Karin Persson Waller

Haptoglobin and serum amyloid A in milk and serum during acute and chronic experimentally induced Staphylococcus aureus mastitis

Local and systemic changes in the acute phase proteins, haptoglobin and serum amyloid A (SAA), were studied in six dairy cows during the acute and chronic phases of experimentally induced Staphylococcus aureus mastitis. Haptoglobin and SAA were measured in serum, and in milk from infected and healthy control udder quarters within each cow. Concentrations of haptoglobin and SAA increased rapidly in both serum and milk during the acute phase of mastitis and followed a similar pattern. Significantly raised milk concentrations of SAA were also found during chronic subclinical mastitis. Serum concentrations of SAA also tended to be higher during the chronic phase than pre-infection. Increases in milk haptoglobin and SAA were specific for the infected udder quarters. In conclusion, measurement of SAA in milk samples could be a useful tool in diagnosing mastitis.

Acute phase proteins as indicators of calf herd health.

Abstract The potential for using acute phase proteins (APPs) in the assessment of herd health was studied by examining the levels of serum haptoglobin, serum amyloid A (SAA) and plasma fibrinogen in relation to clinical findings and leukocyte counts in calves. Two groups of calves from conventional dairy farms were studied. The animals were examined 10 times during the first six weeks after introduction into a new environment. Haptoglobin, SAA and fibrinogen were analysed and weight gain, disease symptoms and treatments were recorded. Analysis of antibodies against viral infections was performed. An acute phase reaction (APR) score was established at each sampling by combining the APP results and total leukocyte counts. The health status differed between the two groups, although no manipulation of health had been performed, except that the group with a higher incidence of disease had a concurrent experimental infection with lungworm as part of another study. In the group with a higher incidence of disease, the mean weight gain was significantly lower, and the number of sampling days with elevated serum concentrations of APPs, and the mean maximum concentrations of haptoglobin and fibrinogen were significantly higher compared to the healthier group. The APR score was significantly higher at days 4 and 8 of the study in the group with a higher incidence of disease. The results indicate that measurement of APPs could be a useful tool for evaluation of health in calf herds.

Mammary gland immunology around parturition. Influence of stress, nutrition and genetics.

Adequate immune functions are essential for the defence against udder infections. Detailed knowledge about the immune response and important defence factors is essential in order to find new ways for the prevention and treatment of udder infections leading to mastitis. Work should be concentrated on ways of minimising the negative influence on immune functions and/or ways of stimulating these functions, especially during periods of immune suppression. A depression of important immune functions has been reported around parturition and there is a higher prevalence of clinical mastitis and other diseases during this period. Immunosuppression is often associated with high levels of glucocorticoids in blood, a common finding around parturition and during stressful conditions. A number of stressors are present around calving, e.g. parturition, onset of lactation and changes in feeding and management regimes. Adequate management including feeding strategies and routines are important for the immune functions. Metabolic stress as well as deficiencies in vitamins and minerals around parturition and during the first month of lactation can have a negative influence on the immune functions and thereby increase the risks for udder infections and mastitis. There seem to be a genetic variability in certain immune functions among periparturient cows. This might indicate a possibility to find markers for genetic selection of individuals with a well-developed immune system without negative effects on milk productivity.

Antimicrobial susceptibility of udder pathogens from cases of acute clinical mastitis in dairy cows.

To investigate occurrence of acquired antimicrobial resistance in udder pathogens MICs in Staphylococcus aureus (n=211), coagulase-negative staphylococci (CNS) (n=56), Streptococcus uberis (n=113), Streptococcus dysgalactiae (n=152), Streptococcus agalactiae (n=6), Escherichia coli (n=163), and Klebsiella spp. (n=42) were determined using microdilution. Isolates were from a nation wide survey employing strict inclusion criteria. Presence of acquired resistance was evaluated by species-specific epidemiological cut-off values issued by EUCAST. Penicillin or methicillin resistance in staphylococci were however evaluated by beta-lactamase production or presence of the mecA gene, respectively. Staphylococci were mostly susceptible to antimicrobials tested but 7.1% of S. aureus and 12.5% of CNS were resistant to penicillin by beta-lactamase production. Methicillin resistance was not found in S. aureus. All Streptococcus dysgalactiae and S. agalactiae were susceptible to penicillin. Bimodal MIC distributions for tetracycline in S dysgalactiae and S. uberis indicate acquired resistance in some isolates. Among E. coli 12.3% of isolates were resistant to one or more antimicrobials. Resistance to streptomycin (11.0%), sulphametoxazole (8.6%), ampicillin (7.4%), or tetracycline (4.9%) were the most common traits. Klebsiella spp. were resistant to ampicillin and some isolates also to tetracycline (7.1%) or sulphonamide (9.5%). The study shows that in Sweden bacteria associated with acute clinical mastitis for the most part are susceptible to antimicrobials used in therapy but resistance to penicillin in S. aureus is not uncommon. Penicillin is recommended for treatment of mastitis caused by gram-positive pathogens and regular monitoring of beta-lactamase production in S. aureus is therefore recommended in herds with udder health problems.

