Isolation and Identification of Brucella Species from Dairy Cattle by Biochemical Tests: the First Report from Ethiopia
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© 2016, Scienceline Publication World’s Veterinary Journal World Vet J, 6(2): 80-88, June 25, 2016 ISSN 2322-4568 License: CC BY 4.0 Isolation and Identification of Brucella Species from Dairy Cattle by Biochemical Tests: The First Report from Ethiopia Minda Asfaw Geresu1*, Gobena Ameni2, Alehegne Wubete3, Angela M Arenas-Gamboa4 and Gezahegne Mamo Kassa5 1School of Agriculture, Animal and Range Sciences Course Team, Madda Walabu University, Bale-Robe, Ethiopia 2Aklilu Lemma Institute of Pathobiology, Addis Ababa University, Addis Ababa, Ethiopia 3National Animal Health Diagnostic and Investigation Center, Sebeta, Ethiopia 4College of Medicine, Texas A and M University, College Station, TX 77845, U.S.A. 5College of Veterinary Medicine and Agriculture, Addis Ababa University, Bishoftu, Ethiopia *Corresponding author`s Email: [email protected] ABSTRACT Isolation of Brucella organism is considered as the gold standard diagnostic method for brucellosis since it is specific and allows biotyping of the isolate, which is relevant for control of brucellosis using vaccination. Serological studies revealed that brucellosis is endemic in bovines in Ethiopia. Even though seroprevalence of brucellosis is established in different species of animals, so far there was no successful attempt to isolate and identify Brucella spp. in dairy cattle at farm level in the country. Therefore, the endeavor of the present study was to isolate Brucella spp. from seropositive cattle with a history of abortion. A total of 570 dairy cattle from 35 herds were ARTICLE ORIGINAL pii: Received: Received: screened serologically by Rose Bengal plate test based on the history of abortion in the farm. Among the tested Accepted: samples 13 (2.28%) were found positive by Rose Bengal plate test screening while 33 samples were found sero S232245681 negative upon serological screening test but were collected from the cattle with history of recent abortion. Forty six 111 clinical samples were cultured which were both from Brucella seropositive and seronegative (dairy cattle with 10 history of abortion) upon Rose Bengal plate test screening. Three (6.52%) samples were Brucella culture positive June May 6 and further characterization of all the three isolates based on biochemical tests result confirmed that the pathogen 000 201 201 was Brucella abortus. Brucella abortus was isolated from placental cotyledon 1/9 (11.1%) and vaginal swab 2/23 12 6 - 6 6 (8.69%) while no isolate was obtained from milk and fetal abomasal contents (abomasal aspirate) of aborted fetus. Our finding revealed the occurrence of B. abortus in dairy cattle of Ethiopia through isolation of the organism for the first time from seropositive dairy cattle with a history of abortion. The organisms were isolated from placental cotyledon (one isolate) and vaginal swab (two isolates) while no isolate was obtained from milk and fetal abomasal contents (abomasal aspirate) of the aborted fetus. Hence, the bacteriological isolation and identification of Brucella abortus from dairy cattle indicates the importance of brucellosis in dairy cattle industry of the area and potential public health implication for human population in the study areas. Key words: Isolation, Dairy cattle, Brucella abortus, Biochemical test, Ethiopia INTRODUCTION Brucellosis is endemic in many developing countries and is caused by Brucella species that affect man, domestic and some wild animals, and marine mammals (Bhatia and Narain, 2010; Seleem et al., 2010 and Geresu et al., 2016). An estimated 500 000 new human Brucella cases were reported annually worldwide (Pappas et al., 2006). Brucellosis is the second most important zoonosis after rabies and has gained prominence over the years since its discovery on the island of Malta (Seleem et al., 2010 and Abubakar et al., 2012). Brucellae species are gram negative cocci bacilli, which are classified into species by various techniques such as growth patterns on media and phage susceptibility. There are six “classical” recognized species; B. abortus, B. melitensis, B. suis, B. ovis, B. canis, and B. neotomae (Godfroid et al., 2005; Hadush and Pal, 2013). Recently, four new Brucella species have been recognized and classified, namely, B. pinnipedialis, B. ceti, B. microti and B. inopinata (Foster et al., 2007; Scholz et al., 2009). The mode of transmission of the bacteria varies with the epidemiological area, the animal reservoir and the occupational exposed groups (Seleem et al., 2010 and Geresu et al., 2016). Sources of infection for the transmission of the bovine brucellosis are aborted fetuses, the fetal membranes after birth, and vaginal discharges and milk from infected animals (Tolosa et al., 