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Serum haptoglobin concentrations in dairy cattle with lameness due to claw disorders
The Veterinary Journal 186 (2010) 162–165
Contents lists available at ScienceDirect
The Veterinary Journal journal homepage: www.elsevier.com/locate/tvjl
Serum haptoglobin concentrations in dairy cattle with lameness due to claw disorders Billy I. Smith *, Johannes Kauffold, Lisa Sherman 1 Department of Clinical Studies, New Bolton Center, School of Veterinary Medicine, University of Pennsylvania, 382 West Street Road, Kennett Square, PA 19348, USA
a r t i c l e
i n f o
Article history: Accepted 11 August 2009
Keywords: Dairy cattle Lameness Claw disorder Haptoglobin Treatment
a b s t r a c t In cattle, elevated blood serum concentrations of haptoglobin, an acute phase protein, have been demonstrated in association with several diseases, but not with lameness. Serum haptoglobin was measured in 60 Holstein dairy cattle diagnosed with lameness due to four claw disorders, pododermatitis septica (PS; n = 41), pododermatitis circumscripta (PC; n = 8), interdigital necrobacillosis (IN; n = 7), papillomatous digital dermatitis (PDD; n = 4). Haptoglobin was measured on day 1 (0–3 days after lameness was observed but before treatment) and on days 3 and 5. A total of 10 healthy cows served as controls (haptoglobin values <1.0 mg/dL). Each of the claw disorders was associated with elevated haptoglobin on day 1 (PS, PC, IN and PDD: 65.9%, 37.5%, 71.4% and 25.0%, respectively). Trimming and antibiotic treatment led to a reduction in the number of PS and IN cows with increased haptoglobin concentrations, respectively (P < 0.05), but trimming did not lead to any reduction in cows with PC. The study showed that lameness due to claw disorders can be associated with a systemic acute phase response and elevated serum haptoglobin in dairy cattle. Based on the course of haptoglobin, treatments seemed effective for all claw disorders except for PC. Published by Elsevier Ltd.
Introduction Haptoglobin is an acute phase protein (APP) synthesized by hepatocytes in response to cytokines released from macrophages and other immune cells as the result of tissue damage, inflammation, infection and bacterial components, and also after stress (Murata et al., 2004; Lomborg et al., 2008). In healthy cattle, serum haptoglobin is either non-detectable or measurable only in very low concentrations, i.e., below 10 mg/dL (Panndorf et al., 1976; Conner et al., 1986). Increased serum haptoglobin has been found in association with, for example, pneumonia, mastitis, metritis and uterine torsions, ketosis, fatty liver syndrome and with stress (Conner et al., 1986; Eckersall and Connor, 1988; Skinner et al., 1991; Young et al., 1996; Smith et al., 1998; Schoenfelder et al., 2006; Stengärde et al., 2008; Lomborg et al., 2008). Lameness is a substantial and costly problem with a high incidence in the dairy industry worldwide (Enevoldsen et al., 1991; Wells et al., 1993; Guard, 1996, 2006a; Hernandez et al., 2007). Inflammatory claw lesions, either due to bacterial infections such as papillomatous digital dermatitis (PDD) and interdigital necrobacillosis (IN), or with a non-infectious genesis such as pododermati* Corresponding author. Tel.: +1 610 925 6310; fax: +1 610 925 8103. E-mail address: [email protected] (B.I. Smith). 1 Present address: 8 Hemlock Lane, Tioga, PA 16946, USA. 1090-0233/$ - see front matter Published by Elsevier Ltd. doi:10.1016/j.tvjl.2009.08.012
tis circumscripta (PC) and pododermatitis septica (PS), are the most prevalent reasons for dairy cattle lameness (Shearer et al., 1996; Bergsten et al., 1998; Hoblet and Weiss, 2001). Increased serum haptoglobin has been described in association with lameness in pigs (van den Berg et al., 2007), horses (Hulten et al., 2002) and most recently also in cattle but only in 14 lame cows subjected to clinic facilities (Jawor et al., 2008). The aim of this study was to gain further knowledge on APP haptoglobin in association with lameness due to claw disorders in dairy cattle on a commercial farm. Since all animals received treatment according to the disorder it was also aimed to test how treatments effect serum haptoglobin and so get an indirect measure of the effectiveness of the treatments (Hulten et al., 2002; Schoenfelder et al., 2006). Materials and methods Farm and management This study was performed at a 600-cow dairy farm in southeast Pennsylvania, USA, over a 6 month period between March and August 2005. Cows were housed in a six-row, open ridge, freestall barn with curtain sidings, canvas mattresses, automatic alley scrapers, open rail feed bunks with rubber flooring. For management purposes, cows were separately kept in five groups according to age and/or milk production (fresh cows; heifers; cows with low, medium or high milk production). They were milked twice a day, fed a total mixed ration according to national recommendations (NRC, 1989) and had ad libitum access to water.
