Validation of FAMACHA System for Detecting Anemic Sheep in Some Regions of Baghdad, Iraq

AENSI Journals Advances in Environmental Biology ISSN-1995-0756 EISSN-1998-1066 Journal home page: http://www.aensiweb.com/aeb/ Validation of FAMACHA System for Detecting Anemic Sheep in Some Regions of Baghdad, Iraq 1 Wathiq Ali Hasson Al-Jbory and 2 Firas Rashad Al-Samarai 1,2 Department of Veterinary Public Health, College of Veterinary Medicine/University of Baghdad, Iraq. Address For Correspondence: Firas Rashad Al-Samarai, Department of Veterinary Public Health, College of Veterinary Medicine/University of Baghdad, Iraq E-mail: firas_rashad@yahoo.com This work is licensed under the Creative Commons Attribution International License (CC BY). http://creativecommons.org/licenses/by/4.0/ Received 22 March 2016; Accepted 28 May 2016; Available online 12 June 2016 ABSTRACT FAMACHA system has been successfully tested in different countries where Haemonchus contortus is the major species that infect the sheep. The present study is the first on the use of the FAMACHA system in Iraq and aimed to evaluate the validation of FAMACHA system for detecting anemic sheep in five regions (Shula, Abu-Ghraib, Mahmoudia, Yousifia, and Alameel) in the West and South of Baghdad during the period from December to February, 2015-2016. A total of 690 animals were used in this study. Fifty animals out of the total number were selected randomly to estimate the proportion of anemic animals and to determine the necessary sample size. According to the pilot study, 140 animals were selected randomly and subjected to general clinical examination and FAMACHA.Each animal take a score of FAMACHA according to identical color of the ocular mucous membrane of the eye with the scores of FAMACHA.The FAMACHA scores (from 1 to 5) were compared to packed cell volume (PCV) and Haemoglobin (Hb). The classification of animals as anemic were determined according to the scores of FAMACHA 3, 4, 5 and 4, 5 in the both of PCV and Hb. Sheep considered anemic at three levels of PCV (PCV 17, PCV 19, PCV 21), or Hb (Hb 6.5, Hb 7, Hb 7.5).The correlation between FAMACHA score and both ofpcv (-0.46) and Hb(-0.26) was significant (P < 0.01). The sensitivity, specificity, predictive negative value (NPV), predictive positive value (PPV) and accuracy of the FAMACHA system were calculated for PCV and Hb. The highest values of sensitivity were 87.5% and 82.3% in the group 3-4-5 for PCV and Hb, respectively. The highest values of specificity were 81.3% and 80.4% in the group 4-5, for PCV and Hb, respectively. The NPV was 68.5% (PCV) and 81.3% (Hb) in the group 3-4-5, while it was 87% (PCV) and 80.4% (Hb) in the group 4-5. FAMACHA 3-5 scores was applied to 500 animals to estimate the apparent prevalence of anemia in sheep.in conclusion, the results of the present study showed that FAMACHA system has a high sensitivity (87%) and (82%) in detecting anemic sheep in Baghdad depending on PCV and Hb respectively. The results strongly support the FAMACHA level 3 to 5 to be used as a safe and reliable tool for detecting anemic sheep and this will lead to delay the development of helminthic resistance. KEYWORDS: FAMACHA, Sheep, packed cell volume, Haemoglobin, Iraq Abbreviations: PCV: packed cell volume, Hb: Haemoglobin, TST: targeted selective treatments, PCV:positive predictive value, NPV: Negative predictive value (PPV INTRODUCTION Sheep has an essential role in the economy of several countries, particularly Middle East countries. However, breeding of sheep faced some difficulties that hampered the progress of this sector, such as the poor nutrition, not adopting modern breeding methods along with parasite diseases [1].Gastrointestinal infection, caused by different genera of nematodes represents one of the main challenges to sheep production in temperate and tropical countries [2]. Among different nematodes, Haemonchus contortus, received a considerable Copyright 2016 by authors and Copyright, American-Eurasian Network for Scientific Information (AENSI Publication).