2010 and Geresu et al., 2016). The most common route of transmission is the gastrointestinal tract following ingestion of contaminated pasture, feed, fodder, or water, and after birth, fetuses, and newborn calves, all of 80 To cite this paper: Geresu MA, Ameni G, Wubete A, Arenas-Gamboa AM, Mamo Kassa G. 2016. Isolation and Identification of Brucella Species from Dairy Cattle by Biochemical Tests: The First Report from Ethiopia. World Vet. J. 6(2): 80-88. Journal homepage: www.wvj.science-line.com which may contain a large number of the organisms and constitute a very important source of infection. The bacteria can be transmitted to humans through direct contact with infected tissue via breaks in skin, ingestion of contaminated tissues or milk products, and inhalation or mucosal exposure to aerosolized bacteria (Radostits et al., 2007). The diagnosis of brucellosis is based on serological, bacteriological, allergic skin reaction, and molecular methods (Simsek et al., 2004). The most important confirmatory method of Brucella infection is bacteriological diagnosis since its specificity is much higher than that of other diagnostic methods and it is used as a gold standard diagnostic method. The existence of different Brucella biotypes among the Brucella spp. and their identification is important to confirm the infection and trace the source of the infection (Guler et al., 2003). Because of the complications involved in the diagnosis of the disease, including the difficulties in distinguishing between infected and vaccinated animals by conventional serological tests, bacteriological isolation and identification of biotypes of the etiological agent are necessary steps in the design of epidemiological and eradication programs (Refai, 2002; Zinstag et al., 2005). Molecular diagnostic methods are also currently being used for the detection of Brucella spp. in various samples (Şahin et al., 2008). Samples for Brucella spp. isolation from cattle include fetal membranes, particularly the placental cotyledons where the number of organisms tends to be very high. In addition, fetal organs such as the lungs, bronchial lymph nodes, spleen and liver, as well as fetal gastric contents, milk, vaginal secretions and semen are samples of choice for isolation (Poester et al., 2006 and Lage et al., 2008). Vaginal secretions should be sampled after abortion or parturition, preferably using a swab with transporter medium, allowing isolation of the organism up to six weeks post parturition or abortion (Poester et al., 2010). Milk samples should be a pool from all four mammary glands. Non pasteurized dairy products can also be sampled for isolation (Lage et al., 2008; Poester et al., 2010). Since the first report of brucellosis in the 1970s in Ethiopia, the disease has been noted as one of the important livestock diseases in the country (Ibrahim et al., 2010; Kebede et al., 2008; Geresu et al., 2016). A large number of studies on bovine have been reporting individual brucellosis seroprevalence ranging from 1.1% to 22.6% in intensive livestock management systems (Tolosa et al., 2010; Tesfaye et al., 2011) and 0.05% -15.2% in extensive management systems (Degefa et al., 2011; Megersa et al., 2011). Though serological survey for Brucella antibodies revealed that brucellosis is known to be endemic in the country, there was no successful attempt to isolate and identify Brucella spp. in dairy cattle at farm level. Therefore, the present study aimed to isolate Brucella spp. from dairy cattle with a history of abortion for the first time in Ethiopia by using standard cultural methods in order to establish an epidemiological base for studies on the control and prevention of brucellosis in the country. MATERIALS AND METHODS Study areas and design The study was conducted in two purposely selected sites in central Ethiopia, Bishoftu, East Shewa zone and Assela, East Arsi zone. These study areas were selected based on the abundance of dairy farms that constituted the known milk sheds (Land O’Lakes Inc, 2010; Geresu et al., 2016). Bishoftu is located at 47kms south east of Addis Ababa. The area is located at 9°N latitude and 40°E longitudes at an altitude of 1850 meters above sea level in the central high land of Ethiopia. It has an annual rainfall of 866mm of which 84% is in the long rainy season (June to September). The dry season extends from October to February. The mean annual maximum and minimum temperatures are 26°C and 14°C respectively, with mean relatively humidity of 61.3% (ADARDO, 2007). Farmers in the vicinity of Bishoftu town use a mixed crop and livestock farming system. Moreover, Bishoftu and its surrounding have variable and yet representative agroecologies of the country. These agroclimatic zones are inhabited with different plant and animal species (Conway and McKenzie, 2007). The second study area was Asella, which