- Email: [email protected]
Relationship between teat-end condition, udder cleanliness and bovine subclinical mastitis
Research in Veterinary Science 93 (2012) 430–434
Contents lists available at ScienceDirect
Research in Veterinary Science journal homepage: www.elsevier.com/locate/rvsc
Relationship between teat-end condition, udder cleanliness and bovine subclinical mastitis Marcela de Pinho Manzi, Diego Borin Nóbrega, Patrícia Yoshida Faccioli, Marcella Zampolli Troncarelli, Benedito Donizete Menozzi, Hélio Langoni ⇑ Department of Veterinary Hygiene and Public Health, São Paulo State University, Botucatu 18618-900, Brazil
a r t i c l e
i n f o
Article history: Received 15 February 2011 Accepted 12 May 2011
Keywords: Bovine mastitis Teat-end condition Udder cleanliness
a b s t r a c t The aims of the present study were to relate intramammary infection (IMI) occurrence and somatic cell count (SCC) with teat-end condition (TEC) and udder cleanliness (UC). Milk samples from 1931 teats were evaluated according to the presence of IMI and SCC. Scores were applied to teats according to the TEC and to UC. Teats ends with a very rough ring had the largest number of IMI when compared to the other three categories, as well as animals with dirtier udders. The change in a TEC score increased by around 30% the chance of IMI. Also, the chance of the animal developing IMI increased by approximately 47% when the UC score increased. No significant association between both scores and quarter SCC was found. It can be concluded that animals with very rough teat end rings and very dirty udders have a greater predisposition to IMI. Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction Mastitis is an inflammation of the mammary gland, which is characterized as the most common infectious disease affecting dairy cattle. It causes extensive economic loss due to decreased milk yield, lower milk quality, higher treatment costs and premature culling of cows with chronic mastitis. Depending on the pathogen, mastitis can be classified into either contagious or environmental (Coffey et al., 2006; Schreiner and Ruegg, 2002). The primary reservoir for contagious pathogens is the udder of infected cows. The most common contagious mastitis pathogens include Staphylococcus aureus and Streptococcus agalactiae (Schreiner and Ruegg, 2002). The environmental pathogens are present in the cows environment. Coliform bacteria and environmental streptococci are the most commonly isolated environmental pathogens. Although the contamination of animals by environmental microorganisms may occur during the milking process (usually due to equipment failure), it is more frequent between milkings. Control is generally difficult because the bacteria are disseminated in the environment of animals (Langoni, 2000). Somatic cell count (SCC) is the most frequently used indicator of subclinical mastitis in dairy cattle, and is recognized as an indicator of cow health and milk quality (Tsenkova et al., 2001). The most important cause of increased SCC is bacterial infection of the mammary gland (de Haas et al., 2004; Olde Riekerink et al., 2007b; Tsenkova et al., 2001). ⇑ Corresponding author. Tel.: +55 14 38116270; fax: +55 14 38116075. E-mail address: [email protected] (H. Langoni). 0034-5288/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.rvsc.2011.05.010
The teat sphincter and teat canal are important primary barriers against pathogen invasion into the udder. Thus, it is essential that such structures be in perfect physical and hygienic conditions to prevent intramammary infection (IMI). The correlation between teat sphincter integrity and milking conditions is not well understood, particularly regarding udder cleanliness (UC) and its involvement with pathogens implicated in mastitis. According to Neijenhuis et al. (2000), lesions in the teat sphincter are frequently colonized by Staphylococcus spp. and Streptococcus spp., which illustrates the correlation between the physical condition of mammary quarters and the presence of microorganisms. The classification of the teat end integrity into scores can serve as an important tool to control bovine mastitis because it allows for the classification of the different types of physical injuries. These irregularities can be related to problems in the management and production system, leading to a greater predisposition toward mastitis in the herd. It is known that cows with clinical mastitis frequently have more pointed teat ends than their paired healthy herd mates, and there is a relationship between teat-end callosity and clinical mastitis (Neijenhuis et al., 2001). Another relevant aspect is udder hygiene, which must be assessed at milking because it influences milk quality and is related to occurrence of pathogens, especially environmental ones. According to item 5.1.2 of ‘‘Instrução Normativa 51’’ [Normative Instruction No. 51] (Mapa, 2002) of the Brazilian Ministry of Agriculture, Livestock and Food Supply, the dairy farm must provide sanitary housing for cows in order to prevent udder contamination. Therefore, there is a concern about the environment where dairy animals are housed in order to produce high quality milk for
M. de Pinho Manzi et al. / Research in Veterinary Science 93 (2012) 430–434
431
human consumption. It is clear that a poor environment should result in dirtier udders. All hygienic factors related to the udder can be classified based on the cleanliness score. There is a relationship between teat or udder contamination and occurrence of mastitis (Galton et al., 1988; Schreiner and Ruegg, 2003). The aims of the present study were to relate IMI occurrence and SCC with teat-end condition (TEC) and UC, and to verify the most frequent teat scores for contagious and environmental pathogens. 2. Materials and methods The study was carried out in seven dairy farms located in São Paulo State, Brazil. The Holstein cows were in herds of different size, and in different stages of lactation and lactation number. The experiment was carried out in two parts (relationship between IMI, TEC and UC; and relationship between SCC, TEC and UC), mainly because of strict farms policies. 2.1. Relationship of IMI to TEC and UC The evaluation included 1931 teats from 499 animals (65 with missing teats were excluded). Loose dirt was brushed of the teats with sanitizing solutions (varies from farm to farm). Teats were dried with disposable towels, then teat ends were scrubbed with cotton pledgets. Foremilk samples were taken after the first streams of milk were discarded. Each animal participated only once in the study. The samples were kept at 4 °C until diagnostic bacteriology. Microbiological culture was done in duplicate according to standard procedures of the National Mastitis Council (NMC, 1999). First, 10 lL of each sample were spread onto 8% ovine blood agar and MacConkey agar media, followed by incubation at 37 °C. Examination of the plates was made after 24, 48 and 72 h of incubation. Isolated colonies were studied morphologically and Gram stained. Biochemical tests were applied according to the bacterial genus. Staphylococci were subjected to coagulase test. Coagulasepositive staphylococci underwent fermentation of sugars (maltose, mannitol and trehalose). Coagulase-negative staphylococci (CNS) were subjected to biochemical identification. Briefly, the method consisted of a set of biochemical tests that determine the utilization of the sugars xylose, arabinose, sucrose, trehalose, maltose, mannitol, lactose, xylitol, ribose, fructose and mannose; production of hemolysin; nitrate reduction; presence of urease and ornithine decarboxylase; and resistance to novobiocin characterized by an inhibition halo of up to 16 mm. Readings of the tests were obtained after 24, 48, and 72 h of incubation at 37 °C in an air incubator (Cunha et al., 2004). The Gram-positive, catalase-negative cocci were identified as belonging to the Streptococcaceae family and subjected to CAMP test and esculin hydrolysis. The Gram-positive, pleomorphic and catalase-positive coccobacilli were classified within the Corynebacterium genus. Blood agar plates containing three or more different colonies were considered contaminated. Major mastitis pathogens included S. aureus, Streptococcus spp., Arcanobacterium pyogenes, or coliform species. Minor pathogens were defined as CNS or Corynebacterium species, as described by other authors (Rodrigues et al., 2009). A quarter was considered infected with a minor pathogen when at least three colonies were observed on the blood agar plate, and one or more colonies for a major pathogen. IMI was considered positive at the animal level when isolation was positive for at least one mammary quarter of the 499 animals. If one of the duplicate samples was contaminated, the results from the uncontaminated duplicate alone was used to diagnose infection. When a quarter had both a major and a minor pathogen isolated at the same time, the quarter was defined as infected with the major pathogen (Compton et al., 2007). Fourteen
Fig. 1. Normal teat (left picture) scored as 1 according to teat end callosity, and teat scored as 4 (right picture), with keratin fronds extending more than 4 mm from the orifice.
