Advances in Biological Research 9 (5): 330-337, 2015 ISSN 1992-0067 IDOSI Publications, 2015 DOI: 10.5829/idosi.abr.2015.9.5.9624 Pasteurellosis in Small Ruminants:biochemical Isolation, Characterization and Prevalence Determinationin Relation to Associated Risk Factors in Fogera Woreda, North-West Ethiopia Tewodros Alemneh Engdaw and Annania Tewodros Alemneh Faculty of Veterinary Medicine, University of Gondar, P.O. Box: 196, Gondar, Ethiopia Abstract: The study was conducted from September 2014 to August 2015 in Fogera Woreda, Amhara Regional State, North-West of Ethiopia with the objectives of isolation and characterization of pasteurella species with culture and biochemical tests,and to determine its prevalence in relation to associated risk factors in apparently pneumonic sheep and goats.out of 988 nasal swabs and blood samples from small ruminants (696 sheep and 292 goats) examined,322 were found positive to pasteurellosis with overall prevalence of 32.6%, of which 180 (55.9%) were from nasal swabs and 142 (44.1%) were from blood samples. Accordingly, 79.5% of the isolates were Mannheimia haemolytica and 20.5% were Pasteurella multocida.species, age, sex, management system and seasons of the year were found risk factors and significantly associated with pasteurella infectionin small ruminants (P<0.05).The prevalence in sheep (37.1%), female (36.42%), young (< 2 years) (52.97%) and extensively managed animals (38.15%) were found higher than the prevalence in goats (21.9%), male (25.29%), adults (= 2 years) (21.26%) and sheep and goats managed under semi-intensive production system (17.18%), respectively.similarly, the frequency of infection was significantly (p<0.05) higher in winter (48.6%) and spring (32.85%) as compared to autumn (23.79%) and summer (19.67%). In conclusion, this finding clearly indicates that the disease is highly prevalent in the study area. Thus, an integrated application of vaccination and overall management measures should be implemented to prevent and control the disease in animals. Key words: Prevalence Pasteurellosis Isolation M. haemolytica P. multocida Fogera Ethiopia INTRODUCTION Disease constraints like respiratory diseases contribute to the great financial losses and the socio- Ethiopia has diverse animal resources and its economic development of poor farmers in the area. These relatively large livestock population (approximately 100 diseases cause a huge mortality and morbidity [4]. The million) is well adapted to and distributed among diverse disease occurs in food animals due to complex factors that ecological conditions and management systems [1]. In often interact to produce disease. Various conditions Ethiopia, like many developing countries, livestock may such as climate, weather, weaning, transportation, poorly play multiple roles. Despite the huge number of cattle, ventilated housing and nutritional deficiencies are known sheep and goats and economic importance, the to play a pre-disposing role as the animal s immunity productivity is low due to the constraints of diseases, weakness. In such conditions, occurrence of the normal nutrition, poor management and poor performance of the flora of the upper respiratory tract and subsequence indigenous breeds. These constraints result in poor infection of the lungs is well documented [5]. reproductive performance of these animals [2]. On the Respiratory tract infections are of common basis of statistics acquired from different sources, occurrence in various species of domestic animals. livestock provides 16% of the total GDP (equivalent to However, pneumonic pasteurellosis, also known as 30% of agricultural GDP) and generates 14% of the respiratory mannheimiosis, is most common example with country s foreign exchange earnings [3]. a wide prevalence in ruminants. The disease in its typical Corresponding Author: Tewodros Alemneh Engdaw (Dr), Faculty of Veterinary Medicine, University of Gondar, P.O. Box: 196, Gondar, Ethiopia. Tel: +251 9 20 49 98 20. 330
