Tomomi Ozawa

Decrease in bovine CD14 positive cells in colostrum is associated with the incidence of mastitis after calving

During the postpartum period there is a high incidence of mastitis in dairy cows. The reason for this increased risk of mastitis still remains unclear. Since leukocytes in colostrum have an important role in preventing the onset of mastitis, we investigated the leukocyte populations, which express CD4, CD8, CD14, CD21 or WC1, in colostrum as well as in blood obtained from 14 Holstein cows. Eight cows developed mastitis within a week after calving and the other 6 remained healthy. The percentage of CD14+ cells in colostrum was significantly lower in mastitic cows than in healthy cows. There were no significant differences in other marker positive cells either in the colostrum or in the blood. The CD14+ cells in colostrum play an important role of defense against invading microorganisms in the mammary glands. Our results suggested that the lower percentage of CD14+ cells in colostrum might predict the incidence of mastitis in the following period.

The cell wall component lipoteichoic acid of Staphylococcus aureus induces chemokine gene expression in bovine mammary epithelial cells

Staphylococcus aureus (SA) is a major cause of bovine mastitis, but its pathogenic mechanism remains poorly understood. To evaluate the role of lipoteichoic acid (LTA) in the immune or inflammatory response of SA mastitis, we investigated the gene expression profile in bovine mammary epithelial cells stimulated with LTA alone or with formalin-killed SA (FKSA) using cap analysis of gene expression. Seven common differentially expressed genes related to immune or inflammatory mediators were up-regulated under both LTA and FKSA stimulations. Three of these genes encode chemokines (IL-8, CXCL6 and CCL2) functioning as chemoattractant molecules for neutrophils and macrophages. These results suggest that the initial inflammatory response of SA infection in mammary gland may be related with LTA induced chemokine genes.

Histopathological study of encephalomalacia in neonatal calves and application of neuronal and axonal degeneration marker

Five calves that had shown neurological symptoms within 9 days after birth were histopathologically diagnosed as encephalomalacia. Two calves showed bilateral laminar cerebrocortical necrosis and neuronal necrosis in the corpus striatum and hippocampus. Since the distributional pattern of the lesions was consistent with that of global ischemia in other species, the lesions were probably hypoxic/ischemic encephalopathy consistent with the history of dystocia and perinatal asphyxia. One calf also showed bilateral laminar cerebrocortical necrosis. However, the lesions were chronic ones, because the calf had survived for long time and necropsied at postnatal day 118. Additionally, the lesions did not involve the corpus striatum and hippocampus. The other two calves showed multifocal necrosis with vascular lesions characterized by fibrin thrombi, perivascular edema and perivascular hyaline droplets in the cerebral cortex, corpus striatum, thalamus, brain stem and cerebellum. Considering the age of onsets and histopathological appearance, it was possible that latter three calves were also hypoxic/ischemic encephalopathy, however, exact cause of them was not revealed. In all calves, degenerated/necrotic neurons showed positive reactions for Fluoro-Jade C and degenerated axons showed immunoreactivity for Alzheimer precursor protein A4. Therefore, these markers were applicable to examination of brain injury in neonatal calves.