milk samples were considered contaminated and excluded from analyses. Scores were applied at the teat level before the pre-milking routine by the same trained technician according to TEC: 1 = no ring, smooth teat end with a small even orifice (Fig. 1); 2 = smooth ring with a raised ring around the orifice (the raised area may be smooth or slightly rough); 3 = rough ring with a raised, roughened ring with fronds of keratin extending 1–3 mm from the orifice; and 4 = very rough ring with keratin extending more than 4 mm from the orifice. Teat-end condition scores were chosen according to (Mein et al., 2001). Cows with missing teats were excluded from analyses. UC was scored according to the udder to which the teat is located (1 = completely free of or has very little dirt; 2 = slightly dirty; and 3 = mostly covered or completely covered in dirt). UC was scored according to Schreiner and Ruegg (2002), grouping the last two scores into one, because score 4 had a low frequency. 2.2. Relationship of SCC to TEC and UC One teat and udder from each of 444 different animals was classified for TEC and UC as described in the first experiment. The animals that participated in the second experiment were from the same farms as the animals that were included in the first experiment. Only one teat per cow was sampled for SCC, mainly because of strict policies of the farms. The teats were selected in a way to include the same number of teats in each position (right front, left front, right rear and left rear). Milk samples (50 mL) were collected from each teat for electronic somatic cell count (eSCC) by using a calibrated Somacount 300 Bentley unit. 2.3. Statistical analysis Before statistical analysis, observations were checked for unlikely values and excluded from the analysis. The analyses were performed according to the considered experiment. 2.3.1. Relationship IMI – TEC and UC Absolute and relative frequency values of IMI, TEC score and UC scores were obtained for all evaluated teats. The logistic regression model was applied at the teat level to assess the effect of changes in TEC and UC, as well as in IMI status (presence or absence). The assessment included all valid teats (1931 teats). Logistic regression model was applied and odds ratio were estimated with respective 95% confidence intervals (CI). The initial model applied was:
Logitðpklmn Þ ¼ b þ farmk þ Tec Scr l þ Uc Scr m þ Tec Scr Uc Scr þ n where p is the probability of existence of IMI (0 = no, 1 = yes), b is the constant, farmk is the effect of the farm to which the animal belongs, Tec_Scrl is the TEC score (1 = Normal ring; 2 = Smooth ring; 3 = Rough ring; and 4 = Very rough ring showing eversion), Uc_Scrm
432
M. de Pinho Manzi et al. / Research in Veterinary Science 93 (2012) 430–434
is the UC score of the udder to which the teat belongs (1 = clean, 2 = dirty, 3 = very dirty), Tec_Scr⁄Uc_Scr is the association term, and en is the residual term. Odds ratio (OR) were estimated with 95% confidence interval (CI). Goodness of fit for the logistic regression model was assessed using the Hosmer and Lemeshow test and graphical analysis of residuals and predicted values, respectively (Field, 2005). The predictors entered the model in a backward conditional method. The final model applied contained only the TEC score as a predictor. Part of its effect was suppressed by the presence of UC score and the interaction effect, showing possible colinearity among variables, which was detected by the correlation matrix. Thus, separate models were used for UC score and TEC score, in which only the latter variable showed contribution. At the animal level, the logistic regression model was also applied, using UC score as predictor and the presence/absence of IMI as outcome. The same parameters previously described were assessed (Field, 2005). In this analysis, 461 animals were included (animals with missing teats were excluded). A specific pathogen was only studied if identified in more than six mastitis cases during the study period. The remaining pathogens were grouped as ‘‘other’’. CNS that did not achieve at least six isolations were grouped as ‘‘Staphylococcus spp.’’. The final classification of the pathogens included: Corynebacterium bovis, Escherichia coli, Staphylococcus spp. (coagulase-negative staphylococci that didn’t achieve six isolations), S. aureus, Staphylococcus epidermidis, Staphylococcus warneri, S. agalactiae, Streptococcus dysgalactiae, and Streptococcus uberis. Relative frequencies were calculated for each valid pathogen. The most frequent TEC score and UC score for each pathogen were obtained (modes). The isolated pathogens were grouped into two categories, contagious and environmental. Teats with IMI caused by contagious and environmental pathogens were compared in terms of TEC. Chi-square test was employed to verify differences among TEC frequencies according to the type of pathogen. 2.3.2. Relationship of SCC to TEC and UC For the second experiment, SCC values were log-converted to base 10 (LogSCC) to achieve homogeneity of variance. Mean values were obtained, as well as lower and upper bounds of the 95% CI, 25 and 75 percentiles and median values for LogSCC according to teat and animal cleanliness scores. Kolmogorov–Smirnov normality tests were applied. Differences in eSCC between TEC and UC scores were evaluated using the General Linear Model (GLM), adopting eSCC as the outcome and TEC score and UC score as the predictors in separate models. The farm entered as a random factor in both models. Variance homogeneity was assessed by means of Levene’s test. Hochberg’s GT2 and Bonferroni tests were used as post hoc tests to verify differences between eSCC in the different teat and udder scores (Field, 2005). In this second experiment, only analyses at the teat level were performed. The level of statistical significance was set at 0.05 for all analyses. 3. Results 3.1. Relationship of IMI to TEC and UC Table 1 shows the variables related to the relationship IMI to TEC and UC for experiment 1. Of 1931 teats evaluated, 1627 (84.3%) did not have IMI. For 571 (29.6%) TEC score = 1 (no ring), for 599 (31.0%) = 2 (smooth or slightly rough ring), for 415 (21.5%) = 3 (rough ring), and for 346 (17.9%) = 4 (very rough ring). Among these, there was a tendency toward the frequency of IMI to increase with increasing TEC score. Score 4 teats had the largest number of IMI when compared to the other three categories. IMI was detected in 58 teat score 1 (10.2%), 98 teat score 2 (16.4%),
73 teat score 3 (17.6%) and 75 teat score 4 (21.7%), respectively. There was a positive effect between higher TEC score and presence of IMI (P < 0.001, B = 0.270, Exp(B) = 1.309 with LB (95%) = 1.169 and UB (95%) = 1.467). With regards to udder scores, 1302 teats (67.4%) were from clean udders, 478 (24.8%) from slightly dirty and 151 (7.8%) from very dirty udders. There appeared to be a tendency for UC to parallel TEC. Intramammary infection was detected in mammary quarters of 195 (15.0%) clean udders, 16.9% of dirty udders and 18.5% of very dirty udders. Although IMI cases tended to increase with a higher UC score, the effect of UC score was not statistically significant (P = 0.097). The interaction between TEC score and UC score was not significant (P = 0.786). At the animal level, 381 animals did not have IMI (82.6%) and 80 animals had IMI (17.4%). A total of 315 udders (68.3%) were classified as clean, 109 (23.6%) as dirty, and 37 (8.0%) as very dirty. IMI cases tended to increase with UC score. Of 80 cases positive for IMI, 46 were in clean udders (14.6% of the total clean udders), 25 in dirty udders (22.9% of the dirty udders) and 9 in very dirty udders (24.3% of the total udders score 3). There was a positive effect between the increase in UC score and the presence of IMI at the animal level (P = 0.03, B = 0.388, Exp(B) = 1.474 with LB (95%) = 1.037 and UB (95%) = 2.093). The most frequent isolated pathogens were C. bovis (46.4%), Streptococcus dysgalactiae (13.5%) and Staphylococcus spp. (8.2%). Table 2 summarizes the results of microbiological tests for the 304 IMI. For almost all isolated bacteria, except S. agalactiae, foremilk samples were from teats of clean udders. Teat scores varied (P < 0.05) among type of pathogen (Table 2). Among the pathogens isolated, 233 were contagious (76.6%) and 71 (23.4%) were environmental (Table 3). There were no differences between isolations of contagious and environmental bacteria according to TEC (P = 0.146) and UC score (P = 0.529).