clinical form, is highly infectious, often fatal and with very farfrom Addis Ababa. Woreta is the capital of Fogeraand serious economic mortality in many animals in which the it is located at 55 kms frombahir Dar (the capital city of disease accounts for approximately 30% of the total cattle Amhara Regional State) in the direction of Gondar. deaths worldwide [6]. Fogerahas an altitude range of1,774 to 2,410 meter at sea The term pasteurellosis was broadly used to level and a latitude and longitude of 11 46' to 11 59'E and designate a number of infections in domestic animals 37 33' to 37 52' N, respectively. It has a minimum and caused by Gram-negative, non-motile, facultative maximum mean annual rain fall of 1,103 to 1,336 mm. The anaerobic rods or coccobacilli formerly grouped under the daily temperature can reach 12.0 c to 27.08 c with relative genus Pasteurella (after Louis Pasteur). humidity of 22% to 80% [11].The weather condition of It is worth mentioning that M. haemolytica and Fogerais predominantly classified as Woinadega. The P.multocida constitute the most important members of the district has a total land area of 117,414 hectare, out of family Pasteurellacae that pose serious hazards in which 9, 602.36 hectare is grazing land. 76%of the zone is livestock industry. These species are commensally Flat land, 11% mountain and hills and the rest 13% is resident in the animal body as normal constituent of the valley bottom. It has bi-modal rainfall. The short rainy naso-pharyngeal microflora and are all capable of causing season being from March 15 to May and long rainy infection when the body defense mechanisms are season similar to the rest part of the country. Mixed cropimpaired. Their presence is mainly confined to ruminants livestock is the farming system in the area. Small ruminant with most adequately characterized strains originating production of the area is an integral component of the from cattle, sheep and goats [7]. traditional farming system. In this area, extensive Pasteurellamultocida is associated with hemorrhagic management system is dominant. Semi-intensive system septicemia and enzootic pneumonic complex in sheep, of production is practiced to a lesser extent [12]. goats and cattle and buffaloes [8]. Concurrent infections According to Fogera Woreda Office of Agriculture [13], of the respiratory tract by viruses, bacteria and and the total number of livestock population in the study area lungworms have been described and such disease were cattle 157,128, goats 27,867, sheep 7,607, chicken conditions are commonly known as respiratory disease 246,496, beehives 21,883, donkey 13,189, mule 339 and complex (RDC), indicating the difficulty to attribute to horse 8. In addition, Fogera woreda has Exotic breeds only one etiology [7, 9]. In the cool central highlands of which include: 22 heifers, 10 young bulls, 22 cows, 3 Ethiopia, respiratory disease complex has been identified calves and 19 improved beehives. Fogera woreda is the as leading. Irregular and insufficient vaccination program home of the Fogera Breed cattle, which is highly for diseases such as pasteurellosis and Pest des Petites productive indigenous animal in the country known for its Ruminants (PPR), lack of strategic mass drenching against milk and meat production as well as traction power. lungworms and occurrence of viral infections may play significant roles in the persistence of respiratory disease Study Population: The study animals were apparently complex in Ethiopia. pneumonicsheep and goats at Woreta city Veterinary Even though pasteurellosis is one of the most Clinic and at the field areas of Fogera kept by individual economically important infectious diseases of sheep and farmers for subsistence and reserved as a means of capital goat in Ethiopia [10], there was lack of information on this for family members. These animals were managed almost regards in South Gondar Zone, especially in the study under extensive farming system mixed with other species. area, Fogera Woreda. Hence, the study was designed to The total number of small ruminant population in the isolate and identify pasteurella species that invade the study area were 35,474 from which 7,607 sheep and 27,867 respiratory tract of sheep and goats causing pneumonic goats.all of the study animals were clinically sick and and septicemic pasteurellosis, to determine the prevalence showed pneumonic signsand were in all age groups. and to assess some of the determinant and risk factors According to Gatenby [14] (as cited in Tewodros and associated with it. Dawit [15]), the study population was grouped into two age groups as young those which are < 2 years and adult MATERIALS AND METHODS those which are= 2 years. Study Area and Population Study Area: The study was conducted on small ruminant pasteurellosis in FogeraWoreda, South Gondar Zone of Amhara Regional State; Ethiopia. Fogera is 625kms Study Design: A cross-sectional study design was conducted to determine the prevalence of small ruminant pasteurellosisin sheep and goatsshowing pneumonic signs.nasal swabs and blood samples were takenfor 331