B.I. Smith et al. / The Veterinary Journal 186 (2010) 162–165 Routine claw trimming was done twice a year on all cows and followed a farm protocol, i.e., at the time the cows were dried off and again at 120 days in milk. Trimming was performed on restrained animals using hoof knives and a chipper wheel. Cows were observed daily for heath status including lameness by the farm crew. Cows found lame were subjected to veterinary supervision. Since supervisions were always done on Tuesdays and Fridays, the interval between detection of lameness by the farm crew and veterinary supervision varied between 0 and 3 days.
Animals Seventy-six cows with lameness were subjected to veterinary supervision during the study interval. The cows were caught in a head catch, the affected limb was then lifted off the ground by using a pulley system. A total of 60 Holstein dairy cows (parity 1–8) were identified as having claw related lameness and diagnosed with PS (n = 41), IN (n = 7), PC (n = 8) or PDD (n = 4). Diagnosis of the claw disorders followed approved procedures (Desrochers et al., 2001). For IN, the severity was additionally determined and graded into ‘moderate’ and ‘severe’. All 60 cows were in good general health and did not have other inflammatory problems such as pneumonia, enteritis, mastitis or any kinds of acute uterine inflammation. Another 10 randomly picked cows all of them in good general health without any obvious disease including lameness as evaluated by routine physical and record analysis of data on the animal’s activity were used as controls to define haptoglobin concentrations in healthy cows without lameness.
Treatments Animals were treated immediately after diagnosis employing classical treatment protocols (Rebhun and Pearson, 1982; Berry, 2001; van Amstel and Shearer, 2001; Guard, 2006b). Lame cows diagnosed with PS were treated by trimming the affected claw to relieve pressure resulting from the accumulation of purulent material between the sole and laminae. Cows diagnosed with PC were treated by trimming the affected claw to relieve pressure resulting from inflamed laminae due to trauma and infection. After the claw was trimmed, a wooden block was glued onto the sole of the unaffected claw. A copper containing topical antimicrobial agent (Kopertox; Fort Dodge Animal Health) was applied to the lesion. No anti-inflammatory or antibiotic drugs were used on these animals. Animals diagnosed with PDD were treated topically with antibiotic claw wraps using tetracycline powder (Tetracycline Soluble Powder 324; Agripharm). Cows with IN were treated systemically with antibiotics according to severity with either ceftiofur sodium (Naxcel; Pfizer) in case of moderate IN (n = 3), or procaine penicillin G (Pen-Aqueous; Agripharm) in case of severe IN (n = 4) over 5 days, respectively. In addition, all IN animals were treated with claw wraps and tetracycline powder (Tetracycline Soluble Powder 324; Agripharm). No anti-inflammatory drugs were used on these animals. Animals that served as controls were not treated at all.
Analysis of haptoglobin in blood serum Blood samples were collected from all 60 animals with lameness and the 10 control animals by coccygeal venepuncture into evacuated tubes (Vacutainer; Fisher Scientific). Lame cows were bled on day 1, i.e., after initial diagnosis but before treatment, and on days 3 and 5 thereafter. Control cows were bled only once and in one session. Blood was allowed to coagulate, was then refrigerated at 4.4 °C (40 °F) and cleaned from clots within 24 h of collection. Serum was recovered by centrifugation at 4500 g and stored frozen at 20 °C ( 4 °F). Haptoglobin concentration was measured in serum by means of radial immunodiffusion (Bovine Hp Plate; code No. P0105-1; Cardiotech Svcs., Inc.) according to the manufacturer’s instructions. This kit is validated for and used with frozen/thawed bovine serum (Conner et al., 1989; Makimura and Usui, 1990; Skinner et al., 1991), and has been described in detail recently (Berry et al., 2004). Briefly, bovine serum samples (100 lL) were treated with an equal amount of 40 mM solution of L-cysteine (24 mg of L-cysteine dissolved in 5 mL of L-cysteine solvent [1 e]), and both were added to a single mixing well. Two standards (A and B) were loaded (5 lL) into specified wells of the plate, and the treated samples then into separate wells, respectively. The plate was covered, incubated at 37 °C for 24–48 h, and the results determined by measuring the external diameter of each precipitin ring to the nearest 0.01 mm with a supplied plastic scale. Diameters of rings obtained with the standard solution were plotted on the vertical axis of semi-logarithmic graph paper versus serum haptoglobin concentration on the horizontal axis. A reference curve was created by drawing a straight line through plotted standard points. The diameters of the precipitin rings for each test sample were used to calculate the haptoglobin concentration using the reference curve. If no precipitin ring appeared, the haptoglobin concentration was below 1.0 mg/dL. Concentrations >100 mg/dL were given as such. The coefficient of variation for the kit was less than 4% for repeated, identical measurements on the same specimen.
