Use of oseltamivir in the treatment of canine parvoviral enteritis
←
→
Page content transcription
If your browser does not render page correctly, please read the page content below
Journal of Veterinary Emergency and Critical Care 20(1) 2010, pp 132–142
Original Study doi:10.1111/j.1476-4431.2009.00404.x
Use of oseltamivir in the treatment of canine
parvoviral enteritis
Michelle R. Savigny, DVM, MS, DACVECC and Douglass K. Macintire, DVM, MS, DACVIM,
DACVECC
Abstract
Objective – To determine if oseltamivir with standard therapy for canine parvoviral enteritis ameliorates
disease morbidity, mortality, or both; to document significant adverse effects associated with its use.
Design – Prospective, randomized, blinded, placebo-controlled clinical trial.
Setting – University veterinary teaching hospital.
Animals – Thirty-five dogs.
Interventions – Standard therapy was administered to all dogs. Treatment dogs also received oseltamivir,
while control dogs received an equivalent volume of placebo.
Measurements and Main Results – Dogs were monitored daily according to a clinical scoring system,
physical parameters, and diagnostic evaluations. Dogs in the treatment group gained a significant percentage
of weight during hospitalization (mean, 12.6%; SD, 7.1%) versus the control dogs (mean, 4.5%; SD, 6.9%)
(P 5 0.006). Treatment dogs did not have any significant changes in their white blood cell (WBC) count, while
control dogs experienced a significant drop in their WBC counts during their initial stay. In addition, it did not
appear that oseltamivir use was associated with any major adverse clinical effects.
Conclusions – While a clear advantage to the use of oseltamivir was not established, a significant weight loss
during hospitalization, as well as a significant decrease in WBC count were documented in the control group.
No major adverse effects were identified that could be associated with oseltamivir administration. Based on
these results, the true role of oseltamivir in the treatment of parvoviral enteritis remains speculative, although
it is believed that further investigation is warranted.
(J Vet Emerg Crit Care 2010; 20(1): 132–142) doi: 10.1111/j.1476-4431.2009.00404.x
Keywords: bacterial translocation, diarrhea, neuraminidase inhibitors
CPV infects and replicates in rapidly dividing cells,
Introduction
most notably the lymphoid organs, latter myeloid pro-
Canine parvovirus (CPV) is a single-stranded DNA genitor cells in the bone marrow, and intestinal epithelial
virus that was first discovered in 1978.1 It is a hardy, cells. Replication results in cell destruction, causing a
highly contagious virus that remains a cause of signifi- clinical disease characterized by severe vomiting, hem-
cant disease in young dogs. It is estimated that over 1 orrhagic diarrhea, dehydration, and neutropenia. This
million dogs are affected each year in the United States2 disease is almost universally fatal without treatment,
despite the availability of an effective vaccine. with reported survival rates of only 9% in an experi-
mental model.3 Treatment increases this figure signifi-
cantly, with reported survival rates ranging from 64%
to 95%.2,4–10 Care given in specialized care settings
From the Department of Clinical Sciences, College of Veterinary Medicine,
Auburn University, Auburn, AL 36849, USA.
has been associated with increased survival rates
(96–100%)2,8 compared with nonspecialized care
Previously presented in abstract form at the 13th IVECCS, 2007, New Or-
leans, LA. settings (67–75%).2,7 It is postulated that this increase
Funding provided by: The Barry and Savannah French-Poodle Memorial may be due to the 24-hour care available, as well as the
Fund.
No financial conflict of interest exists.