bacteriological cultureand biochemical characterizationof appearance of colonies and the ability to ferment lactose pasteurella species beginning from September 2014 to [19]. Thirdly, pure cultures of single colony type from August 2015 in Fogera Woreda. both Blood and MacConkey agars were transferred onto nutrient agar-slants for a series of primary biochemical Sample Size: In this study, the sample size determination tests. Final identification of the bacteria to the species in random sampling for infinite population using expected level was aided by secondary biochemical tests [20, 21]. prevalence of small ruminant pasteurellosis at 5% desired If the organism is able to produce a narrow zone of absolute precision and 95% confidence interval according haemolysis on Blood agar and grow on MacConkey agar, to Thrusfield [16] wasunable to use.this is because of but unable to produce indole, interpreted as M. there is no any previous study conducted in this area on haemolytica. If the organism unable to produce pasteurellosis of ruminants. As a result, a total of 988 haemolysis on Blood agar and cannot grow on small ruminants (696 sheep and 292 goats inclusiveof all MacConkey but able to produce indole, is interpreted as Kebeles of the Woreda)with pneumonic signs were P. multocida [22, 30]. randomly selected from the clinic as well asthe field andexamined. Data Analysis: Data were entered and managed in MS Excel work sheet. The analysis was conducted using SPSS Sampling Procedures 16.0. Prevalence of pasteurellosis at animal was expressed Nasal Swab: Each animal was individually identified and as percentage with 95% confidence interval (CI) by restrained by an assistant and kept fixed. After dividing the total number of animals positive to disinfection of external part of the nose with 70% alcohol, pasteurellosis to the total number of animals examined. An a sterile cotton-tipped swab was inserted in to the nostril animal was considered as positive for pasteurellosis if it and rotated against the wall of the nasal cavity [17]. The was positive either through nasal swab or blood culture. swab was placed in labeled sterile test tube that contains The significance of differences between the prevalence of 3 ml of tryptose Soya broth and then kept in an ice box for pasteurellosis was determined using Fisher s exact test transport to Bahir Dar Regional Veterinary Laboratory when the numbers within the categories were too small for [17]. Chi-square test. Age, sex, species, management system and seasons of the year were considered as risk factors to Blood Sample: Blood samples (for direct culturing and see their association with the prevalence of pasteurellosis. biochemical tests) were taken from the jugular vein of sheep and goats aseptically.after collection,samples were RESULTS kept in an ice box for transportation to the laboratory. Overall Prevalence: Out of the total 988nasal swabs and Isolation and Identification of Pasteurella Species: The blood samplescollected and cultured (696from sheep and isolation and identification of Pasteurella were performed at the Microbiology Department of Bahir Dar Regional Veterinary Laboratory using techniques recommended by Hardy Diagnostics, Santa Maria, CA, USA. The isolation and identification involves the following steps: first, the specimen was incubated for 24 hrs at 37 c. A loop full of the broth cultures were taken and streaked over an identified Petri-plate containing blood agar base supplemented with 7% sheep blood and immediately incubated aerobically at 37 c for 24 hours [18]. Secondly, from culture positive plates, typical colonies were subjected to gram s staining to study staining reactions and cellular morphology. Mixed and gram-negative bacteria were further sub-cultured on both Blood and MacConkey agar plates [18] for further analysis. The growth of typical colonies on both Blood and MacConkey agar was characterized using Blood agar