Trop Anim Health Prod DOI 10.1007/s11250-014-0599-0
REGULAR ARTICLES
Neospora caninum versus Brucella spp. exposure among dairy cattle in Ethiopia: A case control study Kassahun Asmare
Accepted: 17 April 2014 # Springer Science+Business Media Dordrecht 2014
Abstract This case-control study aimed at assessing the relative association of Neospora caninum and Brucella species exposure with reproductive disorders. The study was carried out between October 2011 and June 2012 on 731 dairy cows sampled from 150 dairy farms in selected 17 conurbations of Ethiopia. Two hundred sixty-six of the cows were categorized as cases based on their history of abortion or stillbirth while the remaining 465 were controls. The presence of antibody to N. caninum was screened using indirect ELISA, while Brucella spp. exposure was assayed serially using Rose Bengal Plate Test and Complement Fixation Test. Exposure to N. caninum was more frequently observed among cases (23.8 %) than controls (12.7 %), while no significant difference (p>0.05) was noted for Brucella exposure between the two groups. Moreover, the proportion of cows with disorders like retention of fetal membrane, endometritis and increased inter-calving period were significantly higher (p < 0.05) among Neospora seropositive cows. In conclusion, the finding discloses the strong association of N. caninum with reproductive disorders compared to Brucella spp. exposure. However, neither N. caninum nor Brucella spp. could explain the majority (73.2 %) of the reported abortions and stillbirths in cattle. Hence, this observation underscores the need for more intensive investigation on the identification of causes of the aforementioned disorders in dairy cattle of Ethiopia.
Keywords Abortion . Case-control . Dairy . Stillbirth . Ethiopia
K. Asmare (*) Hawassa University School of Veterinary Medicine, P. O. box 005, Hawassa, Ethiopia e-mail: [email protected]
Introduction Reproductive disorder is one of the major causes of economic losses in the dairy cattle industry (Escamilla et al. 2007; Yoo 2010). Clinically the disorder is exhibited by a range of manifestations comprising abortion, stillbirth, fetal membranes retention, endometritis, metritis, more open days, increased calving interval, and repeat breeding. Abortion and stillbirth are the most prominent clinical manifestations of the reproductive disorder, with significant proportion attributed to infectious agents (Ortega- Mora et al. 2007). Several pathogens, including bacterial, viral, protozoan, and fungal agents, are known to affect the reproductive health of cattle directly (Yoo 2010). Among these agents, Brucella abortus is one of the abortifacient agents in dairy cattle; however, nowadays Neospora caninum infections have been steadily reported from cattle farms, especially from those with a history of high abortion rates (González-Warleta et al. 2008; Kul et al. 2009). In Africa, reports on neosporosis are limited; however, the available information is in line with global understanding of the protozoan that underscores the relevance of the N. caninum to the dairy sector (Ibrahim et al. 2012; Ghalmi et al. 2012). The recent serologic and molecular studies demonstrated that neosporosis is prevalent in the dairy cattle of Ethiopia (Asmare et al. 2013; Asmare et al. 2014). However, the available published information comparing different pathogens exposure vis-à-vis reproductive disorders is limited to a single article based on the data from central and southern part of the country (Asmare et al. 2013). The objectives of this paper are to provide a better picture of exposure of dairy cattle to N. caninum and Brucella spp. in relation to abortion and stillbirth in major milksheds of the country, and address the need for further investigation on unidentified causes of the reproductive disorders in cattle.
Trop Anim Health Prod
Material and methods Study area The study was conducted between October 2011 and June 2012 in 17 major conurbations of Ethiopia. The study areas are located between 3° 24′ and 14° 53′ latitude north and 32° 42′ to 48° 12′ longitude east. Dairy farms established in urban and peri-urban areas using exotic cattle and their crosses were used. The conurbations were selected based on the abundance of dairy farms that constituted the known milksheds in the country (Land O’Lakes Inc. 2010) (Fig. 1). Study animals Dairy cattle of Friesian, Friesian versus local crosses and Jersey breeds for commercial oriented dairy were used in the study. Breeding farms established by the government to supply cross heifers and bulls to expand the rural small-holder dairy production were also included in the study.