likelihood that treatments such as plasma or colloid
therapy are more likely to be used in such settings.2
Address correspondence and reprint requests to
Michelle R. Savigny, Columbia River Veterinary Specialists, 6818 E. Fourth
Therapy for CPV enteritis is supportive and aimed
Plain Blvd., Vancouver, WA 98661, USA. Email: savigmr@gmail.com at controlling the clinical signs of disease. Multiple-
132 & Veterinary Emergency and Critical Care Society 2009Oseltamivir in parvoviral enteritis
directed therapies have been studied, such as human re- gression of CPV enteritis in a nonviral-dependent man-
combinant granulocyte colony stimulating factor,8,9,11 ner. In one report, 90% of dogs that died from CPV
equine antiendotoxin,7,12 recombinant bactericidal/per- enteritis had Escherichia coli cultured from their livers
meability-increasing protein,2 and interferon o13,14 with and lungs.24 Acute respiratory distress syndrome-like
variable or disappointing results. Early enteral nutrition changes in the lungs of these dogs on histopathologic
(EEN) has been the only modality to date to show prom- examination were also present, demonstrating the
ise for a shortened recovery time and decreased disease potential for organ failure secondary to the systemic
morbidity associated with CPV enteritis.10 However, the inflammation associated with this disease.24
need remains for a therapeutic agent that will help ame- The aim of this study was to investigate the efficacy
liorate the morbidity and mortality of this disease, and in and adverse effects of oseltamivir when added to the
doing so also decrease the cost of treatment. While the standard treatment of CPV enteritis. Our hypothesis
survival rate with aggressive treatment is very good, was that oseltamivir would ameliorate the disease mor-
financial constraints often result in suboptimal treatment bidity and mortality associated with naturally occur-
with lower survival, or even euthanasia. This fact dem- ring CPV enteritis, thereby decreasing hospitalization
onstrates the clear need for a directed therapy to make time, as well as the need for colloid and other adjunc-
the appropriate treatment for CPV enteritis more finan- tive therapies.
cially feasible for owners or shelters.
Oseltamivir is a neuraminidase (NA) inhibitor orig-
Materials and Methods
inally designed to treat human influenza virus.15 It has
recently also shown efficacy in the treatment of avian Study design
influenza.16,17 Oseltamivir inhibits the viral NA enzyme The present study was a prospective, randomized,
and thus prevents the cleavage of sialic acid residues. blinded, placebo-controlled clinical trial of the effects of
This cleavage is necessary for liberating newly formed oseltamivir in dogs with naturally occurring parvoviral
virons from the host cell, as well as for preventing the enteritis. Client-owned dogs and dogs from the local
aggregation of viral particles. Both the viron liberation animal shelter with CPV enteritis were recruited be-
and decreased aggregation are mechanisms necessary tween April 2005 and August 2006. A financial incen-
for the spread and dissemination of the virus through- tive was offered to the owner or agent to allow their
out the host in order to further infection. The NA en- dog to participate in the study. Inclusion criteria for
zyme is also needed for the virus to cleave the sialic enrollment included a positive CPV fecal antigen test,a
acid residues in mucin to allow for penetration presence of appropriate clinical signs (vomiting, diar-
of this protective layer and infection of respiratory rhea, lethargy, and anorexia), and lack of any treatment
epithelial tissue. before enrollment. Informed consent was received from
Anecdotal reports of oseltamivir use in veterinary all owners or agents before any treatment at the Au-
medicine claim that it is associated with a less severe burn University Small Animal Teaching Hospital. The
form of disease and a quicker recovery when admin- study protocol was reviewed and approved by the An-
istered to dogs with CPV enteritis. However, unlike the imal Care and Use Committee of Auburn University.
influenza virus, CPV does not rely on NA for effective Following enrollment in the study, dogs were assigned
replication. Therefore, any beneficial effects that may to either the treatment or the control group randomly in a
be present would not be due to a direct antiviral action. block of 20 and then blocks of 10 dogs (equal numbers of
Human studies have shown a significant decrease dogs enrolled in each arm in each block). Group assign-
in the development of bacterial infections secondary ment was selected based on the blind drawing of a
to influenza when oseltamivir is used.15,18–23 This effect folded piece of paper designated for either treatment or
is believed to be due to a decrease in bacterial perme- control. Assignment was allocated by hospital personnel
ation through the mucin layer of the respiratory not directly involved in the clinical aspects of the trial.
epithelial cells, because NA is necessary for this pro- Group assignments were uncovered to the investigators
cess to occur. It is postulated that oseltamivir could also only at the conclusion of the study.
have a similar inhibitory effect on bacterial permeation
through the mucin layer of the gut epithelial cells. This Standard treatment
inhibition would decrease bacterial translocation, A treatment protocol was designed to standardize ther-
resulting in a potentially lower incidence of endo- apy between the 2 groups with the exception of
toxemia, sepsis, systemic inflammatory response, and oseltamivir administration to the treatment group
eventual multisystem failure, which is thought to be the and a placebo to the control group. No deviations
main mechanism of mortality of CPV enteritis. Thus, were made from this protocol for any of the patients.
