Emergence of OXA-48-producing Enterobacter hormaechei in a Swiss companion animal clinic and their genetic relationship to clinical human isolates.

BACKGROUND: Enterobacter hormaechei producing the carbapenemase OXA-48 was identified repeatedly in infections in companion animals hospitalized at a Swiss veterinary clinic where OXA-48-producing Klebsiella pneumoniae was previously reported. OBJECTIVES: To determine the genetic relatedness of animal and human E. hormaechei strains collected in Switzerland during 2017-22 and their mobile genetic elements. METHODS: Hybrid assemblies for phylogenetic and comparative analysis of animal (n = 9) and human (n = 25) isolates were obtained by sequencing with Illumina, PacBio and Oxford Nanopore Technologies. Antimicrobial susceptibility was tested by broth microdilution. RESULTS: The animal strains were identified as E. hormaechei subsp. xiangfangensis ST114 (n = 6) and ST418 (n = 2), and E. hormaechei subsp. hoffmannii ST78 (n = 1). Human E. hormaechei belonged to subspecies steigerwaltii (n = 10), xiangfangensis (n = 13), hoffmannii (n = 1) and hormaechei (n = 1), with a heterogeneous ST distribution differing from the animal strains, except for two ST114. Core-gene SNP analysis confirmed the clonality of the animal ST114 and ST418 isolates (0 to 10 SNPs), and close relatedness of animal and human ST114 strains (80-120 SNPs). The strains harboured the blaOXA-48 gene on ca. 63 kb IncL-type plasmids (n = 27); on ca. 72 kb IncL plasmids co-harbouring blaCTX-M-14 (n = 2); and on ca. 150-180 kb IncFIB (n = 4) or hybrid IncFIB/IncL (n = 1) plasmids. The blaOXA-48-harbouring plasmids and the blaDHA-1-carrying ISCR1 element in one animal ST114 and both ST418 clones were likely acquired from previously spreading K. pneumoniae strains. CONCLUSIONS: Common ecological niches favour the spread of plasmid-borne carbapenemases among Enterobacterales and the emergence of MDR E. hormaechei clones.

Exudative glomerulonephritis associated with acute leptospirosis in dogs.

In the past 20 years in Switzerland, dogs with suspect acute leptospirosis frequently showed severe glomerular changes that had not been previously reported. These features were characterized by abundant extravasated erythrocytes and fewer neutrophils accompanied by marked fibrin exudation into the urinary space that was interpreted as an exudative glomerulonephritis (GN). This retrospective study describes this significant glomerular pathological change and investigates the association with leptospirosis. Tissues from 50 dogs with exudative GN, retrieved from 2 pathology archives in Switzerland were reviewed using hematoxylin and eosin, periodic acid-Schiff, phosphotungstic acid-hematoxylin, and Warthin and Starry stains. Clinical and postmortem data were collected for each case. Immunohistochemistry (IHC) and/or polymerase chain reactions were used as confirmatory tests for leptospirosis. While all 50 cases had clinical and pathological features supporting a diagnosis of leptospirosis, 37 cases were confirmed for the disease. Using a LipL32 antibody in addition to the OMV2177 antibody raised against the lipopolysaccharide of Leptospira interrogans serovar Copenhageni increased the detection rate of Leptospira by IHC in exudative GN from 24% to 62%. Signalment, seasonality, clinical signs, blood results, and pathological changes in dogs with exudative GN were similar to those reported for dogs without GN and confirmed infection by Leptospira spp.. Exudative GN was common among Swiss dogs with leptospirosis where it caused acute severe disease. Leptospirosis should be considered as a cause of this new pathologic feature by the pathologist. The pathogenesis remains unclear, but involvement of a geographic-specific serovar with unique virulence factors is suspected and warrants further investigation.

Parents under stress: Evaluating emergency childcare policies during the first COVID-19 lockdown in Germany.

What are the effects of school and daycare facility closures during the COVID-19 pandemic on parental well-being and parenting behavior? Can emergency childcare policies during a pandemic mitigate increases in parental stress and negative parenting behavior? To answer these questions, this study leverages cross-state variation in emergency childcare eligibility rules during the first COVID-19 lockdown in Germany and draws on unique data from the 2019 and 2020 waves of the German AID:A family panel. Employing a triple-differences approach we identify short- to medium-term intention-to-treat effects and find that while emergency care policies did not considerably affect parents' life satisfaction, partnership satisfaction or mental health, they have been effective in diminishing harsh parenting behavior. We find partly gendered effects, specifically on paternal parenting behavior. Our results suggest that decreasing parental well-being likely constitutes a general effect of the pandemic, whereas the observed increase in negative and potentially harmful parenting behavior is largely directly caused by school and daycare facility closures.

