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Use of plasma samples to assess passive transfer in calves using refractometry: comparison with serum and clinical cut-off point
  1. J. A. MacFarlane, BVSc DBR MRCVS1,
  2. D. H. Grove-White, BVSc MSc DLSHTM DBR PhD DipECBHM FRCVS2,
  3. M. D. Royal, BSc PhD2 and
  4. R. F. Smith, BVSc BSc PhD DipECBHM MRCVS2
  1. 1School of Veterinary Sciences, University of Bristol, Langford, Bristol, Somerset BS40 5DU, UK
  2. 2School of Veterinary Science, University of Liverpool, Leahurst Campus, Neston, Cheshire CH64 7TE, UK
  1. E-mail for correspondence: julie.macfarlane{at}bristol.ac.uk

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Complete or partial failure of passive transfer (FPT) of immunoglobulins in the neonatal calf can result in an increased susceptibility to disease, a higher risk of mortality and decreased growth rates (Robison and others 1988, Tyler and others 1998, Tyler and others 1999, Waldner and others 2009, Furman-Fratczat and others 2011). It is therefore critical to the survival and successful rearing of a calf that adequate passive transfer is achieved. Determining the amount of immunoglobulins a neonatal calf has received is a useful measure for veterinary surgeons and farmers to allow monitoring and standard setting for calf health and husbandry.

Immunoglobulins may be measured directly by means of a radial immunodiffusion assay (Mancini and others 1965), and although this test is the industry gold standard, it is laboratory based, requiring a minimum of 24 hours to be performed and, thus, is not applicable to routine on-farm monitoring of adequacy of passive transfer. Measuring serum total proteins by hand-held refractometry offers a convenient, simple, rapid and inexpensive on-farm tool by which farmers and veterinary surgeons can measure serum immunoglobulin concentrations, thereby allowing assessment of colostrums-feeding protocols. Its use was first described in 1971 by McBeath and ­others. The refractometer measures how much light is refracted by the total proteins in the sample. Because the greatest constituent of total protein in neonatal calf blood is immunoglobulins, and the non-immunoglobulin protein concentration is relatively constant, refractometry provides a close representation of Ig concentrations (McBeath and others 1971, Tyler and others 1996, Vandeputte and others 2011). Published studies (McBeath and others 1971, Tyler and others 1996, Quigley and others 2002, Wallace and others 2006, Vandeputte and others 2011) describe the use of plasma or serum total protein, but no study has directly quantified the agreement between results obtained from each assay method from the same animal. Furthermore, there is no accepted cut-off for plasma total protein (pTP) as measured by refractometry indicating adequacy of passive transfer. Therefore, the aim of the current study was to compare the agreement between refractometer-based measurement techniques for immunoglobulin concentrations in neonatal calves using serum or plasma and derive a plasma protein cut-off value equivalent to the accepted serum TP concentration of 5.2 g/dl (Tyler and others 1996, Calloway and others 2002), which is widely taken as an indicator of FPT at the individual calf level.

This study was part of a larger-scale study investigating neonatal immunoglobulin passive transfer performed under a UK Home Office license for work on living animals with the approval of the University of Liverpool Ethical Review Process.

Jugular venous blood samples were obtained from 61 calves aged between 24 hours and 7 days old; 1 ml of blood was collected into a plain 1.3 ml blood tube and allowed to clot for assessment of serum total protein, while 1 ml was placed into a 1.3 ml heparin blood tube for pTP assessment. The calves were sampled from seven different dairy farms in Cheshire, UK, over a two-month period. Health status of all the calves was assessed at time of sampling by an experienced farm animal veterinary surgeon, and only calves deemed healthy were included in the trial.

Blood samples were analysed at room temperature on the day of collection by one author (JAM). All samples were centrifuged and the plasma or serum taken for assessment using an optical refractometer (Clinical Protein Urine Veterinary Refractometer, Ref 312 Walcom International Industry, Guangdong, China). The refractometer was calibrated with distilled water each day and cleaned between each sample.

The association between pTP measurements and serum total protein measurements were investigated using linear regression analysis, and a Bland-Altman plot was used to compare the agreement between the two measurements (Bland and others 1986).

Sixty-one samples were analysed. Serum total protein (sPT) measurements ranged from 4.4 to 7.6 g/dl and pTP measurements ranged from 4.8 to 8.1 g/dl. The mean and sd serum total protein concentration was 5.7±0.8 g/dl and the mean and sd pTP was 6.1±0.8 g/dl.

A Bland-Altman plot confirmed good agreement between the two total protein measurements for serum and plasma concentrations (see Fig 1). Regression analysis demonstrated a highly significant association (P<0.001) between the two measurements with R2=96.5 per cent (see Fig 2). The regression equation was sTP=–0.226+0.964 (pTP).

FIG 1:

Bland-Altman plot. The difference between measurements of calf serum and plasma total protein drawn against the mean of the serum and plasma total protein measurements in 61 paired samples. With line of best fit and bias. uLOA, upper limits of agreement; lLOA, lower limits of agreement

FIG 2:

Relationship between calf serum and plasma total protein measured by optical refractometer with the line of best fit (n=61, P<0.001)

The results suggest that either plasma or serum total protein measurements can be used for immunoglobulin transfer in neonatal calves, however, different endpoints must be used to assess FPT.

FPT is defined as a serum immunoglobulin concentration below 10 mg/ml (Besser and others 1991, Tyler and others 1996, Godden and others 2008). This cut-off point for FPT has been associated with a serum total protein concentration of 5.2 g/dl in neonatal calves (Tyler and others 1996, Calloway and others 2002). Using the regression equation derived in the current study, a serum protein concentration of 5.2 g/dl equated to a pTP measurement of 5.6 g/dl.

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Footnotes

  • Provenance: not commissioned; externally peer reviewed

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