oseltamivir could have an effect on the disease pro- An intravenous (IV) catheter was placed in all dogs,
& Veterinary Emergency and Critical Care Society 2009, doi: 10.1111/j.1476-4431.2009.00404.x 133M.R. Savigny & D.K. Macintire either a peripheral cephalic catheter or central venous the day of discharge for the purpose of data analysis. catheter in a jugular vein. Balanced electrolyte solu- Postmortem exams were performed on all nonsurviv- tionb, c was administered IV at an initial rate to replace ing dogs. an estimated dehydration deficit over a minimum of 2 hours and a maximum of 4 hours. If the dog was Oseltamivir treatment assessed as being in shock, the crystalloid solution was Dogs assigned to the treatment group received oseltam- bolused at a rate of 10–30 mL/kg over approximately ivirm (2 mg/kg, PO, q 12 h). An equivalent volume of a 20 minutes. Boluses were repeated until physical pa- placebo, consisting of a standard suspension agent with rameter evaluation by the attending clinician indicated color additive, was administered to dogs in the control that shock had resolved, and the remaining fluid deficit group. Prior experience with oseltamivir by the authors estimated to replace dehydration was administered has shown that dogs often react to its taste and fre- as above. No patient required the administration of quently vomit shortly after administration. Dilution of colloidal solutions to treat a state of shock. Assessment the oseltamivir with water (1:1) before administration of hydration and perfusion status, as well as fluid rate seemed to decrease these reactions; therefore, in this choice for all dogs was performed by a single investi- study, both oseltamivir and placebo were diluted (1:1) gator (M.R.S.). After rehydration, the hydration status with water to lessen the risk of adverse reaction to the was reassessed via physical parameters and the fluid oseltamivir. In addition, for dogs receiving chlorprom- rate adjusted as deemed appropriate. Fluids were sup- azine for protracted vomiting, an attempt was made to plemented with potassium chloride,d dextrose,e dose this medication 30–60 minutes before administra- or both, as needed based on laboratory results. tion of the oseltamivir or placebo to minimize the risk Ampicillinf (22 mg/kg, IV, q 8 h), enrofloxacing (5 mg/ of vomiting. Given its propensity for gastric adverse kg, IV, q 12 h), and metoclopromideh (1–2 mg/kg/d, IV effects (vomiting, nausea),15 significant adverse effects infusion) were administered to all patients. If vomiting associated with the administration of oseltamivir, de- persisted at a rate of more than twice per 12-hour pe- spite these precautions, was recorded using in the clin- riod despite a maximum rate of metoclopramide, chlor- ical scoring system used. promazinei (0.5 mg/kg, SQ, q 8 h) was added. Pyrantel pamoatej (10 mg/kg, PO) was administered to all dogs Monitoring/data acquisition within the first 3 days of arrival to eradicate intestinal Historical data, including previous vaccination against parasites. If the total protein (TP) measured using a CPV and the duration of clinical signs as noted by the refractometer fell below 35 g/L (3.5 g/dL), hetastarchk owner or caretaker (rounded to the closest 12-h period was infused at a rate of 10–20 mL/kg/d. If anemia, de- of time), were obtained when this information was fined as a packed cell volume (PCV) 20%, was pres- available at study entry. Signalment for each dog was ent, a blood transfusion was administered. The blood also recorded, as was its estimated percentage of product administered depended on availability at that dehydration at entry. The estimated extracellular fluid time, but was either packed red blood cells (10 mL/kg, deficit was derived from a combination of clinical char- IV, over 4 h) or a fresh whole blood transfusion acteristics such as skin turgor, mucous membrane color (10–20 mL/kg, IV, over 4 h). If vomiting episodes were and capillary refill time, pulse quality and heart rate, o4 per 12-hour period, water and a bland dietl were and ocular position (ie, sunken or not). Laboratory offered. If either appeared to induce nausea by their values, including PCV, TP, lactate, and urine specific presentation, they were immediately removed from the gravity when a voided sample was available, were also animal. Voluntary eating was allowed through mild taken into consideration. Baseline diagnostic testing, vomiting episodes (o4 per 12-h period). Food was not consisting of a CBC, PCV, TP measured using a force fed or tube fed to any dog. refractometer, serum electrolyte concentrations (sodium, Discontinuation of IV fluids was at the discretion of a potassium, and chloride), and blood glucose concentra- single investigator (M.R.S.). This occurred primarily tion, was evaluated for all dogs at entry. These values when the animal was no longer vomiting and consis- were monitored daily for all dogs. With the exception of tently eating and drinking sufficiently to maintain the CBC data, these values were not analyzed statistically hydration. Once fluids were discontinued, all