Susceptibility of Well-Differentiated Airway Epithelial Cell Cultures from Domestic and Wild Animals to Severe Acute Respiratory Syndrome Coronavirus 2.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has spread globally, and the number of worldwide cases continues to rise. The zoonotic origins of SARS-CoV-2 and its intermediate and potential spillback host reservoirs, besides humans, remain largely unknown. Because of ethical and experimental constraints and more important, to reduce and refine animal experimentation, we used our repository of well-differentiated airway epithelial cell (AEC) cultures from various domesticated and wildlife animal species to assess their susceptibility to SARS-CoV-2. We observed that SARS-CoV-2 replicated efficiently only in monkey and cat AEC culture models. Whole-genome sequencing of progeny viruses revealed no obvious signs of nucleotide transitions required for SARS-CoV-2 to productively infect monkey and cat AEC cultures. Our findings, together with previous reports of human-to-animal spillover events, warrant close surveillance to determine the potential role of cats, monkeys, and closely related species as spillback reservoirs for SARS-CoV-2.

Two high-risk clones of carbapenemase-producing Klebsiella pneumoniae that cause infections in pets and are present in the environment of a veterinary referral hospital.

OBJECTIVES: Infections with carbapenem-resistant Enterobacterales (CRE) are an emerging problem in pets and a major threat to public health. We determined the genetic relationships among carbapenemase-producing Klebsiella pneumoniae (CPKp) strains causing infections in hospitalized pets in a veterinary clinic and those found in the environment. METHODS: WGS was performed with both the Illumina and Nanopore platforms. Searches of genetic features were performed using several databases and bioinformatics tools, and phylogeny was assessed by whole-genome MLST (wgMLST) using SeqSphere and SNP calling with Snippy. RESULTS: WGS analysis of the CPKp strains identified all environmental and almost all animal strains as the high-risk clone ST11, with the exception of two strains that belonged to ST307. All CPKp belonged to novel complex types (CTs) and carried a conjugative 63 kb IncL plasmid encoding the carbapenemase gene blaOXA-48, yersiniabactin and other virulence factors. Although all CPKp ST11 strains carried additional similar IncR plasmids harbouring multiple antimicrobial resistance genes (ARGs), such as the plasmid-mediated blaDHA-1 AmpC gene, some structural variations were observed. The two ST307 strains carried identical 156 kb MDR IncFIB(K) plasmids with several ARGs, including the blaCTX-M-15 ESBL gene. Both wgMLST and cgSNP analysis confirmed that CPKp strains of the same ST were genetically highly related independent of the source of isolation. CONCLUSIONS: This study demonstrated that the clinical CPKp strains were highly related to those contaminating the clinical environment. These findings confirmed nosocomial spread and highlight veterinary hospitals as a source of CPKp, which may further spread to animals, the environment and humans.

Cyclic AMP in human preterm infant blood is associated with increased TLR-mediated production of acute-phase and anti-inflammatory cytokines in vitro.

BACKGROUND: Preterm infants are at high risk of infection and have distinct pathogen recognition responses. Suggested mechanisms include soluble mediators that enhance cellular levels of cAMP. The aim of this study was to assess the relationship between blood cAMP concentrations and TLR-mediated cytokine production in infants during the first month of life. METHODS: Cord and serial peripheral blood samples (days of life 1-28) were obtained from a cohort of very preterm (<30 weeks' gestational age) and term human infants. Whole-blood concentrations of cAMP and FSL-1 and LPS in vitro stimulated cytokine concentrations were measured by ELISA and multiplex bead assay. RESULTS: cAMP concentrations were higher in cord than in peripheral blood, higher in cord blood of female preterm infants, and lower at Days 1 and 7 in infants exposed to chorioamnionitis, even after adjusting for leukocyte counts. TLR2 and TLR4-mediated TNF-α, IL-1ß, IL-6, IL-12p70, and IL-10 production in vitro increased over the first month of life in preterm infants and were positively correlated with leukocyte-adjusted cAMP levels and reduced by exposure to chorioamnionitis. CONCLUSIONS: The ontogeny of blood cAMP concentrations and associations with chorioamnionitis and TLR-mediated production of cytokines suggest that this secondary messenger helps shape distinct neonatal pathogen responses in early life.

