Acta Scientiae Veterinariae ISSN: 1678-0345 [email protected] Universidade Federal do Rio Grande do Sul Brasil
Pradieé, Jorgea; da Rosa Moraes, Cristiane; Gonçalves, Michele; Sousa Vilanova, Marcele; Ferreira Corrêa, Gladis; Geter Lauz, Otoniel; Moreira Osório, Maria Teresa; Schmidt, Verônica Somatic Cell Count and California Mastitis Test as a Diagnostic Tool for Subclinical Mastitis in Ewes Acta Scientiae Veterinariae, vol. 40, núm. 2, 2012, pp. 1-7 Universidade Federal do Rio Grande do Sul Porto Alegre, Brasil
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SHORT COMMUNICATION ISSN 1679-9216 (Online) Pub. 1038
Somatic Cell Count and California Mastitis Test as a Diagnostic Tool for Subclinical Mastitis in Ewes*
Jorgea Pradieé1, Cristiane da Rosa Moraes2, Michele Gonçalves1, Marcele Sousa Vilanova1, Gladis Ferreira Corrêa3, Otoniel Geter Lauz4, Maria Teresa Moreira Osório5 & Verônica Schmidt2
ABSTRACT Background: Infectious mastitis has been described as one of the main diseases affecting animals during lactation. The disease in sheep has been studied for many years in countries where mastitis has an economical importance. However, the interest in mastitis studies in animals raised for food production has increased, because the disease may cause a reduction in weight and an increased mortality in lambs. In this study, Somatic Cells Count (SCC) and California Mastitis Test (CMT) were related to bacterial isolation for mastitis diagnosis in Corriedale sheep. Material, Methods & Results: Twenty nine (29) ewes, varying from 2-5 years of age, with different numbers of lactation and born lambs and never before machine-milked were used. Milking was done from October to November, once a week in the morning period, with oxytocin application. Four milk collections were made, at biweekly intervals for bacteriologic, SCC and CMT test, which were analyzed considering each gland as a sampling unit. Low incidence of subclinical mastitis (9.66%) was observed, with the majority (15/17) caused by coagulase negative Staphylococcus. There was no change in milk production related to SCC or bacteria isolation. However changes in milk components occurred in the presence of subclinical mastitis. A poor relation (k = 0.115) was determined between the results obtained in the bacteriological test and the SCC, with low sensitivity (13.33%) and an increased number of false negative results (13%). Comparing the SCC and CMT results as the diagnostic method for subclinical mastitis, a low (r = 0.2319) but significant (P = 0.0209) correlation was observed, as well as a poor concordance (k = 0.152). Using bacterial isolation as the standard test, it was determined that the CMT has low sensitivity (28.57%) as the diagnostic method of mastitis in ewes. N Discussion: Mastitis has been considered an economically important disease in the production of sheep for meat and wool. According to reports, the frequency of its clinical occurrence may range from zero to 50%. In meat-producing herds, a low weight gain in lambs has been associated with subclinical mastitis and the study of mastitis in Corriedale sheep is justi- fied since this is considered a breed of meat sheep with the best milk production. This breed is being crossed with milk- producing breeds, such as Laucane, to form milk-producing herds. Bacterial isolation has been adopted as the diagnostic method of mastitis in all livestock breeds. Similarly to the observation made by this study, Staphylococcus and, in a few cases, Streptococcus, have been the microorganisms most frequently involved in subclinical mastitis in sheep. The milk from ewes with mastitis tends to have a lower fat and lactose content than that of healthy ewes, due to the affected secre- tory function of these animals. The SCC of milk ewes has not yet been established, but its count in a healthy udder may reach up to 1.5x106 cel.mL-1. Similarly, the CMT score to be used in sheep is still controversial, but the maximum score (+++) is adopted to indicate mastitis. The high number of false-negative and false-positive reactions observed in SCC and CMT tests means that healthy and ill animals are incorrectly identified and that no preventive and curative measures are adopted. Since the utilization of only one diagnosis method in sheep mastitis, without confirmation by bacteriologic test is not conclusive, the SCC and CMT should be used cautiously in sheep mastitis diagnosis. Keywords: mastitis, sheep, milk production, diagnosis, milk composition.