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Statistical analyses All 60 animals had complete data sets and were included into statistical analysis. Statistical analysis was performed in SPSS (version 15; SPSS Inc.). To allow statistical analysis, all haptoglobin concentrations >100 mg/dL were transformed into 100.1 mg/dL and all <1.0 mg/dL into 0, and were then transformed back when average haptoglobin concentrations (median and 1st and 3rd quartile) per disorder group and day were given. The Kolmogorov–Smirnov test was then used to test for normal distribution, and the data were found not to be normally distributed. The Friedman test was employed to determine if sets of data recorded for days 1, 3 and 5 for each claw disorder differed, and the Wilcoxon Signed Ranks Test then used to pinpoint the differences. The Pearson’s chi-square test was employed to compare percentages of animals with elevated haptoglobin. A difference of P < 0.05 was considered statistically significant.
Results Concentrations of serum haptoglobin of all healthy cows without lameness were below the detection limit of the assay, i.e., <1.0 mg/dL. Lame cows with any of the claw disorders PS, PC, IN or PDD were found to have either increased serum haptoglobin concentrations (i.e., >1.0 mg/dL: n = 36), or found with concentrations <1.0 mg/dL (n = 24) (Table 1). Animals that tested positive for haptoglobin had concentrations ranging between 37 and >100 mg/dL (Table 1). In PS and IN animals, median haptoglobin concentrations decreased after treatment between days 1 and 5 (P < 0.05), with a concomitant decrease in the number of animals with detectable haptoglobin (P < 0.05; Table 1). Ceftiofur and penicillin G were equally effective in animals with IN (data not detailed). In contrast, treatment did not affect either the median haptoglobin concentration or the number of animals with PC having increased haptoglobin (P > 0.05; Table 1). The one animal with PDD that had increased haptoglobin on day 1 was negative on day 5. Discussion To the best of our knowledge, this study is the first to describe serum haptoglobin concentrations in dairy cows with lameness due to claw disorders on a commercial dairy cattle farm. Haptoglobin is an APP that is released in response to tissue damage, inflammation, infection and bacterial components, as well as stress (Murata et al., 2004; Lomborg et al., 2008), but is absent or almost absent in healthy cows (Panndorf et al., 1976; Conner et al., 1986). This has been shown by the results of this study. Since lame cows were found to have elevated serum haptoglobin regardless of the claw disorder at first testing, our results suggest that any of the disorders PS, PC, IN, PDD can lead to a systemic acute phase response. However, there were simultaneously always lame cows with undetectable serum haptoglobin. This has been similarly observed in a recent study where cows with PS had either high or low serum haptoglobin, respectively (Jawor et al., 2008), or in lame pigs that were found having either high or zero haptoglobin in blood serum (van den Berg et al., 2007). Why animals with the same claw disorder had elevated or undetectable serum haptoglobin at first testing may be due to several reasons. Clearly, animals can vary in their acute phase response to the same exposure (Stengärde et al., 2008; Lomborg et al., 2008). Different disease severity, i.e., the more severe the disease the higher is the serum haptoglobin concentration, might be another reason (Conner et al., 1988; Young et al., 1996; Petersen et al., 2004; Heinonen et al., 2006), but severity was only recorded patchily in this study. Haptoglobin has been shown to increase within 24 h after an animal has been exposed to an acute phase response eliciting agent (Jacobsen et al., 2004) and has a half-life time of about 45 h (Richter, 1975). In this study the first haptoglobin value was obtained between a few hours and 3 days after lameness was observed by the farm crew. It might be thus that
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B.I. Smith et al. / The Veterinary Journal 186 (2010) 162–165
Table 1 Results of measurement of haptoglobin (Hp) in blood serum of lame cows with different claw disorders (n = 60). Day1
