Background: The pathogenesis of postpartum dysgalactia syndrome (PDS) in sows is not fully elucidated and affected sows often present vague clinical signs. Accurate and timely diagnosis is difficult, and PDS is often recognized with a delay once piglets begin to starve. Increased rectal temperature of the sow is an important diagnostic parameter, but it may also be influenced by a number of other parameters and is thus difficult to interpret. Inflammatory markers may be important adjuncts to the clinical assessment of sows with PDS, but such markers have only been studied to a limited extent. The objective was to characterize the inflammatory response in healthy sows and in sows suffering from PDS, and to identify biomarkers that may assist in early identification of PDS-affected sows. Results: Thirty-eight PDS-affected (PDS+) and 38 healthy (PDS-) sows underwent clinical examination and blood sampling every 24 h, from 60 h before the first piglet was born to 36 h after parturition. In both groups, inflammatory markers changed in relation to parturition. Most inflammatory markers changed 12-36 h after parturition [white blood cell counts (WBC), neutrophil counts, lymphocyte counts, tumor necrosis factor alpha (TNF- α), interleukin 6 (IL-6), serum amyloid A (SAA), C-reactive protein (CRP), haptoglobin (Hp), iron (Fe) and albumin (ALB)]. Changes in neutrophil counts, lymphocyte counts, CRP, Fe and ALB were observed -12 to 0 h before parturition. WBC, neutrophil and lymphocyte counts, serum concentrations of TNF-α, IL-6, Hp and Fe differed between PDS+ and PDS- sows. These differences were mainly apparent 12 to 36 h after parturition, but already at 12 h before parturition, PDS+ sows had lower lymphocyte counts than PDS- sows. Conclusions: Parturition itself caused significant inflammatory changes, but PDS+ sows showed a more severe response than PDS- sows. WBC, neutrophil and lymphocyte counts, and concentrations of TNF-α, IL-6, Hp and Fe can be potential biomarkers for PDS. Lymphocyte counts may be used to detect PDS at pre-partum. To assess their diagnostic potential, these markers must be investigated further and most likely combined with assessment of clinical parameters and other biomarkers for improved identification of sows at risk of developing PDS. Keywords: PDS, Inflammatory markers, Postpartum, Dysgalactia, Sow * Correspondence: firstname.lastname@example.org Department of Veterinary Clinical Sciences, Faculty of Health and Medical Sciences, Copenhagen University, Højbakkegård Alle 5, 2630 Taastrup, Denmark Full list of author information is available at the end of the article © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Kaiser et al. BMC Veterinary Research (2018) 14:83 Page 2 of 15 Background may be aided by assessment of biomarkers in blood. The Postpartum dysgalactia syndrome (PDS) is a common objective of our study was to characterize the peripar- disease in sows with recently reported prevalences vary- turient inflammatory response in healthy sows and in ing from 6.0 to 48.2% [1–4]. The pathogenesis of PDS is sows suffering from PDS with the goal of identifying bio- complex and not fully elucidated. Mastitis, metritis and markers that may assist in identification of affected ani- agalactia syndrome (MMA) and coliform mastitis may mals early in the course of disease. all cause dysgalactia, but these disease complexes are considered to be the more readily observable compo- Methods nents of the PDS complex [5, 6]. Therefore, a subgroup Herd and animals of sub-clinically affected sows, which are difficult to de- A case-cohort study was performed from March 2014 to tect in the early stages of the disease, seems to occur. November 2014, including 38 PDS affected (PDS+) and These cases of PDS may only be discovered once piglets 38 healthy (PDS-) multiparous sows. Samples were col- start to lose weight and display milk-searching behav- lected from a Danish sow herd within the Danish Spe- iour. Mortality increases when colostrum intake during cific Pathogen-Free (SPF) system. The farm was chosen the first 24 h of life is less than 200 g per piglet , and based on a treatment rate of at least 20% of all farrowing increased piglet mortality among MMA-affected litters sows. Usually, 20–30% of the sows would receive med- has been reported . Furthermore, increased preva- ical treatment in the periparturient period. The sows lence of stillborn piglets has been found in litters from were housed in confined crates from 1 week before far- sows with fever or elevated rectal temperature before  rowing until 3 weeks after farrowing. Between each and after farrowing . The impact of PDS on the wel- batch of sows, the farrowing units were cleaned and dis- fare of sows and piglets is thus substantial. infected. The farrowing pens had partly slatted floors Currently, elevated rectal temperature seems to be one with 2/3 solid concrete and 1/3 iron bars measuring 1.6 of the more reliable indicators of PDS [4, 11, 12], but as × 2.6 m . The sows were fed 4 times per day with liquid rectal temperature is influenced by metabolic status, par- feed and assigned straw according to the Danish law of