187 Wathiq Ali Hasson Al-Jbory and Firas Rashad Al-Samarai, 2016 attention as a highly pathogenic blood-feeder helminth that could cause higher economic losses in sheep as it causes anemia and reduced productive performance of animals and can lead to death [3]. Traditional management to eliminate gastrointestinal infection in small ruminants is based upon frequent treatments with anthelmintics, which has led to the ineffectiveness of treatments as a result of the development of drug-resistant worm populations throughout the world [4, 5]. To reduce anthelmintic usage and support the maintain populations in refugia, targeted selective treatments (TST) have been proposed which should help to conserve the genes that responsible for the sensitivity to anthelmintics within parasite populations and will lead to delay the development of drug-resistant in worm populations [6].One of the best examples of a TST indicator is FAMACHA proposed by Dr. Faffa Malan as a system developed in South Africa. This chart has been used to detect anemia on the basis of the color of the lower eyelid mucousmembrane in small ruminants. FAMACHA represents a morbidity marker for haemonchosis[6, 7, 8, 9]. FAMACHA has been successfully tested in sheep and goats for the different countries and regions, where H. contortus, is the commonly available parasite. As a field test, FAMACHA has been shown to reduce the number of animals that required treatment and offers the prospect of maintaining a parasite population in refugia in those animals that do not require treatment [5, 6]. Many researchers study the using of FAMACHA in management of H. contortus infected small ruminants in South Africa [10, 11], Kenya [12], Ethiopia [13, 14], United States [3, 5], Brazil[15], Uruguay[16], Germany [17, 18], and Turkey [19].The evaluation of the validity of FAMCHA in most of the studies depends on packed cell volume (PCV) as a golden standard test, despite the correlation between FAMACHA -scores and PCV varied along with different studies and ranged from -0.26 to -0.77 [5, 10, 13, 17]. The management practices must be based on modern recommendations about how to adopt strategies to prevent parasite infections. Several studies confirmed the satisfactory results in maintaining the health and productivity level of sheep through adopting the FAMACHA system. However, the validity of FAMACHA is not confirmed in all countries that adopted this system because when the prevalence of H. contortusis low this system could be insufficient for detecting all anemic animals [3, 5, 18]. However, the present study represents the first attempt to use FAMACHA in Iraq and to investigate the validity of this system for detecting anemic sheep in some regions of Baghdad. MATERIALS AND METHODS The study was carried out in five regions (Shula, Abu-Ghraib, Mahmoudia, Yousifia, and Alameel) West and South of Baghdad during the period from December 2015 to February 2016.Most of the sheep are Awassi breed of different ages and sex. A total number of sheep in the present study was 690 which included a pilot study of 50 animals selected randomly to estimate the proportion of anemic animals and to determine the necessary sample size. Sheep with PCV 21 was considered anemic to maximize the size of the necessary sample. According to the pilot study, the proportion of anemic animals was 10%. Hence, 140 animals were selected randomly and subjected to general clinical examination and FAMACHA system. Blood samples were collected from each animal from jugular vein using a vacutainer containing sodium heparin as an anticoagulant. All the blood samples were subjected to analysis in privet laboratory in