Parameter
PS (n = 41)
PC (n = 8)
IN (n = 7)
PDD (n = 4)
1
Positive n (%)2 Median mg/dL (1st–3rd Quartile)
27 (65.9)A 70.0 (<1.0–>100)a
3 (37.5) 0 (<1.0–41.5)
5 (71.4)A 100.0 (<1.0–>100)a
1 (25.0) 0 (<1.0–75.1)
3
Positive n (%)2 Median mg/dL (1st–3rd Quartile)
24 (58.5)B,C 49.0 (<1.0–>100)a
3 (37.5) 0 (<1.0–78.3)
6 (85.7)A 94.0 (<1.0–>100)a
1 (25.0) 0 (<1.0–75.1)
5
Positive n (%)2 Median mg/dL (1st–3rd Quartile)
17 (41.5)C 0 (<1.0–100)b
4 (50.0) 34.0 (<1.0–94.7)
1 (14.3)B 0 (<1.0)b
0 0 (<1.0)
Claw disorder
PS, pododermatitis septica; PC, pododermatitis circumscripta; IN, interdigital necrobacillosis; PDD, papillomatous digital dermatitis. 1 Day 1 is the day of diagnosis and of treatment. Day 1 samples were taken before treatment. 2 Total number of animals with increased Hp concentration. None of the animals had concentrations below 37.0 mg/dL. (a,b) Concentrations in columns with different superscripts differ significantly (P < 0.05). (A–C) Number of animals in columns with different superscripts differ significantly (P < 0.05).
haptoglobin was yet not elevated in some cows but already decreased in other animals at first testing. As to what sub-clinical diseases of other organs (Stengärde et al., 2008) or pre-exposure to stress (Lomborg et al., 2008) may have had an additional impact on serum haptoglobin elevation is another aspect that requires consideration. The treatments used in this study were all classically recommended procedures for claw disorders in cattle (Rebhun and Pearson, 1982). A direct measure for the control of the treatment’s effectiveness, such as a clinical examination, was not employed and the course of serum haptoglobin over time was instead used as an indirect measure based on the observation that haptoglobin decreases quickly after the acute phase response eliciting agent (such as due to treatment) is removed (Hulten et al., 2002; Petersen et al., 2004; Jawor et al., 2008). Accordingly, trimming of claws with PS (as a primarily non-infectious disorder) led to a decrease in haptoglobin in the test population and is assumed to be effective in diseased cows. In contrast, trimming was ineffective in animals with PC as another primary non-infectious claw disorder. Perhaps the animals needed more time to recover after being treated, or trimming alone was inappropriate. Maybe there was also an infectious agent involved (van Amstel and Shearer, 2006), which would require a systemic antibiotic treatment. Antibiotic treatment with both penicillin G and ceftiofur was shown to be clearly effective in animals with IN and supports standard treatment recommendations for IN (Radostits et al., 2007). The effectiveness of topical antibiotic treatment in cases of PDD cannot be conclusively answered due to the low number of animals involved.
Conclusions The study has shown that lameness due to claw disorders can be associated with a systemic acute phase response and elevated serum haptoglobin in dairy cattle. Why the same disorder elicited a response in only some of the cows requires further investigations. The study suggests that trimming is an effective treatment for pododermatitis septica and that systemic antibiosis is efficient in cases of interdigital necrobacillosis. However, trimming may not be the optimum treatment regimen in case of pododermatitis circumscripta, and the approach may require revision.
Conflict of interest statement None of the authors of this paper has a financial or personal relationship with other people or organisations that could inappropriately influence or bias the concept of the paper.
Acknowledgements The authors thank Bonnnie Vecchiarelli (Section of Animal Production Systems, New Bolton Center, University of Pennsylvania, Kennett Square, PA, USA) for laboratory assistance. We are grateful to Andreas Richter (Large Animal Clinic for Theriogenology and Ambulatory Services, Faculty of Veterinary Medicine, University of Leipzig, Leipzig, Germany) for providing help with the statistical analysis. This project was supported by funding from the Frances Chaney Glover Field Service Endowment Fund, School of Veterinary Medicine, University of Pennsylvania.
