The haemostatic status of dogs with canine parvovirus (CPV) enteritis, within 24 h of admission after initial fluid administration, has been described previously, but the haemostatic status at admission and after standard fluid resuscitation, as well as after initial fluid redistribution, has not been investigated previously. The objective of this study was to characterise the haemostatic status at admission and describe the effect of crystalloid fluid resuscitation on haemostatic variables in dogs with CPV enteritis. Twenty-seven client-owned, hospitalised dogs with confirmed natural CPV infection and 15 healthy age-matched controls were included in a prospective, observational clinical study. The volume of resuscitation fluid, haematocrit (HCT), platelet count, thromboelastography (TEG) variables, antithrombin (AT) activity, fibrinogen- and C-reactive protein (CRP) concentrations were measured in all dogs at admission, after fluid resuscitation and, in 10 dogs, after receiving an additional 3 hours of maintenance-rate crystalloid fluids. For the CPV group at admission, the median TEG reaction time (R) and maximum amplitude (MA) or clot strength, as well as the median HCT, fibrinogen and CRP concentrations, were significantly increased compared to the controls. After fluid resuscitation, median R was significantly shorter, MA significantly increased and HCT and AT activity significantly decreased compared to admission values. The haemostatic variables remained unchanged after 3 h of maintenance-rate crystalloid therapy. The increased clot strength present in dogs with CPV enteritis at admission was exacerbated after fluid resuscitation and persisted for hours after large-volume crystalloid fluid administration.
Canine parvovirus (CPV) is a small, non-enveloped single-stranded deoxyribonucleic acid (DNA) virus that preferentially infects tissues with rapid cell turnover, including the intestinal epithelium and bone marrow (Parrish
Viscoelastic methods of coagulation testing such as thromboelastography (TEG) and thromboelastometry (TEM) combine the evaluation of the traditional plasma components with the cellular components of haemostasis and allow for the evaluation of clot formation, clot strength and fibrinolysis (Donahue & Otto
Hypercoagulability, based on increased maximum amplitude (MA) or clot strength, a TEG variable, increased fibrinogen concentration and decreased antithrombin (AT) activity, has been reported in dogs with CPV enteritis, within 24 hours of admission after initial fluid administration (Otto et al.
The initial standard therapy of dogs with CPV enteritis is the administration of intravenous fluids, usually crystalloids, to correct hypovolaemia and dehydration. Decreased haematocrit (HCT) due to anaemia or haemodilution with crystalloid fluid supplementation has been reported to result in a relative increased clot strength, or MA, while a high HCT generally results in a relatively decreased MA (Bochsen et al.
An
A rapid and large increase in blood volume occurs with rapid administration of isotonic crystalloid fluids, and while the blood volume decreases rapidly after the completion of fluid infusion (the result of redistribution to the interstitium), nearly 30% of the infused fluid remains in the intravascular space 30 min after the cessation of fluid therapy (Silverstein et al.
The haemostatic changes in dogs with CPV enteritis from presentation until after fluid resuscitation, as well as after the initial fluid redistribution, has not been described. The aims of this study were to describe the haemostatic changes in dogs with CPV enteritis at admission compared to controls, and evaluate the effects of crystalloid fluid therapy on haemostatic variables in dogs with CPV enteritis. An additional aim was to determine if the effect of crystalloid fluid resuscitation on haemostasis outlasts fluid redistribution. We hypothesised that (1) dogs with CPV enteritis would have a higher MA at admission compared to control dogs and (2) that fluid resuscitation would result in a further increase in MA that outlasts the expected fluid redistribution.