Effect of intramammary injection of liposomal RbGM-CSF on milk and peripheral blood mononuclear cells of subpopulation in Holstein cows with naturally infected subclinical mastitis

Three related experiments were conducted to gain a better understanding of the physiological responses of teat tissue to machine milking. In the first experiment changes in peak milk flow rate were used as an indicator of congestion of teat end tissues that occur during the milking phase of each pulsation cycle. Teat end congestion was increased by increasing both the b phase of pulsation and the milking vacuum level and was reduced by the application of increasing liner compression. Ultrasound measurements were used to measure changes in teat wall thickness and indicated that increasing vacuum level increased teat wall thickness and that at some critical level of liner compression the recovery rate of teat wall congestion may be reduced. The development of teat end hyperkeratosis was studied for liners with differing compression levels. This experiment confirmed that increasing liner compression increased the development of teat end hyperkeratosis. INTRODUCTION Milking vacuum level and the percentage of time the liner is open during one pulsation cycle are the primary machine factors influencing peak milk flow rate (PMF) and milking speed (Smith and Petersen, 1946; Clough, 1972; Spencer et al. 2007). Increasing milking vacuum level and increasing duration of the b-phase of pulsation have also been shown to increase teat congestion as reflected by changes in teat wall thickness after milking, measured using skin-fold calipers (Hamann et al. 1993), radiographic techniques (McDonald, 1975) or ultrasonic imaging (Gleeson et al. 2004; Neijenhuis et al. 2001a; Vinitchaikul and Suriyasathaporn, 2007; Worstorff et al. 1986), The role of liner compression (LC) in increasing milking speed by reducing teat tissue congestion during milking has become clearer in the last 20 years (Davis et al. 2000; Gleeson et al. 2004; Mein et al. 2003b;). When teats are congested after milking, the defense mechanism of the teat canal to resist invasion and removal of mastitis causing organisms from the canal is compromised (Mein et al., 1987; Hamann, 1990; Zecconi et al., 1992; Gleeson et al., 2004; Vinitchaikul and Suriyasathaporn, 2007). This is probably because the teat canal closes more slowly after milking when teats are congested (Neijenhuis et al., 2001; Mein and Reinemann, 2007). Full tissue recovery after machine milking may take many hours (Gleeson et al., 2002). When teat end thickness changed by > 5%, higher infection rates of quarters and more ducts colonized in teats were observed compared with teats showing less congestion (Zecconi et al., 1992). LC is a critical factor in reducing teat tissue congestion during milking and can also influence peak flow rate and milking speed. At the same time excessive LC contributes to the development of teat-end hyperkeratosis (HK) (Capuco et al., 1994). HK of the skin surrounding the teat canal opening is a result of the stresses applied to skin when the milking liner collapses on the teat end. The duration of milking, as affected by milk production level, milking frequency, and thresholds applied to automatic cluster removal also affect HK (Rasmussen, 1999). HK is also influenced environmental conditions (humidity and temperature) and genetics (teat shape and dimension) (Mein, et. al., 2001). A recent survey of teat-end condition on commercial farms indicated that the percentage of cows with rough or very rough teat ends averaged about 50% with some farms exceeding 70% and some farms less than 20% (Bade et al., 2007b). Teat ends with rough surface is more difficult to clean during pre-milking preparation and provide a site for bacteria colonization. Neijenhuis et al., (2001) found a correlation between increased risk of clinical mastitis and very rough teat-ends. HK is also an undesirable condition also because it may contribute to cow discomfort during milking (Hamann, 2000). Excessive LC may also remove excessive amounts of keratin from the teat canal which makes teats more susceptible to infections. LC equal to mean arterial pressure (about 12 kPa) is thought to be sufficient to relieve congestion with additional LC providing no additional benefit for congestive relief (Mein et al., 1987). More recently it has been speculated that the LC required to relieve congestion increases as milking vacuum level increases (Mein et al., 2003a). While the major milking machine related influences on milking speed and teat tissue condition after milking have been studied previously, most previous studies have either altered only one causal variable and measured only one response variable, or