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Contents lists available at ScienceDirect
The Veterinary Journal journal homepage: www.elsevier.com/locate/tvjl
Serum haptoglobin concentrations in dairy cattle with lameness due to claw disorders Billy I. Smith *, Johannes Kauffold, Lisa Sherman 1 Department of Clinical Studies, New Bolton Center, School of Veterinary Medicine, University of Pennsylvania, 382 West Street Road, Kennett Square, PA 19348, USA
a r t i c l e
i n f o
Article history: Accepted 11 August 2009
Keywords: Dairy cattle Lameness Claw disorder Haptoglobin Treatment
a b s t r a c t In cattle, elevated blood serum concentrations of haptoglobin, an acute phase protein, have been demonstrated in association with several diseases, but not with lameness. Serum haptoglobin was measured in 60 Holstein dairy cattle diagnosed with lameness due to four claw disorders, pododermatitis septica (PS; n = 41), pododermatitis circumscripta (PC; n = 8), interdigital necrobacillosis (IN; n = 7), papillomatous digital dermatitis (PDD; n = 4). Haptoglobin was measured on day 1 (0–3 days after lameness was observed but before treatment) and on days 3 and 5. A total of 10 healthy cows served as controls (haptoglobin values <1.0 mg/dL). Each of the claw disorders was associated with elevated haptoglobin on day 1 (PS, PC, IN and PDD: 65.9%, 37.5%, 71.4% and 25.0%, respectively). Trimming and antibiotic treatment led to a reduction in the number of PS and IN cows with increased haptoglobin concentrations, respectively (P < 0.05), but trimming did not lead to any reduction in cows with PC. The study showed that lameness due to claw disorders can be associated with a systemic acute phase response and elevated serum haptoglobin in dairy cattle. Based on the course of haptoglobin, treatments seemed effective for all claw disorders except for PC. Published by Elsevier Ltd.
Introduction Haptoglobin is an acute phase protein (APP) synthesized by hepatocytes in response to cytokines released from macrophages and other immune cells as the result of tissue damage, inflammation, infection and bacterial components, and also after stress (Murata et al., 2004; Lomborg et al., 2008). In healthy cattle, serum haptoglobin is either non-detectable or measurable only in very low concentrations, i.e., below 10 mg/dL (Panndorf et al., 1976; Conner et al., 1986). Increased serum haptoglobin has been found in association with, for example, pneumonia, mastitis, metritis and uterine torsions, ketosis, fatty liver syndrome and with stress (Conner et al., 1986; Eckersall and Connor, 1988; Skinner et al., 1991; Young et al., 1996; Smith et al., 1998; Schoenfelder et al., 2006; Stengärde et al., 2008; Lomborg et al., 2008). Lameness is a substantial and costly problem with a high incidence in the dairy industry worldwide (Enevoldsen et al., 1991; Wells et al., 1993; Guard, 1996, 2006a; Hernandez et al., 2007). Inflammatory claw lesions, either due to bacterial infections such as papillomatous digital dermatitis (PDD) and interdigital necrobacillosis (IN), or with a non-infectious genesis such as pododermati* Corresponding author. Tel.: +1 610 925 6310; fax: +1 610 925 8103. E-mail address: [email protected] (B.I. Smith). 1 Present address: 8 Hemlock Lane, Tioga, PA 16946, USA. 1090-0233/$ - see front matter Published by Elsevier Ltd. doi:10.1016/j.tvjl.2009.08.012
tis circumscripta (PC) and pododermatitis septica (PS), are the most prevalent reasons for dairy cattle lameness (Shearer et al., 1996; Bergsten et al., 1998; Hoblet and Weiss, 2001). Increased serum haptoglobin has been described in association with lameness in pigs (van den Berg et al., 2007), horses (Hulten et al., 2002) and most recently also in cattle but only in 14 lame cows subjected to clinic facilities (Jawor et al., 2008). The aim of this study was to gain further knowledge on APP haptoglobin in association with lameness due to claw disorders in dairy cattle on a commercial farm. Since all animals received treatment according to the disorder it was also aimed to test how treatments effect serum haptoglobin and so get an indirect measure of the effectiveness of the treatments (Hulten et al., 2002; Schoenfelder et al., 2006). Materials and methods Farm and management This study was performed at a 600-cow dairy farm in southeast Pennsylvania, USA, over a 6 month period between March and August 2005. Cows were housed in a six-row, open ridge, freestall barn with curtain sidings, canvas mattresses, automatic alley scrapers, open rail feed bunks with rubber flooring. For management purposes, cows were separately kept in five groups according to age and/or milk production (fresh cows; heifers; cows with low, medium or high milk production). They were milked twice a day, fed a total mixed ration according to national recommendations (NRC, 1989) and had ad libitum access to water.
