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Theriogenology 72 (2009) 471–476 www.theriojournal.com

Usefulness of C-reactive protein, serum amyloid A component, and haptoglobin determinations in bitches with pyometra for monitoring early post-ovariohysterectomy complications R. Da˛browski a,*, K. Kostro b, U. Lisiecka b, M. Szczubiał a, L. Krakowski a a

Department and Clinic of Animal Reproduction, Faculty of Veterinary Medicine, University of Life Sciences in Lublin, 30, Głe˛boka St., 20-612 Lublin, Poland b Department of Epizootiology and Clinic of Infectious Diseases, Faculty of Veterinary Medicine, University of Life Sciences in Lublin, 30, Głe˛boka St., 20-612 Lublin, Poland Received 27 January 2009; received in revised form 6 March 2009; accepted 22 March 2009

Abstract The aim of this study was to assess changes in serum concentrations of C-reactive protein (CRP), serum amyloid A component (SAA), and haptoglobin (Hp) in bitches with pyometra undergoing ovariohysterectomy that developed postoperative wound infection–related complications. The study revealed that difficulties in postoperative wound healing were induced by infections with Escherichia coli and Staphylococcus spp. leading to re-increased levels of CRP and SAA immediately after surgery and persistently high Hp levels throughout the experiment. Our results indicate that acute-phase proteins in bitches undergoing surgery because of pyometra are useful markers for monitoring the postoperative period. Moreover, they enable prompt therapeutic management once complications develop. # 2009 Elsevier Inc. All rights reserved. Keywords: Acute-phase proteins; CRP; Hp; Postoperative complications; Postoperative monitoring; SAA

1. Introduction The most severe form of endometritis is pyometra, which leads to strong intoxication of the organism and secondary uterine infection, in which Escherichia coli is most commonly isolated [1–4]. The release of lipopolysaccharide (LPS) from the cell wall of E. coli results in activation of neutrophils and macrophages and increased synthesis of proinflammatory cytokines [5–7]. The uncontrollable production of proinflammatory cytokines during pyometra is one of the main factors

* Corresponding author. Tel.: +48 81 445 61 68; fax: +48 81 445 60 99. E-mail address: [email protected] (R. Da˛browski). 0093-691X/$ – see front matter # 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.theriogenology.2009.03.017

provoking irreversible damage to internal organs, in some cases leading to septic shock and death [5]. In general, conservative treatment of pyometra does not bring about desirable therapeutic effects, and therefore ovariohysterectomy remains the treatment of choice [4,8,9]. Numerous reports concerning postoperative complications in dogs are available in the literature; the majority of complications are related to difficult wound healing [10–13]. Determinations of acute-phase proteins (APPs) are increasingly common in monitoring the postoperative period in dogs. Acute-phase proteins as an objective marker of the severity of inflammatory processes enable early detection of complications in patients undergoing surgery and prompt institution of directed therapy [14–16].

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The aim of the study was to establish the usefulness of C-reactive protein (CRP), serum amyloid A component (SAA), and haptoglobin (Hp) determinations for monitoring the severity of postoperative inflammatory responses in bitches undergoing ovariohysterectomy. 2. Materials and methods The study involved 20 randomly chosen mongrel bitches aged 4 to 12 years undergoing ovariohysterectomy due to pyometra at the Department of Animal Reproduction, Veterinary Faculty, University of Life Sciences in Lublin. Bitches were divided into two groups: Group I (10 bitches with the normal postoperative period) and Group II (10 bitches with postoperative complications). All bitches with pyometra showed polydipsia, reduced or complete lack of appetite (anorexia), apathy, enlarged abdominal integuments, and increased pulse and respiration rate; additionally, 60% of them developed purulent discharge from the reproductive organs. In all bitches, abdominal ultrasound examinations with a 5/ 7.5 MHz microprobe (HONDA ELECTRONICS CO. LTD., Japan 1500) revealed an enlarged uterus of diameter ranging from 3 to 6 cm with a hypoechogenic content. Moreover, vaginoscopy showed congested mucous membranes of the vaginal vestibule and vagina. In bitches with open pyometra, the mucous membranes were found to be covered with purulent secretion. Hematologic findings disclosed leukocytosis with shift to the left while biochemical results showed increased activity of hepatic enzymes (AST, ALT). Ovariohysterectomy was performed according to generally accepted surgical guidelines. After surgery and complete recovery from anesthesia, bitches were under observation for 17 days. During the first 3 postoperative days, bitches of both groups received intravenous infusions of 5% glucose, Ringer solution, and compound electrolyte solution in the amount of 10 mL/kg body weight, as well as metaphylactic penicillin and streptomycin (PEN-STREP, ScanVet, Poland). In the group with postoperative complications developing on Day 4, the antibiotic administration was prolonged and Pen-Strep was replaced with enrofloxacine (ENROBIOFLOX, Ve´toquinol, Biowet, Poland) chosen based on drug susceptibility of the bacteria isolated from postoperative wounds. Moreover, the postoperative wounds were washed with hydrogen peroxide and 1% potassium permanganate solutions. The levels of CRP, SAA, and Hp were determined in blood samples collected from the vena saphena at the following points: before surgery (Day 0), and on Day 1, 3, 5, 10, and 17 after ovariohysterectomy. Blood for