Client-owned dogs presenting to the Onderstepoort Veterinary Academic Hospital (OVAH) between January 2016 and January 2017 with naturally occurring CPV enteritis, and healthy control dogs, were enrolled in a prospective, observational study. Dogs in both the CPV and control groups were between 8 weeks and 9 months of age and weighed more than 4.5 kg. Control dogs were presented for vaccination, ovariohysterectomy or castration and were deemed healthy based on the absence of vomiting or diarrhoea in the preceding 14 days, having had no contact with dogs affected by CPV enteritis, a normal clinical examination and a peripheral blood smear negative for blood-borne parasites. Dogs presenting with clinical signs associated with CPV enteritis such as lethargy, anorexia, vomiting, diarrhoea, dehydration and/or hypovolaemic shock were tested for CPV. Dogs that tested positive using CPV antigen ELISA (Anigen Rapid CPV Ag test kit, BioNote Inc., Gyeonggi-do, Republic of Korea or IDEXX Canine Parvovirus Antigen test kit, IDEXX Laboratories Inc., Maine, United States [US]), were preliminarily diagnosed with CPV enteritis and were enrolled in the CPV group. Eligible cases were only enrolled if they were admitted for in-hospital treatment and had not received any treatment prior to admission. Infection with CPV was confirmed by faecal electron microscopy. Dogs that received any medication known to interfere with normal haemostasis – such as corticosteroids, non-steroidal anti-inflammatory drugs or anticoagulant dugs, during hospitalisation or one month prior to hospitalisation – and dogs that had previously received a transfusion of any blood products were excluded from both the control and CPV groups. Dogs were deemed to be in hypovolaemic shock if they showed clinical signs indicative of abnormalities in perfusion such as tachycardia (> 140 beats per minute), peripheral vasoconstriction with cool extremities, hypothermia (< 37.5 °C), a prolonged capillary refill time (> 2 s), poor peripheral pulse quality and mental dullness (Mazzaferro & Powell
Canine parvovirus-affected dogs were cared for according to the standard treatment guidelines of the institution and all dogs received 1 mg/kg maropitant subcutaneously (SC) once daily, 20 mg/kg ampicillin IV every 8 h and 15 mg/kg metronidazole IV every 12 h, during the duration of data collection. Fentanyl constant rate infusion (CRI) was administered at 3 μg/kg/h in dogs with severe abdominal discomfort. Any other required treatment was administered after the last blood sample was collected. The control group did not receive any treatment. None of the dogs received any drugs known to affect haemostasis during the resuscitation period, and 0.9% NaCl without additional heparin was used to flush IV catheters.
Clinical parameters were recorded for each dog in the control and CPV groups, and percentage dehydration or presence of hypovolaemic shock was recorded in the CPV group. The first blood collection was performed at admission, prior to any treatment or procedures for the control and CPV groups. Blood was again collected in the CPV group after fluid resuscitation (hypovolaemic dogs) or 90 min – 110 min of fluid therapy (dehydrated dogs). A third sample was collected from 10 randomly selected dogs in the CPV group, by blinded draw of a card (A or B) from an envelope, 3 h after fluid resuscitation while on a 10 mL/kg/h CRI of LRS. Blood was collected via jugular venipuncture, using a 21G needle into evacuated serum and citrate tubes in the recommended order (Goggs et al.
In dogs with CPV that presented in hypovolaemic shock, intravenous LRS (Fresenius Kabi, Pty Ltd., Midrand, South Africa) boluses of 10 mL/kg bodyweight were given over 10 s – 60 s, and repeated every 15 min, until the normalisation of five out of seven clinical variables (alert mentation, a pink mucous membrane colour, a capillary refill time of 1 s – 2 s, a heart rate of 70–140 beats per min, a strong pulse quality, a respiratory rate of 10–30 breaths per min and a temperature of 37.5 °C – 39.5 °C) was achieved (defined as the completion of fluid resuscitation). In addition, if dehydration was identified in these hypovolaemic dogs, rehydration fluids were initiated at the same time, which entailed the replacement of the estimated percentage dehydration (over 24 h) in addition to maintenance fluid requirements (60 mL/kg/day – 80 mL/kg/day) using LRS. In the hypovolaemic group, blood collection was repeated within 15 min of fluid resuscitation completion, while still on rehydration fluids. For the dehydrated dogs with CPV without signs of hypovolaemia, rehydration entailed the same fluid plan as dehydrated hypovolaemic dogs (excluding the fluid boluses). Repeat blood collection was performed at a pre-determined time of 90 min – 110 min of fluid therapy in all of the dehydrated dogs, while still on rehydration fluids, to be of a similar time period to what was expected for fluid resuscitation in the hypovolaemic dogs. Blood collection was not repeated in the control group.