α-Tocopherol concentration and stereoisomer composition in plasma and milk from dairy cows fed natural or synthetic vitamin E around calving

The aim of this study was to compare the effects of supplementing dairy cows with 1000 IU/day of all-rac-alpha-tocopheryl acetate (SynAc), RRR-alpha-tocopheryl acetate (NatAc), or RRR-alpha-tocopherol (NatAlc), from approximately 3 weeks before estimated calving until 2 weeks after calving, on the concentration of alpha-tocopherol and its stereoisomers (RRR-, RSS-, RRS-, RSR- and the four 2S-forms of alpha-tocopherol) in blood and milk. An unsupplemented group was included as control. Blood samples were collected at 3, 2 and 1 weeks before estimated calving, at calving, and 3, 7 and 14 days after calving, while milk samples were taken twice within 24 h after calving and at 7 and 14 days in milk. Overall, time and treatment had significant effects on plasma alpha-tocopherol with higher concentrations in NatAc than in the other groups. In addition, SynAc had higher concentrations than Control, and NatAlc tended to be higher than Control. The lowest plasma concentrations were observed at calving and 3 days after calving. Independent of treatment, the concentration was higher in colostrum than in milk day 7 and 14 after calving. Analyses of the stereoisomer distribution in plasma and milk showed that, irrespective of dietary treatment, RRR-alpha-tocopherol was the most predominant form, constituting more than 86%, whereas the remaining part of alpha-tocopherol was made up by the three synthetic 2R isomers, while the 2S isomers only contributed less than 1% of the total alpha-tocopherol. In control cows and cows supplemented with natural vitamin E, the proportion of RRR-alpha-tocopherol in plasma and milk constituted more than 98% of the total alpha-tocopherol. In conclusion, the results indicate that daily oral supplementation of dairy cows with RRR-alpha-tocopheryl acetate gives the highest blood concentrations of alpha-tocopherol in the periparturient period. Analyses of the distribution of the individual stereoisomers of alpha-tocopherol further indicate that the bioavailability of RRR-alpha-tocopherol relative to synthetic stereoisomers in cattle is considerably higher than officially accepted until now.

Risk factors for calf mortality in large Swedish dairy herds.

Abstract The aim of this study was to identify possible risk factors for 1–90 day calf mortality in large Swedish dairy herds. Sixty herds with a herd size of ≥160 cows were visited once between December 2005 and March 2006. Thirty herds were known to have low mortality (LM) and 30 were known high mortality herds (HM). Upon the visit, data about housing and management was collected from interviews with personnel responsible for the calves. The herd status regarding the calves’ passive transfer (total protein), levels of α-tocopherol, β-carotene and retinol, and excretion of faecal pathogens (Cryptosporidium spp., Escherichia coli F5, rota and corona virus) was evaluated based on targeted sampling of high risk calf groups; in each herd, blood and faecal samples were collected from calves 1–7 and 1–14 days old, respectively. Similarly, the herd status regarding clinical respiratory disease in calves and history of respiratory virus exposure was evaluated based on lung auscultations and blood samplings of calves 60–90 days old. The median calf mortality risk (in calves 1–90 days of age) among HM herds was 9% (Range: 6–24%) and among LM herds 1% (Range: 0–2%). LM and HM herds were compared using five logistic regression models, covering potential risk factors within different areas: “Disease susceptibility”, “Factors affecting the gastrointestinal tract”, “Factors related to transmission of infectious disease”, “Hygiene” and “Labour management”. The percentage of calves, 1–7 days old, with inadequate serum concentrations of α-tocopherol and β-carotene were significantly higher in HM herds compared to LM herds and also associated with higher odds of being a HM herd (OR=1.02; p =0.023 and OR=1.05; p =0.0028, respectively). The variable “Average number of faecal pathogens in the sampled target group” was significantly associated with higher odds of being a HM herd (OR=4.65; p =0.015), with a higher average in HM herds. The percentage of calves with diarrhoea treated with antibiotics was significantly higher in HM herds and was associated with higher odds of being a HM herd (OR=1.08; p =0.021). The median age at death of calves in the age interval 1–90 days that died during a one-year period was significantly lower among HM herds (13 days) than in LM herds (24 days) (p =0.0013) The results indicate that gastrointestinal disorders may be an important cause of calf mortality in large Swedish dairy herds. Furthermore, our study provides additional indications that fat soluble vitamins might play an important role for calf health.