for the presence of haemolysis, the type of haemolysis, the general 292from goats);pasteurellosis was isolated from 322 samples giving an overall prevalence of 32.6% (322/988) in the population studied. Out of322 positives for pasteurellosis, 180(55.9%) were from nasal swabs and 142 (44.1%) were from blood samples (Table 1). The isolated species of pasteurellosis were M. haemolytica and P. multocidawith prevalence of 79.5%and 20.5%, respectively (Table 3). There was a significance difference on the prevalence of pasteurellosis infection between sheep and goats ( 2= 2.187, p< 0.05). The infection rate was higher in sheep (37.1%) compared to goats (21.9%) (Table 2). Among the 988small ruminants, higher prevalence (29.5%) was observed in younganimals of less than two years of age while lower prevalence (13.9%) was observed in animals of greater than two years. The difference between the prevalence was statistically significant ( 2= 6.360, p < 0.05) (Table 2). 332
Table 1: The isolation of pasteurellosis in nasal swabs and blood samples of sheep and goats: Species No of Positives for Total sample Total Isolates Nasal swabs Blood Sheep 149 109 696 258(37.1%) Goats 31 33 292 64 (21.9%) Prevalence 55.9% 44.1% 988 322(32.6%) Table 2: Prevalence of Pasteurellosis infection in small ruminants in relation to species, age, sex,management system and seasons of the year: No of Animals ----------------------------------------------------- Variable Tested Positive Prevalence (%) Chi-square ( 2) P-Value Species Sheep 696 258 37.1 2.187 p 0.05 Goats 292 64 21.9 Age Group <2 years 353 187 52.97 6.360 p 0.05 2 years 635 135 21.26 Sex of Animals Male 340 86 25.29 13.728 p 0.05 Female 648 236 36.42 Management System Extensive 726 277 38.15 Fisher s exact = 0.021 p 0.05 Semi-intensive 262 45 17.18 Season of the Year Autumn 248 59 23.79 0.428 p < 0.05 Winter 280 136 48.57 Spring 277 91 32.85 Summer 183 36 19.67 Total 988 322 32.6% Table 3: Species of Pasteurellacaeisolated by nasal swabs and blood cultures and their prevalence in small ruminants Species of Pasteurellacae Type of Sample Isolated Nasal swab Blood Total positives Prevalence (%) M. haemolytica 143 113 256 79.50 P. multocida 37 29 66 20.50 Total 988 322 32.6 M. haemolytica:mannheimia haemolytica; P. multocida: Pasteurella multocida Mannheimia haemolytica had higherinfection rate categorized under extensive and semi-intensive (79.50%) compared to that of P. multocida (20.50%). management systems. A prevalence of 38.2% was The difference in the prevalence of pasteurella species observed in animals kept under extensive management insheep and goats was statistically significant system while 17.18% in animals reared under semi- ( 2= 11.370, p< 0.05) (Table 3). intensive system. The difference between the prevalence An assessment was made on pasteurella infection in was statistically significant (Fisher s exact = 0.021, relation to sex of small ruminants and estimated for a p< 0.05) (Table 2). prevalence of 25.3%in males and 36.4% in females. There The prevalence of pasteurellosis was observed was statistically significance difference ( 2= 13.728, among the different seasons of the study period. p< 0.05) between male and female animals in susceptibility The frequency of infection was significantly ( 2 = 0.428, to pasteurellosis infection (Table 2). p< 0.05) higher in winter (48.6%) and spring (32.85%) as To see the effect of management on the prevalence compared to autumn (23.79%) and summer (19.67%), of pasteurellosis infection, sheep and goats were (Table 2). 333
DISCUSSION the prevalence of the two species might be due to the difference in grazing behavior of these species of In the present study, the overall prevalence of small ruminants. Sheep predominantly deep graze; pick up more ruminant pasteurellosis was found to be 32.6% in which bacteria so have higher exposure than goats which mostly 37.1% and 21.9% were recorded in sheep and goats, consume browse. Goats with their browsing behavior respectively.this finding coincides with Yeshwas[23] who consume uncontaminated matter with bacteria, so being reported 33.1% in Farta and Lay Gaint Districts of Amhara less exposed to infection and therefore, have lower Regional State, North-West Ethiopia.However, the current prevalence than sheep [33]. finding was lower than that of Abera et al. [24], Aschalew In