Baghdad to estimate Red blood cells (RBC), Packed Cell Volume (PCV), haemoglobin (Hb). Each animal takes a score of FAMACHA according to the identical color of the ocular mucous membrane of the eye with the scores of FAMACHA.The FAMACHA eye color scores value 3, 4 and 5 or 4 and 5 were considered anemic, while 1 and 2 or 1, 2 and 3 were considered as non-anemic. PCV and Hb data were analyzed according to the classification of anemia severity into three levels (PCV 17, PCV 19, PCV 21), ( Hb 6.5, Hb 7, Hb 7.5). FAMACHA system was applied to 500 animals to estimate the apparent prevalence of anemia in sheep. Statistical Analysis: Data were subjected to analysis using SAS program (Statistical Analysis System) version 9.1. The necessary sample size was determined according to the formula: n=1.96 2 *Pr(1-Pr)/d 2, where, n is required sample size, 1.96 is a confidence interval 95%, Pr is expected prevalence and d is the margin of error ( 0.05). The estimated of sample size was shown below: n=1.96 2 *0.1(1-0.1)/0.05 2 n= 138.3 140 Isaac and Michael [20] and Hill [21] suggested that the appropriate sample size of the pilot study ranged from 10% to 30% of the sample projected for the study. As the sample size of the study was determined to be 500 the pilot study included 50 animals. The data of the pilot study were excluded from the statistical analysis according to Leon et al., [22] who reported that the pilot study data generally should not be combined with data from the subsequent larger scale study along with the data of the animals subjected to general clinical examination (140 animals).the sensitivity, specificity, positive predictive value (PPV), and negative predictive value(npv) of the FAMACHA system were calculated according to Bath et al. [7]. Sensitivity (Sn)%=TP/(TP+FN) x 100

188 Wathiq Ali Hasson Al-Jbory and Firas Rashad Al-Samarai, 2016 Specificity (Sp)%= TN/(TN+FP) x 100 PPV %=TP/(TP+FP) x 100 NPV %=TN/(TN+FN) x 100 Accuracy %=TP+TN/(TP+TN+FP+FN) x 100 Where: TP= True positive test, FN=False negative test, TN= True negative test and FP= False positive test. Spearman s correlations were calculated among PCV, Hb, and FAMACHA. P < 0.05 was considered as statistically significant. The apparent prevalence of 500 animals according to FAMACHA 3-5 scores was estimated. Results: Table (1) shows the means of PCV and Hb for each cut-off point. Results revealed that the accuracy of the group 3-4-5 for PCV 17(58.5%), PCV 19(61.4%) and PCV 21(67.1%) increased gradually along with increasing of cut-off point of PCV(Table 2). However, the accuracy in group4-5 of PCV was higher than group3-4-5but the trend of estimations was on the contrary, as the accuracy decreased along with increasing the cut-off point of PCV(PCV 17(76.4%), PCV 19(75.5%) and PCV 21(72.1%). A similar trend was shown for the FAMACHA (group 3-4-5 or group 4-5) concerning Hb (Table 3). Highest values of sensitivity were 87.5% and 82.3% in the group 3-4-5 for PCV 17 and Hb<6.5 respectively(table 4 and 5),while the highest values of specificity were 81.3% and 80.4% in the group 4-5 of PCV and Hb respectively. In the group 3-4-5, the highest specificity was 68.5% for PCV 21 and 58.5% for Hb 7.5. The values of PPV were 38.5% (PCV) and 51.4% (Hb) in group 3-4-5 and the corresponding values in group 4-5 were 45.2% and 55.5%. The values of NPV were 98.6% (PCV 17) and 95.7%(Hb 6.5) in group 3-4-5, whereas, the values in group 4-5 were 85.7% and 63.4% respectively. The Spearman correlation coefficient was significant (P<0.01) between FAMACHA scores and each of PCV (-0.46) and Hb(-0.26) (Table 6). The prevalence of anemia in 500 animals was 12.2% according to