Antimicrobial prescriptions in cats in Switzerland before and after the introduction of an online antimicrobial stewardship tool.

BACKGROUND: Antimicrobial stewardship activities are essential to improve prudent antimicrobial use. The aim of the present study was to evaluate changes in antimicrobial prescriptions in cats after the introduction of prudent use guidelines promoted by an online antimicrobial stewardship tool (AntibioticScout.ch) in Switzerland. Data from 792 cats presented to two university hospitals and 14 private practices in 2018 were included and compared to 776 cases from 2016. Cats were diagnosed with acute upper respiratory tract disease (aURTD), feline lower urinary tract disease (FLUTD) and abscesses. Clinical history, diagnostic work-up and antimicrobial prescriptions (class, dosage, duration) were assessed. Type and proportions [95% confidence intervals] of antimicrobial prescriptions were compared between the two evaluation periods and a mixed effects logistic regression model was applied to evaluate compliance with Swiss prudent use guidelines. RESULTS: From 2016 to 2018, the proportion of antimicrobial prescription in all included cases decreased from 75.0% [71.8-78.0] to 66.7% [63.3-69.9]; this decrease was most pronounced for treatments at university hospitals (67.1% [59.5-74.0] to 49.3% [40.9-57.8]) and for cats with FLUTD (60.1% [54.6-65.4] to 48.8% [43.2-54.4]). Use of 3rd generation cephalosporins in private practices declined from 30.7% [26.5-35.1] to 22.1% [18.4-26.2], while overall use of non-potentiated aminopenicillins increased from 19.6% [16.4-23.0] to 27.8% [24.1-31.9]. In cases where antimicrobial therapy was indicated, compliance with guidelines did not increase (33.3% [26.6-40.6] to 33.5% [27.2-40.2]), neither at universities nor in private practices. On the other hand, antimicrobial treatment was more often withheld in cases with no indication for antimicrobial therapy (35.6% [30.1-41.4] to 54.0% [47.6-60.4]); this was found for private practices (26.7% [20.8-33.4] to 46.0% [38.4-53.7]) and for aURTD cases (35.0% [26.5-44.2] to 55.4% [44.7-65.8]). CONCLUSIONS: Overall proportions of antimicrobial prescription, unjustified antimicrobial therapy and, in private practices, use of 3rd generation cephalosporins decreased from 2016 to 2018 for the investigated feline diseases. However, overall compliance with Swiss prudent use guidelines was still low, implying that further efforts are required to foster prudent antimicrobial use in cats.

Intravenous pentoxifylline is well tolerated in critically ill preterm infants with sepsis or necrotizing enterocolitis.

Pentoxifylline (PTX) is a candidate adjuvant medication for the treatment of sepsis and necrotizing enterocolitis in preterm infants. There is only limited data on safety and compatibility with other commonly used intravenous medications. This retrospective single-center study of 198 preterm infants (September 2012-September 2018) was performed at a level IV neonatal intensive care unit. Electronic data of all preterm infants who received pentoxifylline for sepsis or necrotizing were extracted from routine databases. We analyzed a total of 1081 PTX treatment days from 217 treatment episodes in 198 preterm infants (mean gestational age 27 weeks; mean birth weight 1060 g). At a mean daily dose of 28 mg/kg, no clinically relevant side effects were observed. PTX therapy was not associated with clinically significant changes of blood biochemistry and hematology parameters. Concomitant infusion of PTX with other common NICU medications was well tolerated, and there was no evidence of incompatibility.Conclusion: Intravenous PTX is compatible with standard NICU drugs and well tolerated in critically ill preterm infants. What is Know: •Currently, there are no evidence-based adjuvant medications available that target the harmful inflammatory host response in neonatal sepsis or necrotizing enterocolitis. •Pentoxifylline (PTX) is a candidate adjuvant medication for the treatment of sepsis and necrotizing enterocolitis in preterm infants; however, safety data are rare and PTX is currently used off-label. What is New: •Here we report on our experience in the pragmatic routine use of PTX as adjuvant therapy in 198 preterm infants with sepsis or NEC. •Concomitant infusion of PTX with other common NICU medications was well tolerated, and there was no evidence of incompatibility. No clinically relevant side effects were observed.