3.2. Relationship of SCC to TEC and UC Even with low SCC (Table 4) for UC score 3 when compared to the remaining ones, there was no difference (P = 0.083) between SCC in the different udder cleanliness scores when the farm effect was controlled (Table 3). The same was not observed for teat score (P = 0.002), especially for lower counts in teats of score 2, relative to score 1, score three and score four (P < 0.001 for all three comparisons, respectively).
Table 1 Absolute (n) and relative frequency (%) of teat-end condition score (TEC score), udder cleanliness score (UC score) and intramammary infection status (IMI) from the animals included in experiment 1. Variable TEC scorea 1 2 3 4 UC scoreb 1 2 3 IMI No Yes
n
%
571 599 415 346
29.60 31.00 21.50 17.90
1302 478 151
67.40 24.80 7.80
1627 304
84.30 15.70
a 1 = no ring; 2 = smooth rough ring; 3 = rough ring; 4 = very rough ring. b 1 = clean udders; 2 = dirty udders; 3 = very dirty udders.
433
M. de Pinho Manzi et al. / Research in Veterinary Science 93 (2012) 430–434 Table 2 Absolute (n) and relative frequency (%) for results of the microbiological test, and most frequent values (modes) of teat-end condition score (TEC score) and udder cleanliness score (UC score) according to the isolated pathogen. Result
n
%
TEC score
UC score
Staphylococcus spp.a Corynebacterium bovis Escherichia coli Staphylococcus aureus Staphylococcus epidermidis Staphylococcus warneri Streptococcus agalactiae Streptococcus dysgalactiae Streptococcus uberis Other pathogensb Total
25 141 6 17 19 13 12 41 20 10 304
8.2 46.4 2 5.6 6.3 4.3 3.9 13.5 6.6 3.3 100
4 2 4 2 2 3,4 2 2 1 3
1 1 1 1 1 1 3 1 1 1,2
eSCC Mean TEC scoreA 1 2.655a 2 2.289b 3 2.716a 4 2.701a UC scoreB 1 2.644a 2 2.496a 3 2.251a
a Staphylococcus captis urealyticus, Staphylococcus haemolyticus, Staphylococcus hyicus, Staphylococcus saprophyticus, Staphylococcus xylosus. b Bacillus spp., Candida albicans, Klebsiella pneumoniae, Micrococcus spp., Prototheca spp., Pseudomonas aeruginosa.
Table 3 Absolute and relative frequencies (%) for contagious or environmental pathogens according to the teat-end condition score (TEC score) and udder cleanliness score (UC score). Contagious
(%)
Environmental
(%)
18 32.2 23.2 26.6
16 23 18 13
22.9 32.9 25.7 18.6
66.1 24 9.9
40 25 5
57.1 35.7 7.1
a
TEC score 1 42 2 75 3 54 4 62 UC scoreb 1 154 2 56 3 23 a b
Table 4 Mean values, lower (LB) and upper bounds (UB) of 95% confidence interval, median values and 25 (P25) and 75 percentiles (P75) of electronic somatic cell count (eSCC – log transformed value 103 somatic cells/mL) according to the teat-end condition score (TEC score) and udder cleanliness score (UC score).
1 = no ring; 2 = smooth rough ring; 3 = rough ring; 4 = very rough ring. 1 = clean udders; 2 = dirty udders; 3 = very dirty udders.
4. Discussion Mammary quarter level risk factors for IMI may include teat position and shape (Breen et al., 2009). In the present study, there was an association between TEC and IMI. The change in a unit of TEC score increased by around 30% the chance of developing IMI, relative to the original category. Neijenhuis et al. (2000) conducted a study where teat-end callosity was evaluated and found out that a hyperplastic stratum corneum, found in teats with an increased teat-end callosity score, leads to a roughened surface to which bacteria can adhere, making disinfection of the teat after milking more difficult and limiting its effectiveness (Neijenhuis et al., 2000). The evaluated TEC scores were positively correlated to the larger number of IMI cases. The incidence of subclinical and clinical mastitis depends on factors such as parity, stage of lactation, type of housing, access to pasture, management, and environmental factors (Olde Riekerink et al., 2007a). Reduced udder hygiene has been associated with intramammary pathogens (Schreiner and Ruegg, 2003). Fecal contamination of stalls is considered a risk factor in the transmission of environmental mastitis (Fregonesi et al., 2009), which suggests that cows with a higher UC score would have a higher prevalence of environmental mastitis, which was not verified in the present study. Udder cleanliness is influenced by external factors (hygiene, season, etc.) and does not necessarily leads to IMI. In addition, many factors can influence the degree of udder cleanliness among cows (Tucker et al., 2001). Rainy seasons, production system (free-stall versus pasture) and the hygiene routine on the farm contribute to udder cleanliness. During rainy seasons, dairy
LB (95%)
UB (95%)
Median
P25
P75
2.486 2.141 2.589 2.560
2.823 2.436 2.843 2.841
2.739 2.361 2.800 2.805
2.185 1.643 2.419 2.235
3.213 2.869 3.180 3.269
2.555 2.339 2.005
2.733 2.654 2.496
2.740 2.623 2.361
2.202 1.748 1.612
3.177 3.196 2.776
A
1 = no ring; 2 = smooth rough ring; 3 = rough ring; 4 = very rough ring. 1 = clean udders; 2 = dirty udders; 3 = very dirty udders. a,b Means within a column of the same classification system (TEC, UC) with different superscripts differ (one-way ANOVA post hoc tests, P < 0.001). B
cows housed in free-stalls will have dirtiers udders compared to other housing systems. The adoption of a suitable pre-milking routine is a preventive measure against occurrence of IMI (Peters et al., 2000), although it was not capable of removing all pathogens from the teat, especially when udders were dirty. A pre-milking routine was not able to remove Klebsiella spp. from teat skin (Munoz et al., 2008). In the present study, the chance of the animal developing IMI in any quarter increased by approximately 47% when UC score was altered by one unit, i.e. animals with dirtier udders clearly have more chance of developing mastitis. There were no differences between pathogen types when considering TEC and UC, because for both contagious and environmental pathogens the assessed variables had similar characteristics. Very rough ring teats and very dirty udders were expected to be more predisposed to develop IMI by environmental pathogens, which was not observed. Bhutto et al. (2010) reported that there is evidence of some association between infection with nearly all major pathogens and CNS and type of teat-end lesions. SCC varied according to TEC. Teats with score 2 (smooth rings teats) had lower SCC when compared to other scores. Because IMI cases tended to increase with increasing teat scores, SCC was expected to follow the same pattern, which did not occur. No relationship between SCC and degree of teat-end callosity was also found in other studies (Shearn and Hillerton, 1996). Animals with UC score = 3 tended to present lower SCC. When the farm effect was controlled, the relationship between UC and SCC was no longer significant, suggesting that UC is highly dependent on external factors and does not necessarily result in increased IMI occurrence. Factors other than IMI that influence SCC include stage of lactation, milk yield and parity. It is known that the SCC is associated with milk loss in lactating cows (Hagnestam-Nielsen et al., 2009). The relationship between SCC and mastitis is also well known. Raising systems, animal productivity and genetic factors also influence SCC. Mastitis is known to increase SCC (ten Napel et al., 2009). It is also known that there is a clear relationship between IMI and clinical mastitis. However, the relationship between IMI and SCC is not always evident, such as in this study. Both UC score and TEC score were positively related to IMI, and a similar relationship to SCC was expected, which did not occur, especially for UC score. In a recent study, udder shape and teat-end lesions did not have a significant association with quarter SCC; while some association existed between IMI and udder shape and teat end lesions (Bhutto et al., 2010). Because of the great variability in SCC, controlled studies can help identify the relationship between udder cleanliness and SCC.