To assess the effect of initial fluid redistribution after the initial fluid resuscitation of 90 min – 110 min of fluid therapy, a blood sample was also collected as previously described, in 10 randomly selected dogs with CPV, 3 h after an additional 10 mL/kg (approximating maintenance-rate fluids and for standardisation across hydration status) of LRS (defined as the CRI group).
Blood samples from admission (CPV and control groups) and after fluid resuscitation (dehydrated and hypovolaemic CPV groups) were analysed. Citrated whole blood was kept at room temperature for 30 min before a tissue factor-activated TEG analysis using the TEG 5000 Thrombelastograph Hemostasis Analyzer system (Haemonetics Corporation, Massachusetts, US) was performed. The principal investigator performed all of the TEG analyses. Machine blood counts (Advia 2120, Siemens, Germany) to determine HCT and platelet count were performed on citrated whole blood at the time of each TEG assay. The HCT and platelet counts were corrected for the dilutional effect of the citrate anticoagulant (Dumont et al.
Statistical analyses were performed using a commercial software package (SPSS Statistics Software version 24, IBM Corp, Armonk, NY, US). Hypovolaemic and dehydrated dogs (subgroups) were grouped together as the CPV group. The Shapiro-Wilk test was used to assess the data for normality. The Chi-Square test was used to compare sex proportions and the Mann-Whitney
After fluid resuscitation, the dogs in the CRI group were evaluated as a whole, and subgroup analysis for the dehydrated and hypovolaemic dogs was performed. The Wilcoxon signed-rank test was also used to determine differences between variables from fluid resuscitation until after the CRI of LRS in the 10 randomly selected CPV dogs, and in the dehydrated and hypovolaemic subgroups. The Mann-Whitney
The study was reviewed and approved by the University of Pretoria Animal Ethics Committee (AEC number V092-15). Informed written consent was obtained from all owners.
Twenty-eight dogs with naturally occurring CPV enteritis and 15 healthy control dogs were enrolled in the study. One dog was excluded after a negative result on faecal electron microscopy. Canine parvovirus-infected dogs consisted of 16 dogs that presented with hypovolaemic shock and 11 with dehydration. All dogs with hypovolaemic shock were also dehydrated and therefore received fluid boluses as well as concurrent rehydration and maintenance rate fluids. The signalment of the CPV and control groups is included in
Signalment of dogs included in the canine parvovirus group and control group.
Variable | Control dogs ( |
CPV dogs ( |
---|---|---|
Median | 3 | 5 |
IQR | 2–7 | 2–6 |
Male | 7 | 14 |
Female | 8 | 13 |
Median | 9.8 | 10.4 |
IQR | 7.2–14.2 | 5.6–14.8 |
CPV, canine parvovirus; IQR, interquartile range.
The principal investigator collected all of the samples. There was no significant difference in the timing of sampling between the dehydrated and hypovolaemic CPV subgroups. The median volume of fluid administered between the first and second blood collection for the hypovolaemic group was 286 mL (246 mL – 470 mL) and was significantly higher compared to the 54 mL (51 mL – 116 mL) for the dehydrated group (
Of the 10 dogs randomly selected to receive a CRI of 10 mL/kg of LRS over 3 h, seven were from the group that presented with initial hypovolaemia and three with dehydration only. No significant difference was identified for age, sex or weight between these two subgroups.
Haemostatic and inflammatory variables in the canine parvovirus group, canine parvovirus subgroups and control group at admission and post fluid resuscitation.