Shedding of OXA-181 carbapenemase-producing Escherichia coli from companion animals after hospitalisation in Switzerland: an outbreak in 2018.

BackgroundCarbapenem-resistant Enterobacteriaceae pose a serious threat to public health worldwide, and the role of companion animals as a reservoir is still unclear.AimsThis 4-month prospective observational study evaluated carriage of carbapenem-resistant Enterobacteriaceae at admission and after hospitalisation in a large referral hospital for companion animals in Switzerland.MethodsRectal swabs of dogs and cats expected to be hospitalised for at least 48 h were taken from May to August 2018 and analysed for the presence of carbapenem-resistant Enterobacteriaceae using selective agar plates. Resistant isolates were further characterised analysing whole genome sequences for resistance gene and plasmid identification, and ad hoc core genome multilocus sequence typing.ResultsThis study revealed nosocomial acquisition of Escherichia coli harbouring the carbapenemase gene bla OXA-181, the pAmpC cephalosporinase gene bla CMY-42 as well as quinolone resistance associated with qnrS1 and mutations in the topoisomerases II (GyrA) and IV (ParC). The bla OXA-181 and qnrS1 genes were identified on a 51 kb IncX3 plasmid and bla CMY-42 on a 47 kb IncI1 plasmid. All isolates belonged to sequence type ST410 and were genetically highly related. This E. coli clone was detected in 17 of 100 dogs and four of 34 cats after hospitalisation (21.6%), only one of the tested animals having tested positive at admission (0.75%). Two positive animals were still carriers 4 months after hospital discharge, but were negative after 6 months.ConclusionsCompanion animals may acquire carbapenemase-producing E. coli during hospitalisation, posing the risk of further dissemination to the animal and human population and to the environment.

Review demonstrates that less invasive surfactant administration in preterm neonates leads to fewer complications.

Surfactant treatment of neonatal respiratory distress syndrome (RDS) was introduced in Europe during the 1990s. Meta-analyses have indicated that using less invasive surfactant administration techniques on preterm neonates receiving continuous positive airway pressure (CPAP) results in improved survival rates without bronchopulmonary dysplasia. Surfactant should be administered early and ventilator settings adapted to changing oxygen requirements and lung mechanics. Side effects including initial bradycardia, oxygen desaturation, tube obstruction and isolated cases of pulmonary haemorrhage have been reported. CONCLUSION: Less invasive surfactant therapy improves pulmonary outcomes in preterm neonates with RDS and should ideally be administered in combination with CPAP.

Pentoxifylline modulates LPS-induced hyperinflammation in monocytes of preterm infants in vitro.

BackgroundPentoxifylline (PTX), a methylxanthine derivate with immunomodulating properties, has been used as adjunctive treatment in severe neonatal sepsis. The aim of the study was to investigate the anti-inflammatory effects of PTX on Lipopolysaccharides (LPS)-stimulated monocytes of preterm neonates in vitro compared with monocytes of term infants and adult controls.MethodsWhole cord blood samples and control adult blood samples were incubated with LPS and PTX. The expression of surface markers, phagocytosis, cytokine secretion, and Toll-like receptor (TLR)4 signaling of monocytes were assessed by flow cytometry. Changes of TLR4-messenger RNA (mRNA) levels were confirmed by reverse-transcriptase PCR.ResultsThe expression of CD14, CD11b, CD64, CD71, and CD80 was downregulated by PTX in a dose-dependent manner; the greatest effect was observed on CD14 and CD11b in preterm infants. PTX markedly downregulated LPS-induced tumor necrosis factor-α, interleukin (IL)-1ß, and IL-6 levels in all age groups. Early IL-10 production was significantly downregulated by PTX in term and preterm neonates, while remaining unchanged in adults. Moreover, PTX downregulated TLR4 expression of monocytes on cellular and mRNA level, decreased signaling, and suppressed phagocytosis.ConclusionPTX downregulated TLR4 expression and signaling, thereby leading to strong anti-inflammatory properties in monocytes. Age-dependent differences were identified for CD14 and CD11b expression and IL-10 production.

