Omp25 in B. ovis Escherichia coli (E. coli) was evaluated as a preparations. Futher, sheep antibodies only diagnostic antigen in an Indirect ELISA (I- reacted to LPS bound proteins and not ELISA) for bovine Brucellosis. The results purified proteins. showed that the rOMP28 of Brucella spp. could be a good candidate for improving In a study by Al-Garadi (2011) CD markers serological diagnostic methods for bovine and their combination in different populations Brucellosis. of peripheral blood mononuclear cells (PMNC) were measured in detail, in different A Brucella protein named CP28, BP26, or stages of B. melitensis infection and in Omp28 has been identified as an experimentally infected mice and goats by immunodominant antigen in infected cattle, using specific monoclonal antibody. The sheep, goats, and humans. In sheep sensitivity of RBPT was 89.04% whilst that experimentally infected with B. melitensis of CFT was 97.02%. The specificity of RBPT H38 the antibody response to BP26 was and CFT was 99.06% and 96.38%, delayed and much weaker than that to O-PS. respectively. Four B. melitensis isolates were Nevertheless, the BP26 protein appears to be obtained from 300 vaginal samples and all a good diagnostic antigen to be used in isolates belonged to B. melitensis biotype 1. confirmatory tests and for serological The real-time PCR was the easier and safer differentiation between infected and B. method for the confirmation of Brucellosis in melitensis Rev.1-vaccinated sheep. Weak goat populations. The CD biomarkers namely; antibody responses to BP26 in some of the CD14, CD4, CD25 markers were identified as latter sheep suggest that a B. melitensis Rev.1 good markers for the different stages of B. bp26 gene deletion mutant should be melitensis infection. A combination of a constructed to ensure this differentiation serological test namely RBPT and a (Cloeckaert et al., 2001). molecular technique, in particular real-time PCR based on the IS711 region of a In a study by Kittelberger et al., (1998), a hypothetical protein, showed promising panel of 45 Brucella ovis serologically results. This combination can be used to positive sera were tested in immunoblots reduce the number of false positive results, against B. ovis outer membrane proteins which can cause severe economical loss Omp31 and Omp25, purified by preparative during the implementation of eradication SDS-gel electrophoresis. Forty-three sera programs. reacted with Omp31, while only 11 reacted with Omp25, suggesting that Omp31 is In a study by Liu et al., (2012) 2D- identical to the previously reported electrophoresis and SDS-PAGE were applied immunodominant 29-kDa protein. Attempts to detect difference in plasma protein

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Int.J.Curr.Microbiol.App.Sci (2018) 7(1): 1848-1877 expression between healthy dairy cows and that differentiate human patients proven to dairy cows suffering from Brucellosis. The have acute Brucellosis from syndromically results showed that 11 protein spots were similar patients. There were 31 cross-reactive found by PDQUest 8.0 Software and 5 of antigens in healthy goats and 20 cross- them were detected by ion trap mass reactive antigens in healthy humans. Only two spectrum. Apoprotein C-III and Serum of the serodiagnostic antigens and eight of the Amyloid protein A (SAA) were acute phase cross-reactive antigens overlap between protein and lipometabolism-related protein humans and goats. Based on these results, a which can serve as the plasma biomarkers of nitrocellulose line blot containing the human Brucellosis for diagnosis. serodiagnostic antigens was fabricated and applied in a simple assay that validated the Ahmed et al., (2015) conducted a study in accuracy of the protein microarray results in which omp25, omp28 and omp31 of B. the diagnosis of human Brucellosis. These melitensis were cloned and expressed using data demonstrate that an experimentally prokaryotic pET-32 Ek/LIC system and their infected natural reservoir host produces a respective rOMPs were combined as one fundamentally different immune response coating antigen to develop rOMPs I-ELISA. than a naturally infected accidental human The production of rOMP25, rOMP28 and host. rOMP31 of B. melitensis were achieved and Western immunoblotting analysis Wareth et al., (2016) carried out a study to demonstrated their reactivity. The RBPT was identify immunodominant proteins of two unable to differentiate the vaccinated mice species of Brucella using antibodies present (group 2) and mice infected with Y. in the serum of naturally infected ruminants to enterocolitica O: 9 (group 3) and categorized gain insight on the mechanism of their them wrongly as positive for Brucellosis. In infection in different hosts. In the study, contrast, the rOMPs I-ELISA was able to whole-cell protein extracts of B. abortus and differentiate the mice infected with B. B. melitensis were separated using SDS- melitensis strain 0331 (group 1) from both PAGE and western blotting was performed group 2 and group 3, and recorded 100% using field sera from cows, buffaloes, sheep sensitivity and 100% specificity. The results and goats. Protein bands that matched with of this study suggested that rOMPs of B. western blot signals were excised, digested melitensis have potential diagnostic ability to with trypsin and subjected to protein differentiate the FPSR in serological identification using MALDI-TOF MS. diagnosis of Brucellosis. Identified proteins included heat shock proteins, enzymes, binding proteins and Liang et al., (2010) conducted a study to hypothetical proteins. Antibodies against the better understand the antibody responses that same set of antigens were found for all develop after B. melitensis infection. A species investigated, except for superoxide protein microarray was fabricated containing dismutase of B. melitensis for which 1,406 predicted B. melitensis proteins. The antibodies were demonstrated only in sheep array was probed with sera from serum. Brucellae appear to express these experimentally infected goats and naturally proteins mainly for their survival in the host infected humans from an endemic region in system during infection. Peru. The assay identified 18 antigens differentially recognized by infected and non- Rolan et al., (2008) studied the vir B proteins infected goats, and 13 serodiagnostic antigens were as potential biomarkers. The Brucella