Incidence of mastitis and bacterial findings at clinical mastitis in Swedish primiparous cows - influence of breed and stage of lactation.

Mastitis is a common disease also among primiparous dairy cows. Identification of the extent and type of problem is important to initiate correct control measures. In Sweden, unique national production and disease databases are available. The main aim of the study was to investigate the occurrence of mastitis, measured by the annual incidence of veterinary-treated clinical mastitis (VTCM) and geometric mean of monthly milk somatic cell count (SCC) recordings in Swedish primiparous cows in relation to older cows during 2002-2006 with emphasis on breed differences. Other aims were to study differences between primiparous and older cows in the distribution of bacterial findings at clinical mastitis, and the occurrence of VTCM and bacterial findings in relation to stage of lactation using data from a Swedish field study performed 2002-2003. Descriptive statistics and univariable analyses were used in the investigations. During 2002-2006 approximately 10% of Swedish primiparous cows experienced VTCM each year, while the geometric mean SCC of Swedish primiparous cows was approximately 65,000 ml(-1). Both parameters were lower than in older cows. Primiparous cows of the Swedish Red (SR) breed had better udder health than cows of the Swedish Holstein (SH) breed. The overall distribution of udder pathogens was similar in primiparous and older cows. In primiparous cows, most VTCM occurred during the first week after calving, and Staphylococcus aureus and Streptococcus dysgalactiae were the most common udder pathogens during this period. Better control measures directed at these infections are warranted around calving to reduce the risk of mastitis in primiparous cows.

Haptoglobin and serum amyloid A in relation to the somatic cell count in quarter, cow composite and bulk tank milk samples

Milk somatic cell count (SCC) is the gold standard in diagnosis of subclinical mastitis, and is also an important parameter in quality programmes of dairy cooperatives. As routine SCC analysis is usually restricted to central laboratories, much effort has been invested in the search for alternative biomarkers of mastitis and milk quality, including the presence in the milk of the acute phase proteins (APP), haptoglobin (Hp) and serum amyloid A (SAA). The aim of this study was to investigate relationships between Hp, SAA and SCC in quarter, cow composite, and bulk tank milk samples. Cows (n=165), without any clinical signs of disease or abnormalities in the milk or udder, from three different dairy farms, were used. Cow composite milk samples from all cows delivering milk at the sampling occasion were taken once in each herd. In one of the farms, representative quarter milk samples (n=103) from 26 cows were also collected. In addition, bulk tank milk samples from 96 dairy farms were included in the study. Samples were analysed for Hp, SAA and SCC, and relationships between the parameters were evaluated at quarter, cow and tank milk levels using Chi-square analysis. Milk samples were categorized according to their SCC, and the presence, or no presence, of SAA and Hp, based on the detection limits of the screening methods (0.3 mg/l and 1.0 mg/l for SAA and Hp, respectively). Hp and SAA were found in milk at quarter, cow composite and bulk tank levels. A large proportion (53%) of the animals had detectable milk concentrations of APP, and SAA was detected more frequently, and at higher concentrations than Hp, regardless of sample type. SAA was detected in as many as 82% of the bulk tank milk samples. Significant relationships were found between Hp, SAA and SCC at quarter and cow composite milk levels, but only between SAA and SCC at bulk tank milk level. Detectable levels of APP were more common at high SCC.