ity, circadian rhythm and days postpartum (p.p.), inter- animal welfare. All sows were of the Danish cross-breed pretation of the measured rectal temperature is not (Landrace/Yorkshire). straight forward . A threshold temperature of 39.5°C is most often applied as a cut-off value when used for Experimental design detection of PDS [11, 13–20]. Other clinical signs, e.g. According to the following and assumptions of the syn- anorexia, inflamed udder, decreased demeanor, and drome , a broad clinical definition of PDS was milk-searching behavior among piglets, have been used adopted. PDS+ sows would have at least two of the fol- in conjunction with rectal temperatures in an attempt to lowing clinically visible characteristics: 1. anorexia, de- identify sows with PDS [13, 18, 19, 21, 22]. fined as “trough not empty 30 minutes after feeding”,2. Systemic inflammation in sows with PDS has only inflammation of the udder, characterized by redness, been investigated to a limited degree. Increased blood swelling and increased skin temperature, 3. rectal levels of haptoglobin (Hp) [8, 23], tumor necrosis factor temperature ≥ 39.5°C. alpha (TNF-α) and interleukin-6 (IL-6)  have been From each of 9 batches, approximately 12 sows were found in sows diagnosed with MMA, and a decrease in randomly selected and monitored when they entered the white blood cell counts (WBC) and neutropenia was farrowing unit. All sows (n = 109) were sampled every 24 observed in sows developing agalactia after experimental h from 60 h before expected parturition and to a max- intramammary infusion of Escherichia (E.) coli . Fur- imum of 36 h p.p. or until PDS occurred. For ethical rea- thermore, increases in concentrations of cytokines and sons, sows were treated as described below and precluded acute phase proteins (APPs) have been demonstrated in from further sampling as soon as PDS was detected. In sows with experimental mastitis elicited by the following order, monitoring prior to parturition in- intramammary infusion of E. coli  or lipopolysac- cluded: 1. samplings of saliva and ear venous blood before charide (LPS) . morning feeding (these data were used for other research Detection of PDS remains elusive, and it is thus pos- purposes and are not shown), 2. veterinary clinical assess- sible that the prevalence of sub-clinically affected sows is ment after feeding with recording of the general de- significant. Consequently, the diagnosis may be delayed meanor, appetite, rectal temperature, respiration until the piglets start expressing hunger, at which time frequency, capillary re-filling time, skin color, eye mucosa the disease has severe consequences on animal welfare color, vaginal mucosa color, vaginal discharges, fecal and production. Early detection of the disease (most consistency, and signs of inflammation (subjective assess- preferably before or during parturition, or at least before ment of skin temperature by palpation, capillary refill piglets show signs of starvation) is thus desirable and time, and hyperemia) of the mammary glands, 3. blood Kaiser et al. BMC Veterinary Research (2018) 14:83 Page 3 of 15 sampling from v. jugularis as described below, and 4. Systems, Minneapolis, USA) as described previously blood sampling from v. epigastrica caudalis superficialis . The absorbance was read at 450 nm using a micro- [v. mammaria cranialis] (these data were used for other titer plate reader (Multiskan EX, Thermo LabSystems, research purposes and are not shown). After parturition, a Massachusetts, USA). Concentrations below the detec- morning and an afternoon milking was included in the tion limit of the assay were set at the detection limit for procedure for other research purposes. the assay in question in the calculations (TNF-α = 23.40 Sows categorized as PDS+ were administered medical pg/mL, IL-1 = 39.10 pg/mL, IL-6 = 18.80 pg/mL). Con- treatment by the farmer immediately after the clinical centrations in serum of the acute phase protein serum examination and sample collection. The medical treat- amyloid A (SAA) was determined by a previously de- ment consisted of systemic antibiotics, either 10.000 IU/ scribed commercial multispecies SAA ELISA kit (Tri- kg bw of benzyl procaine penicillin (Noropen® vet., Scan- delta Development Ltd., Ireland) according to the Vet, Denmark) or 16 mg/kg bw of trimethoprim- manufacturer’s instructions . The absorbance was sulfadiazin (Norodine® vet., ScanVet, Denmark), and 0.4 read at 450 nm using a microtiter plate reader (BIO- mg/kg bw of meloxicam (Loxicom®, ScanVet, Denmark). TEK, Vermont, US). Hp and C-reactive protein (CRP) Sows that farrowed prematurely and sows treated for levels were measured in serum using an automated bio- reasons other than PDS were excluded from the study. chemistry analyzer (Olympus AU600 Automatic Chem- Eventually, 38 sows were defined as PDS+ sows, and istry Analyzer, Olympus Europe GmbH, Germany) with these were retrospectively matched with 38 PDS- sows. commercial quantitative turbidimetric tests produced by The match was done in the following descending order SPINREACT, S.A.U (Spain) and Beckman Coulter® (Cali- of importance: 1. batch, 2. parity, and 