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Int.J.Curr.Microbiol.App.Sci (2018) 7(1): 1848-1877 species type IV secretion system, encoded by B and Omar A R. 2015. Serological diagnostic the virB1-12 locus, is required for potential of recombinant outer membrane intracellular replication and persistent proteins (rOMPs) from Brucella melitensis in mouse model using indirect enzyme-linked infection in vivo. VirB proteins are expressed immunosorbent assay. BMC Veterinary in vivo and may therefore represent Research 11:275. serological markers of infection. Purified Al Dahouk S, Tomaso H, Nöckler K, Neubauer H, recombinant VirB1, VirB5, VirB11, and Frangoulidis D. 2003. Laboratory-based VirB12 were tested for their recognition by diagnosis of brucellosis - a review of the antibodies in sera from experimentally literature. Part I: Techniques for direct infected mice and goats by using an indirect detection and identification of Brucella spp. Clinical Laboratory 49(9-10): 487-505. ELISA. Antibodies to VirB12 but not to Al-Garadi M A M. 2011. Detection of infection and VirB1, VirB5, or VirB11 were detected in determination of biomarkers for Brucella 20/20 mice experimentally inoculated with melitensis infection in goats. PhD thesis, Brucella abortus and 12/12 goats Universiti Putra Malaysia. experimentally infected with Brucella Alonso-Urmeneta B, Marin C M, Aragon V, Blasco melitensis. The potential use of VirB12 as a J M, Diaz R and Moriyon I. 1998. Evaluation serological tool for the diagnosis of of lipopolysaccharides and polysaccharides of different epitopic structures in the enzyme- Brucellosis was evaluated in the natural linked immunosorbent assay for diagnosis of bovine host. One hundred two out of 145 Brucellosis in small ruminants and cattle. cattle samples (70.3%) were positive for Clinical Diagnostic and Laboratory antibodies to VirB12, while 43 samples were Immunology 5: 749–754. negative (29.7%). A positive serological Alton G G and Forsyth J R L. 1996. Brucella. In th response to VirB12 correlated with positive Baron S, editor. Medical Microbiology. 4 ed. serology to whole B. abortus antigen in 99% Churchill Livingstone, New York. Alton G G, Jones L M, Angus R D and Verger J M. of samples tested. They suggested that 1988. Techniques for the Brucellosis VirB12 may be a useful serodiagnostic Laboratory. INRA, Paris. marker for Brucellosis. Alton G G. 1977. Report to the government of the United Arab Republic on the control of References Brucellosis. FAO Report No. 1633, FAO, Rome. Adams L G and Schutta C. 2010. Natural resistance Alton G G. 1990a. Brucella melitensis. In: Nielsen K against Brucellosis: A review. The Open and Duncan J R. (eds.) Animal Brucellosis. Veterinary Science Journal 4(1): 61-71. CRC Press Inc., Boca Raton, Florida pp 383– Adel E G, Mohamed E B, Mahmoud A E H, Fatma 409. E G and Mona M E D. 2015. In vitro Alton G G. 1990b. Brucella suis. In: Nielsen K and evaluation of commonly used disinfectants and Duncan J R. (eds.) Animal Brucellosis. CRC antiseptics in Veterinary practice against Press Inc., Boca Raton, Florida, 411–422. Brucella abortus. Advances in Veterinary and Araj G F. 1989. Profiles of Brucella-specific Animal Sciences 2(4): 77-85. immunoglobulin G subclasses in sera of Adosinda M C, Pinto M L, García Díez J and patients with acute and chronic Brucellosis. Coelho A C. 2015. Impact of B. melitensis Serodiagnosis and Immunotherapy of Rev-1 vaccination on Brucellosis prevalence. Infectious Diseases 2: 401-410. Turkish Journal of Veterinary and Animal Ashraf M A, Ahmed K A, Torad F A and Marouf S Sciences 39: 261-270. A. 2015. Ultrasonographic and Agasthya A S, Isloor S and Prabhudas K. 2007. histopathological findings in rams with Brucellosis in high risk group individuals. epididymo-orchitis caused by Brucella Indian Journal of Medical Microbiology 25: melitensis. Pakistan Veterinary Journal 35(4): 28-31. 456-460. Ahmed I M, Khairani-Bejo S, Hassan L, Abdul Gani Avinash Reddy D, Kumari G, Rajagunalan S, Singh

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How to cite this article:

Neharika Saxena, Balbir Bagicha Singh and Hari Mohan Saxena. 2018. Brucellosis in Sheep and Goats and Its Serodiagnosis and Epidemiology. Int.J.Curr.Microbiol.App.Sci. 7(01): 1848- 1877. doi: https://doi.org/10.20546/ijcmas.2018.701.225

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