FAMACHA scores in group 3-4-5 and 7.2% in group 4-5 (Table 7). Discussion: Across several studies, it is usually, to find different estimates of sensitivity and specificity. These differences could be attributed to several factors such as the criteria employed, breed, the management system, the evaluators experience and the prevalence of anemia [15]. In the present study three cut-off points for PCV and Hb were used for calculating the sensitivity and specificity of the FAMACHA system because there is no specific cut-off point for PCV and Hb that could use to classify the animals to healthy and anemic [3]. Different PCV cut-off points for anemia confirmation were used and ranged between 15-27 [5, 15, 23]. On the other hand, a few studies used Hb to evaluate the FAMACHA system. Hence, the defining of cut-off points of Hb was determined according to the previous study conducted by Badawi and AL-Hadithy, [24] who found that the mean of Hb in clinically healthy Iraqi Awassi sheep was10.8 g/dl and ranged from 8.6 to 15.1g/dl. Hence, three cut-off points of Hb were considered as they were lowered than the Hb minimum level of healthy sheep. The high variation in cut-off points led to great variations in the results of different studies. Thus, three cut-off values of PCV ( 19%, 18%, and 15%) and Hb ( 7.5, 7, and 6.5) were selected for defining anemic animals in this study. The estimates of sensitivity and specificity in group 3-4-5 for PCV and Hb were higher than the corresponding estimates in group 4-5. These results in agreement with results of Kaplan et al. [5], Burke et al. [3] and Sotomaior et al., [15] for PCV and Di Loria et al., [23] for Hb. However, the estimates of sensitivity in groups 3-4-5 and 4-5 for PCV were higher than corresponding estimates of Hb but the estimates of specificity for the same groups were lowered than the estimates of specificity for Hb. These results are partially consistent with Di Loria et al., [23] who found that estimates of sensitivity in group 3-4-5 and 4-5 for PCV were 66% and 9% while the estimates of specificity were 60% and 98%. In the same study, the estimates of the sensitivity of Hb were 60% and 8% in group 3-4-5 and 4-5, whereas the estimates of specificity were 62% and 98% respectively. The high sensitivity for PCV (87.5%) indicated that the cut-off point 17 is more appropriated to identify the anemic animals. In this regard, Kaplan et al., [5] stated that higher sensitivity estimates are more important than higher specificity estimates in the FAMACHA system because kept the animals that are truly in need of treatment (false negatives) without treating will exposes them to the risk of death, while treating animals that do not need treatment (false positives) does not cause serious problems. The reciprocal relationship between sensitivity and the false negatives are very important to take into our consideration. When FAMACHA score 3 considered as anemic this could lead to an increase in the proportion of false positive (40.7%) as compared with excluding score 3 (22.1%). The reduction in drug use is very important to delay helminthic resistance because treating an entire flock can lead to a great selection pressure for resistance in nematode populations, depending on the levels of refugia at the time [15].Therefore, the programs used to control nematode should be designed to maintain the maximum amount of refugia (the portion of the population that is not exposed to the drug)[6]. However, the inclusion of FAMACHA score 3 will increase the number of treating healthy animals but even with such increase, there is still a considerable