purposely based on farm record or owners information. At each farm, animals with the history of abortion and or stillbirth were considered as cases while those cows or heifers that had no history of such disorders were considered as controls. In order to minimize confounding effect, for all cases, the respective controls were picked from the same farm. Dairy farms were further classified into two broad categories based on the herd size, i.e., small and large. In small herds, a maximum of 2 cases and 4 controls were included, while a maximum of 6 cases and 12 controls were enrolled from larger herds. Matching of individual case with control was intentionally avoided to increase the chance of control availability, especially for small herds. In farms where large numbers of cases or controls were available, individual animals were picked randomly using a lottery method after registration of cows’ ear tags or given name in their respective category. Eventually, a total of 731 cows or heifers were involved in the study. Two hundred sixty-six of them were cases, while the remaining 465 were controls. Laboratory analysis
Study design and sampling Brucella spp. exposure This study is a part of large scale study where more than 2,300 dairy cows were involved from 273 herds. In this case control investigation 150 dairy farms with the presence of cow/s with a history of abortion and stillbirth in the farm were selected Fig. 1 Major conurbations where studied dairy farms were located. Conurbations: 1 Mekele, 2 Gondar, 3 Bahirdar, 4 Jimma, 5 Hosaena, 6 Wolitasoddo, 7 Arsinegele, 8 Shashemene, 9 Hawassa, 10 Yergalem, 11 Holleta, 12 Sebeta, 13 Debrezeit, 14 Nazreth, 15 Diredawa, 16 Harar, 17 AddisAbaba
Blood samples collected from jugular vein aseptically using vacutainer needle and syringe were allowed to stand overnight and centrifuged at 1,000×g for 10 min. to obtain the serum.
Trop Anim Health Prod
The sera were identified and transported to the National Veterinary Institute (NVI), Ethiopia in ice packs and stored at −20 °C until been screened for antibodies against Brucella exposure using Rose Bengal Plate Test (RBPT). Thirty microliters of RBPT antigen (Lillidale Diagnostics, Windborne, UK) was added to equal volumes of test sera dropped on glass slide. The antigen and test serum were mixed thoroughly by a plastic applicator, shaken for 4 min, and the occurrence of agglutination was recorded immediately. The test was repeated when test results were ambiguous. Positive sera were further subjected to the Complement Fixation Test (CFT) conducted complying with the standard protocol (OIE 2012). The control sera and complement were both obtained from the Federal Institute for Health Protection of Consumers, Germany. As there has never been history of vaccination for brucellosis, all positive results were attributed to natural infection. N. caninum exposure The presence of antibodies to N. caninum was determined on all preceding samples using the IDEXX Neospora X2 Ab ELISA test kit (IDEXX Laboratories). The screening was performed according to the manufacturer’s test protocol (Westbrook, ME 04092, USA) supplied with the kits, using the recommended test sample dilution (1:100) and positive and negative controls provided with the kits. Results were expressed as sample to positive ratios (S/P). A serum with absorbance value (S/P) with a cut-off level of 0.50 was considered to be Neospora positive as per the manufacturer’s recommendation. Epidemiological information Biologically relevant information of the animal and farm was obtained from farm records where available. In a farm where records were lacking, a semi-structured questionnaire was used to get information from the dairy farmer. Data collected included breed, herd size, origin of the animal and history of maternal reproductive disorders. Climate related information about the study locality was obtained from national meteorology agency website (http://www.ema.gov.et/). The reproductive disorder information included abortion, stillbirth, presence or absence of fetal membrane retention, endometritis, birth of congenitally defective calf or weak calf, length of inter-calving period and number of services perpregnancy. A cow was considered as repeat breeder if it required three or more services per pregnancy. Similarly, inter-calving period is assumed to be longer if the farmer could not obtain calf crop every other year, i.e., two calves per 3 years. A herd was also categorized as small herd, if the number of animals per herd was 10 or less. Those herds with
more than 10 animals per herd were considered as large herd in the analysis.
