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Prevalence of contagious caprine pleuropneumonia in the Borana pastoral areas of Ethiopia
Small Ruminant Research 70 (2007) 131–135
Prevalence of contagious caprine pleuropneumonia in the Borana pastoral areas of Ethiopia A. Gelagay a,∗ , S. Teshale b , W. Amsalu c , G. Esayas a b
a National Veterinary Institute (NVI), P.O. Box 19, Debre Zeit, Ethiopia Faculty of Veterinary Medicine, Addis Ababa University, P.O. Box 34, Debre Zeit, Ethiopia c Hashim Export Abattoir, Debre Zeit, Ethiopia
Received 12 April 2005; received in revised form 18 October 2005; accepted 3 February 2006 Available online 29 March 2006
Abstract The study was conducted in selected districts of Borana pastoral areas of Ethiopia, namely Yabello, Dire, Moyale and Liban, during June–September 2004 to determine the status of contagious caprine pleuropneumonia (CCPP). The study includes a retrospective study, fieldwork, serology, an abattoir investigation and isolation of the causative Mycoplasma. Outbreaks of CCPP were reported from almost all regions of the country, especially from lowland areas, which are known goat-rearing regions, with the highest in 2002. The predominant clinical findings observed in affected flocks were coughing, nasal discharge, weakness, reluctance to move, abduction of elbow and laboured breathing. A total of 35 sheep and goats, of 183 examined, were sero-positive for MCCP F38 antibodies using CFT. From the total of 217 goats examined in the abattoir for the presence of CCPP lesions, 21 (9.7%) showed pathological lesions, inflamed lungs with marbled appearance, fibrous pleuropneumonia, yellowish-coloured pleural fluid and swollen bronchial and mediastinal lymph nodes. The causative agent (Mycoplasma capricolum sp. capripneumoniae) was isolated and identified from tissue samples and thoracic fluid treated bacteriologically. In conclusion, the study indicates that CCPP is becoming a very important goat disease in Borana pastoral areas. © 2006 Elsevier B.V. All rights reserved. Keywords: Contagious caprine pleuropneumoniae; Prevalence; Goat; Sheep; Borana; Southern Ethiopia
1. Introduction Africa hosts 31% of the world’s goat population, which is around 174 million head, and 12% of them are found in Ethiopia (Ademosun, 1992). Ethiopia stands second in Africa and fifth in the world in goat population, with 75% of the national goat flock being raised in the lowland pastoral areas. The importance of goats in pastoral and semi-pastoral areas of Ethiopia cannot be overstated.
∗
Corresponding author. E-mail address: [email protected] (A. Gelagay).
0921-4488/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.smallrumres.2006.02.001
Although goats represent a great national resource, their productivity is sub-optimal. Among the various factors responsible for this low productivity, diseases remain a significant problem. Contagious caprine pleuropneumonia (CCPP) and peste des petite ruminant (PPR) are the most important infectious diseases of goats, causing huge economic losses through morbidity and mortality. Contagious caprine pleuropneumonia is a severe disease of goats occurring in many countries in Africa and in some Asian countries where the total goat population is more than 500 million (Acharya, 1992). Classical, acute CCPP is caused by Mycoplasma capricolum subsp. capripneumoniae (Mccp) (Rurangirwa et
132
A. Gelagay et al. / Small Ruminant Research 70 (2007) 131–135
al., 1987), originally known as the F38 biotype. This organism was first isolated and shown to cause CCPP in Kenya; it has subsequently been isolated in the Sudan, Tunisia, Oman, Turkey, Chad, Uganda, Ethiopia, Niger, Tanzania, Eritrea and the United Arab Emirates (Rurangirwa et al., 1987; Macowam and Minette, 1976; Thiaucourt et al., 2000; Jones and Wood, 1988). A disease, indistinguishable from naturally occurring CCPP, has been experimentally reproduced with Mccp by several research groups. Typical cases of CCPP are characterised by extreme fever (41–43 ◦ C), high morbidity and mortality rates in susceptible herds affecting all ages and both sexes, and abortions in pregnant goats. After approximately 2–3 days of high fever, respiratory signs become apparent: respiration is accelerated and painful, and, in some cases, is accompanied by a grunt. Coughing is frequent, violent and productive. In the terminal stages, animals are unable to move, they stand with their front legs wide apart, the neck is stiff and extended, and sometimes saliva continuously drips from the mouth. Postmortem examination reveals fibrinous pleuropneumonia with massive lung hepatisation and pleurisy, accompanied by accumulation of straw-coloured pleural fluid (OIE, 2000). In Ethiopia, the presence of CCPP has been suspected for a long period, especially in areas in the immediate vicinity of endemic regions of Kenya and Sudan. It has been confirmed to be present in Ethiopia since the 1980s (Thiaucourt et al., 2000). An exact epidemiological picture of the disease has not yet been established; however, outbreaks of CCPP has been recorded in different regions of the country, such as Tigray, Afar, Dire Dawa, southern nations and nationalities people, Oromia, Benishangul-Gumz and Amhara regional states (Nesru, 2003). These reports were mostly based on clinical manifestations of the disease. The current study was conducted in the Borana pastoral area, the largest pastoral area in Ethiopia, to determine the prevalence of the disease and provide confirmative diagnosis by isolation of the causative mycoplasma. 2. Material and methods 2.1. Field investigation 2.1.1. Study area and animals The study was conducted in selected districts of Borana pastoral areas of Ethiopia, namely Yabello, Dirre, Moyale and Liban during June–September 2004.
