Acute phase protein concentration in pharyngeal swabs from clinically healthy commercial dairy calves

doi:10.21203/rs.3.rs-3124016/v1

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Acute phase protein concentration in pharyngeal swabs from clinically healthy commercial dairy calves

https://doi.org/10.21203/rs.3.rs-3124016/v1

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Journal Publication

published 25 Nov, 2023

Read the published version in Acta Veterinaria Scandinavica →

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Background

Early diagnosis of disease in calves are crucial for fast recovery and prudent use of antibiotics. Acute phase proteins (APP) are up- or downregulated in response to tissue injury and have been studied widely in human medicine. There is growing interest to use APPs as biomarkers for different diseases and as a tool to initiate and monitor treatment in veterinary medicine too. Pharyngeal swabs, tracheal aspirate (TA) and blood samples were collected from 84 clinically healthy commercial dairy calves and analyzed for the APPs serum amyloid A (SAA), haptoglobin (Hp) and lipopolysaccharide binding protein (LBP).

Results

We found detectable concentrations of SAA, Hp and LBP in pharyngeal swabs from calves, as well as in TA and serum. We found no correlations between APP concentrations in serum and TA nor pharyngeal swabs.

Conclusions

SAA, Hp and LBP can be measured in saliva and TA from calves. However, a considerably technical variation in the sampling method for both pharyngeal swab and TA exist, and further validation of the methods are needed.

acute phase proteins

dairy calves

saliva

serum

tracheal aspirate

Acute phase proteins (APP) are up- or downregulated as part of the acute phase response in response to tissue injury. They are unspecific, as they increase or decrease when there is inflammation and as such, does not differentiate between the agent responsible for inflammation or type of tissue injury [1]. APPs have been studied widely in human medicine as biomarkers for a variety of diseases and as a tool to initiate and monitor treatment, and in veterinary medicine there is growing interest in using APPs.

Serum amyloid A (SAA), haptoglobin (Hp) and lipopolysaccharide binding protein (LBP) have been correlated to different diseases and disease states in cows and calves [1]. Reference values for some serum APPs in calves have also been established to distinguish between healthy and diseased animals [2–4].

SAA, Hp and LBP have different characteristics and are expressed in different quantities in different body fluids and depending on health state [5–7]. There are differences in the speed of which the APP rises, and serum LBP and SAA seem to rise earlier in the disease course than Hp [8, 9]. Prohl et al. found that LBP and Hp, amongst other APPs, can be measured in broncho-alveolar fluid in both healthy calves and experimentally infected calves with respiratory disease [10]. LBP changes in BALF were both associated with changes in respiratory score and systemic LBP response, and LBP seems to be a very promising candidate for a local marker of respiratory disease in calves [10]. The concentration of Hp and SAA in BALF in calves with bronchopneumonia were increased compared to clinically healthy calves and the intensity in the increase of APP was higher in BALF than serum [11]. This together indicates that detection of APPs in BALF can be used to detect respiratory disease in calves.

An easier and less invasive method of detecting APPs from the respiratory tract, would be to measure APPs in saliva in e.g., pharyngeal fluid. In pigs, Hp and C-reactive protein (CRP) have been measured in saliva, and the concentration of Hp in saliva mimicked the concentration in serum, just at a lower concentration [12, 13]. In addition, Hp and CRP in saliva were able to differentiate between healthy and diseased pigs and were in fact superior to serum [13]. In cows, it is possible to measure SAA in saliva and there is a positive, but weak, correlation between somatic cell count and salivary SAA [14]. SAA, Hp and LBP can be measured in saliva in calves [15], however the authors does not state much about the sampling method or the age of the calves. With the overall purpose of establishing a calf side test for detection of respiratory inflammation, concentrations of APPs in the bovine pharynx needs to be explored.

The aim of this study was therefore to investigate if SAA, Hp and LBP can be measured in saliva from pharyngeal swabs and tracheal aspirate (TA) from healthy calves.