this study, higher rate of infection was associated [25] and Tesfaye [26] who reported 50.2%, 63.8% and with young age groups (52.97%) of sheep and goats as 67.6%, respectively.on the other hand, the currentresult compared to adults (21.26%) (p< 0.05). This finding was slightly higher than that of Tilaye [27] who reported coincides with Maru et al. [30]. This might be due to the 28.4%. This might be due to the different ways of taking immune status of the animals being able to predispose to samples from purely pneumonic sheep and goats, the bacterial infection and other pre-disposing etiological improved health facilities, laboratory facilities, ecologyof agents [24]. Similarly, this result is also in agreement with the study areas and predisposing factors [24]. the findings of Gilmour and Gilmour [34], that elucidates Comparing the two pasteurella species, pneumonic pasteurellosis occur in all ages of sheep and M. haemolytica constituted 79.5% of the total positives goats, with the most susceptible in lambs and kids during indicated that M. haemolytica was the major causative first life and dams at lambing. agent involved in small ruminant pneumonic In the current study, a significant association pasteurellosis. Although the infection rate varies, this between pneumonic pasteurellosis and sex of apparently finding is consistent with previous reports of Abera et al. sick ovine and caprine irrespective of pasteurella species [24], Aschalew [25], Daniel et al. [28], Eshetu [29], Maru was observed. A prevalence of 25.29% and 36.42% was et al. [30], Mohammed [31], Tesfaye [26] and Asefa et al. found in male and female small ruminants, respectively. [37].Mannheimiahaemolytica, which is a normal flora of This result; however, contradicts to the findings of Maru the upper respiratory tract, may play a secondary role et al. [30] in Haramaya District who reported that sex has after the primary initiating agent suppressed the host no any association in pasteurella infection in sheep. This defense mechanism and favors the multiplication of difference in prevalence between female and male animals pasteurella species leading to bronchopneumonia in is probably due to the fact that resistance to infection is purely pneumonic animals [1]. Although the percentage abrogated at the time of parturition and during early of isolation was relatively low (20.5%), the possible role of lactation. This pre-parturient relaxation of resistance P. multocida in the etiology and pathogenesis of sheep results in the females inability to resist the inhabitant and goat pneumonia should not be under estimated [30]. bacteriawhich cause higher level of infection [35]. The Concerning to the rate of pasteurellaspecies way that males and females treated in terms of nutrition isolation in sheep and goats, it was 32.6% (322 out of may also attribute for such differences. Males are kept for 988), 258 positive isolates with recovery rate of 37.1% fattening to be sold later, except some which are kept for in sheepwhile it was 64 positive isolates with breeding, receive more attention by small ruminant recovery rate of 21.9% in goats. In comparison producers. Crop leftovers and remnants after human between sheep and goats, the isolation percentage of consumption, for instance, are provided primarily for pasteurellosiswassignificantly higher rate in sheep than males [36]. that of goats (p< 0.05). These results; however, are not In the present study, the level of prevalence was inlinewith that obtained by Rasha et al. [32] who compared between animals kept under extensive and semirecovered pasteurella species from sheep and goats with intensive management systems. The prevalence was recovery rate of 56% and 44%, respectively.this higher (38.15%) in small ruminants kept under extensive deference might be due to the difference in sampling system of production and the difference between the procedures and sample takingfrom apparently healthy and prevalence was statistically significant (p<0.05).the purely pneumonic sheep and goats. The other possible difference in the prevalence rate that were found between difference might be the epidemiological and ecological the management systems might be due to the proportion differences of the study areas. Similarly, the difference in of number of animals tested for semi-intensive system was 334