patients but were monitored for guiding treatment decisions only. were monitored for at least 12 hours before discharge For white blood cell (WBC) values, the following vari- from the hospital in order to ensure no relapse of clin- ables were evaluated: initial total WBC, neutrophil ical signs. The day of discharge was assigned as the day (NEUT), and lymphocyte (LYMPH) counts; the absolute when it was deemed the dog was healthy enough to be nadir value and the day of hospitalization (with day of discharged. In those animals that did not survive, the presentation being Day 1) on which the nadir occurred day of death was considered an endpoint equivalent to for each of the WBC, NEUT, and LYMPH counts; and the 134 & Veterinary Emergency and Critical Care Society 2009, doi: 10.1111/j.1476-4431.2009.00404.x
Oseltamivir in parvoviral enteritis
number of days for which the counts for each value were of the 4 categories were totaled for each dog per day,
considered to show a clinically relevant decrease. These with a possible category score ranging from 0 (normal)
decreases were defined by a WBC o3.0 109/L, NEUT to 6 (most severely affected) per 24-hour period. In
o2.0 109/L, and LYMPH o1.0 109/L. In addition, addition, a cumulative score consisting of a total of all
the WBC count change from presentation (Day 1) to Days 4 categories was calculated for each dog per day, with a
2, 3, 4, and 5 was analyzed for each group. The WBC data possible score ranging from 0 (normal) to 24 (most se-
in conjunction with clinical scores on presentation and verely affected). Occurrence of adverse events associ-
Day 4 were also analyzed. Day 4 was chosen for this ated with medication administration was a subjective
comparison to represent a period in time that should observation based on the timing of vomiting or in-
have encompassed NEUT and clinical recovery for most creased nausea in the immediate period following
dogs based on the expected timeline of disease progres- drug/placebo dosing. Other indicators of possible
sion as described previously.11 Body weight was recorded adverse reactions to oseltamivir administration could
twice daily, with the same scale used to monitor each dog also be indicated by the routine monitoring of other
during its stay for consistency. In addition, vital param- standard clinicopathologic data and physical exam
eters (heart rate, respiratory rate, rectal temperature, mu- characteristics throughout the study.
cous membrane color, and capillary refill time) were
assessed a minimum of twice daily, as were hydration
status and mentation. Days on which dogs demonstrated Statistical Analysis
systemic inflammatory response syndrome (SIRS) criteria
The Shapiro-Wilks test was used to evaluate the distri-
were also calculated as a percentage of days of their total
bution of continuous variables. Continuous variables
stay (days with positive SIRS criteria/total days in hos-
not normally distributed are described as median (min-
pital 100%). SIRS was defined as the presence of at
imum, maximum) and normally distributed continuous
least 2 of the following 4 criteria: (1) temperature 439.21C
variables are described as mean (standard deviation).
(102.51F) or o37.81C (100.01F), (2) heart rate 4140/min,
The Wilcoxon rank sum test was used to test for differ-
(3) respiratory rate 440/min, or (4) total WBC count
ences in not normally distributed continuous variables,
419.0 109/L or o6.0 109/L. These criteria were ex-
while the t test was used for normally distributed vari-
trapolated from a recent study of dogs with CPV enteri-
ables. Categorical variables were described using per-
tis,25 with the exception of a modification of respiratory
centage and the Fisher’s exact test was used to test for
rate parameters from 420/min to 440/min. This mod-
differences between the treatment and control groups.
ification was done in an effort to increase the specificity
For all comparisons, a Po0.05 was considered signifi-
of the criteria, as it was noted that most dogs in this study
cant. All statistical analyses were performed using a
had respiratory rates in the range of 21–36/min.
statistical software program.n
Clinical scoring system
Results
A previously published clinical scoring system10 was
used to evaluate 4 clinical attributes of each patient: A total of 35 dogs were enrolled: 19 in the treatment
attitude, appetite, vomiting, and feces. A score of 0 group and 16 in the control group. Power analysis was
represented a clinically normal parameter, with in- not performed as part of the design of the study, but
creasing severity of signs as the score increased up to a post hoc analysis revealed that 47 dogs would be
maximum of 3 for each variable (Table 1). Scores were needed in each group to give the study a power of 0.80
assigned twice daily, to encompass the previous at a significance level of 0.05 based on measurement of
12-hour period, and were assigned by the same inves- outcome. Only 1 dog that was eligible during the trial
tigator for all dogs (M.R.S.). The clinical scores for each period was not included due to lack of owner consent.