Geographically, the Borana zone is located between 3◦ 36 and 6◦ 38 N latitude and 36◦ 43 and 41◦ 40 E longitude. The area is characterised by a climate with bimodal and erratic rainfall. The mean annual rainfall ranges from 250 to 700 mm. The annual mean temperature varies from 19 to over 25 ◦ C. Extensive pastoralism is the main means of livelihoods for the Borana people. Nomadic pastoralism is the rational livestock system in the area. The experimental animals were sheep and goats raised by the Borana pastoralists in the study area. The sheep were typically black head Ogaden sheep, while the goats belong to the local Borana breeds (which have not yet been fully characterised). A total of 183 sheep and goats were involved in the field investigation. Due to difficult working conditions in the pastoral area, it was not possible to use real random sampling of flocks and individual animals within flocks. 2.1.2. Clinical examination and sample collection Flocks with recent outbreak of respiratory disease were selected deliberately. Acutely infected subjects, displaying clinical episodes of CCPP, were selected and examined in depth. A detailed physical examination was performed on each selected animal. Samples were collected from nasal cavities of these animals with swabs moistened with serum. The swabs were placed in test tubes containing Hayflick’s medium with antibiotics. Each of these tubes was labelled, packed and transported to the laboratory under refrigeration. Blood samples (5 ml) were collected from the jugular vein using plain vaccutainer tubes and allowed to clot at room temperature overnight. Sera were collected from each tube with sterile pipettes and transferred to haemolytic tubes. The tubes were labelled, packed and placed in an icebox containing preformed blocks of ice for transportation to the laboratory. Two goats manifesting typical symptoms of classical, acute CCPP were purchased from the pastoralists and transported to the Southern Rangeland Development Unit (SORDU) clinic, Yabello District. They were sacrificed and post-mortem gross pathological lesions recorded. Sections of affected lung tissue, at the interface between the inflamed and the adjacent healthy tissue and mediastinal lymph nodes, were collected with sterile Universal bottles. The bottles were labelled and placed in an icebox containing ice blocks. All specimens were preserved frozen (−20 ◦ C) in the laboratory until processing. Laboratory investigations were carried out at the National Veterinary Institute, Debre Zeit, Ethiopia.
A. Gelagay et al. / Small Ruminant Research 70 (2007) 131–135
2.2. Abattoir investigation A total of 217 goats, which originated from the Borana pastoral area, were randomly selected and slaughtered at the Debre Zeit export abattoir, located about 45 km southeast of the capital, Addis Ababa, for detailed necropsy examination. Gross pathological lesions were recorded and portions of affected lung tissues were excised, collected in Universal bottles and transported to the laboratory under refrigeration. 2.3. Laboratory investigation 2.3.1. Serology Collected sera were decomplemented in a waterbath at 60 ◦ C for 30 min before execution of the test proper. Standard complement fixation test (CFT) was used to determine the prevalence of antibodies against Mycoplasma capricolum capripneumoniae strain F38 , as recommended by OIE (2000). Briefly, collected sera were decomplemented in a water-bath at 56 ◦ C for 30 min. Then, 25 l of veronal buffer was dispensed to each well of a U-bottomed micro-plate, and 1/5 diluted sera were added to first column and diluted serially in two-fold dilutions (1/10, 1/20, . . ., 1/160). A 25-l aliquot of antigen was added to each well with 25 l of complement, which was agitated and incubated for 45 min at 37 ◦ C. Finally, 25 l of sensitised sheep red blood cells (RBC + haemolysin) was added, mixed well and incubated at 37 ◦ C for 45 min, and kept at 4 ◦ C for 1 h to allow the unlysed cells to settle. More than 50% haemolysis was considered as positive (OIE, 2000). 2.3.2. Isolation of the causative agent Isolation and identification of Mycoplasma from infected lung, pleural exudates, fibrinous mass and nasal swabs was carried out according to the standard procedure (Lefevre et al., 1987). The basal culture media used were heart infusion broth (2.5%) and modified Hayflick Bacto PPLO 2.1% containing 0.2% glucose, bactoneopeptone and bacto-casitone 0.25% (w/v). Solidification of the basal broth media was done by adding 1.4% agar (Difco). Liquid and solid basal media were sterilised by autoclaving at 121 ◦ C for 15 min. Before inoculation, the sterile basal media were enriched with 20% horse serum, NVI produce (v/v), fresh yeast extract 10% (v/v) and kept overnight at 37 ◦ C to verify absence of any contamination. Penicillin at a final concentration of 200 IU/ml and 10% Thalous acetate 0.12% (v/v) were used as inhibitors against bacterial and fungal contaminants in the pathological samples. Finally, 4 mg/ml sodium pyruvate and 0.2% calf thymus DNA, in a pro-
133