Variable | Reference interval for adult dogs | Control dogs ( |
Admission |
Post fluid resuscitation |
||||
---|---|---|---|---|---|---|---|---|
CPV ( |
Hypovolaemic CPV ( |
Dehydrated CPV ( |
CPV ( |
Hypovolaemic CPV ( |
Dehydrated CPV ( |
|||
Median | 0.37–0.55 | 0.35 | 0.44* | 0.44 | 0.43 | 0.37******** | 0.39*********** | 0.35**** |
IQR | - | 0.22–0.43 | 0.35–0.48 | 0.35–0.52 | 0.35–0.48 | 0.34–0.42 | 0.32–0.42 | 0.33–0.42 |
Median | 200–500 | 325 | 296 | 296 | 292 | 301****** | 309 | 301 |
IQR | - | 276–463 | 230–427 | 222–477 | 253–402 | 235–383 | 206–409 | 235–318 |
Median | 2.0–11.0 | 4.9 | 8.9** | 9.2**** | 7.9 | 7.4****** | 7.6 | 7.3 |
IQR | - | 2.4–7.6 | 6.9–10.4 | 7.4–10.9 | 5.7–9.6 | 5.8–9.3 | 6.2–10.1 | 5.4–7.9 |
Median | 1.0–5.0 | 1.4 | 2.2* | 2.5*** | 2.0 | 2.0****** | 2.0 | 1.8 |
IQR | - | 0.8–2.6 | 1.8–3.1 | 2.1–3.3 | 1.5–2.7 | 1.4–2.6 | 1.5–3.1 | 1.2–2.3 |
Median | 34.0–77.0 | 70.7 | 61.5* | 58.4 | 62.6 | 65.3****** | 64.8 | 65.3 |
IQR | - | 56.6–78.4 | 53.2–65.9 | 54.3–65.5 | 53.2–67.5 | 60.5–71.2 | 53.5–70.4 | 61.8–73.8 |
Median | 42.0–7.1 | 67.0 | 77.0** | 77.2**** | 76.0*** | 78.3******* | 80.8********** | 78.1 |
IQR | - | 56.3–70.1 | 72.1–80.3 | 73.4–80.0 | 69.2–81.7 | 75.9–82.2 | 76.2–82.7 | 75.9–80.1 |
Median | 80–100 | 80.2 | 81.2 | 82.1 | 80.8 | 76.3******** | 74.7********** | 82.1********* |
IQR | - | 71.5–88.9 | 77.2–88.6 | 77.4–90.1 | 76.3–88.6 | 70.3–83.0 | 70.2–79.3 | 71.5–83.3 |
Median | 200–300 | 276 | > 700** | > 700***** | > 700***** | > 700***** | > 700 | > 700 |
IQR | - | 250–360 | - | - | - | - | - | - |
Median | 0–10.0 | 6.43 | 135.10** | 135.60***** | 133.40***** | 117.9****** | 96.4********* | 88.8********** |
IQR | - | 4.74–12.76 | 98.95–155.07 | 105.00–155.00 | 76.00–155.50 | 103.38–148.23 | 75.4–136.6 | 52.8–106.9 |
Note: CPV subgroups at admission and after fluid resuscitation.
CPV, canine parvovirus; IQR, interquartile range; HCT, haematocrit; R, reaction time; K, clot formation time; MA, maximum amplitude; AT, antithrombin; α-angle, rapidity of fibrin build-up and cross-linking.
Significant difference: *,
Significant difference: ***,
Significant difference: ******,
Significant difference: *********,
After fluid resuscitation, the median R and K were significantly shorter (
No significant changes in the TEG variables were seen in the CPV group that received the CRI from the end of the initial fluid resuscitation up until 3 h of the CRI at 10 mL/kg, or between the hypovolaemic and dehydrated subgroups that received the CRI (
Haemostatic and inflammatory variables in the 10 canine parvovirus dogs at the end of the initial fluid resuscitation and after 3 h of a lactated ringer solution constant rate infusion. No significant differences were found between canine parvovirus groups or subgroups at fluid resuscitation or after 3 h of a lactated ringer solution constant rate infusion.