Table 1: Clinical scoring system
Score Attitude Appetite Vomiting Feces
0 Normal Normal Absent Well formed or absent
1 Mild to moderate depression Voluntarily eats small amounts Mild; once per 12 hours Soft or pasty feces
2 Severe depression No interest in food Moderate; 2–5 times per 12 hours Watery diarrhea, nonbloody
3 Collapsed or moribund Not offered Severe; 46 times per 12 hours Watery, bloody diarrhea
Scores for each category were assigned to each dog twice daily to encompass the previous 12-hour period.
& Veterinary Emergency and Critical Care Society 2009, doi: 10.1111/j.1476-4431.2009.00404.x 135M.R. Savigny & D.K. Macintire
All dogs that entered the study completed the trial. 20
There were 3 shelter dogs included in the study, all of
which were in the control group. Most dogs (30/35)
10
% Weight Change
were of the mixed-breed variety, with the purebreds
consisting of 2 American Pit Bull Terriers and 1 each of
Dachshund, Beagle, and Labrador Retriever. The 0
median age of dogs in the control group was 14 weeks
(8, 36 w), while that of the treatment group was 12
weeks (8, 44 w) (P 5 0.50). Twenty-one of 35 (60%) dogs –10
were female, with 10 of 21 (48%) randomized to the
control group and 11 of 21 (52%) randomized to the
–20
treatment group. Fourteen of 35 (40%) dogs were male,
Control Treatment
with 6 of 14 (43%) randomized to the control group and
8 of 14 (57%) to the treatment group (P 5 1.0). All dogs Figure 1: Boxplots of percent weight change during hospital stay
were sexually intact. Vaccination status was known for control and treatment dogs. A significant difference (P 5 0.006)
for 10 of 16 (62%) dogs in the control group and 13 of was found in the change in weight from entry until discharge
between the control and treatment groups. The control group lost
19 (68%) dogs in the treatment group. Of these, 5 of 10
a mean of 4.5% (6.9%) of their body weight, and the treatment
(50%) dogs in the control group had received at least 1
dogs gained a small amount with the mean change of 2.6% (7.1%)
vaccination against CPV, whereas 6 of 19 (32%) in the of their body weight. Percent weight change was calculated as
treatment group had been vaccinated at least once (discharge weight entry weight)/entry weight 100.
against CPV (P 5 1.0). The duration of clinical signs
before presentation was known for 15 of 16 (94%) con-
trol dogs and 16 of 19 (84%) treatment dogs. Mean days the new calculation without these dogs resulted in a
sick before presentation for the control group was 1.4 mean percent weight change of 5.2% (7.5%), compared
days (0.9 d), while that for the treatment group was 1.8 with the mean percent weight change of 2.6% (7.1%) for
days (1.0 d) (P 5 0.31). No statistically significant differ- the treatment dogs (P 5 0.006).
ences were found between groups in the baseline char- The percentage of days in the hospital that SIRS
acteristics of age, sex, vaccination status, or duration of criteria were met was calculated for each dog. There
clinical signs before presentation. was no significant difference between groups, with
There was no significant difference between groups control dogs meeting SIRS criteria a mean of 52% of
in the degree of estimated dehydration at entry. The days, versus 54% for treatment dogs (P 5 0.91).