Data analysis The established databases in Microsoft Excel spreadsheet were coded and edited for tabulation and preliminary descriptive analyses. The data were transferred to Stata SE/11 for further analysis. The associations of covariates, reproductive disorders with exposure to infectious agents were assessed using univariable logistic regression analysis after cluster or herd adjustment for sampling weight using sampling fraction. The predictor covariate variables (p
REGULAR ARTICLES
Neospora caninum versus Brucella spp. exposure among dairy cattle in Ethiopia: A case control study Kassahun Asmare
Accepted: 17 April 2014 # Springer Science+Business Media Dordrecht 2014
Abstract This case-control study aimed at assessing the relative association of Neospora caninum and Brucella species exposure with reproductive disorders. The study was carried out between October 2011 and June 2012 on 731 dairy cows sampled from 150 dairy farms in selected 17 conurbations of Ethiopia. Two hundred sixty-six of the cows were categorized as cases based on their history of abortion or stillbirth while the remaining 465 were controls. The presence of antibody to N. caninum was screened using indirect ELISA, while Brucella spp. exposure was assayed serially using Rose Bengal Plate Test and Complement Fixation Test. Exposure to N. caninum was more frequently observed among cases (23.8 %) than controls (12.7 %), while no significant difference (p>0.05) was noted for Brucella exposure between the two groups. Moreover, the proportion of cows with disorders like retention of fetal membrane, endometritis and increased inter-calving period were significantly higher (p < 0.05) among Neospora seropositive cows. In conclusion, the finding discloses the strong association of N. caninum with reproductive disorders compared to Brucella spp. exposure. However, neither N. caninum nor Brucella spp. could explain the majority (73.2 %) of the reported abortions and stillbirths in cattle. Hence, this observation underscores the need for more intensive investigation on the identification of causes of the aforementioned disorders in dairy cattle of Ethiopia.
Keywords Abortion . Case-control . Dairy . Stillbirth . Ethiopia
K. Asmare (*) Hawassa University School of Veterinary Medicine, P. O. box 005, Hawassa, Ethiopia e-mail: [email protected]
Introduction Reproductive disorder is one of the major causes of economic losses in the dairy cattle industry (Escamilla et al. 2007; Yoo 2010). Clinically the disorder is exhibited by a range of manifestations comprising abortion, stillbirth, fetal membranes retention, endometritis, metritis, more open days, increased calving interval, and repeat breeding. Abortion and stillbirth are the most prominent clinical manifestations of the reproductive disorder, with significant proportion attributed to infectious agents (Ortega- Mora et al. 2007). Several pathogens, including bacterial, viral, protozoan, and fungal agents, are known to affect the reproductive health of cattle directly (Yoo 2010). Among these agents, Brucella abortus is one of the abortifacient agents in dairy cattle; however, nowadays Neospora caninum infections have been steadily reported from cattle farms, especially from those with a history of high abortion rates (González-Warleta et al. 2008; Kul et al. 2009). In Africa, reports on neosporosis are limited; however, the available information is in line with global understanding of the protozoan that underscores the relevance of the N. caninum to the dairy sector (Ibrahim et al. 2012; Ghalmi et al. 2012). The recent serologic and molecular studies demonstrated that neosporosis is prevalent in the dairy cattle of Ethiopia (Asmare et al. 2013; Asmare et al. 2014). However, the available published information comparing different pathogens exposure vis-à-vis reproductive disorders is limited to a single article based on the data from central and southern part of the country (Asmare et al. 2013). The objectives of this paper are to provide a better picture of exposure of dairy cattle to N. caninum and Brucella spp. in relation to abortion and stillbirth in major milksheds of the country, and address the need for further investigation on unidentified causes of the reproductive disorders in cattle.
Trop Anim Health Prod
Material and methods Study area The study was conducted between October 2011 and June 2012 in 17 major conurbations of Ethiopia. The study areas are located between 3° 24′ and 14° 53′ latitude north and 32° 42′ to 48° 12′ longitude east. Dairy farms established in urban and peri-urban areas using exotic cattle and their crosses were used. The conurbations were selected based on the abundance of dairy farms that constituted the known milksheds in the country (Land O’Lakes Inc. 2010) (Fig. 1). Study animals Dairy cattle of Friesian, Friesian versus local crosses and Jersey breeds for commercial oriented dairy were used in the study. Breeding farms established by the government to supply cross heifers and bulls to expand the rural small-holder dairy production were also included in the study.