3. date of partur- fornia, USA), respectively. Assays for Hp and CRP were ition. Data from the remaining 33 sows were not in- performed as reported before . The APP assays all cluded in the study. had intra-run and inter-run coefficients of variation < 10%, and the limits of detection were 10 mg/L for Hp, Sampling 0.6 mg/L for CRP and 3.06 mg/L for SAA. Where con- Blood samples were collected from v. jugularis in tubes centrations were higher than the upper limit of the with no additive for preparation of serum (BD, New assay, concentrations were set at the concentrations of Jersey, US) and with EDTA for haematological analyses the highest calibrator (SAA, 500 mg/L, n = 7 samples). (BD, New Jersey, US). To reduce stress and build up confidence, the sows were fed small sugar cubes after Statistical analyses handling. All blood samples were kept at room Retrospectively, the exact sampling times (date:hour:- temperature for a maximum of 30 minutes before being min.) were determined relative to the exact time of far- processed. The additive-free tubes were centrifuged for rowing of the first piglet (date:hour:min.) which was 10 min. at 3,000 × g and the serum was separated and recorded by videos. Sampling times were grouped into stored at – 80°C until analysis, which was performed time intervals where 0 h was the parturition time of the within 15 months from first sampling date. The EDTA first piglet: A. -60 to -36 h; B. -36 to -24 h; C. -24 to -12 tubes were carefully mixed and blood smears prepared h; D. -12 to 0 h; E. 0 to 12 h; F. 12 to 24 h, and G. 24 to within 2 h. Blood smears underwent cytological examin- 36 h. The number of observations (n) within each inter- ation. Blood samples were stored at 5°C for a maximum val varied because of variation in the individual sampling of 48 hours before they were shipped to the Veterinary times relative to parturition (0 h) [For illustrative explan- Diagnostic Laboratory at University of Copenhagen for ation, see Additional file 1]. determination of WBC count, hematocrit (Ht), For statistic evaluation, two autoregressive linear re- hemoglobin (Hb), iron (Fe), Albumin (ALB) and total gression models (A and B) were performed in the PROC protein (TP), which were performed by the Hematology MIXED procedure of Statistic Analytical Software, En- System Complete Blood Count method using an auto- terprise Guide 7.1 (SAS® Institute, Cary, North Carolina, mated biochemistry analyzer (ADIVA 2120/2120i, Sie- USA). Least-squares means (LSMEANS) and standard mens Healthcare A/S, Denmark). Blood smears were deviation (SD) were included in the statistic model A. stained with modified Wright stain (Siemens AG, Model A was OUTCOME PARAMETER = μ + TIME ij i Germany), and differential count of WBC was per- + GROUP + TIME*GROUP + ε where OUTCOME j ij formed as described . Concentrations of the cyto- PARAMETER was the measured value of the inflamma- ij kines interleukin-1 (IL-1), IL-6 and TNF-α in serum tory parameter, μ was the observations value at time 0, were determined by commercially available pig-specific TIME was explanatory variable “time intervals A-G”, ELISAs (Porcine IL-1 beta/IL-1F2 Quantikine ELISA GROUP the explanatory variable “PDS+/PDS-”, TIME*- Kit, Porcine IL-6 Quantikine ELISA Kit, and Porcine GROUP the interaction between the two groups and ij TNF-alpha Quantikine ELISA Kit, all from R&D change over time and ε was the random residual error Kaiser et al. BMC Veterinary Research (2018) 14:83 Page 4 of 15 term. When significant interaction was identified using after the first piglet was born. Obstetric aid was per- model A, differences between the relevant groups and formed in 18 PDS+ sows and 11 PDS- sows. Reduced time intervals were accepted. In case of non-significant appetite was observed in 10 PDS+ sows and 5 PDS- interaction, model A were replaced with model B which sows at 0-12 h, while 12 PDS+ sows and 12 PDS- was OUTCOME PARAMETER = μ + TIME + showed reduced appetite at 12-24 h (with 1 sow having ij i GROUP + ε. If a non-significant change in TIME occur complete anorexia). Mean parity, relevant production re- j i in model B, the OUTCOME PARAMETER was consid- sults, and clinical findings are shown in Table 1. ij ered non-significant. For significant TIME value, differ- ences in the relevant groups were still accepted and Inflammatory markers recorded from the model A output. In case of a signifi- Change in level of inflammatory markers in relation to cant effect of GROUP , an overall effect of group was ac- parturition cepted. Significance was considered if p<0.05. Parity and In both PDS+ and PDS- sows, inflammatory markers body condition score were included as explanatory vari- changed in relation to parturition. Developments over ables. By preliminary analyses, obstetric aid and farrow- time for both groups are illustrated by raw data for ing length were found not to be associated with any of WBC, neutrophils, lymphocytes, TNF-α,IL-6,SAA, the outcome variables. Natural logarithm was performed CRP, Hp, Fe and ALB (Figs. 1, 2, 3, 4, 5, 6, 7, 8, 9 for lymphocytes, IL-6, TNF-α, CRP, SAA and TP be- and 10). Some markers (IL-1, TP, Hb, and Ht) did