control dogs were estimated to have a mean fluid The WBC values that were evaluated (initial counts
deficit of 6.3% (1.5%), while the treatment dogs were for WBC, NEUT, and LYMPH; the nadir values and day
estimated at 6.9% (1.7%) (P 5 0.35). In addition, there of nadir; and clinically relevant decreases in counts for
was no statistical difference among the weights at entry WBC, NEUT, and LYMPH) were compared between
or the weights at discharge between the 2 groups. Dogs groups. No significant differences were found for any
in the control group had a median entry weight of of these values between groups. When entry (Day 1)
6.7 kg (1.8, 28.2 kg), and those of the treatment group WBC counts were compared with counts on Days 2, 3,
4 kg (1.6, 25 kg) (P 5 0.21). At discharge, the median 4, and 5, significant differences were found for the con-
weight of control dogs was 6.5 kg (1.8, 27.3 kg) and that of trol group but not the treatment group. The control
the treatment group was 4.4 kg (1.6, 28.6 kg) (P 5 0.42). dogs, with a median WBC count on entry of 7.83 (0.59,
However, a significant difference was found in the 18.3) 109/L, showed a significant decline on Day 2
weight change from entry until discharge. Dogs in the (6.1 [0.39, 14.6] 109/L) (P 5 0.04), Day 3 (6.3 [0.5,
control group experienced a median change of 0.21 kg 10.9] 109/L) (P 5 0.04), and most notably on Day 4
( 2.8, 0.5 kg), while those in the treatment group had a (2.49 [0.41, 13.6] 109/L) (P 5 0.009). No difference
median change of 0.07 kg ( 1, 3.6 kg) (P 5 0.01). This from entry to Day 5 (3.33 [0.4, 11.0] 109/L) was found
correlates also to a significant difference in the percentage (P 5 0.08). The treatment dogs, on the other hand, had no
of change in body weight ([discharge weight entry significant changes in their median WBC counts from
weight/entry weight] 100) (Figure 1). Dogs in the con- entry (4.95 [0.5, 24.1] 109/L) to Day 2 (2.75 [0.46,
trol group had a mean change of 4.5% (6.9%), and 14.5] 109/L) (P 5 0.08), Day 3 (3.46 [0.46, 11.9] 109/L)
those in the treatment group a mean change of 2.6% (P 5 0.10), Day 4 (4.7 [0.71, 16,28] 109/L) (P 5 0.55), or
(7.1%) (P 5 0.006). When this analysis was repeated for Day 5 (4.79 [0.78, 24.4] 109/L) (P 5 0.97) (Figure 2).
survivors only, the results were still found to be signifi- It was noted that an approximately equal proportion
cant. The control group contained all 3 nonsurvivors, and of dogs in each group experienced a severe decline in
136 & Veterinary Emergency and Critical Care Society 2009, doi: 10.1111/j.1476-4431.2009.00404.xOseltamivir in parvoviral enteritis
9 Table 2: Comparison of values between groups across time
8 Treatment Control
Initial Day 4 Day 5 Initial Day 4 Day 5
7
WBC 4.95 4.71 4.79 7.83 2.49n 3.28
Median WBC (x1000/ul)
6 Attitude 2 1 1 2 2 2
Appetite 5 2 2 6 4 3
5 Vomit 2 1 1 2 3 2
Fecal 3 2 2 2 3 3
Cumulative 12 7 6 13 11 9
4
Median values for white blood cells (WBC) ( 109/L), and average daily
3
total clinical scores at presentation and on Days 4 and 5 of hospitalization.
There was a significant decrease in WBC count from entry to Day 4 in the
2 control group, but no significant changes over time in WBC count in the
treatment group.
1 Control n
Significant difference from initial count; P 5 0.009.
Treatment
0
24 h 48 h 72 h 96 h 120 h
Time of hospitalization chlorpromazine was necessary in 10 of 19 (53%) dogs in
the treatment group, and 9 of 16 (56%) dogs in the
Figure 2: Change in median white blood cell (WBC) count.
control group. Dogs in the treatment group that did
Control dogs showed a significant decrease in WBC count from
entry (0 h) to hours 48, 72, and 96. nSignificant decrease from
receive chlorpromazine did so an average of 21% of
presentation. their days in the hospital, while the dogs requiring it in
the control group were given chlorpromazine an aver-
age of 30% of their days in the hospital. The overall
WBC, with nadirs occurring at o0.1 109/L in 7 of 16 survival rate was 91% (32/35). Three dogs died, all
(44%) control dogs and 8 of 19 (42%) treatment dogs. Of from the control group, giving this group a survival
these, 2 of the control dogs did not survive (1 died, rate of 81% (13/16) compared with a survival rate of
1 euthanized) while there were no deaths in the treat- 100% in the treatment group. The difference between
ment group. Because of the small number of dogs the 2 groups, however, was not significant (P 5 0.09).
involved, statistical analysis was not performed. Only 1 of the dogs that died was from the animal shel-
When the clinical scores were compared day by day ter, the others were client owned. One animal was eu-
for each category as well as the cumulative total for that thanized after severe progression of clinical signs
day, no significant differences were found between despite treatment, and it was deemed that the dog
groups with the exception of Day 6 for the appetite was suffering and would not recover, while the other 2
score (P 5 0.02), when the treatment group scores were had natural deaths.
significantly lower (more normal). The trends in WBC Postmortem examinations were performed on all
and clinical scores for both groups are presented in nonsurviving dogs. Findings were consistent with a
Table 2. While there were no significant differences diagnosis of CPV enteritis. All dogs had diffuse, severe,
found in scores between groups, it can be seen that necrotizing enteritis. Bone marrow was examined in 2
there was a slight trend for lower scores over time in of 3 dogs, both showing sections of moderate hypo-
the treatment group. Vomiting, when it did occur, did cellularity. While 2 dogs were noted to have diffuse
not appear to be associated with administration of congestion and edema in their lungs, the third dog was
oral medications. found to have a mild interstitial pneumonia.