portion of 1% (v/v), were added to enhance growth of fastidious Mycoplasma, such as Mccp (Thiaucourt, 1994). The seeded agar plates were incubated at 37 ◦ C in a CO2 incubator for 14 days. Evidence of micro-colonies was checked daily for growth. Typical colonies with a fried-egg appearance were taken and biochemically tested for break down of glucose, arginine hydrolysis, reduction of tetrazolium chloride, phosphates activity and digitonin sensitivity. Identities of the isolated Mycoplasma were confirmed by growth inhibition and dot blot tests (OIE, 2000; Thiaucourt, 1994). 2.4. Dot blot immuno-assay A 5-l aliquot of mycoplasma culture was dropped on pieces of hybridisation transfer membrane, left to dry at 37 ◦ C for 10 min, washed three times with Tris buffer solution (TBS), incubated with blocking buffer (TBS + 10% horse serum) for 30 min and later with Mycoplasma capricolum subsp. capripneumoniaespecific monoclonal antibody. After three washings, immuno-enzymatic antigen antibody reaction was determined using anti-mice immunoglobulin G conjugate with horseradish peroxidase (HRP, DAKO), which gave a brown staining following reaction with orthophenylenediamine (OPD) chromogen and hydrogen peroxide substrate (Thiaucourt, 1994). 2.4.1. Retrospectives data collection Annual livestock disease reports of the Federal Animal Health Department of the Ministry of Agriculture were used to gain insight into recent CCPP outbreaks and areas. There were five reports covering the period from 1999 to 2003. Areas that had experienced a CCPP outbreak in the year proceeding the investigation were identified. 3. Results 3.1. Clinical and pathological findings The predominant clinical findings observed in affected flocks were coughing, nasal discharge, weakness, reluctance to move, abduction of elbow and labour breathing. Few of them displayed signs of mouth breathing, carrying their head low, beating and lying down behind the rest of flock. Gross pathological examination of the scarified goats revealed inflamed lungs with marbled appearance, yellowish-coloured pleural exudate and swollen bronchial and mediastinal lymph nodes.
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A. Gelagay et al. / Small Ruminant Research 70 (2007) 131–135
Table 1 Seroprevalence of CCPP using CFT District
Sheep n
Dire Liban Yabello Moyalle Total
Table 2 Pathological finding of CCPP in export abattoir
Goat
+
%
8 0 4 2
1 0 0 0
12.5 0 0 0
14
1
7.14
n
Total +
%
37 78 19 35
7 12 4 11
18.92 15.38 21.05 31.43
169
34
20.12
n
Age (Years) +
%
45 78 23 37
8 12 4 11
17.78 15.38 17.39 29.73
183
35
19.13
n, number of samples tested; +, number positive; %, percent positive.
3.2. Serology A total of 35 of the 183 examined serum samples were positive for MCCP F38 antibodies using CFT. In all, 14 sheep, raised with an affected goat flock, were examined for the presence F38 antibodies and one (7.14%) was found positive. From 169 goat sera tested, 34 (20.12%) were positive. A higher prevalence rate (29.73%) was recorded in the Moyale district, while lower rate was observed in Liban for goats and both species combined (Table 1). 3.2.1. Mycoplasma isolation and identification Growth on solid medium revealed the presence of typical fried-egg colonies of Mycoplasma after 7 days of inoculation. The colonies were examined by various biochemical tests and were positive for glucose fermentation, reduction of tetrazolium chloride and phosphatase activity, and negative for hydrolysis of arginine and digitonin sensitivity. Growth of the isolates was inhibited with MCCP hyperimmune. Also the isolates were positive for MCCP with the monoclonal antibody-based dot blot test. 3.2.2. Retrospective study Outbreaks of CCPP were reported from almost all regions of the country, especially from the lowland areas, which are known goat-rearing regions. The highest number of outbreaks was reported in 2002, i.e. 25, of which 17 were from the southern nations and nationalities people (SNNP). Half of total reports were also from this region, which is near Kenya and Sudan. 3.2.3. Pathological findings In all, 21 (9.7%) of the total of 217 goats showed typical CCPP lesions during pathological investigation in the abattoir. Gross pathological examination of the slaughtered goats revealed swollen lungs, yellowishcoloured pleural fluid and swollen bronchial and medi-
Positive
Negative
Total
%CCPP+
<1
9
85
94
9.5
1–2
8
52
60
13.3
>2
4
59
63
6.3
21
196
217
9.7
Total
[95% CI] Fisher’s exact [0.1379336– 2.431079] [0.0922814– 1.771602] –
astinal lymph nodes. From the age of 1–2 years, the prevalence rate was 13.3% (Table 2). 4. Discussion Goat production plays an important role in the economy and livelihood of Ethiopia, especially in pastoral areas where goats are raised in large number. The major health problem of goats in pastoral areas is contagious caprine pluropneumonia. Outbreaks of CCPP have been reported from almost all regions of the country, especially from the lowland areas, which are known goat-rearing regions. The highest number of outbreaks was reported in 2002 (Table 3), i.e. 25, of which 17 were from the southern nations and nationalities people, and half of the total, within the 5-year survey, were also from this region, which is near Kenya and Sudan. The current study area is also included in this region. Since pastoralism is the main livelihood in Borana, there has been regular mixing of flocks at watering points and communal grazing areas, which is likely to spread the infection between flocks. Seasonal movement of flocks, which has been adopted by pastoralists as an efficient means of resource utilization, can introduce the Table 3 Reported number of CCPP outbreaks between 1999 and 2003 by regional state Region
No. of districts Number of outbreaks of CCPP reported 1999 2000 2001 2002 2003 Total
Afar 10 Amhara 6 B/Gumuz 3 D/Dawa 2 Oromia 3 SNNP 21
– 3 1
2 1 –
– 4
Total
8
45
– 3
8 1 1 1 – 3
3 1 – 2 2 17
1 – 1 – 3 4
14 6 3 3 5 31
6
14
25
9
62
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References
Fig. 1. Seroprevalence of CCPP using CFT.