have introduced confounding into experimental designs by lack of independent control of several causal variables. The primary objective of our studies was to quantify the milking machine effects of milking vacuum level, bphase and LC on milk flow rates and to gain a better understanding of the physiological responses of teat tissues to machine milking. Our studies were designed to control these three causal variables independently over a broad range so that both main and interactive effects could be estimated. MATERIALS AND METHODS Experiment I. Effects of milking vacuum, b-phase and LC on PMF and teat end congestion: The main and interactive effects of vacuum level, b-phase duration, and LC on PMF of 88 Holstein cows were studied by independently controlling these causal variables over a wide range of settings (42 to 53 kPa system vacuum, 220 to 800 milliseconds of b-phase, and LC from 8 to 14 kPa) using an inscribed central composite experimental design (Bade, 2007a). Pulsation rate and ratio were adjusted so that the d-phase of pulsation was maintained at 250 ms for all treatments. Automatic cluster removers were set at a flow threshold of 0.6 kg/min and a detachment delay of 3 seconds. PMF was defined as the maximum milk yield from all four teats in any 11.2 s interval during the milking session. Average milk flow (AMF) was defined as the total milk yield divided by the total cups-on time for the milking session. Experiment II. Ultrasonic measurement of teat wall thickness: Ultrasounic scans of teat wall thickness were performed on six Holstein cows using the method described by Spanu et al. (2008). Scans were performed immediately prior to milking, immediately after milking, one hour after milking, two hours after milking and four hours after milking. Measurements of teat-wall thickness 1 cm above the top of the teat canal were taken from each scan. Teat wall thickness was expressed as percentage change compared to the pre-milking values. The following four treatments selected as a subset from the 15 treatments from experiment I were applied to each cow [A: Vacuum = 44.2 kPa, b-phase = 322 ms, LC = 9 kPa; B: Vacuum = 47.5 kPa, b-phase = 500 ms, LC = 11 kPa; C: Vacuum = 50.8 kPa, b-phase = 678 ms, LC = 9 kPa; D: Vacuum = 50.8 kPa, bphase = 678 ms, LC = 13 kPa]. Experiment III. Effect of LC on HK: This study was conducted on 75 Holstein cows, milked twice per day. A quarter-udder experiment was performed by installing four different types of liners on each of the four clusters in the milking parlor so that each quarter of every cow was milked with the same liner for a period of one month (Zucali, et al, 2008). Liners L1, L2, and L3 were round Nitrile rubber that had similar dimensions but varying wall thickness. Liner L4 was a silicon liner that is round in the open position and triangular shaped when collapsed, achieved by fixing the outer walls of the liner to the inner walls of the shell at three points. Liner L2 was used on all clusters for several months prior to the start of this experiment. LC was measured using the Start-of-Milk-Flow Method as described by Mein et al. (2003a) [ L1 = 18 kPa, L2 = 15 kPa, L3 = 13 kPa, L4 = 9 kPa]. All teats were visually scored using the N, S, R, VR method recommended by the Teat Club International (Mein et al, 2001) as well as photographed before the experiment began and once each week for the 4 week duration of the study. RESULTS Experiment I. Effects of milking vacuum, b-phase and LC on PMF and teat end congestion: A physical model was used as the basis for statistical analysis of PMF data. The Bernoulli equation for incompressible fluid flow through a tube (neglecting static pressure) can be written as: u = C1 + C2*V, where u is the velocity of fluid flow and V is negative pressure – or vacuum difference across the tube ( in this model the average claw vacuum at the peak milk flow rate) and the C2 accounts for fluid density. The volumetric flow of milk when the liner is open can be calculated using the cross sectional average fluid velocity and effective area diameter of the teat canal. The volumetric flow rate of milk, PMF, is further proportional to the fraction of the pulsation cycle in which the liner is open (F, or milk fraction): PMF = F * A * (C1 + C2*V) 1⁄2 The teat canal opens at some critical vacuum difference across it and continues to open further as this vacuum level is increased until the canal is fully unfolded and the skin has reached its elastic limit. Congestion of tissue surrounding the canal will act to decrease its effective diameter while LC will act to increase canal diameter by reducing congestion. Increasing the bphase (B) may also act to increase congestion but is likely interactive with milking vacuum (e.g. the