Received: October 2011 www.ufrgs.br/actavet Accepted: December 2011 *Suporte financeiro recebido pela Fundação de Amparo à Pesquisa (FAPERGS - Processo nº 0619726-Edital casadinho). 1Programa de Pós-graduação em Zootecnia Faculdade de Agronomia Eliseu Maciel, UFPEL, RS, Brazil. 2Programa de Pós-graduação em Ciências Veterinárias, Faculdade de Veterinária, UFRGS. 3Universidade Federal do Pampa, Campus Dom Pedrito, RS, Brazil. 4Instituto Federal de Educação Ciência e Tecnologia Farroupilha, RS. Brazil. 5Departamento de Zootecnia, Faculdade de Agronomia Eliseu Maciel. UFPEL, RS, Brazil. 6Departamento de Medicina Veterinária Preventiva, Faculdade de Veterinária, UFRGS. Porto Alegre, RS, Brazil. CORRESPONDENCE: V. Schmidt [[email protected] - Fax: +55 (51) 3308-7305]. Departa- mento de Medicina Veterinária Preventiva, Faculdade de Veterinária, UFRGS. Av. Bento Gonçalves, n. 9090. CEP 91540-000 Porto Alegre, RS, Brazil.
1 J. Pradieé, C. R. Moraes, M. Gonçalves, et al . 2012. Somatic Cell Count and California Mastitis Test as a Diagnostic Tool for Subclinical Mastitis in Ewes. Acta Scientiae Veterinariae. 40(2): 1038.
INTRODUCTION lambs. After being milked, the ewes and their lambs would remain together in the same fenced meadow. The milk production of small ruminants is Consequently, the milk production and composition based on three closely related aspects: adequate nutri- data presented and discussed in this study refer to a tional, sanitary and productive management. 15 h period. Infectious mastitis has been described as one Considering that the purpose of the herd was of the main diseases affecting animals during lacta- the production of lambs, the milk was collected 60 tion. The most used methods to determine the health days after birth, when the lambs no longer depended of the udder of lactating ewes are: California Mastitis on the ewe’s milk. Four milk collections were made Test (CMT), also known as indirect method, whose every 15 days, when the end of milk production was principle is the utilization of a detergent that acts verified. The udders were cleaned with water and on the external membrane of the cells (lipoprotein dried with paper towels. Once dried, the udders were membrane), exposing the gel-like DNA and, for that physically examined [25] in order to detect clinical reason, the higher its consistency the higher will be the mastitis, which is characterized by visible signs, such somatic cell count (SCC) which, in turn, is known as as edema and increased sensitivity and temperature of direct method, in which an electronic device, through the mammary halves [29], fever, inappetence, dehydra- optical and infrared filter systems, determines the tion, etc. Then the teats were disinfected using cotton quantity of somatic cells and other components in the wool soaked in alcohol 70ºGL. The first jets of milk milk, as well as the causal agent of mastitis. were discarded onto a paddle in order to perform the Ovine mastitis has been known and studied California Mastitis Test (CMT) [18,28]. Then indi- for many years in countries where the production of vidual milk samples from each teat were aseptically ewe’s milk is economically important [34]. The inter- collected [28], of which 0.01 mL were streaked onto est in mastitis studies has also increased in relation to 5% sheep blood agar and incubated at 37ºC during 24 meat-producing herds, because the disease may lead to 48 h. After incubation, the colonies were counted to a weight reduction and an increase in mortality of and characterized with regard to their morphology and lambs [12,14]. staining characteristics [26] and then maintained at The milk production in meat sheep breeds can -20ºC in brain heart infusion added by 20% glycerol, be considered an important source of animal protein. until the moment of their identification [19]. Samples We therefore aimed at characterizing the CMT and with a growth of 5 or more identical colonies [10] SCC tests as diagnostic methods of subclinical mas- were considered positive for subclinical mastitis. The titis in Corriedale sheep, comparing them to bacterial growth of two or