disease into disease-free flocks by expanding its geographical distribution. The clinico-pathological manifestations recorded during this investigation were consistent with those of Radostitis et al. (2000), Kusiluka et al. (2000), Wesonga et al. (1998) and Quinn et al. (2002). From the total of 217 goats examined in the export abattoir for the presence of lesions, 21 (9.7%) showed CCPP-like lesions. The seroprevalence encountered in this study agrees with previous reports from Ethiopia (Roger and Berket, 1996) and India (Shaheen et al., 2001). However, the current prevalence is lower than other studies, so far performed. For example, it is far lower than that reported by Sharew et al. (2005), Gezahegn (1993) Singh et al. (1999), Mekonnen (1996) and Berket (1995). These variations could arise from differences in sample size, sampling techniques and the type of tests used to evaluate the seroprevalence of CCPP. The sample size of this study was very small compared to that considered by others. In addition, the samples collected during this study were mostly from goats that were acutely infected during outbreak. In these animals, the antibodies are eclipsed with the antigens from the infecting Mycoplasma and most of the sera collected during this time are likely to give negative result. Similarly, serum samples collected from animals long after the outbreak could give negative results. All of the studies reporting higher prevalence of antibodies to Mccp used ELISA tests, while we used CFT. B-ELCSA is found to be more sensitive than CFT in detecting the presence of antibodies to Mccp (Sharew et al., 2005); however, the prevalence encountered during this investigation was greater than that of Garg and Kaur (1997) (Fig. 1). This study has also shown a 7.14% prevalence of antibodies to Mccp in sheep. This agrees with the report of Mekonnen (1996), who has recorded a 5% proprevalence in sheep. Roger and Berket (1996) reported a seroprevalence of 25% from the Konso district of southern Ethiopia.
Acharya, R.M., 1992. Goat production. In: Recent Advances in Goat Production. Fifth International Conference on Goats, New Delhi, India, March, pp. 49–94. Ademosun, A.A., 1992. Constraints and prospects for small ruminants research and development in Africa. Small Rumin. Dev. Afr., 1–5. Berket Z. 1995. Contagious caprine pleuropneumonia prevalence in konso, southern-Ethiopia. DVM Thesis, A.AU, FVM, Debre Zeit, Ethiopia. Garg, D.N., Kaur, C., 1997. Sero-detection of CCPP antibodies in caprine and bovine population by ELISA and LAT. Indian J. Comp. Microbiol. Immunol. Infect. Dis. 18 (2), 153–156. Gezahegn, M. 1993. Preliminary study of CCPP in selected sites of East Shewa. DVM Thesis, A.AU, FVM, Debre Zeit, Ethiopia. Jones, G.E., Wood, A.R., 1988. Microbiological and serological studies on caprine pleuropneumonia in Oman. Res. Vet. Sci. 44, 125–131. Kusiluka, L.T.M., Semuguraka, W.D., Kazwala, R.R., Fris, N.F., 2000. Demonstration of Mccp and Mmmsc type in outbreaks of caprine pleuropneumnia in Eastern Tanzania. Acta Vet. Scand. 41 (3), 311–319. Lefevre, P.C., Breard, A., Alfaroukh, I., Buron, S., 1987. Mycoplasma species F 38 isolated in Chad. Vet. Rec. 121, 575–576. Macowam, K.J., Minette, J.E., 1976. A Mycoplasma from acute contagious caprice pleuropneumonia in Kenya. Trop. Anim. Health Prod. 8, 91–95. Mekonnen Lemma. 