cause assumptions concerning residual plots and test for not change significantly over time in any group [See normality were unlikely. Additional file 2, Additional file 3, Additional file 4 and Additional file 5]. A leukocyte left shift was de- Results tected in one time interval in 5 PDS+ sows and 1 Clinical findings PDS- sow. Mild neutrophilic toxic changes were ob- The 38 sows categorized as PDS+ constituted 34.9% of served in one time interval in 5 PDS+ sows and 7 the farrowings in the randomly selected 109 sows. PDS+ PDS- sows. Reactive lymphocytes in low numbers sows were on average treated 18.6 h (range 6-26.9 h) were observed in 2 PDS+ sows and in 7 PDS- sows. Table 1 Parity and productions results for 38 sows with postpartum dysgalactia syndrome (PDS+) and 38 healthy sows (PDS-) together with key clinical parameters in time interval A, E and F. The number of observations (n) in each interval varies because of variation in individual sampling times relative to parturition (0 h) [For further explanation see Additional file 1] Sow related observations PDS+ PDS- n Mean Min Max n Mean Min Max Parity and production Parity, no. 38 4.26 2 7 38 4.12 2 7 Liveborn, no. 37 16.9 10 23 38 17.3 6 24 Stillborn, no. 38 0.18 0 3 38 0.32 0 3 Clinical findings time point A (-60 to -36 h p.p.) Rectal temp, °C 18 38.1 37.4 38.7 22 38.2 37.6 38.8 Increased mammary skin temperature, no. of glands/sow 18 0.1 0 1 22 0.9 0 14 Heart rate, beats/min. 18 122 96 152 22 112 82 160 Respiratory rate, breaths/min. 18 46 22 86 22 45 22 88 Clinical findings time point E (0 to12 h p.p.) Rectal temp, °C 15 38.9 38.0 40.1 10 38.6 37.3 39.3 Increased mammary skin temperature, no. of glands/sow 14 4.4 0 16 10 1.5 0 15 Heart rate, beats/min. 14 112 68 144 10 117 92 144 Respiratory rate, breaths/min. 15 31 13 112 10 37 12 84 Clinical findings time point F (12 to 24 h p.p.) Rectal temp., °C 23 39.5 38.1 40.5 25 39.0 38.1 39.3 Increased mammary skin temperature, no. of glands/sow 23 10.4 0 17 24 6.0 0 16 Heart rate, beats/min. 23 117 84 158 25 101 74 144 Respiratory rate, breaths/min. 23 25 12 38 24 27 12 66 Kaiser et al. BMC Veterinary Research (2018) 14:83 Page 5 of 15 Fig. 1 White blood cell (WBC) counts (10 cells/L) in sows with postpartum dysgalactia syndrome (PDS+, red) and healthy sows (PDS-, blue) sampled from 60 h before until 36 h after parturition (time interval A-G). Each dot represents the exact sample time of each observation relative to the exact birth of the first piglet (0 h). The lines show the mean value (Normal range; 11.3-22.8 × 10 cells/L) Fig. 2 Neutrophils counts (10 cells/L) in sows with postpartum dysgalactia syndrome (PDS+, red) and healthy sows (PDS-, blue) sampled from 60 h before until 36 h after parturition (time interval A-G). Each dot represents the exact sample time of each observation relative to the exact birth of the first piglet (0 h). The lines show the mean value (Normal range; 3.1-9.6 × 10 cells/L) Kaiser et al. BMC Veterinary Research (2018) 14:83 Page 6 of 15 Fig. 3 Lymphocyte counts (10 cells/L) in sows with postpartum dysgalactia syndrome (PDS+, red) and healthy sows (PDS-, blue) sampled from 60 h before until 36 h after parturition (time interval A-G). Each dot represents the exact sample time of each observation relative to the exact birth of the first piglet (0 h). The lines show the mean value (Normal range; 4.6 – 10.0 × 10 cells/L) Statistically significant changes of the inflammatory neutrophil counts (Fig. 2), lymphocyte counts (Fig. 3), markers in relation to parturition for both PDS- and TNF-α (Fig. 4), IL-6 (Fig. 5), SAA (Fig. 6), CRP (Fig. 7), PDS+ sows are summarized in Fig. 11. Most inflamma- Hp (Fig. 8), Fe (Fig. 9) and ALB (Fig. 10)], while changes tory markers were changed relative to baseline (time in neutrophil counts (Fig. 2), lymphocyte counts (Fig. 3), interval A) between 12 and 36 h p.p. [WBC (Fig. 1), CRP (Fig. 7), Fe (PDS+ sows only, Fig. 9), ALB (PDS- -9 Fig. 4 Tumor necrosis factor alpha (TNF-α) concentration (10 g/L) in sows with postpartum dysgalactia syndrome (PDS+, red) and healthy sows (PDS-, blue) sampled from 60 h before until 36 h after parturition (time interval A-G). Each dot represents the exact sample time of each observation relative to the exact birth of the first piglet (0 h). The lines show the mean value Kaiser et al. BMC Veterinary Research (2018) 14:83 Page 7 of 15 -9 Fig. 5 Interleukin 6 (IL-6) concentration (10 g/L) in sows with postpartum dysgalactia syndrome (PDS+, red) and healthy sows (PDS-, blue) sampled from 60 h before until 36 h after parturition (time interval A-G). Each dot represents the exact sample time of each observation relative to the exact birth of the first piglet (0 h). The lines show the mean value -3 Fig. 6 Serum amyloid A (SAA) (10 g/L) in sows with postpartum dysgalactia syndrome (PDS+, red) and healthy sows (PDS-, blue) sampled from 60 h before until 36 h after parturition (time interval A-G). Each dot represents the exact sample time of each observation relative to the exact birth of the first piglet (0 h). The lines show the mean value Kaiser et al. BMC Veterinary Research (2018) 14:83 Page 8 of 15 -2 Fig. 7 