effect of b-phase on congestion is likely to be greater as the milking vacuum increases). The following physically based model is assumed for the cross sectional area of the teat canal as a function of milking vacuum, LC and b-phase: A = C6 + C7*V + C8*LC + C9*B + C10*V*LC + C11*V*B + C12*V + C13*LC*V + C14*B*V + C15*B*LC*V. This expression was substituted into the PMF equation and fitted to the data using the SAS GLM procedure eliminating insignificant terms (p>0.05) to yield the following final model: PMF = 1.499 + (0.5202 * F * V1/2) + (0.02826 * F * V1/2 * LC) + (0.001025 * F * V1/2 * B) –(0.00019 * F * V * B). Response surfaThe main aim of the present study was to examine the economic consequences of a reduction of the incidence of clinical mastitis (CM) at herd level under current Swedish farming conditions. A second aim was to ask whether the estimated cost of CM alters depending upon whether the model reflects the fact that in different stages of lactation CM gives rise to different yield-loss patterns or postulates just one type of yield-loss pattern irrespective of when, during lactation, CM occurs. A dynamic and stochastic simulation model, SimHerd, was used to study the effects of CM in a herd with 150 cows. Technical and economic results given the initial incidence of CM (25.6 per 100 cow/year) were studied together with the consequences of reducing the initial risk of CM by 50% and 90% throughout lactation and the consequences of reducing the initial risk by 50% and 90% before peak yield. A conventional way of modelling yield losses-i.e. one employing a single yield-loss pattern irrespective of when, during the lactation period, the cow develops CM-was compared with a new modelling strategy in which CM was assumed to affect production differently depending on its lactational timing. The yearly cost of CM at herd level was estimated at 14,504, corresponding to 6.9% of the net return given the initial incidence of CM. Expressed per cow/year, the cost was 97. The cost per case of CM was estimated at 427. There were no major differences in the results obtained using the new and the conventional modelling strategy, with the exception of the yield loss per case of CM. The study, consequently, suggests that it is not worthwhile in decision making in CM prevention to put effort into deriving specific yield-loss patterns for different periods in lactation. (Less)

Effect of intramammary injection of RbGM-CSF on neutrophils function of blood and milk in Holstein cows with subclinical mastitis

Three related experiments were conducted to gain a better understanding of the physiological responses of teat tissue to machine milking. In the first experiment changes in peak milk flow rate were used as an indicator of congestion of teat end tissues that occur during the milking phase of each pulsation cycle. Teat end congestion was increased by increasing both the b phase of pulsation and the milking vacuum level and was reduced by the application of increasing liner compression. Ultrasound measurements were used to measure changes in teat wall thickness and indicated that increasing vacuum level increased teat wall thickness and that at some critical level of liner compression the recovery rate of teat wall congestion may be reduced. The development of teat end hyperkeratosis was studied for liners with differing compression levels. This experiment confirmed that increasing liner compression increased the development of teat end hyperkeratosis. INTRODUCTION Milking vacuum level and the percentage of time the liner is open during one pulsation cycle are the primary machine factors influencing peak milk flow rate (PMF) and milking speed (Smith and Petersen, 1946; Clough, 1972; Spencer et al. 2007). Increasing milking vacuum level and increasing duration of the b-phase of pulsation have also been shown to increase teat congestion as reflected by changes in teat wall thickness after milking, measured using skin-fold calipers (Hamann et al. 1993), radiographic techniques (McDonald, 1975) or ultrasonic imaging (Gleeson et al. 2004; Neijenhuis et al. 2001a; Vinitchaikul and Suriyasathaporn, 2007; Worstorff et al. 1986), The role of liner compression (LC) in increasing milking speed by reducing teat tissue congestion during milking has become clearer in the last 20 years (Davis et al. 2000; Gleeson et al. 2004; Mein et al. 2003b;). When teats are congested after milking, the defense mechanism of the teat canal to resist invasion and removal of mastitis causing organisms from the canal is compromised (Mein et al., 1987; Hamann, 1990; Zecconi et al., 1992; Gleeson et al., 2004; Vinitchaikul and Suriyasathaporn, 2007). This is probably because the teat canal closes more slowly after milking when