1996. CCPP study on Economic significance and vaccine trials. DVM Thesis, A.AU, FVM, Debre Zeit, Ethiopia. Nesru, H., 2003. A monthly report of animal diseases out break, January 2001 to January 2002. MoA, Addis Ababa, Ethiopia. OIE, 2000. Manual of Standards for Diagnostic Tests and Vaccines. Contagious caprine pleuropneumonia. OIE Paris, pp. 503–514. Quinn, P.J., Markkey, B.K., Carter, M.E., Donnelly, W.J., Leonard, F.C., 2002. Veterinary Microbiology and Microbial Disease, first ed. Blackwell Science, Oxford, pp. 194. Radostitis, O.M., Gay, C.C., Blood, D.C., 2000. A Textbook of Cattle, Sheep, Pigs, Goats and Horses. Disease Caused by Mycoplasma, ninth ed. Brailliere Tindal, pp. 748–785. Roger, F., Berket, Z. 1996. CCPP in Ethiopia. Lab. Investigation, station and field studies (1994–1995), Preliminary report, CIRADEMVT. March 1996, No. 5035. Rurangirwa, F.R.M.C., Guire, J.C., Kibor, A., Chema, S., 1987. An inactive vaccine for contagious caprine pleuropneumonia. Vet. Rec. 121, 397–402. Shaheen, M., Hague, S., Thakar, D.K., 2001. CCPP in kids in Ranch. Indian J. Anim. Sci. 71 (2), 136–137. Sharew, A.D., Staak, C., Thiaucourt, F., Roger, F., 2005. A serological investigation into CCPP in Ethiopia. Trop. Anim. Health Prod. 37 (1), 11–19. Singh, R.K., Srivastara, N.C., Singh, Y.P.S., 1999. Serosurveollance of CCPP using ELISA. Indian J. Anim. Sci. 69 (12), 999– 1001. Thiaucourt, F., 1994. La Pleuropneumonie Contagiouse Caprine, De l’observation clinique a la mise au point de technique diagnostique. Ph.D. Thesis. Thiaucourt, F., Johansson, K.E., Smith, D.G., Wesonga, H., Yigezu, L., 2000. Contagious pleuropneumonia recent advances. In: Seventh International Conference on Goats, France, 15–21 May 2000. Wesonga, H.O., Lindberg, R.J., Litamoi, J.K., Bolske, G., 1998. Late lesions of experimental CCPP caused by Mccp. J. Vet. Med. Ser. B 45 (2), 105–114.
Prevalence of contagious caprine pleuropneumonia in the Borana pastoral areas of Ethiopia A. Gelagay a,∗ , S. Teshale b , W. Amsalu c , G. Esayas a b
a National Veterinary Institute (NVI), P.O. Box 19, Debre Zeit, Ethiopia Faculty of Veterinary Medicine, Addis Ababa University, P.O. Box 34, Debre Zeit, Ethiopia c Hashim Export Abattoir, Debre Zeit, Ethiopia
Received 12 April 2005; received in revised form 18 October 2005; accepted 3 February 2006 Available online 29 March 2006
Abstract The study was conducted in selected districts of Borana pastoral areas of Ethiopia, namely Yabello, Dire, Moyale and Liban, during June–September 2004 to determine the status of contagious caprine pleuropneumonia (CCPP). The study includes a retrospective study, fieldwork, serology, an abattoir investigation and isolation of the causative Mycoplasma. Outbreaks of CCPP were reported from almost all regions of the country, especially from lowland areas, which are known goat-rearing regions, with the highest in 2002. The predominant clinical findings observed in affected flocks were coughing, nasal discharge, weakness, reluctance to move, abduction of elbow and laboured breathing. A total of 35 sheep and goats, of 183 examined, were sero-positive for MCCP F38 antibodies using CFT. From the total of 217 goats examined in the abattoir for the presence of CCPP lesions, 21 (9.7%) showed pathological lesions, inflamed lungs with marbled appearance, fibrous pleuropneumonia, yellowish-coloured pleural fluid and swollen bronchial and mediastinal lymph nodes. The causative agent (Mycoplasma capricolum sp. capripneumoniae) was isolated and identified from tissue samples and thoracic fluid treated bacteriologically. In conclusion, the study indicates that CCPP is becoming a very important goat disease in Borana pastoral areas. © 2006 Elsevier B.V. All rights reserved. Keywords: Contagious caprine pleuropneumoniae; Prevalence; Goat; Sheep; Borana; Southern Ethiopia
1. Introduction Africa hosts 31% of the world’s goat population, which is around 174 million head, and 12% of them are found in Ethiopia (Ademosun, 1992). Ethiopia stands second in Africa and fifth in the world in goat population, with 75% of the national goat flock being raised in the lowland pastoral areas. The importance of goats in pastoral and semi-pastoral areas of Ethiopia cannot be overstated.
∗
Corresponding author. E-mail address: [email protected] (A. Gelagay).