C-reactive protein (CRP) concentration (10 g/L) in sows with postpartum dysgalactia syndrome (PDS+, red) and healthy sows (PDS-, blue) sampled from 60 h before until 36 h after parturition (time interval A-G). Each dot represents the exact sample time of each observation relative to the exact birth of the first piglet (0 h). The lines show the mean value -3 Fig. 8 Haptoglobin (Hp) concentration (10 g/L) in sows with postpartum dysgalactia syndrome (PDS+, red) and healthy sows (PDS-, blue) sampled from 60 h before until 36 h after parturition (time interval A-G). Each dot represents the exact sample time of each observation relative to the exact birth of the first piglet (0 h). The lines show the mean value Kaiser et al. BMC Veterinary Research (2018) 14:83 Page 9 of 15 -6 Fig. 9 Iron (Fe) concentration (10 mol/L) in sows with postpartum dysgalactia syndrome (PDS+, red) and healthy sows (PDS-, blue) sampled from 60 h before until 36 h after parturition (time interval A-G). Each dot represents the exact sample time of each observation relative to the -6 exact birth of the first piglet (0 h). The lines show the mean value (Normal range is 9.0 – 30.4 10 mol/L) Fig. 10 Albumin (ALB) concentration (10 g/L) in sows with postpartum dysgalactia syndrome (PDS+, red) and healthy sows (PDS-, blue) sampled from 60 h before until 36 h after parturition (time interval A-G). Each dot represents the exact sample time of each observation relative to the exact birth of the first piglet (0 h). The lines show the mean value (Normal range is 32 – 48 10 g/L) Kaiser et al. BMC Veterinary Research (2018) 14:83 Page 10 of 15 Fig. 11 Changes in levels of inflammatory markers over time in 38 sows with postpartum dysgalactia syndrome (PDS+) and 38 healthy (PDS-) sows, with asterisk symbols indicating significant differences between time interval A and the subsequent time intervals (D to G). Upward pointing arrows indicate increasing concentrations. Downward pointing arrows indicate decreasing concentrations sows only and Fig. 10) were observed also before partur- antepartum levels 48-72 h and 24-96 h p.p., respectively ition (Fig. 11). . Several factors may cause periparturient changes in inflammatory markers. Hormonal changes with fast ante- Differences between PDS+ and PDS- sows partum cortisol increase have been suggested to be the WBC, neutrophil counts, lymphocyte counts, TNF-α,IL- cause of periparturient leukocytosis and neutrophilia . 6, Hp and Fe differed between PDS+ and PDS- sows Release of cytokines and APPs after parturition could be (Table 2). Lymphocyte counts differed between PDS- and caused by tissue trauma to the birth canal, elicited by the PDS+ sows pre-partum, with lymphocyte counts being foetus as well as by obstetric interventions. Understanding lower in PDS+ than in PDS- sows from -12 h to 36 h. periparturient inflammation in otherwise healthy sows is While WBC and neutrophil counts tended to be higher in important for differentiating physiology from pathology PDS+ than in PDS- sows immediately around parturition when using inflammatory markers for assessing peripar- (-12 to 12 h p.p.), WBC and neutrophil counts dropped in turient disease, as inflammatory markers are non-specific PDS+ sows and were significantly lower than in PDS- in nature and will be released in infectious as well as non- sows 12-24 h p.p. After parturition, concentrations of infectious inflammation. Limiting periparturient inflam- TNF-α, IL-6 and Hp were significantly higher in PDS+ mation may be beneficial to avoid inflammatory-induced than in PDS- sows at 12-36 h. Concentrations of Fe were physiological and behavioural changes such as malaise, significantly lower in PDS+ than in PDS- sows 24-36 h pain, fatigue and anorexia, which may affect the sow’sabil- p.p. Concentrations of SAA, CRP, ALB, TP, Hb and Ht did ity to provide for the piglets and thus impact welfare of not differ between PDS+ and PDS- sows. both sow and piglets. PDS+ as well as PDS- sows had in- creased serum concentrations of TNF-α and IL-6 12-36 h Discussion p.p., and these two cytokines have been shown to cause a Periparturient inflammation wide range of clinical signs [39, 40]. The link between In the present study, hematological and blood biochemical parturition-induced inflammation and disease is unex- changes indicate the presence of systemic inflammation in plored, but sows with larger litters and sows receiving both healthy and PDS-affected sows. Periparturient in- birth interventions, and thus potentially more trauma to flammation in healthy individuals has previously been de- the birth canal, were more likely to develop coliform mas- scribed in other species [33, 34]aswellasinsows . In titis . Pre-existing inflammation may also be a problem horses, WBC  and SAA concentrations were in- if animals are subsequently affected by an infectious agent. creased within 2 and 36 h, respectively, after foaling. In It has been shown that exposure to exogenous LPS exac- healthy primiparous sows, blood neutrophil counts in- erbates tissue injury and mortality in experimentally in- creased significantly between day 3 before parturition and duced ischemia and reperfusion