relatively small as compared to extensive system. An extensive management system (free grazing) which allows unrestricted contact between animals might also have contribution to the spread of pasteurellosis in animals in the extensive system. In case of semi-intensive management system it could be associated with better management practices like introducing sheep and goats after being vaccinated for pasteurellosis and the routine cleaning of the house may decrease the establishment of infection in the semi-intensive production system and this makes small ruminants not being exposed more by pasteurellosis [15]. In the current study, the seasonal variation of the isolates of pasteurella was also determined. The frequency of infection was significantly (p< 0.05) higher in winter (48.6%) and spring (32.85%) as compared to autumn (23.79%) and summer (19.67%) which is in agreement withrasha et al. [32]. This is due to the high rain fall of the area in winter and spring which exposes the animals to cold stress. In addition, during spring and winter there is prevailing of most parasites. These parasites predominantly affect the gastrointestinal tract and respiratory system of sheep and goats causingimmunocompromization. This in turn favors the multiplication and expansion of the normal inhabitant bacteria, pasteurella, leading to infection of the host (sheep and goats). CONCLUSIONS At the most end, pasteurellosis was the major disease of sheep and goat in the study area in which M. haemolytica is the most common cause of infection.females, young animals and extensively managed animals were the risk factors of the disease. It also demonstrated that pneumonic pasteurellosis is a highly complex multifactorial disease particularly in sheep and goats which could be associated with stress, immune compromization, adverse environmental condition, previous illness or co-infection and misuse of traditional medicines. All these and other factors made the control and prevention of pasteurellosis more difficult.measures such as, regular vaccination, rearing of sheep and goats separately and improving management practices by providing optimal sanitation and air quality in housing, minimizing transportation stress, providing good quality hay and water and supplement feeds should be taken into account to reduce the risk of the diseasein animals. ACKNOWLEDGEMENTS The Authors Gratitude to Bahir Dar Regional Veterinary Laboratory, Fogera Woreda Office of Agriculture and Rural Developments, Woreta City Office of Agriculture and Environmental Sanitation and Small ruminants producers. REFERENCES 1. Aiello, S.E. and A. May, 1998. The Merck Veterinary th Manual. 8 edition. New Jersey. USA: Merck and Co, Inc., pp: 1053-1057. 2. Lobago, F., B. Bekana, H. Gustafsso and H. Kindahl, 2006. Reproductive performance of dairy cows in small holder production system in Selalle, central Ethiopia. Trop. Anim. Prod. Health, 38: 333-342. 3. Central Statistical Agency, 2009. The Federal Democratic Republic of Ethiopia central statistical agency, agriculture in figure key findings of the 2008/9-2010/11 agricultural sample surveys for all sectors and seasons country summary, FDRECSA Addis Ababa, Ethiopia, pp: 14-18. 4. Zewdie, S., 2004. Current status of veterinary services in Ethiopia. In Proceedings of a national work shop on managing animal health constraint to export marketing of meat and livestock. 5. Radostits, O.M., C. Gay, D.C. Blood and K.W. Hinchclift, 2000. Diseases the Respiratory system. In: Veterinary Medicine, a text book of the disease of cattle, sheep, pigs, goatsand horse. th 9 ed. Saunders, London, pp: 1550-1600. 6. Baundreaux, C.M., 2004. A novel strategy of controlling bovine pneumonic pasteurellosis transfecting the upper respiratory tract of cattle with a gene coding for the antimicrobial peptide cecropin. MSc Thesis Louisianastate University, USA. 7. Biberstin, L. and C. Hirsh, 1999. Pasteurella. In: Veterinary Microbiology. Black science Inc. USA., pp: 135-139. 8. Jones, T.C., R.D. Hunt and M.W. King, 1997. Pasteurella multocida pneumonia seminar in Respiratory infections, 12: 54-56. 9. Quninn, P.J., B.K. Markey, M.E. Carter, W.J. Donnelly and F.C. Leonard, 2002. Veterinary Microbiology and Microbial Diseases. st 1 edition. Blackwell Science, USA., pp: 137-143. 335
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