189 Wathiq Ali Hasson Al-Jbory and Firas Rashad Al-Samarai, 2016 reduction in drug use, thus, making it an important tool for maintaining a population of H. contortus in refugia [3, 5, 25].Although, the recommendation of inclusion or exclusion FAMACHA score 3 depend on the status of anemia in a given flock but applying this system still represents an excellent alternative to treatment of all animals [6, 11]. The advantage of the application of FAMACHA system is not limited to slow the developing of genetic resistance of parasites but also to reduce the cost of the breeding resulting from the useful use of drugs and then increasing the profits. For instance, in the light of the results of this study, the number of animals that must be given treatment is 8 out of 140 animals which representing about 6% of the total cost of treatment for all animals. The Spearman correlation between FAMACHA scores and each of PCV and Hb were -0.46 and-0.26 respectively. The correlation between FAMACHA and each of PCV and Hb was significant (P< 0.01).The correlation between FAMACHA and PCV was closed to -0.52 obtained by Kaplan et al., [5] but lower than - 0.72 and -0.77 reported by Sissay et al., [13] and Bisset et al., [10] respectively and higher than -0.26 recorded by Gauly et al., [17]. The apparent prevalence of anemia according to FAMACHA 3-4-5 score was 12.2% (Table 7) which was closed to 13.3% recorded in Brazil [18] and lower than 53.5% recorded in Ethiopia [26]. Table 1: Mean±SE of PCV and Hb according to FAMACHA scores Score of FAMACHA PCV Hb 1 28.25±1.04 8.56±0.28 2 26.89±0.60 8.24±0.21 3 23.20±0.68 7.71±0.19 4 21.88±0.76 7.53±0.26 5 0 0 Mean 24.99±43 8.05±0.12 Table 2: The number of false-negative, false-positive, true-negative, true-positive and accuracy results of the FAMACHA system, according to the different cut-off point of PCV, in sheep. Scores False-Negative False-Positive True-Negative True-Positive Accuracy PCV 17 1(0.7%) 57(40.7%) 75(53.5%) 7(5.0%) 82(58.5%) PCV 19 3(2.1%) 51(36.4%) 74(52.8%) 12(8.5%) 86(61.4%) PCV 21 11(7.8%) 35(25.0%) 72(51.4%) 22(15.7%) 94(67.1%) PCV 17 2(1.4%) 31(22.1%) 101(72.1%) 6(4.2%) 107(76.4%) PCV 19 5(3.5%) 29(20.7%) 96(68.5%) 10(7.1%) 106(75.7%) PCV 21 14(10.0%) 23(16.4%) 84(60.0%) 19(12.1%) 101(72.1%) Table 3: The number of false-negative, false-positive, true-negative, true-positive and accuracy results of the FAMACHA system, according to different cut-off points of Hb in sheep Scores False-Negative False-Positive True-Negative True-Positive Accuracy Hb 6.5 3(%2.1) 56(%40.0) 67(%47.8) 14(%10.0) 81(57.8%) Hb 7 12(%8.5) 46(%32.8) 58(%41.4) 24(%17.1) 82(58.5%) Hb 7.5 22(%15.7) 34(%24.2) 48(%34.2) 36(%25.7) 84(60.0%) Hb 6.5 9(%6.4) 28(%20.0) 95(%67.8) 8(%5.71) 103(73.5%) Hb 7 21(%15.0) 21(%15.0) 83(%59.2) 15(%10.7) 98(70.0%) Hb 7.5 38(%27.1) 16(%11.4) 66(%47.1) 20(%14.2) 86(61.4%) Table 4: The sensitivity and specificity of the FAMACHA system, according to different cut-off points of PCV in sheep Scores Sensitivity a Specificity b (a+b)/2 x 100 PPV% NPV% Prevalence% PCV 17 87.5 56.8 72.1 10.9 98.6 5.7 PCV 19 80 58.4 70.4 17.1 95.7 10.7 PCV 21 66.6 68.5 67.5 38.5 86.7 23.5 PCV 17 75.0 76.5 75.7 16.2 98.0 5.7 PCV 19 66.6 76.8 71.1 25.6 95.0 10.7 PCV 21 57.5 81.3 69.0 45.2 85.7 23.5 Table 5: The sensitivity and specificity of the FAMACHA system, according to different cut-off points of Hb in sheep Scores Sensitivity a Specificity b (a+b)/2 x 100 PPV% NPV% Prevalence% Hb 6.5 82.3 54.4 68.3 20.0 95.7 12.1 Hb 7 66.6 55.7 61.1 34.2 82.8 25.7 Hb 7.5 62.0 58.5 60.2 51.4 68.5 41.4 Hb 6.5 47.0 77.2 62.1 22.2 91.3 12.1 Hb 7 41.6 79.8 60.7 41.6 79.8 25.7 Hb 7.5 34.4 80.4 57.4 55.5 63.4 41.4