434
M. de Pinho Manzi et al. / Research in Veterinary Science 93 (2012) 430–434
5. Conclusion There is a clear relationship between IMI and TEC. Teat ends with very rough rings with keratin extending more than 4 mm from the orifice (score 4) had the highest chance of developing IMI when compared to the other three categories, as well as animals with dirtier udders. UC is dependent on external factors (season, stall hygiene) and was related to IMI. Controlled studies can help identify an actual relationship between udder cleanliness and SCC. It can be concluded that animals with very rough ring teats and very dirty udders have greater predisposition to IMI, which can be used as an important parameter in the monitoring of mastitis control.
References Bhutto, A.L., Murray, R.D., Woldehiwet, Z., 2010. Udder shape and teat-end lesions as potential risk factors for high somatic cell counts and intra-mammary infections in dairy cows. The Veterinary Journal 183, 63–67. Breen, J.E., Green, M.J., Bradley, A.J., 2009. Quarter and cow risk factors associated with the occurrence of clinical mastitis in dairy cows in the United Kingdom. Journal of Dairy Science 92, 2551–2561. Coffey, T.J., Pullinger, G.D., Urwin, R., Jolley, K.A., Wilson, S.M., Maiden, M.C., Leigh, J.A., 2006. First insights into the evolution of Streptococcus uberis: a multilocus sequence typing scheme that enables investigation of its population biology. Applied Environmental Microbiology 72, 1420–1428. Compton, C.W., Heuer, C., Parker, K., McDougall, S., 2007. Epidemiology of mastitis in pasture-grazed peripartum dairy heifers and its effects on productivity. Journal of Dairy Science 90, 4157–4170. Cunha, M.L.R.S., Sinzato, Y.K., Silveira, L.V.A., 2004. Comparison of methods for the identification of coagulase-negative Staphylococci. Memórias do Instituto Oswaldo Cruz 99, 855–860. de Haas, Y., Veerkamp, R.F., Barkema, H.W., Grohn, Y.T., Schukken, Y.H., 2004. Associations between pathogen-specific cases of clinical mastitis and somatic cell count patterns. Journal of Dairy Science 87, 95–105. Field, A., 2005. Discovering Statistics Using SPSS, second ed. Sage Publications Ltd, London, UK, 781p. Fregonesi, J.A., von Keyserlingk, M.A., Tucker, C.B., Veira, D.M., Weary, D.M., 2009. Neck-rail position in the free stall affects standing behavior and udder and stall cleanliness. Journal of Dairy Science 92, 1979–1985. Galton, D.M., Peterson, L.G., Merrill, W.G., 1988. Evaluation of udder preparations on intramammary infections. Journal of Dairy Science 71, 1417–1421.
Hagnestam-Nielsen, C., Emanuelson, U., Berglund, B., Strandberg, E., 2009. Relationship between somatic cell count and milk yield in different stages of lactation. Journal of Dairy Science 92, 3124–3133. Langoni, H., 2000. [Modernization trends in the industry: milk quality monitoring by somatic cell counts]. Revista de Educação Continuada 3, 57–64. Mein, G.A., Neijenhuis, F., Britt, J.S., Reinemann, D.J., Hillerton, J.E., Farnsworth, R., Baines, J.R., Ohnstad, I., Morgan, W.F., Rasmussen, M.D., Timms, L., Cook, N.B., 2001. Evaluation of bovine teat condition in commercial dairy herds: 1. NonInfectious Factors, 2nd International Mastitis and Milk Quality Symposium, Vancouver, Canada. Munoz, M.A., Bennett, G.J., Ahlstrom, C., Griffiths, H.M., Schukken, Y.H., Zadoks, R.N., 2008. Cleanliness scores as indicator of Klebsiella exposure in dairy cows. Journal of Dairy Science 91, 3908–3916. Neijenhuis, F., Barkema, H.W., Hogeveen, H., Noordhuizen, J.P., 2000. Classification and longitudinal examination of callused teat ends in dairy cows. Journal of Dairy Science 83, 2795–2804. Neijenhuis, F., Barkema, H.W., Hogeveen, H., Noordhuizen, J.P., 2001. Relationship between teat-end callosity and occurrence of clinical mastitis. Journal of Dairy Science 84, 2664–2672. National Mastitis Council, 1999. Laboratory Handbook on Bovine Mastitis. National Mastitis Council Incorporation, Madison, WI. Olde Riekerink, R.G., Barkema, H.W., Stryhn, H., 2007a. The effect of season on somatic cell count and the incidence of clinical mastitis. Journal of Dairy Science 90, 1704–1715. Olde Riekerink, R.G., Barkema, H.W., Veenstra, W., Berg, F.E., Stryhn, H., Zadoks, R.N., 2007b. Somatic cell count during and between milkings. Journal of Dairy Science 90, 3733–3741. Peters, R.R., Komaragiri, S., Paape, M.J., Douglass, L.W., 2000. Evaluation of 1.6% phenol as a premilking and postmilking teat dip in preventing new bovine intramammary infections. Journal of Dairy Science 83, 1750–1757. Rodrigues, A.C., Cassoli, L.D., Machado, P.F., Ruegg, P.L., 2009. Short communication: evaluation of an on-farm test to estimate somatic cell count. Journal of Dairy Science 92, 990–995. Schreiner, D.A., Ruegg, P.L., 2002. Effects of tail docking on milk quality and cow cleanliness. Journal of Dairy Science 85, 2503–2511. Schreiner, D.A., Ruegg, P.L., 2003. Relationship between udder and leg hygiene scores and subclinical mastitis. Journal of Dairy Science 86, 3460–3465. Shearn, M.F., Hillerton, J.E., 1996. Hyperkeratosis of the teat duct orifice in the dairy cow. Journal of Dairy Research 63, 525–532. Ten Napel, J., De Haas, Y., De Jong, G., Lam, T.J., Ouweltjes, W., Windig, J.J., 2009. Characterization of distributions of somatic cell counts. Journal of Dairy Science 92, 1253–1264. Tsenkova, R., Atanassova, S., Kawano, S., Toyoda, K., 2001. Somatic cell count determination in cow’s milk by near-infrared spectroscopy: a new diagnostic tool. Journal of Animal Science 79, 2550–2557. Tucker, C.B., Fraser, D., Weary, D.M., 2001. Tail docking dairy cattle: effects on cow cleanliness and udder health. Journal of Dairy Science 84, 84–87.