Animals and sample collection

The study protocol was approved by the Danish Animal Experiments Inspectorate (Approval number, 2021-15-0201-01099). Samples were collected during autumn 2018 and winter 2019. Nine Danish dairy herds were enrolled in the study. All single housed calves were taken into consideration, and both bulls and heifers were included. All enrolled calves had to be clinically healthy and to determine the health-status, each calf undervent a clinical examination (CE) and thoracic ultrasonography at the day of the visit. Based on the CE, calves were only included if they were bright, alert and responsive, and did not show any of the following clinical signs: nasal and/or ocular discharge, a rectal temperature above 39,0°C, abnormal breathing or abnormal lung sounds during auscultation and were given a thoracic ultrasonography score (TUS) of 0 or 1 as described by Ollivett and Buczinski [16]. Thoracic ultrasonography was performed on non-sedated calves, the hair coat stayed untouched and ethanol was used as contact medium. The entire lung field was scanned from the 12th intercostal space cranial to the first intercostal space on both sides.

After CE and TUS, the calves were lightly sedated with Xylazine (0,06 mg/kg) intravenous. A non-guided TA sample was collected with a calf flush catheter (proVETnordic, Ref.: 20096). The catheter was placed in the trachea until resistance. Fifty mL isotonic NaCl were flushed into the lungs and as much as possible was aspirated. Two mL were transferred to a 2 mL EDTA tube and the rest was transferred to a 50 mL plain tube (Sarstedt AG & Co. KG, Germany). It was possible to aspirate 22–38 mL, with an average of 32 mL. Thereafter, four long cotton swabs were placed as far in pharynx as possible and rotated until soaked in saliva. The cotton swabs were placed in cryotubes containing 1 mL phosphate buffer saline (PBS). Finally, blood was drawn from v. jugularis into three serum tubes (8,5mL, SST^TMII Advance).

Preparation of samples

The serum tubes were kept at room temperature for at least 2 hours, centrifuged at 4327 Rpm for 10 min and the supernatant were distributed into cryotubes, which were kept at -80° Celsius until further analysis.

The pharyngeal swabs were stored in PBS solution for 24 hours at room temperature. Then the cotton swabs were removed, and the PBS liquid was stored at -80° Celsius until further analysis. A cotton swab can contain approximately 100–110 µL of saliva and concentrations of acute phase proteins are therefore multiplied by 11, as the cotton swab was stored in 1 mL of PBS.

The TA liquid was kept at 5° C for maximum 2 days, then it was centrifuged at 1410 Rpm for 10 min at 4°C and the supernatant were kept at -80° C until further analysis.

Laboratory analysis

Serum, pharyngeal swabs, and TA samples were analysed for SAA, Hp and LBP. All analysis were performed at Veterinary Diagnostic Laboratory, Frederiksberg, Denmark. The pharyngeal swabs and TA samples were run undiluted, but besides this according to the manufacturer´s manual for serum. None of the test kits are validated for either saliva or TA and as both sample types were already diluted, we were concerned about getting below the detection limit, if we diluted the samples more. The serum samples were run according to the manufacturer´s manual. The SAA were analysed with multispecies SAA ELISA (Tridelta Development Ltd., Maynooth, County Kildare, Ireland). The Hp with a colorimetric assay (phase, haptoglobin assay, Tridelta Development Ltd., Maynooth, County Kildare, Ireland). Calibration was performed using a calibrator set from the same manufacturer using ADVIA 1800 (Siemens Healthineers, Ballerup, Denmark). Analysis for LBP were done with the ELISA kit HK503 (Hycult Biotech, Uden, The Netherlands).

Statistical analysis

All data management and analyses were done in R version 3.2.2 [17]. When modelling the correlation between serum LBP and age, serum LBP concentration was log-transformed to improve the normality of the residuals. The goodness-of-fit of the model was estimated using r². The correlation between serum and pharyngeal swabs, and pharyngeal swabs, TA and age were assessed visually.

Eighty-four healthy commercial Holstein-Friesian dairy calves aged 2–29 days were enrolled in the study. They originated from nine Danish dairy herds. The minimum and maximum number of calves from a herd was one and 27, respectively, with a mean of 9. The mean, median, minimum, and maximum concentrations of SAA, Hp and LBP in pharyngeal swabs, TA and serum are shown in Table 1.