Biosensor assay for determination of haptoglobin in bovine milk

Despite more than 30 years of research into mastitis diagnostics, there are few alternatives to the somatic cell count (SCC) in practical use for identification of cows with subclinical mastitis. Mastitis is not only an animal welfare problem, but also affects the yield, composition and technological properties of milk. Hence, dairy cooperatives give farmers a premium quality payment to encourage low SCC although there is no clear scientific data defining the level of SCC in bulk tank milk that is associated with additional benefits in terms of milk quality. Recent research on alternative markers for inflammatory reactions in the lactating cow, e.g. in mastitis, includes investigations of the acute phase protein, haptoglobin (Hp). So far, the content of Hp in milk has mainly been studied in relation to mastitis diagnostics, with little attention given to its importance for milk composition and technological properties. At present, Hp in milk is measured using ELISA, but this technique is not suitable for routine large-scale analysis. In recent years, optical biosensor technology has been used for automated and rapid quantitative analysis of different components in milk, but so far not for analysis of acute phase proteins. The aim of the present study was to develop a rapid and sensitive biosensor method to determine Hp in milk. An affinity sensor assay based on the interaction between Hp and haemoglobin was developed using surface plasmon resonance (SPR) biosensor technology. The assay was used to analyse Hp in composite milk samples from cows without any clinical signs of mastitis and quarter milk samples with a weak to strong reaction in the California Mastitis Test (CMT). A commercial ELISA for determination of Hp in milk was used for comparison. The limit of detection (LOD) of the biosensor assay was determined as 1.1 mg/l. Within-assay and between-day variations were determined both with bulk tank milk spiked with human Hp and with composite milk samples containing bovine Hp. Coefficients of variation varied between 3.6 and 8.6% at concentrations between 4.0 and 12 mg/l, respectively. Agreement between the results obtained by the biosensor assay and the ELISA was satisfactory; however, the results obtained by the biosensor were generally lower than the results obtained by the ELISA. Possible explanations for this observation are discussed.

Risk factors for antimicrobial resistance in fecal Escherichia coli from preweaned dairy calves.

The primary objective of this study was to investigate calf and farm factors associated with antimicrobial-resistant Escherichia coli in the feces of preweaned dairy calves in Sweden. In particular, we investigated the effects of feeding calves colostrum and milk from cows treated with antimicrobials. The secondary objective was to describe the prevalence of resistant E. coli in feces of preweaned dairy calves in Sweden. Fecal samples from 3 calves, aged 7 to 28d, from 243 farms were analyzed for the within-sample prevalence of E. coli resistant to nalidixic acid, streptomycin, and cefotaxime using selective agars supplemented with antimicrobials. In addition, resistance to 12 antimicrobials was tested in one randomly selected E. coli isolate per calf. Information was collected from the farmers via questionnaires regarding the use of colostrum and milk from cows treated with antimicrobials as calf feed and other uses of antimicrobials in the herd. Multivariable zero-inflated negative binomial and logistic regression models were used to assess the effect of various risk factors for shedding of resistant E. coli. Escherichia coli resistant to streptomycin, nalidixic acid, or cefotaxime were isolated from 90, 49, and 11% of the calves, respectively. Resistance to at least one antimicrobial was found in a random isolate of E. coli from 48% of the calves. Feeding colostrum from cows treated with antimicrobials at drying off did not affect the prevalence of resistant E. coli. In contrast, feeding milk from cows treated with antimicrobials during lactation resulted in significantly more nalidixic acid- and streptomycin-resistant E. coli than when such milk was discarded; no significant effect was seen for other resistance traits. Furthermore, an interaction was found between feeding milk from cows treated with antimicrobials and use of fluoroquinolones in cows. In general, the prevalence of resistance was lower for older calves and calves on small farms. Other factors that were associated with the shedding of resistant E. coli were administration of oral dihydrostreptomycin to calves, administration of systemic tetracycline and ceftiofur to cows and calves, housing of the calves, predominant breed of the herd, and geographic location of the farm. The presence of resistant E. coli in calves was clearly due to multiple factors, but minimizing the feeding of milk from cows treated with antimicrobials during lactation should lower the prevalence of resistant E. coli in the gastrointestinal tract of the calves.