Contents lists available at ScienceDirect
Research in Veterinary Science journal homepage: www.elsevier.com/locate/rvsc
Relationship between teat-end condition, udder cleanliness and bovine subclinical mastitis Marcela de Pinho Manzi, Diego Borin Nóbrega, Patrícia Yoshida Faccioli, Marcella Zampolli Troncarelli, Benedito Donizete Menozzi, Hélio Langoni ⇑ Department of Veterinary Hygiene and Public Health, São Paulo State University, Botucatu 18618-900, Brazil
a r t i c l e
i n f o
Article history: Received 15 February 2011 Accepted 12 May 2011
Keywords: Bovine mastitis Teat-end condition Udder cleanliness
a b s t r a c t The aims of the present study were to relate intramammary infection (IMI) occurrence and somatic cell count (SCC) with teat-end condition (TEC) and udder cleanliness (UC). Milk samples from 1931 teats were evaluated according to the presence of IMI and SCC. Scores were applied to teats according to the TEC and to UC. Teats ends with a very rough ring had the largest number of IMI when compared to the other three categories, as well as animals with dirtier udders. The change in a TEC score increased by around 30% the chance of IMI. Also, the chance of the animal developing IMI increased by approximately 47% when the UC score increased. No significant association between both scores and quarter SCC was found. It can be concluded that animals with very rough teat end rings and very dirty udders have a greater predisposition to IMI. Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction Mastitis is an inflammation of the mammary gland, which is characterized as the most common infectious disease affecting dairy cattle. It causes extensive economic loss due to decreased milk yield, lower milk quality, higher treatment costs and premature culling of cows with chronic mastitis. Depending on the pathogen, mastitis can be classified into either contagious or environmental (Coffey et al., 2006; Schreiner and Ruegg, 2002). The primary reservoir for contagious pathogens is the udder of infected cows. The most common contagious mastitis pathogens include Staphylococcus aureus and Streptococcus agalactiae (Schreiner and Ruegg, 2002). The environmental pathogens are present in the cows environment. Coliform bacteria and environmental streptococci are the most commonly isolated environmental pathogens. Although the contamination of animals by environmental microorganisms may occur during the milking process (usually due to equipment failure), it is more frequent between milkings. Control is generally difficult because the bacteria are disseminated in the environment of animals (Langoni, 2000). Somatic cell count (SCC) is the most frequently used indicator of subclinical mastitis in dairy cattle, and is recognized as an indicator of cow health and milk quality (Tsenkova et al., 2001). The most important cause of increased SCC is bacterial infection of the mammary gland (de Haas et al., 2004; Olde Riekerink et al., 2007b; Tsenkova et al., 2001). ⇑ Corresponding author. Tel.: +55 14 38116270; fax: +55 14 38116075. E-mail address: [email protected] (H. Langoni). 0034-5288/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.rvsc.2011.05.010
The teat sphincter and teat canal are important primary barriers against pathogen invasion into the udder. Thus, it is essential that such structures be in perfect physical and hygienic conditions to prevent intramammary infection (IMI). The correlation between teat sphincter integrity and milking conditions is not well understood, particularly regarding udder cleanliness (UC) and its involvement with pathogens implicated in mastitis. According to Neijenhuis et al. (2000), lesions in the teat sphincter are frequently colonized by Staphylococcus spp. and Streptococcus spp., which illustrates the correlation between the physical condition of mammary quarters and the presence of microorganisms. The classification of the teat end integrity into scores can serve as an important tool to control bovine mastitis because it allows for the classification of the different types of physical injuries. These irregularities can be related to problems in the management and production system, leading to a greater predisposition toward mastitis in the herd. It is known that cows with clinical mastitis frequently have more pointed teat ends than their paired healthy herd mates, and there is a relationship between teat-end callosity and clinical mastitis (Neijenhuis et al., 2001). Another relevant aspect is udder hygiene, which must be assessed at milking because it influences milk quality and is related to occurrence of pathogens, especially environmental ones. According to item 5.1.2 of ‘‘Instrução Normativa 51’’ [Normative Instruction No. 51] (Mapa, 2002) of the Brazilian Ministry of Agriculture, Livestock and Food Supply, the dairy farm must provide sanitary housing for cows in order to prevent udder contamination. Therefore, there is a concern about the environment where dairy animals are housed in order to produce high quality milk for
M. de Pinho Manzi et al. / Research in Veterinary Science 93 (2012) 430–434
431
human consumption. It is clear that a poor environment should result in dirtier udders. All hygienic factors related to the udder can be classified based on the cleanliness score. There is a relationship between teat or udder contamination and occurrence of mastitis (Galton et al., 1988; Schreiner and Ruegg, 2003). The aims of the present study were to relate IMI occurrence and SCC with teat-end condition (TEC) and UC, and to verify the most frequent teat scores for contagious and environmental pathogens. 2. Materials and methods The study was carried out in seven dairy farms located in São Paulo State, Brazil. The Holstein cows were in herds of different size, and in different stages of lactation and lactation number. The experiment was carried out in two parts (relationship between IMI, TEC and UC; and relationship between SCC, TEC and UC), mainly because of strict farms policies. 2.1. Relationship of IMI to TEC and UC The evaluation included 1931 teats from 499 animals (65 with missing teats were excluded). Loose dirt was brushed of the teats with sanitizing solutions (varies from farm to farm). Teats were dried with disposable towels, then teat ends were scrubbed with cotton pledgets. Foremilk samples were taken after the first streams of milk were discarded. Each animal participated only once in the study. The samples were kept at 4 °C until diagnostic bacteriology. Microbiological culture was done in duplicate according to standard procedures of the National Mastitis Council (NMC, 1999). First, 10 lL of each sample were spread onto 8% ovine blood agar and MacConkey agar media, followed by incubation at 37 °C. Examination of the plates was made after 24, 48 and 72 h of incubation. Isolated colonies were studied morphologically and Gram stained. Biochemical tests were applied according to the bacterial genus. Staphylococci were subjected to coagulase test. Coagulasepositive staphylococci underwent fermentation of sugars (maltose, mannitol and trehalose). Coagulase-negative staphylococci (CNS) were subjected to biochemical identification. Briefly, the method consisted of a set of biochemical tests that determine the utilization of the sugars xylose, arabinose, sucrose, trehalose, maltose, mannitol, lactose, xylitol, ribose, fructose and mannose; production of hemolysin; nitrate reduction; presence of urease and ornithine decarboxylase; and resistance to novobiocin characterized by an inhibition halo of up to 16 mm. Readings of the tests were obtained after 24, 48, and 72 h of incubation at 37 °C in an air incubator (Cunha et al., 2004). The Gram-positive, catalase-negative cocci were identified as belonging to the Streptococcaceae family and subjected to CAMP test and esculin hydrolysis. The Gram-positive, pleomorphic and catalase-positive coccobacilli were classified within the Corynebacterium genus. Blood agar plates containing three or more different colonies were considered contaminated. Major mastitis pathogens included S. aureus, Streptococcus spp., Arcanobacterium pyogenes, or coliform species. Minor pathogens were defined as CNS or Corynebacterium species, as described by other authors (Rodrigues et al., 2009). A quarter was considered infected with a minor pathogen when at least three colonies were observed on the blood agar plate, and one or more colonies for a major pathogen. IMI was considered positive at the animal level when isolation was positive for at least one mammary quarter of the 499 animals. If one of the duplicate samples was contaminated, the results from the uncontaminated duplicate alone was used to diagnose infection. When a quarter had both a major and a minor pathogen isolated at the same time, the quarter was defined as infected with the major pathogen (Compton et al., 2007). Fourteen
Fig. 1. Normal teat (left picture) scored as 1 according to teat end callosity, and teat scored as 4 (right picture), with keratin fronds extending more than 4 mm from the orifice.