0921-4488/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.smallrumres.2006.02.001
Although goats represent a great national resource, their productivity is sub-optimal. Among the various factors responsible for this low productivity, diseases remain a significant problem. Contagious caprine pleuropneumonia (CCPP) and peste des petite ruminant (PPR) are the most important infectious diseases of goats, causing huge economic losses through morbidity and mortality. Contagious caprine pleuropneumonia is a severe disease of goats occurring in many countries in Africa and in some Asian countries where the total goat population is more than 500 million (Acharya, 1992). Classical, acute CCPP is caused by Mycoplasma capricolum subsp. capripneumoniae (Mccp) (Rurangirwa et
132
A. Gelagay et al. / Small Ruminant Research 70 (2007) 131–135
al., 1987), originally known as the F38 biotype. This organism was first isolated and shown to cause CCPP in Kenya; it has subsequently been isolated in the Sudan, Tunisia, Oman, Turkey, Chad, Uganda, Ethiopia, Niger, Tanzania, Eritrea and the United Arab Emirates (Rurangirwa et al., 1987; Macowam and Minette, 1976; Thiaucourt et al., 2000; Jones and Wood, 1988). A disease, indistinguishable from naturally occurring CCPP, has been experimentally reproduced with Mccp by several research groups. Typical cases of CCPP are characterised by extreme fever (41–43 ◦ C), high morbidity and mortality rates in susceptible herds affecting all ages and both sexes, and abortions in pregnant goats. After approximately 2–3 days of high fever, respiratory signs become apparent: respiration is accelerated and painful, and, in some cases, is accompanied by a grunt. Coughing is frequent, violent and productive. In the terminal stages, animals are unable to move, they stand with their front legs wide apart, the neck is stiff and extended, and sometimes saliva continuously drips from the mouth. Postmortem examination reveals fibrinous pleuropneumonia with massive lung hepatisation and pleurisy, accompanied by accumulation of straw-coloured pleural fluid (OIE, 2000). In Ethiopia, the presence of CCPP has been suspected for a long period, especially in areas in the immediate vicinity of endemic regions of Kenya and Sudan. It has been confirmed to be present in Ethiopia since the 1980s (Thiaucourt et al., 2000). An exact epidemiological picture of the disease has not yet been established; however, outbreaks of CCPP has been recorded in different regions of the country, such as Tigray, Afar, Dire Dawa, southern nations and nationalities people, Oromia, Benishangul-Gumz and Amhara regional states (Nesru, 2003). These reports were mostly based on clinical manifestations of the disease. The current study was conducted in the Borana pastoral area, the largest pastoral area in Ethiopia, to determine the prevalence of the disease and provide confirmative diagnosis by isolation of the causative mycoplasma. 2. Material and methods 2.1. Field investigation 2.1.1. Study area and animals The study was conducted in selected districts of Borana pastoral areas of Ethiopia, namely Yabello, Dirre, Moyale and Liban during June–September 2004.
Geographically, the Borana zone is located between 3◦ 36 and 6◦ 38 N latitude and 36◦ 43 and 41◦ 40 E longitude. The area is characterised by a climate with bimodal and erratic rainfall. The mean annual rainfall ranges from 250 to 700 mm. The annual mean temperature varies from 19 to over 25 ◦ C. Extensive pastoralism is the main means of livelihoods for the Borana people. Nomadic pastoralism is the rational livestock system in the area. The experimental animals were sheep and goats raised by the Borana pastoralists in the study area. The sheep were typically black head Ogaden sheep, while the goats belong to the local Borana breeds (which have not yet been fully characterised). A total of 183 sheep and goats were involved in the field investigation. Due to difficult working conditions in the pastoral area, it was not possible to use real random sampling of flocks and individual animals within flocks. 2.1.2. Clinical examination and sample collection Flocks with recent outbreak of respiratory disease were selected deliberately. Acutely infected subjects, displaying clinical episodes of CCPP, were selected and examined in depth. A detailed physical examination was performed on each selected animal. Samples were collected from nasal cavities of these animals with swabs moistened with serum. The swabs were placed in test tubes containing Hayflick’s medium with antibiotics. Each of these tubes was labelled, packed and transported to the laboratory under refrigeration. Blood samples (5 ml) were collected from the jugular vein using plain vaccutainer tubes and allowed to clot at room temperature overnight. Sera were collected from each tube with sterile pipettes and transferred to haemolytic tubes. The tubes were labelled, packed and placed in an icebox containing preformed blocks of ice for transportation to the laboratory. Two goats manifesting typical symptoms of classical, acute CCPP were purchased from the pastoralists and transported to the Southern Rangeland Development Unit (SORDU) clinic, Yabello District. They were sacrificed and post-mortem gross pathological lesions recorded. Sections of affected lung tissue, at the interface between the inflamed and the adjacent healthy tissue and mediastinal lymph nodes, were collected with sterile Universal bottles. The bottles were labelled and placed in an icebox containing ice blocks. All specimens were preserved frozen (−20 ◦ C) in the laboratory until processing. Laboratory investigations were carried out at the National Veterinary Institute, Debre Zeit, Ethiopia.