B.I. Smith et al. / The Veterinary Journal 186 (2010) 162–165 Routine claw trimming was done twice a year on all cows and followed a farm protocol, i.e., at the time the cows were dried off and again at 120 days in milk. Trimming was performed on restrained animals using hoof knives and a chipper wheel. Cows were observed daily for heath status including lameness by the farm crew. Cows found lame were subjected to veterinary supervision. Since supervisions were always done on Tuesdays and Fridays, the interval between detection of lameness by the farm crew and veterinary supervision varied between 0 and 3 days.
Animals Seventy-six cows with lameness were subjected to veterinary supervision during the study interval. The cows were caught in a head catch, the affected limb was then lifted off the ground by using a pulley system. A total of 60 Holstein dairy cows (parity 1–8) were identified as having claw related lameness and diagnosed with PS (n = 41), IN (n = 7), PC (n = 8) or PDD (n = 4). Diagnosis of the claw disorders followed approved procedures (Desrochers et al., 2001). For IN, the severity was additionally determined and graded into ‘moderate’ and ‘severe’. All 60 cows were in good general health and did not have other inflammatory problems such as pneumonia, enteritis, mastitis or any kinds of acute uterine inflammation. Another 10 randomly picked cows all of them in good general health without any obvious disease including lameness as evaluated by routine physical and record analysis of data on the animal’s activity were used as controls to define haptoglobin concentrations in healthy cows without lameness.
Treatments Animals were treated immediately after diagnosis employing classical treatment protocols (Rebhun and Pearson, 1982; Berry, 2001; van Amstel and Shearer, 2001; Guard, 2006b). Lame cows diagnosed with PS were treated by trimming the affected claw to relieve pressure resulting from the accumulation of purulent material between the sole and laminae. Cows diagnosed with PC were treated by trimming the affected claw to relieve pressure resulting from inflamed laminae due to trauma and infection. After the claw was trimmed, a wooden block was glued onto the sole of the unaffected claw. A copper containing topical antimicrobial agent (Kopertox; Fort Dodge Animal Health) was applied to the lesion. No anti-inflammatory or antibiotic drugs were used on these animals. Animals diagnosed with PDD were treated topically with antibiotic claw wraps using tetracycline powder (Tetracycline Soluble Powder 324; Agripharm). Cows with IN were treated systemically with antibiotics according to severity with either ceftiofur sodium (Naxcel; Pfizer) in case of moderate IN (n = 3), or procaine penicillin G (Pen-Aqueous; Agripharm) in case of severe IN (n = 4) over 5 days, respectively. In addition, all IN animals were treated with claw wraps and tetracycline powder (Tetracycline Soluble Powder 324; Agripharm). No anti-inflammatory drugs were used on these animals. Animals that served as controls were not treated at all.
Analysis of haptoglobin in blood serum Blood samples were collected from all 60 animals with lameness and the 10 control animals by coccygeal venepuncture into evacuated tubes (Vacutainer; Fisher Scientific). Lame cows were bled on day 1, i.e., after initial diagnosis but before treatment, and on days 3 and 5 thereafter. Control cows were bled only once and in one session. Blood was allowed to coagulate, was then refrigerated at 4.4 °C (40 °F) and cleaned from clots within 24 h of collection. Serum was recovered by centrifugation at 4500 g and stored frozen at 20 °C ( 4 °F). Haptoglobin concentration was measured in serum by means of radial immunodiffusion (Bovine Hp Plate; code No. P0105-1; Cardiotech Svcs., Inc.) according to the manufacturer’s instructions. This kit is validated for and used with frozen/thawed bovine serum (Conner et al., 1989; Makimura and Usui, 1990; Skinner et al., 1991), and has been described in detail recently (Berry et al., 2004). Briefly, bovine serum samples (100 lL) were treated with an equal amount of 40 mM solution of L-cysteine (24 mg of L-cysteine dissolved in 5 mL of L-cysteine solvent [1 e]), and both were added to a single mixing well. Two standards (A and B) were loaded (5 lL) into specified wells of the plate, and the treated samples then into separate wells, respectively. The plate was covered, incubated at 37 °C for 24–48 h, and the results determined by measuring the external diameter of each precipitin ring to the nearest 0.01 mm with a supplied plastic scale. Diameters of rings obtained with the standard solution were plotted on the vertical axis of semi-logarithmic graph paper versus serum haptoglobin concentration on the horizontal axis. A reference curve was created by drawing a straight line through plotted standard points. The diameters of the precipitin rings for each test sample were used to calculate the haptoglobin concentration using the reference curve. If no precipitin ring appeared, the haptoglobin concentration was below 1.0 mg/dL. Concentrations >100 mg/dL were given as such. The coefficient of variation for the kit was less than 4% for repeated, identical measurements on the same specimen.