Table 1

Concentrations of serum amyloid A (SAA), haptoglobin (Hp) and lipopolysaccharide binding protein (LBP) in serum, pharyngeal swabs, and tracheal aspirate fluid from 84 clinical healthy dairy calves, divided age in days. Mean (median, min, max), N = number of calves.
	N	SAA mg/L	Hp mg/L	LBP mg/L
Overall serum	84	118.3 (131.7, 32.9, 180.9)	100.6 (77.6, 45.1, 788.8)	2.05 (1.90, 0.73, 5.63)
0–7 days	16	154.1 (153.5, 135, 180.9)	88.8 (78.5, 53.9, 230.3)	2.35 (2.47, 1.33, 3.30)
8–14 days	37	122.2 (134.5, 36.2, 179.8)	125.7 (77.6, 56.8, 788.8)	2.24 (1.93, 0.93, 5.63)
15–21 days	22	97 (84.5, 33.0, 178.0)	76.5 (77.2, 45.1, 99.7)	1.82 (1.63, 0.75, 4.41)
22–30 days	9	90.6 (87, 39.2, 164.4)	77.4 (77.6, 58.6, 87.5)	1.31 (1.10, 0.72, 2.25)
		SAA mg/L	Hp mg/L	LBP ng/mL
Overall Pharyngeal swab	84	2.18 (1.97, 0.00, 8.01)	20.1 (18.1, 0.0, 60.8)	6.06 (0.00, 0.00, 47.94)
0–7 days	16	2.6 (2.3, 0.9, 6.7)	15.7 (12, 0, 58.6)	8.8 (0.0, 0.0, 47.9)
8–14 days	37	2.2 (1.9, 0, 8)	20 (16.6, 0, 55.9)	5.6 (0, 0, 33.4)
15–21 days	22	1.7 (1.2, 0, 4.4)	22.7 (22.6, 0, 43.1)	6 (0, 0, 42.6)
22–30 days	9	2.7 (2.9, 1.3, 4.7)	22.2 (19.9, 6.4, 60.8)	3 (0, 0, 21.4)
		SAA mg/L	Hp mg/L	LBP ng/mL
Overall Tracheal aspirate	84	0.055 (0.042, 0.004, 0.240)	0.71 (0.39, 0.00, 5.89)	3.03 (2.72, 0.00, 13.22)
0–7 days	16	0.07 (0.053, 0.007, 0.143)	0.47 (0.29, 0.00, 2.15)	3.63 (3.12, 0.00, 13.22)
8–14 days	37	0.055 (0.04, 0.006, 0.24)	0.59 (0.37, 0.00, 2.55)	2.82 (2.61, 0.00, 11.52)
15–21 days	22	0.05 (0.043, 0.004, 0.164)	0.93 (0.71, 0.00, 3.05)	2.73 (1.47, 0.00, 8.99)
22–30 days	9	0.041 (0.042, 0.012, 0.07)	1.08 (0.43, 0.00, 5.89)	3.52 (3.49, 0.00, 8.78)

There was a statistically significant correlation (p < 0.05, r ²=0.30) between serum SAA and age, with decreasing concentration with increasing age (Fig. 1 - top). This age-related correlation was not found for SAA in pharyngeal swabs or TA. The concentration level of SAA was highest in serum, then pharyngeal swabs and least in TA. There were three calves with no detectable SAA concentration in pharyngeal swab.

The serum Hp concentration was generally below the accepted cut-off of 200 mg/L, despite 5 calves having higher serum Hp concentrations than the rest of the calves. The concentration in pharyngeal swabs varied from 0–60 mg/L, and the TA Hp concentration between 0–3 mg/L. Hp concentration was not statistically significantly associated with age in any of the fluids (Fig. 2). The concentration level of Hp was highest in serum, then pharyngeal swabs and least in TA. There were five and 31 calves with no detectable Hp concentration in pharyngeal swabs and TA, respectively.

There was a statistically significant correlation (p < 0.05, r² = 0.20) between serum LBP and age, with decreasing concentration with increasing age (Fig. 3 - top). This age-related correlation was not found for LBP in pharyngeal swabs or TA. There were 59 and 9 calves with no detectable LBP concentration in pharyngeal swab and TA, respectively.

There was no statistically significant correlation between the concentration of SAA, Hp and LBP, in serum, pharyngeal swab or TA (Fig. 4). For some of the correlations, e.g., SAA in TA and serum (lower left corner) there was a tendency towards a correlation, but it is not consistent.

In this study we investigated the concentration of SAA, Hp and LBP in pharyngeal swabs, TA and serum from 84 healthy dairy calves. We found that the APPs could be detected in all the investigated body fluids, but in different concentrations. We found no direct correlations between serum and pharyngeal swabs or TA, neither between TA nor pharyngeal swabs.