A. Gelagay et al. / Small Ruminant Research 70 (2007) 131–135
2.2. Abattoir investigation A total of 217 goats, which originated from the Borana pastoral area, were randomly selected and slaughtered at the Debre Zeit export abattoir, located about 45 km southeast of the capital, Addis Ababa, for detailed necropsy examination. Gross pathological lesions were recorded and portions of affected lung tissues were excised, collected in Universal bottles and transported to the laboratory under refrigeration. 2.3. Laboratory investigation 2.3.1. Serology Collected sera were decomplemented in a waterbath at 60 ◦ C for 30 min before execution of the test proper. Standard complement fixation test (CFT) was used to determine the prevalence of antibodies against Mycoplasma capricolum capripneumoniae strain F38 , as recommended by OIE (2000). Briefly, collected sera were decomplemented in a water-bath at 56 ◦ C for 30 min. Then, 25 l of veronal buffer was dispensed to each well of a U-bottomed micro-plate, and 1/5 diluted sera were added to first column and diluted serially in two-fold dilutions (1/10, 1/20, . . ., 1/160). A 25-l aliquot of antigen was added to each well with 25 l of complement, which was agitated and incubated for 45 min at 37 ◦ C. Finally, 25 l of sensitised sheep red blood cells (RBC + haemolysin) was added, mixed well and incubated at 37 ◦ C for 45 min, and kept at 4 ◦ C for 1 h to allow the unlysed cells to settle. More than 50% haemolysis was considered as positive (OIE, 2000). 2.3.2. Isolation of the causative agent Isolation and identification of Mycoplasma from infected lung, pleural exudates, fibrinous mass and nasal swabs was carried out according to the standard procedure (Lefevre et al., 1987). The basal culture media used were heart infusion broth (2.5%) and modified Hayflick Bacto PPLO 2.1% containing 0.2% glucose, bactoneopeptone and bacto-casitone 0.25% (w/v). Solidification of the basal broth media was done by adding 1.4% agar (Difco). Liquid and solid basal media were sterilised by autoclaving at 121 ◦ C for 15 min. Before inoculation, the sterile basal media were enriched with 20% horse serum, NVI produce (v/v), fresh yeast extract 10% (v/v) and kept overnight at 37 ◦ C to verify absence of any contamination. Penicillin at a final concentration of 200 IU/ml and 10% Thalous acetate 0.12% (v/v) were used as inhibitors against bacterial and fungal contaminants in the pathological samples. Finally, 4 mg/ml sodium pyruvate and 0.2% calf thymus DNA, in a pro-
133
portion of 1% (v/v), were added to enhance growth of fastidious Mycoplasma, such as Mccp (Thiaucourt, 1994). The seeded agar plates were incubated at 37 ◦ C in a CO2 incubator for 14 days. Evidence of micro-colonies was checked daily for growth. Typical colonies with a fried-egg appearance were taken and biochemically tested for break down of glucose, arginine hydrolysis, reduction of tetrazolium chloride, phosphates activity and digitonin sensitivity. Identities of the isolated Mycoplasma were confirmed by growth inhibition and dot blot tests (OIE, 2000; Thiaucourt, 1994). 2.4. Dot blot immuno-assay A 5-l aliquot of mycoplasma culture was dropped on pieces of hybridisation transfer membrane, left to dry at 37 ◦ C for 10 min, washed three times with Tris buffer solution (TBS), incubated with blocking buffer (TBS + 10% horse serum) for 30 min and later with Mycoplasma capricolum subsp. capripneumoniaespecific monoclonal antibody. After three washings, immuno-enzymatic antigen antibody reaction was determined using anti-mice immunoglobulin G conjugate with horseradish peroxidase (HRP, DAKO), which gave a brown staining following reaction with orthophenylenediamine (OPD) chromogen and hydrogen peroxide substrate (Thiaucourt, 1994). 2.4.1. Retrospectives data collection Annual livestock disease reports of the Federal Animal Health Department of the Ministry of Agriculture were used to gain insight into recent CCPP outbreaks and areas. There were five reports covering the period from 1999 to 2003. Areas that had experienced a CCPP outbreak in the year proceeding the investigation were identified. 3. Results 3.1. Clinical and pathological findings The predominant clinical findings observed in affected flocks were coughing, nasal discharge, weakness, reluctance to move, abduction of elbow and labour breathing. Few of them displayed signs of mouth breathing, carrying their head low, beating and lying down behind the rest of flock. Gross pathological examination of the scarified goats revealed inflamed lungs with marbled appearance, yellowish-coloured pleural exudate and swollen bronchial and mediastinal lymph nodes.
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A. Gelagay et al. / Small Ruminant Research 70 (2007) 131–135
Table 1 Seroprevalence of CCPP using CFT District
Sheep n
Dire Liban Yabello Moyalle Total
Table 2 Pathological finding of CCPP in export abattoir
Goat
+
%
8 0 4 2
1 0 0 0
12.5 0 0 0
14
1
7.14
n
Total +
%
37 78 19 35
7 12 4 11
18.92 15.38 21.05 31.43
169
34
20.12
n
Age (Years) +
%
45 78 23 37
8 12 4 11
17.78 15.38 17.39 29.73
183
35
19.13
n, number of samples tested; +, number positive; %, percent positive.