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Statistical analyses All 60 animals had complete data sets and were included into statistical analysis. Statistical analysis was performed in SPSS (version 15; SPSS Inc.). To allow statistical analysis, all haptoglobin concentrations >100 mg/dL were transformed into 100.1 mg/dL and all <1.0 mg/dL into 0, and were then transformed back when average haptoglobin concentrations (median and 1st and 3rd quartile) per disorder group and day were given. The Kolmogorov–Smirnov test was then used to test for normal distribution, and the data were found not to be normally distributed. The Friedman test was employed to determine if sets of data recorded for days 1, 3 and 5 for each claw disorder differed, and the Wilcoxon Signed Ranks Test then used to pinpoint the differences. The Pearson’s chi-square test was employed to compare percentages of animals with elevated haptoglobin. A difference of P < 0.05 was considered statistically significant.
Results Concentrations of serum haptoglobin of all healthy cows without lameness were below the detection limit of the assay, i.e., <1.0 mg/dL. Lame cows with any of the claw disorders PS, PC, IN or PDD were found to have either increased serum haptoglobin concentrations (i.e., >1.0 mg/dL: n = 36), or found with concentrations <1.0 mg/dL (n = 24) (Table 1). Animals that tested positive for haptoglobin had concentrations ranging between 37 and >100 mg/dL (Table 1). In PS and IN animals, median haptoglobin concentrations decreased after treatment between days 1 and 5 (P < 0.05), with a concomitant decrease in the number of animals with detectable haptoglobin (P < 0.05; Table 1). Ceftiofur and penicillin G were equally effective in animals with IN (data not detailed). In contrast, treatment did not affect either the median haptoglobin concentration or the number of animals with PC having increased haptoglobin (P > 0.05; Table 1). The one animal with PDD that had increased haptoglobin on day 1 was negative on day 5. Discussion To the best of our knowledge, this study is the first to describe serum haptoglobin concentrations in dairy cows with lameness due to claw disorders on a commercial dairy cattle farm. Haptoglobin is an APP that is released in response to tissue damage, inflammation, infection and bacterial components, as well as stress (Murata et al., 2004; Lomborg et al., 2008), but is absent or almost absent in healthy cows (Panndorf et al., 1976; Conner et al., 1986). This has been shown by the results of this study. Since lame cows were found to have elevated serum haptoglobin regardless of the claw disorder at first testing, our results suggest that any of the disorders PS, PC, IN, PDD can lead to a systemic acute phase response. However, there were simultaneously always lame cows with undetectable serum haptoglobin. This has been similarly observed in a recent study where cows with PS had either high or low serum haptoglobin, respectively (Jawor et al., 2008), or in lame pigs that were found having either high or zero haptoglobin in blood serum (van den Berg et al., 2007). Why animals with the same claw disorder had elevated or undetectable serum haptoglobin at first testing may be due to several reasons. Clearly, animals can vary in their acute phase response to the same exposure (Stengärde et al., 2008; Lomborg et al., 2008). Different disease severity, i.e., the more severe the disease the higher is the serum haptoglobin concentration, might be another reason (Conner et al., 1988; Young et al., 1996; Petersen et al., 2004; Heinonen et al., 2006), but severity was only recorded patchily in this study. Haptoglobin has been shown to increase within 24 h after an animal has been exposed to an acute phase response eliciting agent (Jacobsen et al., 2004) and has a half-life time of about 45 h (Richter, 1975). In this study the first haptoglobin value was obtained between a few hours and 3 days after lameness was observed by the farm crew. It might be thus that
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B.I. Smith et al. / The Veterinary Journal 186 (2010) 162–165
Table 1 Results of measurement of haptoglobin (Hp) in blood serum of lame cows with different claw disorders (n = 60). Day1