APP determinations was collected into aseptic silicone tubes (Vakuette 9 ml; Greiner Labortechnik GmbH, Kremsmu¨nster, Austria) with a coagulator, and blood for biochemical examinations was collected into tubes containing 20 IU/mL heparin. Serum was prepared by centrifugation with the coagulant (10 min, 3000  g), placed in ampoules in 1-mL amounts, and stored at– 80 8C until use. The levels of CRP and SAA were determined using ELISA and commercial C-Reactive Protein Assay and Serum Amyloid A Assay kits (Tridelta Development Ltd, Kildare, Ireland). The serum levels of Hp were determined using the colorimetric method and a commercial Haptoglobin Assay kit (Tridelta Development Ltd). Absorption was recorded using the ELISA ELX 800 (BIO-TEK Instruments Inc., Highland Park, Winooski, Vermont, USA) at 450 nm. To confirm the infections of postoperative wounds in Group II, bacteriological cultures of wound discharge were performed. The material was cultured on agar medium with 5% ram blood; after 24 h intubation at 37 8C under oxygen conditions, the microorganisms were initially identified by evaluating the morphology of colonies and Gram-stained microscopic specimens. The isolated bacterial strains were finally identified using commercial Analytical Profile Index (API) tests (API-Staph, API-Strep, BioMerieux, Marcy l’Etoile, France). The data were statistically analyzed by calculating the mean, standard deviation, and significance of differences. Intragroup and intergroup means were compared with baseline values using the Statistica 5.0 software. The significance of differences was set at P  0.05 and P  0.01. 3. Results In Group II, postoperative complications were observed on postoperative Day 4 (i.e., reddened postoperative wounds, elevated temperature of the adjacent tissues and sutures, and serous-bloody discharge). The bacteriological tests showed pure E. coli culture in 80% of cases and Staphylococcus spp. in the remaining 20%. Positive culture results were the basis for the administration of enrofloxacine at a dosage of 1 mL/10 kg body weight for 7 days. On Day 3 (postoperative Day 7) after the application of enrofloxacine, the postoperative wound started gradually to heal; on Day 11 (Day 7 after antibiotic application), a marked growth of granulation tissue was observed, and skin sutures were removed. On the last observation day, postoperative wounds were healed in all Group II bitches. In Group I, skin sutures

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Fig. 1. Serum CRP levels in bitches with normal (Group I) and complicated (Group II) post-ovariohysterectomy postoperative period. A, intragroup differences compared with baseline value (P  0.01); D, intergroup differences at an individual determination point (P  0.05).

were removed on postoperative Day 8 and wounds healed by first intention. The levels of CRP at individual determination points in bitches with the normal or the complicated post-

operative period are presented in Fig. 1. The mean preoperative serum CRP levels in both groups of bitches were comparable: 85.4 mg/mL in Group I and 87.7 mg/ mL in Group II. In both groups, significant increases in

Fig. 2. Serum SAA levels in bitches with normal (Group I) and complicated (Group II) post-ovariohysterectomy period. A, intragroup differences compared with baseline value (P  0.01); C, intergroup differences at an individual determination point (P  0.01); D, intergroup differences at an individual determination point (P  0.05).

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Fig. 3. Serum Hp levels in bitches with normal (Group I) and complicated (Group II) post-ovariohysterectomy period. A, intragroup differences compared with baseline value (P  0.01); C, intergroup differences at an individual determination point (P  0.01); D, intergroup differences at an individual determination point (P  0.05).