3.2. Serology A total of 35 of the 183 examined serum samples were positive for MCCP F38 antibodies using CFT. In all, 14 sheep, raised with an affected goat flock, were examined for the presence F38 antibodies and one (7.14%) was found positive. From 169 goat sera tested, 34 (20.12%) were positive. A higher prevalence rate (29.73%) was recorded in the Moyale district, while lower rate was observed in Liban for goats and both species combined (Table 1). 3.2.1. Mycoplasma isolation and identification Growth on solid medium revealed the presence of typical fried-egg colonies of Mycoplasma after 7 days of inoculation. The colonies were examined by various biochemical tests and were positive for glucose fermentation, reduction of tetrazolium chloride and phosphatase activity, and negative for hydrolysis of arginine and digitonin sensitivity. Growth of the isolates was inhibited with MCCP hyperimmune. Also the isolates were positive for MCCP with the monoclonal antibody-based dot blot test. 3.2.2. Retrospective study Outbreaks of CCPP were reported from almost all regions of the country, especially from the lowland areas, which are known goat-rearing regions. The highest number of outbreaks was reported in 2002, i.e. 25, of which 17 were from the southern nations and nationalities people (SNNP). Half of total reports were also from this region, which is near Kenya and Sudan. 3.2.3. Pathological findings In all, 21 (9.7%) of the total of 217 goats showed typical CCPP lesions during pathological investigation in the abattoir. Gross pathological examination of the slaughtered goats revealed swollen lungs, yellowishcoloured pleural fluid and swollen bronchial and medi-
Positive
Negative
Total
%CCPP+
<1
9
85
94
9.5
1–2
8
52
60
13.3
>2
4
59
63
6.3
21
196
217
9.7
Total
[95% CI] Fisher’s exact [0.1379336– 2.431079] [0.0922814– 1.771602] –
astinal lymph nodes. From the age of 1–2 years, the prevalence rate was 13.3% (Table 2). 4. Discussion Goat production plays an important role in the economy and livelihood of Ethiopia, especially in pastoral areas where goats are raised in large number. The major health problem of goats in pastoral areas is contagious caprine pluropneumonia. Outbreaks of CCPP have been reported from almost all regions of the country, especially from the lowland areas, which are known goat-rearing regions. The highest number of outbreaks was reported in 2002 (Table 3), i.e. 25, of which 17 were from the southern nations and nationalities people, and half of the total, within the 5-year survey, were also from this region, which is near Kenya and Sudan. The current study area is also included in this region. Since pastoralism is the main livelihood in Borana, there has been regular mixing of flocks at watering points and communal grazing areas, which is likely to spread the infection between flocks. Seasonal movement of flocks, which has been adopted by pastoralists as an efficient means of resource utilization, can introduce the Table 3 Reported number of CCPP outbreaks between 1999 and 2003 by regional state Region
No. of districts Number of outbreaks of CCPP reported 1999 2000 2001 2002 2003 Total
Afar 10 Amhara 6 B/Gumuz 3 D/Dawa 2 Oromia 3 SNNP 21
– 3 1
2 1 –
– 4
Total
8
45
– 3
8 1 1 1 – 3
3 1 – 2 2 17
1 – 1 – 3 4
14 6 3 3 5 31
6
14
25
9
62
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References
Fig. 1. Seroprevalence of CCPP using CFT.
disease into disease-free flocks by expanding its geographical distribution. The clinico-pathological manifestations recorded during this investigation were consistent with those of Radostitis et al. (2000), Kusiluka et al. (2000), Wesonga et al. (1998) and Quinn et al. (2002). From the total of 217 goats examined in the export abattoir for the presence of lesions, 21 (9.7%) showed CCPP-like lesions. The seroprevalence encountered in this study agrees with previous reports from Ethiopia (Roger and Berket, 1996) and India (Shaheen et al., 2001). However, the current prevalence is lower than other studies, so far performed. For example, it is far lower than that reported by Sharew et al. (2005), Gezahegn (1993) Singh et al. (1999), Mekonnen (1996) and Berket (1995). These variations could arise from differences in sample size, sampling techniques and the type of tests used to evaluate the seroprevalence of CCPP. The sample size of this study was very small compared to that considered by others. In addition, the samples collected during this study were mostly from goats that were acutely infected during outbreak. In these animals, the antibodies are eclipsed with the antigens from the infecting Mycoplasma and most of the sera collected during this time are likely to give negative result. Similarly, serum samples collected from animals long after the outbreak could give negative results. All of the studies reporting higher prevalence of antibodies to Mccp used ELISA tests, while we used CFT. B-ELCSA is found to be more sensitive than CFT in detecting the presence of antibodies to Mccp (Sharew et al., 2005); however, the prevalence encountered during this investigation was greater than that of Garg and Kaur (1997) (Fig. 1). This study has also shown a 7.14% prevalence of antibodies to Mccp in sheep. This agrees with the report of Mekonnen (1996), who has recorded a 5% proprevalence in sheep. Roger and Berket (1996) reported a seroprevalence of 25% from the Konso district of southern Ethiopia.
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