milk samples were considered contaminated and excluded from analyses. Scores were applied at the teat level before the pre-milking routine by the same trained technician according to TEC: 1 = no ring, smooth teat end with a small even orifice (Fig. 1); 2 = smooth ring with a raised ring around the orifice (the raised area may be smooth or slightly rough); 3 = rough ring with a raised, roughened ring with fronds of keratin extending 1–3 mm from the orifice; and 4 = very rough ring with keratin extending more than 4 mm from the orifice. Teat-end condition scores were chosen according to (Mein et al., 2001). Cows with missing teats were excluded from analyses. UC was scored according to the udder to which the teat is located (1 = completely free of or has very little dirt; 2 = slightly dirty; and 3 = mostly covered or completely covered in dirt). UC was scored according to Schreiner and Ruegg (2002), grouping the last two scores into one, because score 4 had a low frequency. 2.2. Relationship of SCC to TEC and UC One teat and udder from each of 444 different animals was classified for TEC and UC as described in the first experiment. The animals that participated in the second experiment were from the same farms as the animals that were included in the first experiment. Only one teat per cow was sampled for SCC, mainly because of strict policies of the farms. The teats were selected in a way to include the same number of teats in each position (right front, left front, right rear and left rear). Milk samples (50 mL) were collected from each teat for electronic somatic cell count (eSCC) by using a calibrated Somacount 300 Bentley unit. 2.3. Statistical analysis Before statistical analysis, observations were checked for unlikely values and excluded from the analysis. The analyses were performed according to the considered experiment. 2.3.1. Relationship IMI – TEC and UC Absolute and relative frequency values of IMI, TEC score and UC scores were obtained for all evaluated teats. The logistic regression model was applied at the teat level to assess the effect of changes in TEC and UC, as well as in IMI status (presence or absence). The assessment included all valid teats (1931 teats). Logistic regression model was applied and odds ratio were estimated with respective 95% confidence intervals (CI). The initial model applied was:
Logitðpklmn Þ ¼ b þ farmk þ Tec Scr l þ Uc Scr m þ Tec Scr Uc Scr þ n where p is the probability of existence of IMI (0 = no, 1 = yes), b is the constant, farmk is the effect of the farm to which the animal belongs, Tec_Scrl is the TEC score (1 = Normal ring; 2 = Smooth ring; 3 = Rough ring; and 4 = Very rough ring showing eversion), Uc_Scrm
432
M. de Pinho Manzi et al. / Research in Veterinary Science 93 (2012) 430–434
is the UC score of the udder to which the teat belongs (1 = clean, 2 = dirty, 3 = very dirty), Tec_Scr⁄Uc_Scr is the association term, and en is the residual term. Odds ratio (OR) were estimated with 95% confidence interval (CI). Goodness of fit for the logistic regression model was assessed using the Hosmer and Lemeshow test and graphical analysis of residuals and predicted values, respectively (Field, 2005). The predictors entered the model in a backward conditional method. The final model applied contained only the TEC score as a predictor. Part of its effect was suppressed by the presence of UC score and the interaction effect, showing possible colinearity among variables, which was detected by the correlation matrix. Thus, separate models were used for UC score and TEC score, in which only the latter variable showed contribution. At the animal level, the logistic regression model was also applied, using UC score as predictor and the presence/absence of IMI as outcome. The same parameters previously described were assessed (Field, 2005). In this analysis, 461 animals were included (animals with missing teats were excluded). A specific pathogen was only studied if identified in more than six mastitis cases during the study period. The remaining pathogens were grouped as ‘‘other’’. CNS that did not achieve at least six isolations were grouped as ‘‘Staphylococcus spp.’’. The final classification of the pathogens included: Corynebacterium bovis, Escherichia coli, Staphylococcus spp. (coagulase-negative staphylococci that didn’t achieve six isolations), S. aureus, Staphylococcus epidermidis, Staphylococcus warneri, S. agalactiae, Streptococcus dysgalactiae, and Streptococcus uberis. Relative frequencies were calculated for each valid pathogen. The most frequent TEC score and UC score for each pathogen were obtained (modes). The isolated pathogens were grouped into two categories, contagious and environmental. Teats with IMI caused by contagious and environmental pathogens were compared in terms of TEC. Chi-square test was employed to verify differences among TEC frequencies according to the type of pathogen. 2.3.2. Relationship of SCC to TEC and UC For the second experiment, SCC values were log-converted to base 10 (LogSCC) to achieve homogeneity of variance. Mean values were obtained, as well as lower and upper bounds of the 95% CI, 25 and 75 percentiles and median values for LogSCC according to teat and animal cleanliness scores. Kolmogorov–Smirnov normality tests were applied. Differences in eSCC between TEC and UC scores were evaluated using the General Linear Model (GLM), adopting eSCC as the outcome and TEC score and UC score as the predictors in separate models. The farm entered as a random factor in both models. Variance homogeneity was assessed by means of Levene’s test. Hochberg’s GT2 and Bonferroni tests were used as post hoc tests to verify differences between eSCC in the different teat and udder scores (Field, 2005). In this second experiment, only analyses at the teat level were performed. The level of statistical significance was set at 0.05 for all analyses. 3. Results 3.1. Relationship of IMI to TEC and UC Table 1 shows the variables related to the relationship IMI to TEC and UC for experiment 1. Of 1931 teats evaluated, 1627 (84.3%) did not have IMI. For 571 (29.6%) TEC score = 1 (no ring), for 599 (31.0%) = 2 (smooth or slightly rough ring), for 415 (21.5%) = 3 (rough ring), and for 346 (17.9%) = 4 (very rough ring). Among these, there was a tendency toward the frequency of IMI to increase with increasing TEC score. Score 4 teats had the largest number of IMI when compared to the other three categories. IMI was detected in 58 teat score 1 (10.2%), 98 teat score 2 (16.4%),
73 teat score 3 (17.6%) and 75 teat score 4 (21.7%), respectively. There was a positive effect between higher TEC score and presence of IMI (P < 0.001, B = 0.270, Exp(B) = 1.309 with LB (95%) = 1.169 and UB (95%) = 1.467). With regards to udder scores, 1302 teats (67.4%) were from clean udders, 478 (24.8%) from slightly dirty and 151 (7.8%) from very dirty udders. There appeared to be a tendency for UC to parallel TEC. Intramammary infection was detected in mammary quarters of 195 (15.0%) clean udders, 16.9% of dirty udders and 18.5% of very dirty udders. Although IMI cases tended to increase with a higher UC score, the effect of UC score was not statistically significant (P = 0.097). The interaction between TEC score and UC score was not significant (P = 0.786). At the animal level, 381 animals did not have IMI (82.6%) and 80 animals had IMI (17.4%). A total of 315 udders (68.3%) were classified as clean, 109 (23.6%) as dirty, and 37 (8.0%) as very dirty. IMI cases tended to increase with UC score. Of 80 cases positive for IMI, 46 were in clean udders (14.6% of the total clean udders), 25 in dirty udders (22.9% of the dirty udders) and 9 in very dirty udders (24.3% of the total udders score 3). There was a positive effect between the increase in UC score and the presence of IMI at the animal level (P = 0.03, B = 0.388, Exp(B) = 1.474 with LB (95%) = 1.037 and UB (95%) = 2.093). The most frequent isolated pathogens were C. bovis (46.4%), Streptococcus dysgalactiae (13.5%) and Staphylococcus spp. (8.2%). Table 2 summarizes the results of microbiological tests for the 304 IMI. For almost all isolated bacteria, except S. agalactiae, foremilk samples were from teats of clean udders. Teat scores varied (P < 0.05) among type of pathogen (Table 2). Among the pathogens isolated, 233 were contagious (76.6%) and 71 (23.4%) were environmental (Table 3). There were no differences between isolations of contagious and environmental bacteria according to TEC (P = 0.146) and UC score (P = 0.529).