lesions [41, 42]. day 1 p.p. [37, 38], followed by a decrease back towards In sow mammary tissue, lymphocytes are located in antepartum levels on day 3 p.p. . In healthy sows, SAA the interalveolar tissue, in the epithelium and, during and Hp concentrations were increased relative to lactation within the alveolar lumen [43, 44]. At the end Kaiser et al. BMC Veterinary Research (2018) 14:83 Page 11 of 15 Table 2 Least-squares means (LSMEANS) and standard deviation (SD) for white blood cell counts (WBC), neutrophil counts, lymphocyte counts, tumor necrosis factor alpha (TNF-α), interleukin 6 (IL-6), serum amyloid A (SAA), C-reactive protein (CRP), hapto- globin (Hp), iron (Fe) and albumin (ALB) in 38 sows with postpartum dysgalactia syndrome (PDS+) and 38 healthy sows (PDS-) A. (-60 to -36 h) B. (-36 to -24 h) C. (-24 to -12 h) D. (-12 to 0 h) E. (0 to 12 h) F. (12 to 24 h) G. (24 to 36 h) Parameter Group n LSMEANS ±SD LSMEANS ±SD LSMEANS ±SD LSMEANS ±SD LSMEANS ±SD LSMEANS ±SD LSMEANS ±SD **** White blood PDS+ 110 11.46 0.69 11.29 0.58 12.62 1.04 12.14 0.58 15.45 1.04 8.74 0.66 6.15 1.45 cells **** PDS- 126 10.77 0.58 11.22 0.57 11.68 0.97 11.09 0.55 13.94 1.16 12.20 0.60 9.24 1.18 ** Neutrophils PDS+ 110 6.35 0.59 6.36 0.49 7.93 0.90 8.34 0.49 11.53 0.89 5.22 0.56 3.35 1.25 ** PDS- 126 5.57 0.49 6.39 0.49 6.43 0.83 7.09 0.48 8.9 1.00 7.46 0.51 4.35 1.01 * **** * Lymphocytes PDS+ 110 3.96 1.07 3.81 1.05 3.69 1.10 2.74 1.05 2.43 1.10 2.22 1.06 1.90 1.14 * **** * PDS- 126 3.95 1.05 3.75 1.05 3.83 1.01 3.20 1.05 3.23 1.12 3.30 1.06 2.69 1.12 **** * Tumor PDS+ 121 30.04 1.15 31.81 1.13 32.27 1.21 31.02 1.13 34.35 1.21 130.43 1.14 130.36 1.26 necrosis **** * PDS- 119 31.53 1.15 32.80 1.13 29.87 1.20 31.71 1.13 34.23 1.22 55.21 1.14 58.99 1.25 factor-α *** Interleukin 6 PDS+ 121 19.10 1.13 22.28 1.11 19.94 1.20 24.61 3.03 24.76 1.20 39.20 1.13 39.54 1.25 *** PDS- 119 20.50 1.13 22.23 1.12 21.15 1.20 22.81 1.11 20.70 1.22 22.06 1.12 21.47 1.24 Serum PDS+ 120 15.01 1.27 12.81 1.22 13.33 1.42 14.84 1.23 54.20 1.42 187.41 1.26 234.58 1.53 amyloid A PDS- 122 18.45 1.26 20.63 1.24 19.81 1.41 21.36 1.23 69.06 1.46 145.13 1.24 292.72 1.52 C-reactive PDS+ 122 10.03 1.18 8.71 1.13 13.72 1.26 14.86 1.16 24.60 1.26 56.30 1.17 71.52 1.32 protein PDS- 122 9.23 1.18 11.80 1.16 11.05 1.25 14.65 1.16 29.14 1.27 48.52 1.17 53.02 1.31 * * Haptoglobin PDS+ 122 2.24 0.08 2.26 0.08 2.35 0.10 2.30 0.08 2.31 0.10 2.73 0.08 2.92 0.12 * * PDS- 122 2.11 0.08 2.15 0.08 2.16 0.10 2.15 0.08 2.23 0.10 2.46 0.08 2.56 0.11 Iron PDS+ 122 24.36 1.51 23.40 1.23 22.16 2.23 18.30 1.27 16.75 2.22 20.86 1.38 17.97 2.73 PDS- 125 23.30 1.44 21.22 1.30 23.42 2.20 21.12 1.25 21.28 2.32 24.08 1.30 26.40 2.68 Albumin PDS+ 122 37.31 1.09 37.38 0.90 37.82 1.60 37.74 0.93 38.38 1.59 41.99 1.00 40.15 1.95 PDS- 125 36.67 1.04 38.52 0.94 37.17 1.57 39.73 0.91 37.79 1.66 39.88 0.95 42.83 1.91 Total protein PDS+ 122 72.29 1.03 71.20 1.02 72.76 1.04 70.74 1.02 70.73 1.04 76.14 1.03 76.50 1.05 PDS- 125 69.30 1.03 72.54 1.02 68.87 1.04 73.29 1.02 68.92 1.04 72.93 1.02 77.71 1.05 Hemoglobin PDS+ 110 6.51 0.12 6.32 0.10 6.31 0.17 6.31 0.10 6.02 0.17 6.41 0.11 6.50 0.23 PDS- 126 6.44 0.10 6.34 0.10 6.38 0.16 6.29 0.10 6.46 0.18 6.22 0.10 6.39 0.19 Hematocrit PDS+ 110 0.35 0.01 0.34 0.01 0.34 0.01 0.34 0.01 0.32 0.01 0.34 0.01 0.34 0.01 PDS- 126 0.34 0.01 0.34 0.01 0.35 0.01 0.33 0.01 0.35 0.01 0.33 0.01 0.34 0.01 **** *** ** * Asterisk symbols indicating significant differences between PDS+ and PDS- sows: p<0.0001, p< 0.001, p<0.01, p<0.05 of gestation, the total number of lymphocytes in the association between increased serum cortisol and de- mammary tissue increases but at parturition the amount creased numbers of mononuclear cells are found in decreases temporarily, followed by a second increase healthy sows during parturition [37, 38]. Magnusson and from the day of parturition [43, 44]. Moreover, increased Fossum  therefore concluded that variations in num- opsonic activity in mammary secretion are found p.p. bers of mononuclear cells could be influenced by corti- . The observed decrease in lymphocyte counts ob- sol. This could also be the case in the present study. served between -12 and 36 h in PDS+ sows, and to some extent in PDS- sows, could therefore be explained by re- Inflammatory changes in sows with PDS cruitment of lymphocytes into the mammary tissue for The prevalence of PDS+ sows (34.9 %) in the herd indi- colostrum production. Further, Salmon  demon- cates that high herd health and SPF status did not pro- strated a temporal association between lymphocyte accu- tect the sows from developing PDS. mulation and expression of prolactin-receptors on the Leukocyte counts (WBC, neutrophil and lymphocyte epithelial cells which indicate that prolactin plays a role counts), serum concentrations of the proinflammatory directing lymphocytes to the mammary tissue. Though, cytokines (TNF-α and IL-6) and acute phase reactants lymphocytopenia also occur in response to glucocortic- (Hp and Fe) differed between healthy and PDS affected oid administration in humans [44–46] and an sows. These changes suggest that PDS+ sows suffered Kaiser et al. BMC Veterinary