Variable | End of initial fluid resuscitation |
Post CRI (3 hours) |
||||
---|---|---|---|---|---|---|
CPV ( |
Hypovolaemic CPV ( |
Dehydrated CPV ( |
CPV ( |
Hypovolaemic CPV ( |
Dehydrated CPV ( |
|
Median | 0.35 | 0.37 | 0.33 | 0.34 | 0.36 | 0.32 |
IQR | 0.31–0.39 | 0.29–0.40 | 0.32 – - | 0.30–0.39 | 0.25–0.40 | 0.31– - |
Median | 305 | 295 | 315 | 296 | 292 | 301 |
IQR | 246–330 | 250–351 | 235– - | 249–367 | 249–410 | 202– - |
Median | 6.9 | 6.0 | 7.7 | 7.6 | 7.4 | 7.8 |
IQR | 5.8–11.6 | 5.6–11.5 | 5.8– - | 6.7–10.2 | 6.2–9.5 | 7.2– - |
Median | 1.7 | 1.6 | 1.8 | 1.8 | 1.8 | 1.7 |
IQR | 1.2–3.1 | 1.2–4.0 | 1.2– - | 1.6–3.7 | 1.6–3.6 | 1.6– - |
Median | 69.4 | 70.4 | 68.3 | 68.9 | 68.5 | 69.2 |
IQR | 58.0–73.3 | 49.7–73.1 | 60.7– - | 52.0–70.1 | 52.4–70.8 | 50.9– - |
Median | 79.2 | 80.5 | 78.3 | 81 | 82.8 | 79.1 |
IQR | 77.8–82.6 | 77.8–82.7 | 75.9– - | 77.1–83.1 | 76.4–83.1 | 77.9– - |
Median | 76.7 | 77.5 | 68.8 | 75.1 | 77.2 | 61.6 |
IQR | 68.4–80.0 | 72.9–79.3 | 59.8– - | 64.4–82.3 | 68.0–81.6 | 60.0– - |
CPV, canine parvovirus; IQR, interquartile range; HCT, haematocrit; R, reaction time; K, clot formation time; MA, maximum amplitude; AT, antithrombin; α-angle, rapidity of fibrin build-up and cross-linking.
After fluid resuscitation, for the CPV group the median HCT was significantly lower (
For the CPV group at admission, R had a moderate negative correlation with AT activity (rs = −0.405,
Post fluid resuscitation, MA retained a moderate positive correlation with platelet count (rs = 0.448,
The haemostatic status of dogs hospitalised with CPV enteritis at admission and after fluid resuscitation in this study was characterised by an increased clot strength (MA) and delayed clot initiation (R, K, α-angle) compared to healthy control dogs. The MA increased, clot initiation time shortened and AT activity decreased after crystalloid fluid resuscitation in dogs that received large volumes of fluids. The increased MA and decreased AT activity persisted up to 3 h after resuscitation with crystalloid fluid therapy, while still receiving a constant rate of fluids.