teats are congested (Neijenhuis et al., 2001; Mein and Reinemann, 2007). Full tissue recovery after machine milking may take many hours (Gleeson et al., 2002). When teat end thickness changed by > 5%, higher infection rates of quarters and more ducts colonized in teats were observed compared with teats showing less congestion (Zecconi et al., 1992). LC is a critical factor in reducing teat tissue congestion during milking and can also influence peak flow rate and milking speed. At the same time excessive LC contributes to the development of teat-end hyperkeratosis (HK) (Capuco et al., 1994). HK of the skin surrounding the teat canal opening is a result of the stresses applied to skin when the milking liner collapses on the teat end. The duration of milking, as affected by milk production level, milking frequency, and thresholds applied to automatic cluster removal also affect HK (Rasmussen, 1999). HK is also influenced environmental conditions (humidity and temperature) and genetics (teat shape and dimension) (Mein, et. al., 2001). A recent survey of teat-end condition on commercial farms indicated that the percentage of cows with rough or very rough teat ends averaged about 50% with some farms exceeding 70% and some farms less than 20% (Bade et al., 2007b). Teat ends with rough surface is more difficult to clean during pre-milking preparation and provide a site for bacteria colonization. Neijenhuis et al., (2001) found a correlation between increased risk of clinical mastitis and very rough teat-ends. HK is also an undesirable condition also because it may contribute to cow discomfort during milking (Hamann, 2000). Excessive LC may also remove excessive amounts of keratin from the teat canal which makes teats more susceptible to infections. LC equal to mean arterial pressure (about 12 kPa) is thought to be sufficient to relieve congestion with additional LC providing no additional benefit for congestive relief (Mein et al., 1987). More recently it has been speculated that the LC required to relieve congestion increases as milking vacuum level increases (Mein et al., 2003a). While the major milking machine related influences on milking speed and teat tissue condition after milking have been studied previously, most previous studies have either altered only one causal variable and measured only one response variable, or have introduced confounding into experimental designs by lack of independent control of several causal variables. The primary objective of our studies was to quantify the milking machine effects of milking vacuum level, bphase and LC on milk flow rates and to gain a better understanding of the physiological responses of teat tissues to machine milking. Our studies were designed to control these three causal variables independently over a broad range so that both main and interactive effects could be estimated. MATERIALS AND METHODS Experiment I. Effects of milking vacuum, b-phase and LC on PMF and teat end congestion: The main and interactive effects of vacuum level, b-phase duration, and LC on PMF of 88 Holstein cows were studied by independently controlling these causal variables over a wide range of settings (42 to 53 kPa system vacuum, 220 to 800 milliseconds of b-phase, and LC from 8 to 14 kPa) using an inscribed central composite experimental design (Bade, 2007a). Pulsation rate and ratio were adjusted so that the d-phase of pulsation was maintained at 250 ms for all treatments. Automatic cluster removers were set at a flow threshold of 0.6 kg/min and a detachment delay of 3 seconds. PMF was defined as the maximum milk yield from all four teats in any 11.2 s interval during the milking session. Average milk flow (AMF) was defined as the total milk yield divided by the total cups-on time for the milking session. Experiment II. Ultrasonic measurement of teat wall thickness: Ultrasounic scans of teat wall thickness were performed on six Holstein cows using the method described by Spanu et al. (2008). Scans were performed immediately prior to milking, immediately after milking, one hour after milking, two hours after milking and four hours after milking. Measurements of teat-wall thickness 1 cm above the top of the teat canal were taken from each scan. Teat wall thickness was expressed as percentage change compared to the pre-milking values. The following four treatments selected as a subset from the 15 treatments from experiment I were applied to each cow [A: Vacuum = 44.2 kPa, b-phase = 322 ms, LC = 9 kPa; B: Vacuum = 47.5 kPa, b-phase = 500 ms, LC = 11 kPa; C: Vacuum = 50.8 kPa, b-phase = 678 ms, LC = 9 kPa; D: Vacuum = 50.8 kPa, bphase = 678 ms, LC = 13 kPa]. Experiment III. Effect of LC on HK: This study was conducted on 75 Holstein cows, milked twice per day. A