CRP levels compared with baseline values were observed already on postoperative Day 1. At this determination point, these were the highest serum levels of CRP in Group I. At subsequent determinations in Group I, the serum level of CRP markedly decreased, and on Day 17 its lowest mean levels were observed. In Group II, on postoperative Day 3, the CRP level decreased slightly; on Day 4 another increase was found, which was the highest one throughout the experiment (296.6 mg/mL). After the application of enrofloxacine, the level of CRP in these bitches decreased gradually although its mean value was substantially higher compared with baseline values throughout the experiments and with those in Group I. The levels of SAA in bitches after ovariohysterectomy are presented in Fig. 2. Serum amyloid A component changes in individual groups and at individual determinations were similar to CRP changes (Fig. 1). As shown in Fig. 3, on Day 0, the mean levels of Hp in both groups were similar. In Group I, a significant Hp increase compared with the baseline value was observed already on postoperative Day 1. On successive days, the level of Hp increased further reaching its highest value (8.4 mg/mL) on postoperative Day 4. During the subsequent days, the mean Hp values considerably decreased and on Day 10 and 17 were significantly lower than baseline values and those in Group II. In Group II, the mean Hp levels were similar to baseline values throughout the experiment; at the remaining

determination points, except for the first one, they were significantly different from those in Group I. 4. Discussion Determinations of changes in APPs are useful tools to monitor the severity of inflammatory responses in animals and humans exposed to harmful factors [2,16– 21]. Our findings indicate that inflammatory responses developing in bitches undergoing surgery due to pyometra may be identified based on serum levels of CRP, SAA, and Hp. Moreover, determination of levels of those APPs in bitches is a good prognostic marker enabling early detection of bacterial infections of postoperative wounds. The lesions developing in the uterus with secondary bacterial infections induce the inflammatory response and increased biosynthesis of APPs in hepatic cells mediated by IL-1 and IL-6 released by activated macrophages [22]. Thus, elevated levels of CRP, SAA, and Hp in bitches with pyometra before surgery result from chronic inflammation, which is confirmed by Krzyz˙anowski et al. [3], Yamamoto et al. [23], and Da˛browski et al. [2]. On the other hand, significant increases in mean CRP, SAA, and Hp levels compared with baseline values observed on postoperative Day 1 result from tissue discontinuity and reflect the severity of inflammatory processes [14,24]. In bitches without complications, the highest mean levels of CRP and SAA were observed on postoperative Day 1 but that of Hp on Day 4, which confirms that CRP

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and SAA in dogs react rapidly whereas Hp reacts slowly [22,25]. Serum levels of APPs examined normalized on postoperative Day 17, which indicates the abating local inflammatory process and proper postoperative wound healing [2]. It cannot be excluded that an increase or decrease in CRP, SAA, and Hp levels in Group I observed at individual measurement points results from their involvement in modulation of the inflammatory immune response and repair processes of damaged tissues, by, for example, binding and facilitating phagocytosis of dead and damaged tissues and stimulation of angiogenesis, which is essential for wound healing and chronic inflammatory processes. In bitches with postoperative complications, changes in CRP, SAA, and Hp levels were different. Persistent high levels of CRP and SAA indicate the inflammatory process resulting from infection of postoperative wounds. Thus, it may be supposed that infections of surgical wounds with E. coli and Staphylococcus spp. observed in our study were the direct cause of complications in bitches undergoing surgery because of pyometra. A relevant factor triggering the development of infection is immunosuppression accompanying pyometra [2]. Proper healing of postoperative wounds depends largely on proper immune response, particularly synergistic effects of proinflammatory cytokines [12,26]. The results of our study confirm earlier literature reports demonstrating that postoperative infections are still relevant despite improved surgical techniques and better sterilization methods of surgical materials as well as metaphylactic use of antibiotics [11,13,16,27–29]. The highest CRP and SAA increases observed in Group II on Day 4 and persistent high levels of Hp are likely to result from the inflammatory process and effects of LPS, which activates the macrophages and release of IL-1 and Il-6-the main inducers of increased biosynthesis of APPs in the liver [28]. Proinflammatory cytokines, especially IL-6, are actively involved in proper wound healing [26]. Excessive secretion of Il-6 caused by, for example, infections may lead to difficult wound healing [30] by delaying the apoptosis of polimorfonuclear (PMN) cells [31]. In conclusion, serum levels of CRP, SAA, and Hp in bitches undergoing surgery because of pyometra are useful markers for monitoring the postoperative period, and their determination facilitates early detection of postoperative complications.

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