3.2. Relationship of SCC to TEC and UC Even with low SCC (Table 4) for UC score 3 when compared to the remaining ones, there was no difference (P = 0.083) between SCC in the different udder cleanliness scores when the farm effect was controlled (Table 3). The same was not observed for teat score (P = 0.002), especially for lower counts in teats of score 2, relative to score 1, score three and score four (P < 0.001 for all three comparisons, respectively).
Table 1 Absolute (n) and relative frequency (%) of teat-end condition score (TEC score), udder cleanliness score (UC score) and intramammary infection status (IMI) from the animals included in experiment 1. Variable TEC scorea 1 2 3 4 UC scoreb 1 2 3 IMI No Yes
n
%
571 599 415 346
29.60 31.00 21.50 17.90
1302 478 151
67.40 24.80 7.80
1627 304
84.30 15.70
a 1 = no ring; 2 = smooth rough ring; 3 = rough ring; 4 = very rough ring. b 1 = clean udders; 2 = dirty udders; 3 = very dirty udders.
433
M. de Pinho Manzi et al. / Research in Veterinary Science 93 (2012) 430–434 Table 2 Absolute (n) and relative frequency (%) for results of the microbiological test, and most frequent values (modes) of teat-end condition score (TEC score) and udder cleanliness score (UC score) according to the isolated pathogen. Result
n
%
TEC score
UC score
Staphylococcus spp.a Corynebacterium bovis Escherichia coli Staphylococcus aureus Staphylococcus epidermidis Staphylococcus warneri Streptococcus agalactiae Streptococcus dysgalactiae Streptococcus uberis Other pathogensb Total
25 141 6 17 19 13 12 41 20 10 304
8.2 46.4 2 5.6 6.3 4.3 3.9 13.5 6.6 3.3 100
4 2 4 2 2 3,4 2 2 1 3
1 1 1 1 1 1 3 1 1 1,2
eSCC Mean TEC scoreA 1 2.655a 2 2.289b 3 2.716a 4 2.701a UC scoreB 1 2.644a 2 2.496a 3 2.251a
a Staphylococcus captis urealyticus, Staphylococcus haemolyticus, Staphylococcus hyicus, Staphylococcus saprophyticus, Staphylococcus xylosus. b Bacillus spp., Candida albicans, Klebsiella pneumoniae, Micrococcus spp., Prototheca spp., Pseudomonas aeruginosa.
Table 3 Absolute and relative frequencies (%) for contagious or environmental pathogens according to the teat-end condition score (TEC score) and udder cleanliness score (UC score). Contagious
(%)
Environmental
(%)
18 32.2 23.2 26.6
16 23 18 13
22.9 32.9 25.7 18.6
66.1 24 9.9
40 25 5
57.1 35.7 7.1
a
TEC score 1 42 2 75 3 54 4 62 UC scoreb 1 154 2 56 3 23 a b
Table 4 Mean values, lower (LB) and upper bounds (UB) of 95% confidence interval, median values and 25 (P25) and 75 percentiles (P75) of electronic somatic cell count (eSCC – log transformed value 103 somatic cells/mL) according to the teat-end condition score (TEC score) and udder cleanliness score (UC score).
1 = no ring; 2 = smooth rough ring; 3 = rough ring; 4 = very rough ring. 1 = clean udders; 2 = dirty udders; 3 = very dirty udders.
4. Discussion Mammary quarter level risk factors for IMI may include teat position and shape (Breen et al., 2009). In the present study, there was an association between TEC and IMI. The change in a unit of TEC score increased by around 30% the chance of developing IMI, relative to the original category. Neijenhuis et al. (2000) conducted a study where teat-end callosity was evaluated and found out that a hyperplastic stratum corneum, found in teats with an increased teat-end callosity score, leads to a roughened surface to which bacteria can adhere, making disinfection of the teat after milking more difficult and limiting its effectiveness (Neijenhuis et al., 2000). The evaluated TEC scores were positively correlated to the larger number of IMI cases. The incidence of subclinical and clinical mastitis depends on factors such as parity, stage of lactation, type of housing, access to pasture, management, and environmental factors (Olde Riekerink et al., 2007a). Reduced udder hygiene has been associated with intramammary pathogens (Schreiner and Ruegg, 2003). Fecal contamination of stalls is considered a risk factor in the transmission of environmental mastitis (Fregonesi et al., 2009), which suggests that cows with a higher UC score would have a higher prevalence of environmental mastitis, which was not verified in the present study. Udder cleanliness is influenced by external factors (hygiene, season, etc.) and does not necessarily leads to IMI. In addition, many factors can influence the degree of udder cleanliness among cows (Tucker et al., 2001). Rainy seasons, production system (free-stall versus pasture) and the hygiene routine on the farm contribute to udder cleanliness. During rainy seasons, dairy
LB (95%)
UB (95%)
Median
P25
P75
2.486 2.141 2.589 2.560
2.823 2.436 2.843 2.841
2.739 2.361 2.800 2.805
2.185 1.643 2.419 2.235
3.213 2.869 3.180 3.269
2.555 2.339 2.005
2.733 2.654 2.496
2.740 2.623 2.361
2.202 1.748 1.612
3.177 3.196 2.776
A
1 = no ring; 2 = smooth rough ring; 3 = rough ring; 4 = very rough ring. 1 = clean udders; 2 = dirty udders; 3 = very dirty udders. a,b Means within a column of the same classification system (TEC, UC) with different superscripts differ (one-way ANOVA post hoc tests, P < 0.001). B
cows housed in free-stalls will have dirtiers udders compared to other housing systems. The adoption of a suitable pre-milking routine is a preventive measure against occurrence of IMI (Peters et al., 2000), although it was not capable of removing all pathogens from the teat, especially when udders were dirty. A pre-milking routine was not able to remove Klebsiella spp. from teat skin (Munoz et al., 2008). In the present study, the chance of the animal developing IMI in any quarter increased by approximately 47% when UC score was altered by one unit, i.e. animals with dirtier udders clearly have more chance of developing mastitis. There were no differences between pathogen types when considering TEC and UC, because for both contagious and environmental pathogens the assessed variables had similar characteristics. Very rough ring teats and very dirty udders were expected to be more predisposed to develop IMI by environmental pathogens, which was not observed. Bhutto et al. (2010) reported that there is evidence of some association between infection with nearly all major pathogens and CNS and type of teat-end lesions. SCC varied according to TEC. Teats with score 2 (smooth rings teats) had lower SCC when compared to other scores. Because IMI cases tended to increase with increasing teat scores, SCC was expected to follow the same pattern, which did not occur. No relationship between SCC and degree of teat-end callosity was also found in other studies (Shearn and Hillerton, 1996). Animals with UC score = 3 tended to present lower SCC. When the farm effect was controlled, the relationship between UC and SCC was no longer significant, suggesting that UC is highly dependent on external factors and does not necessarily result in increased IMI occurrence. Factors other than IMI that influence SCC include stage of lactation, milk yield and parity. It is known that the SCC is associated with milk loss in lactating cows (Hagnestam-Nielsen et al., 2009). The relationship between SCC and mastitis is also well known. Raising systems, animal productivity and genetic factors also influence SCC. Mastitis is known to increase SCC (ten Napel et al., 2009). It is also known that there is a clear relationship between IMI and clinical mastitis. However, the relationship between IMI and SCC is not always evident, such as in this study. Both UC score and TEC score were positively related to IMI, and a similar relationship to SCC was expected, which did not occur, especially for UC score. In a recent study, udder shape and teat-end lesions did not have a significant association with quarter SCC; while some association existed between IMI and udder shape and teat end lesions (Bhutto et al., 2010). Because of the great variability in SCC, controlled studies can help identify the relationship between udder cleanliness and SCC.