No difference was found in the duration of hospital-
ization between the 2 groups. Dogs in the control group
Discussion
had a mean stay of 5.9 days (2.6 d), and those of the
treatment group 6.0 days (2.3 d) (P 5 1.0). Colloid ther- The use of oseltamivir in addition to standard therapy
apy was not required often, as the median number for naturally occurring CPV enteritis did not result in a
of days on colloids for the control group was 0 days significant decrease in hospitalization time, treatments
(0, 3 d) and also 0 days (0, 5 d) for the treatment group needed, clinical scores, morbidity, or mortality. Dogs in
(P 5 0.5). None of the 16 dogs in the control group the treatment group, however, gained significantly
received a blood transfusion, while 2 of 19 (10%) dogs more weight than those in the control group, which
in the treatment group did (P 5 0.5). The addition of on average, lost weight. Initial dehydration was not
& Veterinary Emergency and Critical Care Society 2009, doi: 10.1111/j.1476-4431.2009.00404.x 137M.R. Savigny & D.K. Macintire different between groups and therefore was unlikely to dogs receiving oseltamivir. While a direct link between influence observed weight gain. In addition, it was preservation of weight and outcome was not found, noted that the control group experienced a significant these findings were both present with oseltamivir decrease in WBC count from entry (Day 1) to Days 2, 3, administration and not in the control subjects. and 4 of hospitalization, while those dogs in the treat- A veterinary study investigating the effect of EEN on ment group had no significant decline in WBC count for dogs with parvoviral enteritis10 found that dogs in the any of the initial 5 days of hospitalization. EEN group had a significant increase in weight from CPV does not rely on NA for replication. However, entry on all days of the study, while the conventional anecdotal reports of the use of oseltamivir in dogs with group had no significant change in weight. In addition, CPV enteritis have claimed decreased morbidity and dogs in the EEN group showed a more rapid clinical shortened recovery time in the treated dogs. It is spec- improvement, based on normalization of clinical scores, ulated that the drug may inhibit bacterial translocation than did the conventional group. These results also that subsequently leads to endotoxemia, sepsis, suggest that the change in weight might be a marker SIRS, and death. Bacterial adherence and colonization of disease severity. Dogs with less severe disease, as of respiratory epithelial cells is potentiated in the pres- interpreted based on clinical scores, are more apt to ence of viral NA, and inhibited with NA blocking maintain their body weight. The mechanism for this agents.19–21 It is believed that the bacteria that com- effect, while yet to be truly elucidated, is thought to be monly invade the lower respiratory tract express their associated with improved enterocyte health secondary own NA, thus enabling them to penetrate the protective to the trophic effects of enternal nutrition, resulting in mucin layer and infect the epithelial cells.19 Although less bacterial translocation. Oseltamivir is postulated to unproven, a similar mechanism may exist in the gas- also help decrease bacterial translocation across the trointestinal tract. Oseltamivir may exert a beneficial gut endothelial cells by blocking the action of NA. effect by inhibiting NA on enteric bacteria, preventing Decreased bacterial adherence and subsequent bacterial their translocation across the gastrointestinal mucosal translocation would result in less cytokine release from barrier. In CPV enteritis, the mucosal barrier is already the gut-associated lymphatic tissue. By blocking bacte- impaired, allowing easier passage of bacteria. If bacte- rial translocation through NA inhibition, oseltamivir rial NA plays a role similar to that in the lungs, the NA could decrease disease severity both locally in the gas- would cleave sialic acid residues on the gut epithelium, trointestinal tract, and systemically. Reduced severity of exposing receptor sites for bacterial adherence and disease would likely contribute to weight gain in further encouraging translocation. In addition, CPV treated animals. Although the treatment group in suppresses the dog’s immune system, both humoral the present study showed an increase in weight dur- and cell-mediated factors, allowing for systemic spread ing hospitalization, suggesting a less severe form of of bacteria and the resultant deleterious effects. Further disease, this improvement in disease morbidity was not studies are needed to accurately define the actual demonstrated with our clinical scoring system. This mechanism behind the observed anecdotal benefits of discrepancy could be due to an inherent limitation of the use of oseltamivir to treat CPV enteritis. sensitivity with the scoring system utilized, or the pos- The importance and implications of weight gain sibility that there really was no effect. Although weight associated with oseltamivir treatment are unknown at gain in treated animals appears to be a beneficial effect