Pharyngeal swabs

We explored for the first time the concentrations of SAA, Hp and LBP in pharyngeal swabs from healthy calves. Hp concentrations in saliva from calves have been preliminary reported [15] and together we find the results promising for the use of APP in saliva as biomarker for respiratory disease in calves.

For all APPs, there were samples with APP concentration below the detection limit. In particularly LBP, where 59 of 84 samples had a concentration below the detection limit. For SAA and Hp, it was only 3 and 5, respectively. Saliva is a diluted liquid, containing more than 99% water [18], and we then dilute it even more in PBS, so it is not surprising that the APP concentration is low. This was also the reason for not diluting the samples further during analysis. When collecting the pharyngeal swabs, we noticed that the amount and viscosity of pharyngeal saliva varied a lot between calves, which has probably caused the amount of saliva in the samples to vary. Even though we corrected the APP concentration for the dilution effect of the PBS, the explanation for some of the variance in APP concentration, could be a technical difference introduced by the sampling method. For LBP the amount, of samples with very low concentrations were too high to be explained by technical issues alone, also since there were detectable concentrations of SAA and Hp in nearly all these samples. There might be very low concentrations of LBP in pharyngeal swabs in healthy calves, but if this increases significantly with inflammation, LBP in saliva might still be a potential indicator of airway inflammation. However, LBP in saliva needs to be explored further to conclude on the potential as a biomarker.

The pharyngeal swabs were collected under light sedation. This was done because we also collected TA samples. However, the collection of pharyngeal swabs can also be done without sedation, as a non-invasive procedure. Caplen and Held collected saliva from cows using a cotton swab developed for saliva collection in children without sedation of the cows [14], which would presumably also work in calves.

The enrolled calves were young and still milk-fed. This could be a potential bias, as there could be traces of APP from milk in their pharynx. This was probably not a problem, since all samples were sampled at least 2 hours after milk feeding.

TA

The SAA concentration in TA ranged from 0.004–0.240 mg/L, with an average of 0.055 mg/L, Hp ranged from 0.00-5.89 mg/L, with an average of 0.71 mg/L and LBP ranged from 0-13.22 ng/mL, with an average of 3.03 ng/mL. Prohl et al. found a median Hp concentration in TA of healthy calves of 0.1073 mg/L, ranging from 0.003–0.347 mg/L [10] and Coskun et al. found a mean of 0.03 mg/L and standard deviation of 0.007 mg/L [11]. The median LBP concentration in healthy calves were 9.5 ng/mL, ranging from 1.7–23.6 ng/mL [10]. We believe our TA results overall agrees with the findings by other authors. Especially, because there is an unknown dilution factor of the TA samples. Even though all calves were flushed with the same amount of NaCl it was different how much fluid it was possible to aspirate again, and we could not take account of this because we don’t know the exact concentration of the TA.

Based on this study, we don’t know how the APP concentrations changes in pharyngeal swabs during respiratory disease. SAA, Hp and LBP in TA increases in calves with respiratory disease [10, 11], but a validation of the used assays in this material have not been performed. Rahman et al. found that saliva Hp increased mildly 24 hours after dehorning [15], indicating that there is a potential for APP in saliva to differentiate between healthy and diseased calves. As mentioned, for both pharyngeal swabs and TA there is a considerably technical variation in the sampling methods, but if the true difference in APPs between healthy and diseased are large enough, this will overcome the technical variation and still be promising for detecting respiratory disease in calves.

For our analysis of the APP concentration in pharyngeal swabs and TA, we used ELISA kits not validated in these materials. Full interpretation of the data and calculation of reference intervals for APPs in TA and pharyngeal swabs requires a validation of the assays using the biological materials TA and saliva.

Serum

The serum SAA concentrations agrees with other studies of serum SAA from healthy calves, where the concentrations range from 80–200 mg/L [2, 4]. We also found an association between age and serum SAA level, with a higher level in young calves less than 15 days old [3, 4]. The majority of our serum samples were below the generally accepted cut-off of 200 mg/L Haptoglobin and the few calves with higher values are discussed in detail in a later section.

The concentration of serum LBP ranged from 0.72–5.63 mg/L, with an average of 2.05 mg/L. Serum LBP in a group of slightly older healthy calves before inoculation was found to be 1.5-3 mg/L [8] and 1-69.8 mg/L [10]. This seems to be close to our samples, even though Prohl et al. found a wider range than our samples [10]. However, it can be difficult to compare, as the same test kits have not been used and there can be differences between laboratories. Based on the results, there is a considerable variation in healthy calves. Orro et al. also investigated serum LBP in calves less than 21 days of age using the same ELISA kit, as used in our study [3]. They found a higher mean serum LBP concentration ranging from 16–32 mg/L and with a decreasing serum LBP with increasing age up to approximately 20 days of age [3].