The TLR-specific adjuvants R-848 and CpG-B endorse the immunological reaction of neonatal antigen-presenting cells.

BACKGROUND: Preterm neonates display an impaired vaccine response. Neonatal antigen-presenting cells (APCs) are less effective to induce an adaptive immune response and to promote the development of immunological memory. Efficient adjuvantal toll-like receptor (TLR)-triggering may overcome the neonatal immunological impairment. Accordingly, the aim of this study was to investigate the immunostimulatory action of R-848 and CpG-B on neonatal APCs. METHODS: Surface marker and cytokine secretion of APCs were evaluated after incubation of cord blood and peripheral blood mononuclear cells with the indicated adjuvants and were analyzed using flow cytometry. RESULTS: TLR-specific stimulation resulted in a significant induction of costimulatory molecules on neonatal APCs. Stimulation with R-848 resulted in significant higher secretion of TNFα, IL-6, IL-10, IL-12/IL-23p40, IL-12p70, and IFN-γ. Interestingly, CpG-B resulted in significant higher secretion of TNFα and IL-6. CONCLUSION: In summary, the incubation of TLR-agonists induced activation and maturation of neonatal APCs. These data show that modern TLR-specific adjuvants achieve a direct effect and potent upregulation of activation and maturation markers and cytokines in preterm neonates. We thus conclude that agents triggering TLRs might possibly overcome neonatal lack of vaccine responses.

Hemophagocytic lymphohistiocytosis triggered by Gaucher disease in a preterm neonate.

OBJECTIVE: To present the diagnostic workup in an extremely low birth weight infant patient with signs of both sepsis and hemophagocytosis. PARTICIPANTS: A preterm infant presented with clinical and laboratory signs of early-onset sepsis including hepatosplenomegaly, thrombocytopenia, direct hyperbilirubinemia, and elevated liver enzymes. METHODS: Despite extensive septic workup, no underlying infection was detected. Additional hyperferritinemia and other elevated inflammatory parameters raised the suspicion of a primary or secondary hemophagocytic lymphohistiocytosis (HLH). RESULTS: However, further metabolic analysis yielded a positive result for Gaucher disease (GD) type 2, a rare, but possible trigger of HLH. CONCLUSIONS: Our case shows that GD may lead to the picture of a secondary HLH and that a metabolic workup should always be performed in patients in whom primary HLH has been excluded.

Comparison between a Flash Glucose Monitoring System and a Portable Blood Glucose Meter for Monitoring of Cats with Diabetic Ketosis or Ketoacidosis.

Cats with diabetic ketosis or ketoacidosis DK(A) require intensive glucose monitoring. The aim of this study was to assess the agreement between a portable blood glucose meter (PBGM) and a flash glucose monitoring system (FGMS; FreeStyle Libre 2.0 Abbott®) measuring interstitial glucose in cats with DK(A). Ten client-owned cats with naturally occurring DK(A) were prospectively enrolled. Glucose concentrations were assessed with both methods every 1-3 h until resolution of DK(A), and every 4-8 h thereafter. While the median FGMS measured glucose concentration (14.3 mmol/L) was significantly lower than the median PBGM measured glucose concentration (19 mmol/L) (p < 0.001), the overall correlation between the FGMS and PBGM was high (r = 0.88; p < 0.001). In the Parkes error grid analysis, 98.3% of measurements fell in zones A and B. Bland-Altman plot analysis demonstrated that in the low glycaemic range (BG < 5.5 mmol/L), 50% of FGMS measurements deviated more than ±0.83 mmol/L, and in the high glycaemic range (BG > 5.5 mmol/L), 81% of results deviated >15% from the PBGM measurements. There was significant inter-individual variation in the difference between glucose concentrations measured by the FGMS and PBGM (p < 0.001). In spite of being more easily tolerated and easier to use, currently this method cannot be recommended for routine monitoring of cats with DK(A).

Immune profiling of age and adjuvant-specific activation of human blood mononuclear cells in vitro.