190 Wathiq Ali Hasson Al-Jbory and Firas Rashad Al-Samarai, 2016 Table 6: Spearman correlations among FAMACHA, PCV and Hb Hb PCV FAMACH -0.26** -0.46** PCV 0.64** ** (P< 0.01) Table 7: The prevalence of anemia in sheep FAMACH Total No. +ive Prevalence % Confidence limit 95% scores 3-4-5 500 61 12.2 9.6-15.4 Conclusions: The results of the present study strongly support the FAMACHA level 3 to 5 to be used as a safe and reliable tool for detecting anemic sheep and this will lead to delay the development of helminthic resistance. The detection of anemic animals by the FAMACHA system could be affected by the anemic status of a sheep, the local epidemiology, the treatments, the breed, and the physiological status of animals. Therefore, further studies are needed in Iraq to confirm the validity of the FAMACHA system as TST indicator of anemia in sheep. Conflict Of Interest: The authors declare that there is no conflict of interest regarding this study. Authors Contribution: Firas R. Al-Samarai designed the study and planned research methodology and gave final approval of the version to be published. Wathiq Ali Al-Jbory has accomplished the study and collected the data. ACKNOWLEDGEMENTS This research is non-funded. It is a part of the MSc thesis for the first author submitted to the College of Veterinary Medicine, University of Baghdad, Iraq. REFERENCES [1] Park, Y.W., and G.F.W. Haenlein, 2006. In: Park, Y.W., and Haenlein, G.F.W. (Eds.). Handbook of Milk of Non-bovine Mammals. Blackwell Publishing Professional, Oxford, UK. [2] Rinaldi, L., V. Veneziano and G. Cringoli, 2007. Dairy goat production and the importance of gastrointestinal strongyle parasitism.trans. R. Soc. Trop. Med. Hyg., 101: 745-746. [3] Burke, J.M., R.M. Kaplan, J.E. Miller, T.H. Terrill, W.R. Getz, S. Mobini, E. Valencia, M.J., Williams, L.H. Williamson, and A.F. Vatta, 2007.Accuracy of the FAMACHA system for on-farm use by sheep and goat producers in the southeastern United States. Vet. Parasitol., 147: 89-95. [4] Fernanda, S., F.S. Fortes and M.B. Molento, 2013. Anthelmintic resistance in gastrointestinal nematodes of small ruminants: advances and limitations for diagnosis. Pesq. Vet. Bras., 33(12): 1391-1402. [5] Kaplan, R.M., J.M. Burke, T.H. Terrill, J.E. Miller, W.R. Getz, S. Mobini, E. Valencia, M. Williams, L.H. Williamson, M. Larsen and A.F. Vatta, 2004.Validation of the FAMACHA eye color chart for detecting clinical anemia on sheep and goat farms in the southern United States. Vet. Parasitol., 123: 105-120. [6] Van Wyk, J.A., and G.F. Bath, 2002.The FAMACHA system for managing haemonchosis in sheep and goats by clinically identifying individual animals for treatment. Vet. Res., 33: 509-529. [7] Bath, G.F., F.S. Malan and J.A.Van Wyk, 1996. The FAMACHA ovine anemia guide to assist with the control of haemonchosis. In: Proceedings of the 7 th Annual Congress of the Livestock Health and Production Group of the South African Veterinary Association, Port Elizabeth, South Africa, 5 7 June 1996, p: 5. [8] Malan, F.S., J.A. VanWyk and C. Wessels, 2001. Clinical evaluation of anaemia in sheep: early trials. Onderstepoort J. Vet. Res., 68: 165-174. [9] Zeryhun, T., 2011. Helminthosis of sheep and goats in and around Haramaya, Southeastern Ethiopia. J. Vet. Med. Anim. Health, 4: 48-55. [10] Bisset, S.A., J.A.VanWyk, G.F. Bath, C.A. Morris, M.O. Stenson and, F.S. Malan, 2001. Phenotypic and genetic relationships amongst FAMACHA score, faecal egg count and performance data in Merino sheep exposed to Haemonchus contortus infection in South Africa. In: Proceedings of the 5 th International Sheep Veterinary Congress, Stellenbosch, South Africa, 22-25.

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