quarter-udder experiment was performed by installing four different types of liners on each of the four clusters in the milking parlor so that each quarter of every cow was milked with the same liner for a period of one month (Zucali, et al, 2008). Liners L1, L2, and L3 were round Nitrile rubber that had similar dimensions but varying wall thickness. Liner L4 was a silicon liner that is round in the open position and triangular shaped when collapsed, achieved by fixing the outer walls of the liner to the inner walls of the shell at three points. Liner L2 was used on all clusters for several months prior to the start of this experiment. LC was measured using the Start-of-Milk-Flow Method as described by Mein et al. (2003a) [ L1 = 18 kPa, L2 = 15 kPa, L3 = 13 kPa, L4 = 9 kPa]. All teats were visually scored using the N, S, R, VR method recommended by the Teat Club International (Mein et al, 2001) as well as photographed before the experiment began and once each week for the 4 week duration of the study. RESULTS Experiment I. Effects of milking vacuum, b-phase and LC on PMF and teat end congestion: A physical model was used as the basis for statistical analysis of PMF data. The Bernoulli equation for incompressible fluid flow through a tube (neglecting static pressure) can be written as: u = C1 + C2*V, where u is the velocity of fluid flow and V is negative pressure – or vacuum difference across the tube ( in this model the average claw vacuum at the peak milk flow rate) and the C2 accounts for fluid density. The volumetric flow of milk when the liner is open can be calculated using the cross sectional average fluid velocity and effective area diameter of the teat canal. The volumetric flow rate of milk, PMF, is further proportional to the fraction of the pulsation cycle in which the liner is open (F, or milk fraction): PMF = F * A * (C1 + C2*V) 1⁄2 The teat canal opens at some critical vacuum difference across it and continues to open further as this vacuum level is increased until the canal is fully unfolded and the skin has reached its elastic limit. Congestion of tissue surrounding the canal will act to decrease its effective diameter while LC will act to increase canal diameter by reducing congestion. Increasing the bphase (B) may also act to increase congestion but is likely interactive with milking vacuum (e.g. the effect of b-phase on congestion is likely to be greater as the milking vacuum increases). The following physically based model is assumed for the cross sectional area of the teat canal as a function of milking vacuum, LC and b-phase: A = C6 + C7*V + C8*LC + C9*B + C10*V*LC + C11*V*B + C12*V + C13*LC*V + C14*B*V + C15*B*LC*V. This expression was substituted into the PMF equation and fitted to the data using the SAS GLM procedure eliminating insignificant terms (p>0.05) to yield the following final model: PMF = 1.499 + (0.5202 * F * V1/2) + (0.02826 * F * V1/2 * LC) + (0.001025 * F * V1/2 * B) –(0.00019 * F * V * B). Response surfaThe main aim of the present study was to examine the economic consequences of a reduction of the incidence of clinical mastitis (CM) at herd level under current Swedish farming conditions. A second aim was to ask whether the estimated cost of CM alters depending upon whether the model reflects the fact that in different stages of lactation CM gives rise to different yield-loss patterns or postulates just one type of yield-loss pattern irrespective of when, during lactation, CM occurs. A dynamic and stochastic simulation model, SimHerd, was used to study the effects of CM in a herd with 150 cows. Technical and economic results given the initial incidence of CM (25.6 per 100 cow/year) were studied together with the consequences of reducing the initial risk of CM by 50% and 90% throughout lactation and the consequences of reducing the initial risk by 50% and 90% before peak yield. A conventional way of modelling yield losses-i.e. one employing a single yield-loss pattern irrespective of when, during the lactation period, the cow develops CM-was compared with a new modelling strategy in which CM was assumed to affect production differently depending on its lactational timing. The yearly cost of CM at herd level was estimated at 14,504, corresponding to 6.9% of the net return given the initial incidence of CM. Expressed per cow/year, the cost was 97. The cost per case of CM was estimated at 427. There were no major differences in the results obtained using the new and the conventional modelling strategy, with the exception of the yield loss per case of CM. The study, consequently, suggests that it is not worthwhile in decision making in CM prevention to put effort into deriving specific yield-loss patterns for different periods in lactation. (Less)