434
M. de Pinho Manzi et al. / Research in Veterinary Science 93 (2012) 430–434
5. Conclusion There is a clear relationship between IMI and TEC. Teat ends with very rough rings with keratin extending more than 4 mm from the orifice (score 4) had the highest chance of developing IMI when compared to the other three categories, as well as animals with dirtier udders. UC is dependent on external factors (season, stall hygiene) and was related to IMI. Controlled studies can help identify an actual relationship between udder cleanliness and SCC. It can be concluded that animals with very rough ring teats and very dirty udders have greater predisposition to IMI, which can be used as an important parameter in the monitoring of mastitis control.
References Bhutto, A.L., Murray, R.D., Woldehiwet, Z., 2010. Udder shape and teat-end lesions as potential risk factors for high somatic cell counts and intra-mammary infections in dairy cows. The Veterinary Journal 183, 63–67. Breen, J.E., Green, M.J., Bradley, A.J., 2009. Quarter and cow risk factors associated with the occurrence of clinical mastitis in dairy cows in the United Kingdom. Journal of Dairy Science 92, 2551–2561. Coffey, T.J., Pullinger, G.D., Urwin, R., Jolley, K.A., Wilson, S.M., Maiden, M.C., Leigh, J.A., 2006. First insights into the evolution of Streptococcus uberis: a multilocus sequence typing scheme that enables investigation of its population biology. Applied Environmental Microbiology 72, 1420–1428. Compton, C.W., Heuer, C., Parker, K., McDougall, S., 2007. Epidemiology of mastitis in pasture-grazed peripartum dairy heifers and its effects on productivity. Journal of Dairy Science 90, 4157–4170. Cunha, M.L.R.S., Sinzato, Y.K., Silveira, L.V.A., 2004. Comparison of methods for the identification of coagulase-negative Staphylococci. Memórias do Instituto Oswaldo Cruz 99, 855–860. de Haas, Y., Veerkamp, R.F., Barkema, H.W., Grohn, Y.T., Schukken, Y.H., 2004. Associations between pathogen-specific cases of clinical mastitis and somatic cell count patterns. Journal of Dairy Science 87, 95–105. Field, A., 2005. Discovering Statistics Using SPSS, second ed. Sage Publications Ltd, London, UK, 781p. Fregonesi, J.A., von Keyserlingk, M.A., Tucker, C.B., Veira, D.M., Weary, D.M., 2009. Neck-rail position in the free stall affects standing behavior and udder and stall cleanliness. Journal of Dairy Science 92, 1979–1985. Galton, D.M., Peterson, L.G., Merrill, W.G., 1988. Evaluation of udder preparations on intramammary infections. Journal of Dairy Science 71, 1417–1421.
Hagnestam-Nielsen, C., Emanuelson, U., Berglund, B., Strandberg, E., 2009. Relationship between somatic cell count and milk yield in different stages of lactation. Journal of Dairy Science 92, 3124–3133. Langoni, H., 2000. [Modernization trends in the industry: milk quality monitoring by somatic cell counts]. Revista de Educação Continuada 3, 57–64. Mein, G.A., Neijenhuis, F., Britt, J.S., Reinemann, D.J., Hillerton, J.E., Farnsworth, R., Baines, J.R., Ohnstad, I., Morgan, W.F., Rasmussen, M.D., Timms, L., Cook, N.B., 2001. Evaluation of bovine teat condition in commercial dairy herds: 1. NonInfectious Factors, 2nd International Mastitis and Milk Quality Symposium, Vancouver, Canada. Munoz, M.A., Bennett, G.J., Ahlstrom, C., Griffiths, H.M., Schukken, Y.H., Zadoks, R.N., 2008. Cleanliness scores as indicator of Klebsiella exposure in dairy cows. Journal of Dairy Science 91, 3908–3916. Neijenhuis, F., Barkema, H.W., Hogeveen, H., Noordhuizen, J.P., 2000. Classification and longitudinal examination of callused teat ends in dairy cows. Journal of Dairy Science 83, 2795–2804. Neijenhuis, F., Barkema, H.W., Hogeveen, H., Noordhuizen, J.P., 2001. Relationship between teat-end callosity and occurrence of clinical mastitis. Journal of Dairy Science 84, 2664–2672. National Mastitis Council, 1999. Laboratory Handbook on Bovine Mastitis. National Mastitis Council Incorporation, Madison, WI. Olde Riekerink, R.G., Barkema, H.W., Stryhn, H., 2007a. The effect of season on somatic cell count and the incidence of clinical mastitis. Journal of Dairy Science 90, 1704–1715. Olde Riekerink, R.G., Barkema, H.W., Veenstra, W., Berg, F.E., Stryhn, H., Zadoks, R.N., 2007b. Somatic cell count during and between milkings. Journal of Dairy Science 90, 3733–3741. Peters, R.R., Komaragiri, S., Paape, M.J., Douglass, L.W., 2000. Evaluation of 1.6% phenol as a premilking and postmilking teat dip in preventing new bovine intramammary infections. Journal of Dairy Science 83, 1750–1757. Rodrigues, A.C., Cassoli, L.D., Machado, P.F., Ruegg, P.L., 2009. Short communication: evaluation of an on-farm test to estimate somatic cell count. Journal of Dairy Science 92, 990–995. Schreiner, D.A., Ruegg, P.L., 2002. Effects of tail docking on milk quality and cow cleanliness. Journal of Dairy Science 85, 2503–2511. Schreiner, D.A., Ruegg, P.L., 2003. Relationship between udder and leg hygiene scores and subclinical mastitis. Journal of Dairy Science 86, 3460–3465. Shearn, M.F., Hillerton, J.E., 1996. Hyperkeratosis of the teat duct orifice in the dairy cow. Journal of Dairy Research 63, 525–532. Ten Napel, J., De Haas, Y., De Jong, G., Lam, T.J., Ouweltjes, W., Windig, J.J., 2009. Characterization of distributions of somatic cell counts. Journal of Dairy Science 92, 1253–1264. Tsenkova, R., Atanassova, S., Kawano, S., Toyoda, K., 2001. Somatic cell count determination in cow’s milk by near-infrared spectroscopy: a new diagnostic tool. Journal of Animal Science 79, 2550–2557. Tucker, C.B., Fraser, D., Weary, D.M., 2001. Tail docking dairy cattle: effects on cow cleanliness and udder health. Journal of Dairy Science 84, 84–87.