purposely based on farm record or owners information. At each farm, animals with the history of abortion and or stillbirth were considered as cases while those cows or heifers that had no history of such disorders were considered as controls. In order to minimize confounding effect, for all cases, the respective controls were picked from the same farm. Dairy farms were further classified into two broad categories based on the herd size, i.e., small and large. In small herds, a maximum of 2 cases and 4 controls were included, while a maximum of 6 cases and 12 controls were enrolled from larger herds. Matching of individual case with control was intentionally avoided to increase the chance of control availability, especially for small herds. In farms where large numbers of cases or controls were available, individual animals were picked randomly using a lottery method after registration of cows’ ear tags or given name in their respective category. Eventually, a total of 731 cows or heifers were involved in the study. Two hundred sixty-six of them were cases, while the remaining 465 were controls. Laboratory analysis
Study design and sampling Brucella spp. exposure This study is a part of large scale study where more than 2,300 dairy cows were involved from 273 herds. In this case control investigation 150 dairy farms with the presence of cow/s with a history of abortion and stillbirth in the farm were selected Fig. 1 Major conurbations where studied dairy farms were located. Conurbations: 1 Mekele, 2 Gondar, 3 Bahirdar, 4 Jimma, 5 Hosaena, 6 Wolitasoddo, 7 Arsinegele, 8 Shashemene, 9 Hawassa, 10 Yergalem, 11 Holleta, 12 Sebeta, 13 Debrezeit, 14 Nazreth, 15 Diredawa, 16 Harar, 17 AddisAbaba
Blood samples collected from jugular vein aseptically using vacutainer needle and syringe were allowed to stand overnight and centrifuged at 1,000×g for 10 min. to obtain the serum.
Trop Anim Health Prod
The sera were identified and transported to the National Veterinary Institute (NVI), Ethiopia in ice packs and stored at −20 °C until been screened for antibodies against Brucella exposure using Rose Bengal Plate Test (RBPT). Thirty microliters of RBPT antigen (Lillidale Diagnostics, Windborne, UK) was added to equal volumes of test sera dropped on glass slide. The antigen and test serum were mixed thoroughly by a plastic applicator, shaken for 4 min, and the occurrence of agglutination was recorded immediately. The test was repeated when test results were ambiguous. Positive sera were further subjected to the Complement Fixation Test (CFT) conducted complying with the standard protocol (OIE 2012). The control sera and complement were both obtained from the Federal Institute for Health Protection of Consumers, Germany. As there has never been history of vaccination for brucellosis, all positive results were attributed to natural infection. N. caninum exposure The presence of antibodies to N. caninum was determined on all preceding samples using the IDEXX Neospora X2 Ab ELISA test kit (IDEXX Laboratories). The screening was performed according to the manufacturer’s test protocol (Westbrook, ME 04092, USA) supplied with the kits, using the recommended test sample dilution (1:100) and positive and negative controls provided with the kits. Results were expressed as sample to positive ratios (S/P). A serum with absorbance value (S/P) with a cut-off level of 0.50 was considered to be Neospora positive as per the manufacturer’s recommendation. Epidemiological information Biologically relevant information of the animal and farm was obtained from farm records where available. In a farm where records were lacking, a semi-structured questionnaire was used to get information from the dairy farmer. Data collected included breed, herd size, origin of the animal and history of maternal reproductive disorders. Climate related information about the study locality was obtained from national meteorology agency website (http://www.ema.gov.et/). The reproductive disorder information included abortion, stillbirth, presence or absence of fetal membrane retention, endometritis, birth of congenitally defective calf or weak calf, length of inter-calving period and number of services perpregnancy. A cow was considered as repeat breeder if it required three or more services per pregnancy. Similarly, inter-calving period is assumed to be longer if the farmer could not obtain calf crop every other year, i.e., two calves per 3 years. A herd was also categorized as small herd, if the number of animals per herd was 10 or less. Those herds with
more than 10 animals per herd were considered as large herd in the analysis.
Data analysis The established databases in Microsoft Excel spreadsheet were coded and edited for tabulation and preliminary descriptive analyses. The data were transferred to Stata SE/11 for further analysis. The associations of covariates, reproductive disorders with exposure to infectious agents were assessed using univariable logistic regression analysis after cluster or herd adjustment for sampling weight using sampling fraction. The predictor covariate variables (p