more morphological types (>5 UFC isolation and variations in milk composition of these per type) was considered as contamination and the animals suffering from this disease. result was excluded from the analysis [2,15]. MATERIALS AND METHODS After milk collection of bacteriological ex- amination, the udders were emptied and the milk was The experiment was carried out at Centro Ag- homogenized. One sample per animal was produced ropecuário da Palma/UFPEL, in the town of Capão do in order to analyze fat, raw protein, lactose and total Leão, RS, Brazil (31o 52’ S and 52o 29’ W) between solid contents through infrared absorption, using the October and November 2007. The herd was composed Bentley 2000® device and the somatic cell count tech- of Corriedale and Texel sheep specimens for the pro- nique, which was carried out by an electronic counting duction of lambs. Twenty-nine Corriedale ewes were device based on flow cytometry, at Embrapa Terras randomly selected, having their date of parturition as Baixas’ milk analysis laboratory. Samples presenting a selection factor. The ewes were between 2 and 5 years mastitis-positive counting higher than 1,000,000 cells. old, with different number of lactations and number mL-1 [4] were considered. of born lambs, and none of them had been mechani- The statistical analysis was made using the cally milked before. They were milked once a week in software GraphPad Prism 4.0, applying a significance the morning, upon application of oxytocin [27]. The level of 5%. The CMT, SCC and bacteriological data ewes would be separated from their herd the night were analyzed considering each gland as a sample before, remaining approximately 15 h apart from their unit [23]. For the purpose, bacterial isolation was
2 J. Pradieé, C. R. Moraes, M. Gonçalves, et al. 2012. Somatic Cell Count and California Mastitis Test as a Diagnostic Tool for Subclinical Mastitis in Ewes. Acta Scientiae Veterinariae. 40(2): 1038. considered in at least one of the teats and the highest cal mastitis was detected [10] in 17 (9.66%) samples. CMT result was considered as the result of the animal. Coagulase-negative Staphylococcus was identified in The sensitivity, specificity, as well as the positive and 15 samples, Streptococcus sp. was identified in one negative predictive values of the SCC and CMT were sample and a Gram-negative bacteria was identified in determined by the Fischer’s exact test and kappa index, another. This latter was not identified because it could using bacterial isolation as the standard test. The as- not be retrieved after being frozen. sociation between the CMT and SCC was calculated Among 29 females examined, bacteria could by the McNemar test and by the Kappa index and the be isolated from 6 in at least one collection and from correlation was calculated by the Spermann’s test. 4, in two or more collections. Only two ewes had The correlation index was classified as high (r > 0.7), subclinical mastitis in the two mammary halves at medium (0.5 < r < 0.7) and low (r < 0.5) [9]. The value the same time. scale of the kappa index was used [1]. The comparisons There was no significant difference in the pro- of production medians were made by the T-test and duction of animals with subclinical mastitis (0.4750 those of milk constituent medians, with or without L), in comparison to those without bacterial growth mastitis, were made by the Mann-Whitney’s test. in their milk (0.4370 L). With regard to milk constituents, a significant RESULTS increase in the levels of protein (P = 0.0299), total This present study did not show evidence of solids (P = 0.0106) and fat (P = 0.018) was verified females whose symptoms were compatible with those in animals with subclinical mastitis (Table 1). of clinical mastitis. One hundred seventy-six individual Considering that animals which showed bacte- samples were collected from each teat, and subclini- rial growth in at least one teat had subclinical mastitis,
Table 1. Median values of Corriedale ewes’ milk in the presence of subclinical mastitis or in a healthy udder, considering bacterial isolation as the diagnostic method.