Parameter
PS (n = 41)
PC (n = 8)
IN (n = 7)
PDD (n = 4)
1
Positive n (%)2 Median mg/dL (1st–3rd Quartile)
27 (65.9)A 70.0 (<1.0–>100)a
3 (37.5) 0 (<1.0–41.5)
5 (71.4)A 100.0 (<1.0–>100)a
1 (25.0) 0 (<1.0–75.1)
3
Positive n (%)2 Median mg/dL (1st–3rd Quartile)
24 (58.5)B,C 49.0 (<1.0–>100)a
3 (37.5) 0 (<1.0–78.3)
6 (85.7)A 94.0 (<1.0–>100)a
1 (25.0) 0 (<1.0–75.1)
5
Positive n (%)2 Median mg/dL (1st–3rd Quartile)
17 (41.5)C 0 (<1.0–100)b
4 (50.0) 34.0 (<1.0–94.7)
1 (14.3)B 0 (<1.0)b
0 0 (<1.0)
Claw disorder
PS, pododermatitis septica; PC, pododermatitis circumscripta; IN, interdigital necrobacillosis; PDD, papillomatous digital dermatitis. 1 Day 1 is the day of diagnosis and of treatment. Day 1 samples were taken before treatment. 2 Total number of animals with increased Hp concentration. None of the animals had concentrations below 37.0 mg/dL. (a,b) Concentrations in columns with different superscripts differ significantly (P < 0.05). (A–C) Number of animals in columns with different superscripts differ significantly (P < 0.05).
haptoglobin was yet not elevated in some cows but already decreased in other animals at first testing. As to what sub-clinical diseases of other organs (Stengärde et al., 2008) or pre-exposure to stress (Lomborg et al., 2008) may have had an additional impact on serum haptoglobin elevation is another aspect that requires consideration. The treatments used in this study were all classically recommended procedures for claw disorders in cattle (Rebhun and Pearson, 1982). A direct measure for the control of the treatment’s effectiveness, such as a clinical examination, was not employed and the course of serum haptoglobin over time was instead used as an indirect measure based on the observation that haptoglobin decreases quickly after the acute phase response eliciting agent (such as due to treatment) is removed (Hulten et al., 2002; Petersen et al., 2004; Jawor et al., 2008). Accordingly, trimming of claws with PS (as a primarily non-infectious disorder) led to a decrease in haptoglobin in the test population and is assumed to be effective in diseased cows. In contrast, trimming was ineffective in animals with PC as another primary non-infectious claw disorder. Perhaps the animals needed more time to recover after being treated, or trimming alone was inappropriate. Maybe there was also an infectious agent involved (van Amstel and Shearer, 2006), which would require a systemic antibiotic treatment. Antibiotic treatment with both penicillin G and ceftiofur was shown to be clearly effective in animals with IN and supports standard treatment recommendations for IN (Radostits et al., 2007). The effectiveness of topical antibiotic treatment in cases of PDD cannot be conclusively answered due to the low number of animals involved.
Conclusions The study has shown that lameness due to claw disorders can be associated with a systemic acute phase response and elevated serum haptoglobin in dairy cattle. Why the same disorder elicited a response in only some of the cows requires further investigations. The study suggests that trimming is an effective treatment for pododermatitis septica and that systemic antibiosis is efficient in cases of interdigital necrobacillosis. However, trimming may not be the optimum treatment regimen in case of pododermatitis circumscripta, and the approach may require revision.
Conflict of interest statement None of the authors of this paper has a financial or personal relationship with other people or organisations that could inappropriately influence or bias the concept of the paper.
Acknowledgements The authors thank Bonnnie Vecchiarelli (Section of Animal Production Systems, New Bolton Center, University of Pennsylvania, Kennett Square, PA, USA) for laboratory assistance. We are grateful to Andreas Richter (Large Animal Clinic for Theriogenology and Ambulatory Services, Faculty of Veterinary Medicine, University of Leipzig, Leipzig, Germany) for providing help with the statistical analysis. This project was supported by funding from the Frances Chaney Glover Field Service Endowment Fund, School of Veterinary Medicine, University of Pennsylvania.
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