Research (2018) 14:83 Page 12 of 15 from more severe systemic inflammation than did the sensitive marker of inflammation , did not differ be- PDS- sows. The cause of this inflammation was not de- tween the groups. In a previous study, higher blood con- termined. A previous study in gilts demonstrated a de- centrations of SAA were detected in sows undergoing crease in WBC and neutrophils 12-36 h after LPS intra-mammary inoculation with E. coli as compared to infusion at parturition  and a non-significant de- healthy controls . The discrepancy between studies crease of WBC and neutrophils was found among sows may be related to the timing of sampling, as different with clinical signs of agalactia day 1 and 2 p.p. . The APPs have different response patterns, with some in- leukopenia and low neutrophil counts that developed in creasing more slowly than others. In the study by Zhu PDS+ sows 12-36 h p.p. could be related to overwhelm- et al.  sows were sampled until 96 h p.p., and the ing infection or exposure to LPS, as LPS may cause pro- study showed that SAA concentrations peaked at 48 h found leukopenia in several species including pigs . p.p. following intramammary infusion of E. coli, whereas E. coli has been found in larger amount among agalactic concentrations of proinflammatory cytokines peaked at sows , which indicate that LPS could be involved in 24 h. To the authors knowledge, CRP has not been in- the pathogenesis of PDS. Leukocyte left shift has been vestigated in sows at parturition. However, CRP has well demonstrated among sows affected with porcine agalac- described APP properties in the porcine species, as in- tia  after exposure to LPS  and E. coli , so re- creased concentrations have been found in pigs with lease of this substance from the mammary gland or clinical signs of disease , pigs with lesions at slaugh- intestine may have resulted in the cytological changes ter  and after experimental injection of LPS or observed in the PDS+ sows. turpentine . Similar to SAA, CRP could not distin- There was a tendency (p<0.1) for transient elevated neu- guish PDS+ from PDS- sows. trophil counts in PDS+ sows from -12 to 12 h compared Iron is a so-called negative acute phase reactant, as its to PDS- sows (Fig. 2). This could reflect an endocrine concentration decreases during inflammation. In accord- stress response with increased blood cortisol, as cortisol ance with this, PDS+ sows had lower serum Fe concen- causes leukocytosis and neutrophilia by preventing migra- trations than PDS- sows. A decrease in Fe and an tion of leukocytes to the extracellular space . increase in Hp has been shown previously in pigs follow- Concentrations of the pro-inflammatory cytokines ing experimental inoculation with Actinobacillus . TNF-α and IL-6 were higher in PDS+ than in PDS- Odink et al.  found lower serum Fe concentrations sows. A previous study demonstrated higher serum in slaughter pigs with abscesses and other inflammatory TNF-α and IL-6 concentrations in sows affected with processes than in pigs with no postmortem findings. In MMA compared to healthy sows for up to 72 h p.p. ruminants and horses, Fe have been shown to be a fast . Experimental induction of udder inflammation by reacting marker, with concentrations decreasing within intra-mammary infusion of LPS or E. coli inocula- few hours following an inflammatory stimulus, and tion  has been shown to cause increased blood con- returning to normal values within 48 h [59, 60]. In centrations of TNF-α and IL-6. Based on these findings, horses, assessment of serum Fe was more useful for de- previous studies have suggested that TNF-α and IL-6 tection of acute inflammation than was the assessment could be potential biomarkers for detection of sows with of fibrinogen . The results of the present study indi- coliform mastitis or MMA [24, 26], thus corroborating cate that Fe may be used for distinguishing PDS+ and the results of the present study. PDS- sows 12-36 h p.p. Proinflammatory cytokines are responsible for the in- duction of hepatic synthesis of APPs. Concentrations of Conclusions the three measured APPs, CRP, SAA and Hp, increased This study suggests that inflammation is part of the significantly peripartum in both groups of sows, but only pathogenesis in PDS. WBC, neutrophil and lymphocyte Hp concentrations differed in PDS- and PDS+ sows. counts, and serum concentrations of TNF-α, IL6, Hp While p.p. concentrations of Hp were similar in healthy and Fe differed between PDS+ and PDS- sows and may sows and sows receiving intramammary administration potentially serve as diagnostic adjuncts for detection of of E. coli in one study , another study detected PDS. However, considering the inflammatory changes higher Hp concentrations in sows with MMA than in found also in PDS- sows, the diagnostic value of measur- healthy sows 1-10 days p.p. . Based on our results, Hp ing inflammatory markers needs further investigation in seems to be the most useful of the three APPs measured. larger study populations, where diagnostic capacity Considering the modest difference in Hp concentrations (positive and negative predictive value) of the investi- in PDS+ and PDS- sows (peak Hp concentration was 14 gated markers may be fully assessed. Combining mea- % higher in PDS+ sows than in PDS- sows), the diagnos- surements of inflammatory markers with assessment of tic potential of Hp may be limited. It is not clear why clinical and behavioral parameters may improve identifi- concentrations of SAA, which is considered to be a very cation of sows at risk of developing PDS. Development Kaiser et al. BMC Veterinary Research (2018) 14:83 Page 13 of 15 of assay systems that allow farmers and veterinarians to was approved by SEGES and IFD. SEGES contributed with technical support during data collection and statistical analysis. measure relevant inflammatory markers sow-side may enhance future use of clinical-chemical parameters in Availability of data and materials porcine herd health medicine markedly. Early (antepar- The datasets supporting the conclusions of this article are included within the article and its Additional files. tum) detection of sows at risk of developing PDS is de- sirable, but only lymphocyte counts were different in Authors’ contributions PDS+ and PDS- sows before parturition. The study was designed by MK, MJ, PHA, PB and SJ. Data and samples were collected by MK and MJ. JC, DE and MK performed laboratory analyses. MK, JD and SJ performed statistical analyses. MK and SJ drafted the manuscript. All authors interpreted, read, revised, and approved the final manuscript. Additional files Ethics approval Additional file 1: Three fictive examples of sampling points. The figure The experimental protocol was approved in advance by the Danish Animal illustrates how the number of observations (n) in each time interval differ Experiments Inspectorate (permit 2013−15−2934−00970). All procedures between variables because of individual sampling times relative to were carried out in agreement with the Danish Animal Testing Act. The parturition (0 h). (PPTX 46 kb) animals were derived from a private owner, who in writing gave his consent -9 Additional file 2: Interleukin 1 (IL-1). IL-1 concentration (10 g/L) in for the use of the animals for scientific purposes. sows with postpartum dysgalactia syndrome (PDS+, red) and healthy sows (PDS-, blue) sampled from 60 h before until 36 h after parturition Consent for publication (time interval A-G). Each dot represents the exact sample time of each Not applicable. observation relative to the exact birth of the first piglet (0 h). The lines show the mean value. (DOCX 30 kb) Competing interests The authors declare that they have no competing interests. Additional file 3: Total protein (TP). TP concentration (10 g/L) in sows with postpartum dysgalactia syndrome (PDS+, red) and healthy sows (PDS-, blue) sampled from 60 h before until 36 h after parturition (time Publisher’sNote interval A-G). Each dot represents the exact sample time of each observa- Springer Nature remains neutral with regard to jurisdictional claims in tion relative to the exact birth of the first piglet (0 h). The lines show the published maps and institutional affiliations. mean value. (DOCX 33 kb) -3 Additional file 4: Hemoglobin (Hb). Hb concentration (10 mol/L) in Author details sows with postpartum dysgalactia syndrome (PDS+, red) and healthy Department of Veterinary Clinical Sciences, Faculty of Health and Medical sows (PDS-, blue) sampled from 60 h before until 36 h after parturition Sciences, Copenhagen University, Højbakkegård Alle 5, 2630 Taastrup, (time interval A-G). Each dot represents the exact sample time of each Denmark. Department of Clinical Sciences, Faculty of Veterinary Medicine observation relative to the exact birth of the first piglet (0 h). The lines and Animal Science, Swedish University of Agricultural Sciences, p.o. Box -3 show the mean value. Normal range is 6.2 – 9.4 × 10 mol/L. 7054, SE-750 07 Uppsala, Sweden. SEGES, Danish Pig Research Center, (DOCX 31 kb) Danish Agriculture & Food Council, Agro Food Park 15, 8200 Aarhus N, Denmark. Department of Animal Medicine and Surgery, Regional “Campus Additional file 5: Hematocrit (Ht). Ht (L/L) in sows with postpartum of Excellence Mare Nostrum”, University of Murcia, 30100 Espinardo, Murcia, dysgalactia syndrome (PDS+, red) and healthy sows (PDS-, blue) sampled Spain. Danish Agriculture and Food Council, Axelborg, Axeltorv 3, 1709 from 60 h before until 36 h after parturition (time interval A-G). Each dot Copenhagen V, Denmark. represents the exact sample time of each observation relative to the exact birth of the first piglet (0 h). The lines show the mean value. Normal Received: 26 May 2017 Accepted: 20 February 2018 range is 0.31 – 0.46 L/L. (DOCX 41 kb) References Abbreviations 1. Kemper N, Gerjets I. Bacteria in milk from anterior and posterior mammary ALB: Albumin; APP’s: Acute phase proteins; CRP: C-reactive protein; E. glands in sows affected and unaffected by postpartum dysgalactia coli: Escherichia coli; Fe: Iron; Hb: Hemoglobin; Hp: Haptoglobin; syndrome (PPDS). Acta Vet Scand. 2009;51:26. 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BMC Veterinary Research – Springer Journals
Published: Mar 12, 2018
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