N
Different letters in the same line indicates a significant statistic difference (P < 0.05). there was no significant difference (P = 0.2176) in the high number of false negative results (13%). Consider-
SCC median of animals with (5.1 Log10) and without ing bacterial isolation as the standard method, its was
(4.8 Log10) mastitis (Table 2). determined that the SCC, as the diagnostic method of It was verified that at least six females had a mastitis in sheep, had low sensitivity (13.33%) and a cell count above one million (1,250,000 to 9,342,000 Positive Predictive Value (33.33%), and high specific- cel.mL-1) in one single collection. For the purpose of ity (95.29%) and Negative Predictive Value (86.17%). comparison between the SCC /other diagnostic meth- Furthermore, there was no significant differ- ods and the milk constituents and production, samples ence (P = 0.6121) in the milk production (0.4371 L) with a SCC > 500,000 cel.mL-1 were considered posi- of animals with high SCC (> 500,000 cel.mL-1) and tive in the diagnosis of mastitis. A cell count above other animals (0.4684 L). However, the protein (P = this number was observed in 10 females and, of them, 0.0278), lactose (P < 0.0001), total solids (P = 0.0056) only one had a SCC count above 500,000 cel.mL-1) in and fat (P = 0.0009) values were significantly different three collections. (Table 3). This study used score > 2 (++ or +++) in A poor relation (k = 0.115) was determined the CMT [18] as the presumptive diagnosis of mastitis between the results obtained in the bacteriological test in this species. and the SCC. Comparing the results of the bacterial Comparing the SCC and CMT results as the isolation and of the SCC, it was observed concordance diagnostic method for subclinical mastitis, a low (r = in 83 and discordance in 17 observations, resulting in a 0.2319) but significant (P = 0.0209) correlation and 3 J. Pradieé, C. R. Moraes, M. Gonçalves, et al . 2012. Somatic Cell Count and California Mastitis Test as a Diagnostic Tool for Subclinical Mastitis in Ewes. Acta Scientiae Veterinariae. 40(2): 1038.
Table 2. Somatic Cell Count (SCC) in ewes with and without subclinical mastitis, considering bacterial isolation as the diagnostic method.
Table 3. Median values of Corriedale ewes’ milk in the presence (infected ud- der) or not (healthy udder) of Somatic Cell Counts (SCC) indicating mammary infection (>500.000 cel.mL-1).
Different letters in the same line indicates a significant statistic difference (P < 0.05). poor concordance (k = 0.152) were observed between gain in lambs has been associated with the presence of the two diagnostic methods of mastitis in ewes. subclinical mastitis [12]. Differently in milk-producing Even though 78 of 99 results observed cor- breeds, mastitis has been observed more frequently, respond to the CMT and the bacterial isolation, there affecting up to 41% of the samples analyzed [2]. The was no association between the results of these two study made with the Corriedale breed is justified since methods in the mastitis diagnosis in sheep. Consider- this is considered the breed of meat sheep with the best ing bacterial isolation as the standard method, it was milk production [11] and, for that reason, it has been determined that the CMT has low sensitivity (28.57%) crossed with milk-producing breeds, such as Laucane, and a Positive Predictive Value (26.67%), and high to form milk-producing herds. specificity (87.06%) and Negative Predictive Value Bacterial isolation has been adopted as the (88.10%). It was observed that the number of false diagnostic method of mastitis in all livestock breeds. positive and false negative results was similar. Similarly to the observation made by this study, Sta- phylococcus and, in a few cases, Streptococcus, have DISCUSSION been the microorganisms most frequently involved in Mastitis has been considered an economically subclinical mastitis in sheep [2,24]. Mastitis caused important disease in the production of sheep for meat by Gram-negative microorganisms is not frequent and and wool [22]. According to reports, the frequency the fact that the sample isolated in this study could not of its clinical occurrence may range from zero to be recovered is supported by the scientific literature, 50% [20]. In sheep herds in Spain [2], mastitis was where there is a loss of up to 40% of bacterial samples observed in only 0.4% of the samples analyzed and a during the laboratorial identification process [5] or higher number of subclinical mastitis was observed. after being frozen [32]. On the other hand, in Brazil, subclinical mastitis was The fact that a higher mastitis frequency was observed in Santa Inês females (11%) [35] during the observed in the right teat could be explained by the first lactation week and clinical mastitis (6%) was number or offspring, considering that nine females observed at birth; in Hampshire Down ewes [13], a with subclinical mastitis gave birth to one lamb and clinical mastitis outbreak was observed in 10% of a only two gave birth to multiple lambs. Since the lambs herd. However, the diagnostic methods used in each were kept with their mothers during the experiment, record must be considered, using the bacterial isolation there is a tendency of the lamb to suck more frequently in most cases. In meat-producing herds, a low weight from the same teat in case of single births, due to