The R and K variables were significantly longer and the α-angle significantly smaller for the CPV group at admission, indicating delayed clot initiation and amplification compared to the healthy control group. The prolonged R and K were also present in the hypovolaemic subgroup at admission compared to the controls, but not in the dehydrated subgroup. The R, K and α-angle variables measure the time to, and rate of, clot generation (Donahue & Otto
Maximum amplitude reflects maximal clot strength and is influenced by the fibrin and fibrinogen concentration, platelet count, platelet function, thrombin concentration, factor XIII and HCT (Donahue & Otto
The changes noted in the CPV group of significantly shorter clot initiation (shorter R and K, and larger α-angle) and increased clot strength (larger MA) after fluid resuscitation compared to admission may be as a result of the crystalloid fluid therapy, the progression of disease or a combination of these two factors. In the CPV group, the magnitude of decrease in HCT after fluid resuscitation correlated moderately with the volume of fluid administered, as would be expected. The significant decrease in HCT after fluid therapy may have contributed to the increased MA, as reported in an
Within the subgroups, MA only increased significantly in the hypovolaemic group, while the R, K and α-angle did not change significantly for either subgroup. It is possible that the dehydrated group did not receive enough fluids to affect haemostasis, particularly MA, as these dogs received only 90 min – 120 min of a 24-h rehydration fluid plan. However, the volume of fluids administered was not correlated to the change in MA in the CPV group or either subgroup. The median volume of LRS received in the hypovolaemic group approached the 33%
The change in AT activity from admission to the end of fluid resuscitation had a strong correlation with the volume of fluid administered in the CPV group, as well as a strong correlation in the dehydrated subgroup and a moderate correlation in the hypovolaemic subgroup. A possible explanation for the correlation between the magnitude of reduction in AT activity and the volume of fluid administered could be that the reduction of AT activity after fluid therapy may, in part, be due to haemodilution. An
The lack of significant changes in haemostatic variables after 3 h of approximate maintenance-rate fluids in the CPV group, including subgroups, suggests that the shortened clot initiation time and increase in clot strength after fluid resuscitation is not transient and is unlikely to be exclusively a result of the effect of dilution by fluids.
Further investigation is required to determine the clinical relevance of a hypercoagulable state in dogs with CPV enteritis. The effect of crystalloids on TEG is significantly less than the effect of colloids in dogs with inflammation (Gauthier et al.
Several limitations inherent to clinical studies were present in this study. Disease severity and duration of illness could not be standardised. The determination of illness severity scores and severity prediction index would have allowed a more accurate comparison between the subgroups of dogs with CPV. All dogs diagnosed with CPV enteritis at the OVAH are offered hospitalisation and treatment, regardless of the severity of illness, and although a bias towards sicker dogs in this study is likely, it cannot be confirmed. The findings of the study can therefore not be extrapolated to dogs with milder disease treated on an outpatient basis. The extent of dehydration was estimated based on subjective guidelines. The relatively small sample size may have affected the statistical analysis, particularly in the CRI group. There were small variations in the time (5 min – 15 min) from admission blood sampling to the initiation of fluid therapy, the speed at which fluid boluses were given and the time between fluid boluses, although all of these were case-appropriate. The objective of the study was to determine haemostatic changes after fluid resuscitation, and the true effect of the minor time differences could not be determined from the study. The lack of a CPV group that did not receive fluids did not allow for determination of the cause of the increase in the hypercoagulable state after fluid administration, but would not have been ethically acceptable. The measurement of the D-dimer concentration and fibrin degradation products were not performed in this study. No measurable D-dimer concentrations were found in a previous study evaluating TEG in CPV (Otto et al.
In conclusion, this study showed that although clot initiation is delayed at admission in dogs hospitalised with CPV enteritis, clot strength is increased compared to healthy control dogs. Crystalloid fluid resuscitation resulted in shortened clot initiation (shorter R and K, and larger α-angle) and further increased clot strength, as evidenced by larger MA in dogs with CPV that were hypovolaemic at presentation. Increased clot strength persisted for hours after large-volume crystalloid fluid administration.
The authors gratefully acknowledge Carien Muller, Sr. Marizelle DeClerque and the staff at the Onderstepoort Veterinary Academic Hospital (OVAH) Clinical Pathology laboratory for their technical assistance as well as Lizette du Plessis and Antoinette Buys for performing the electron microscopy.
The authors have declared that no competing interests exist.
Z.W., P.P., A.G. and W.J.B. were responsible for the experimental and project design. Z.W. performed the data collection and TEG., Z.W. and P.P. performed the statistical analysis. Z.W., P.P., W.J.B. and A.G. drafted the manuscript and approved the final draft.
This study was funded, in part, by a grant from the Health and Welfare Sector Education and Training Authority (HWSETA).
Data sharing is not applicable to this article as no new data were created or analysed in this study.
The views and opinions expressed in this article are those of the authors and do not necessarily reflect the official policy or position of any affiliated agency of the authors.