Vaccination reduces morbidity and mortality due to infections, but efficacy may be limited due to distinct immunogenicity at the extremes of age. This raises the possibility of employing adjuvants to enhance immunogenicity and protection. Early IFNγ production is a hallmark of effective vaccine immunogenicity in adults serving as a biomarker that may predict effective adjuvanticity. We utilized mass cytometry (CyTOF) to dissect the source of adjuvant-induced cytokine production in human blood mononuclear cells (BMCs) from newborns (~39-week-gestation), adults (~18-63 years old) and elders (>65 years of age) after stimulation with pattern recognition receptors agonist (PRRa) adjuvants. Dimensionality reduction analysis of CyTOF data mapped the BMC compartment, elucidated age-specific immune responses and profiled PRR-mediated activation of monocytes and DCs upon adjuvant stimulation. Furthermore, we demonstrated PRRa adjuvants mediated innate IFNγ induction and mapped NK cells as the key source of TLR7/8 agonist (TLR7/8a) specific innate IFNγ responses. Hierarchical clustering analysis revealed age and TLR7/8a-specific accumulation of innate IFNγ producing γδ T cells. Our study demonstrates the application of mass cytometry and cutting-edge computational approaches to characterize immune responses across immunologically distinct age groups and may inform identification of the bespoke adjuvantation systems tailored to enhance immunity in distinct vulnerable populations.

Updated ACVIM consensus statement on leptospirosis in dogs.

Since publication of the last consensus statement on leptospirosis in dogs, there has been revision of leptospiral taxonomy and advancements in typing methods, widespread use of new diagnostic tests and vaccines, and improved understanding of the epidemiology and pathophysiology of the disease. Leptospirosis continues to be prevalent in dogs, including in small breed dogs from urban areas, puppies as young as 11 weeks of age, geriatric dogs, dogs in rural areas, and dogs that have been inadequately vaccinated for leptospirosis (including dogs vaccinated with 2-serovar Leptospira vaccines in some regions). In 2021, the American College of Veterinary Internal Medicine (ACVIM) Board of Regents voted to approve the topic for a revised Consensus Statement. After identification of core panelists, a multidisciplinary group of 6 experts from the fields of veterinary medicine, human medicine, and public health was assembled to vote on the recommendations using the Delphi method. A draft was presented at the 2023 ACVIM Forum, and a written draft posted on the ACVIM website for comment by the membership before submission to the editors of the Journal of Veterinary Internal Medicine. This revised document provides guidance for veterinary practitioners on disease in dogs as well as cats. The level of agreement among the 12 voting members (including core panelists) is provided in association with each recommendation. A denominator lower than 12 reflects abstention of ≥1 panelists either because they considered the recommendation to be outside their scope of expertise or because there was a perceived conflict of interest.

Plasma procalcitonin kinetics in healthy dogs and dogs undergoing tibial plateau leveling osteotomy.

BACKGROUND: Procalcitonin (PCT) is a well-established biomarker for bacterial infection in human patients. OBJECTIVES: We aimed to analyze the kinetics of plasma PCT (pPCT) in healthy dogs and dogs with canine cranial cruciate ligament (CCL) rupture undergoing tibial plateau leveling osteotomy (TPLO). METHODS: This prospective, longitudinal study included 15 healthy dogs and 25 dogs undergoing TPLO. Hematology, pPCT, and C-reactive protein (CRP) were assessed on 3 consecutive days in healthy dogs and 1 day preoperatively and days 1, 2, 10, and 56 postoperatively. Inter- and intraindividual variability of pPCT were assessed in healthy dogs. Median pPCT concentrations of dogs with CCL rupture preoperatively were compared with healthy controls, and median pPCT concentrations, as well as percentage change post anesthesia, arthroscopy, and TPLO, were compared with baseline. For the correlation analysis, the Spearman rank correlation test was used. RESULTS: Inter- and intraindividual variabilities of pPCT in healthy dogs were 36% and 15%, respectively. Median baseline pPCT concentrations were not significantly different between healthy dogs (118.9 pg/mL; IQR: 75.3-157.3 pg/mL) and dogs undergoing TPLO (95.9 pg/mL; IQR: 63.8-117.0 pg/mL). Plasma PCT concentrations were significantly lower immediately post- than preoperatively (P < 0.001). CRP, WBC, and neutrophil concentrations increased significantly on post-OP day 2 and had normalized by day 10. CONCLUSIONS: These results indicate that CCL rupture, as well as anesthesia, arthroscopy, and TPLO combined, are not associated with increased pPCT concentrations in dogs with uncomplicated recovery. Considering the high intraindividual variability, individual serial measurements rather than a population-based reference interval should be considered.