4 J. Pradieé, C. R. Moraes, M. Gonçalves, et al. 2012. Somatic Cell Count and California Mastitis Test as a Diagnostic Tool for Subclinical Mastitis in Ewes. Acta Scientiae Veterinariae. 40(2): 1038. physiological and anatomical characteristics. As a tion will have a SCC above 1 million cells, in at least consequence, residual milk may occur in the teats, two consecutive samplings [4]. leading to the proliferation of microorganisms. Other method to diagnose mastitis widely used With regard to the milk components, it was ob- in bovines is the CMT. This test is used worldwide served a tendency for an increase in the fat percentage to diagnose subclinical mastitis and, additionally, it in ewes with subclinical mastitis [16] and for the reduc- can be carried out in the herd when the animals are tion of these contents in experimental clinical mastitis being milked. The interpretation of the CMT is based [29]. Animals with mastitis would tend to have a lower on the visual inspection of the milk after the reagent fat and lactose content than that of healthy ewes, due is mixed in. The reaction takes place between the to the affected secretory function of these animals [4]. reagent and the genetic material from somatic cells In ewes with infectious mastitis, a high found in the milk, forming a gel whose concentration percentage of protein was observed in their milk is proportional to the number of somatic cells [6]. in comparison to healthy ewes, and the fact can be However, in small ruminants, this test still generates explained by the presence of blood cells in the milk, controversies, because the amount of physiological which remains with the same production volume [33]. somatic cells from these animals is very large, causing In addition to the hygiene issue, the economi- a false-positive result. cal aspect of mastitis is important due to the reduction The CMT score to be used in sheep is still it causes in the milk production [29,30]. However, controversial. A CMT score of 1 [14] is recommended this fact was not observed in this study. Similarly, an for the subclinical mastitis diagnosis, and the maxi- increase in somatic cells [30], which was not observed mum score (+++) is recommended for the diagnosis in this study, has been reported. of infectious mastitis of sheep [8]. The high number The microbiology of the milk is associated of false-negative and false-positive reactions observed with the SCC, which is increased in pathological in diagnostic tests means that healthy and ill animals and inflammatory processes of the mammary gland, are incorrectly identified and that no preventive and reducing the fat, casein and total solids contents and curative measures are adopted. increasing the total nitrogen, non-protein nitrogen and The economical losses caused by mastitis Nin milk protein contents [3]. In milk-producing cows, the small ruminants are extremely high, particularly in SCC has been widely used as the diagnostic method of milk-producing herds. A reduction in the milk produc- mastitis, being employed as a qualitative milk evalu- tion due to clinical and subclinical mastitis, as well as ation method, also with regard to the price paid to a reduction in the milk quality and the consequences it the producer. However, in small ruminants, there is a has on animals and on food safety are aspects associ- considerable epithelial desquamation of the mammary ated with said losses. For that reason, it is necessary apparatus, i.e., presence of cytoplasmic corpuscles to study and develop strategies to control this disease resulting from the apocrine milk secretion process [24] in sheep. [7], thus increasing the amount of SCC. Differently CONCLUSIONS from the observation made in this study, a reduction has been noticed in the production of ewe’s milk with A low occurrence of subclinical mastitis a high SSC count [20]. (9.66%) was observed, and the majority (15/17) was
SCC values in healthy sheep (4.86 Log10), caused by coagulase-negative Staphylococcus. There which are lower than the SCC values observed in ewes was no alteration in the production of milk associated with mastitis (5.9 Log10) [2], similarly as observed in with the SCC or bacterial isolation. However, there this study, have been reported. However, the somatic was an alteration in the milk constituents when sub- cell count (SCC) of a healthy udder in milk-producing clinical mastitis was present. SCC and CMT have low ewes has not yet been established. Studies shows that sensitivity when compared to the bacterial isolation the SCC levels vary greatly, reaching a count up to technique and, as such, they must be carefully used 1.5x106.mL-1 in a healthy udder [20]. However, a in the diagnosis of mastitis in sheep. count limit above 250,000 cells.mL-1 [17,21] or below Declaration of interest. The authors report no conflicts of -1 500,000 cells.mL [4] has been suggested for healthy interest. The authors alone are responsible for the content and udders. On the other hand, ewes with mammary infec- writing of the paper.
5 J. Pradieé, C. R. Moraes, M. Gonçalves, et al . 2012. Somatic Cell Count and California Mastitis Test as a Diagnostic Tool for Subclinical Mastitis in Ewes. Acta Scientiae Veterinariae. 40(2): 1038.
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