Correlations

We did not find a linear correlation or relationship between the APP concentration in serum and either pharyngeal swab or TA, neither between TA nor swab. A linear correlation between serum and pharyngeal swabs or TA, would indicate a direct reflection of the APP concentration in serum in the bronchial fluid and the mucous membranes of the pharynx. Rahman et al, found a moderate correlation between serum and saliva SAA in calves 24 hours after dehorning [15], but this to our best knowledge the only previous investigation. Coskun et al. and Prohl et al. investigated concentrations of SAA, Hp and LBP in paired serum and TA samples, but none of the authors presents the results for individual calves [10, 11], therefore it is not possible to figure out if they found a correlation in the APPs between the different fluids.

The correlation between serum and TA or pharyngeal swab could also be time-shifted, e.g., first there is a rise in serum, which is followed in pharyngeal swabs or TA after some time. Such a correlation would require repeated measurements on the same calves, and as we have cross-sectional data, this cannot be investigated in this study.

Another explanation could be that the APP concentration in pharyngeal swab or TA, only rises in case of respiratory disease. Others have shown that the concentration of SAA, Hp and LBP in TA increases with respiratory disease [10, 11] and salivary Hp increases after dehorning [15]. We might have seen a detectable association if we had investigated diseased calves.

Clinical status

Although only clinically healthy calves were enrolled in the study, concentrations of serum Hp was above 200 mg/L in five calves. Hp has a longer duration in serum than other APPs [9] and the Hp concentration can possibly be above the cut-off in a clinically healthy calf, because of previous disease, not evident anymore. All but one of the five calves with a higher serum Hp concentration, were observed with mild diarrhoea and no signs of dehydration, which could explain the high Hp concentration [19, 20]. Other authors have had the same experiences and highlight the differences in getting a homogenous sample of calves in commercial herds for evaluation of APP concentrations [3].

This study showed that concentrations of the APPs SAA, Hp and LBP can be detected in pharyngeal swabs and TA from healthy dairy calves. We found no correlations between APP concentration in serum and pharyngeal TA nor swab. A considerably technical variation in the chosen sampling method for both pharyngeal swab and TA exist, and further validation of the methods are needed. However, there is a great potential for using pharyngeal swabs as a less invasive method for detection of respiratory disease in calves.

APP acute phase protein

Hp haptoglobin

LBP lipopolysaccharide binding protein

SAA serum amyloid A

TA tracheal aspirate

Author contributions

NC and MBP designed the study. MBP performed the data collection, the statistical analyses, and drafted the manuscript. Both authors participated in the discussion of data and finalizing the manuscript. All authors have read and approved the final version of the manuscript.

Acknowledgements

We wish to thank all staff at the “Robust calves”-project and in particular, veterinarians Anne Marie Michelsen, Nina Dam Otten, Jesper Kjærgaard Davidsen and Thomas Damm Poulsen for technical assistance.

Competing interests

The authors declare that they have no competing interests.

Availability of data and materials

The datasets used and analyzed during the current study are available from the corresponding author on reasonable request.

Consent for publication

Not applicable

Ethics approval

The study protocol was approved by the Danish Animal Experiments Inspectorate (Approval number, 2021-15-0201-01099) and the department’s local ethics committee.

Prior publication

Preliminary results were presented as an Abstract at the 31^st World Buiatrics Congress, Madrid, 4^th-8^th September 2022.

Funding

This study was funded by a grant from the Danish Milk Levy and Danish Cattle Levy Funds.

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Journal Publication

published 25 Nov, 2023

Read the published version in Acta Veterinaria Scandinavica →

Editorial decision: Major revision
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You are reading this latest preprint version

Acute phase protein concentration in pharyngeal swabs from clinically healthy commercial dairy calves

Status:

Journal Publication

Version 1

Abstract

Background

Results

Conclusions

Figures

Background

Methods

Animals and sample collection

Preparation of samples

Laboratory analysis

Statistical analysis

Results

Discussion

Pharyngeal swabs

TA

Serum

Correlations

Clinical status

Conclusions

Abbreviations

Declarations

References

Status:

Journal Publication

Version 1