this time. Other studies have shown that a significant associated with oseltamivir administration, the inter- change in weight in study subjects is associated with an pretation of the weight change and assignment of as- improved outcome from infectious disease. In 1 inves- sociations with this finding must be done with caution tigation,20 mice treated prophylactically with oseltami- because the actual mechanism is unknown and inter- vir lost an average of only 5% of their body weight, and pretations are speculative. had a survival of 100% after an influenza and bacterial A decline in WBCs is a considered a hallmark sign of challenge. In contrast, the control mice lost an average CPV infection. This decline is postulated to be due to of 25% of their body weight, and no mice survived the multiple effects, including lymphoid necrosis, a direct same viral/bacterial challenge. Weight loss was corre- toxic effect of CPV on neutrophil precursors in the bone lated with increased severity of disease and decreased marrow, as well as overwhelming consumption sec- survival rate in the mice in the influenza study. In our ondary to acute inflammation of the gut. The decline in study, an increased survival rate and increased weight WBC count has been associated with clinical signs in gain were seen in dogs receiving oseltamivir. While the CPV enteritis, both in timeline11 and outcome.9 Neutro- actual mechanisms behind the weight gain in the dogs phil nadir has been shown to coincide with the period in the treatment group are unknown, one possible when clinical signs are at their worst, and leukopenic explanation is that disease severity was decreased in dogs have been demonstrated to have a decreased sur- 138 & Veterinary Emergency and Critical Care Society 2009, doi: 10.1111/j.1476-4431.2009.00404.x
Oseltamivir in parvoviral enteritis vival. This decreased survival is speculated to be due to that adverse effects of oseltamivir were minimal, as were the impaired ability of the immune system to fight off a beneficial effects on decreasing disease morbidity. systemic infection and its associated inflammatory The intensity of treatment required and the expected effects secondary to bacterial translocation. A higher cost of treatment were inferred based on additional WBC count could mean a stronger immune system and therapy, such as colloid infusion or blood transfusions, an increased ability to protect against the negative as well as prolonged hospitalization times. Colloids effects of sepsis development. The control dogs in this were not frequently used, in contrast to a previous re- study showed a significant decline in WBC count from port.25 Indications for the institution of colloid therapy entry to Days 2, 3, and most notably to Day 4. The dogs were not elucidated in the previous study. Protocol in receiving oseltamivir had no significant changes in their this study required decrease in total protein to 35 g/L. WBC count from entry to any of the initial 5 days of Much more modest decreases, often around 40–45 g/L, hospitalization. Although clinical scores did not show a are frequently used in the clinic setting as indication for difference in disease severity, and survival rates, while treatment with colloids. A higher cutoff value as a trig- different, were not significantly so, it can be argued that ger for colloid infusion likely would have increased the this lack of WBC decline could be a protective effect of incidence of its use in this study. Blood transfusions oseltamivir. Given a more sensitive clinical scoring sys- were also rarely indicated. The 2 cases that did require tem and a larger number of dogs, this protective effect a transfusion received fresh whole blood, rather than could potentially be associated with a decreased dis- packed red blood cells. Packed red blood cell availabil- ease morbidity and mortality. Further studies are ity was very limited at the time of the first required needed to more fully define this relationship. transfusion. In order to minimize differences in treat- Although the treatment dogs had a slightly lower ment between dogs, when the second transfusion be- median WBC count on entry than the control dogs, this came necessary, although packed red blood cells were difference was not found to be significant. Being that available, fresh whole blood from the previous donor there was no difference in entry values for WBC count, was again administered. The total volume of crystall- nor in the reported duration of clinical signs before en- oids as well as colloids administered between the 2 try, both groups should have been at approximately groups may have been helpful to evaluate. Dogs expe- equivalent stages of their disease process and the time- riencing greater fluid loss from vomiting or diarrhea as line of disease progression should not have differed well as having less voluntary intake per os would have significantly between the groups. Thus, it appears that a greater IV fluid need. This would correlate with an the lack of WBC count decline in the treatment group as increased severity of clinical signs and manifestation of opposed to the control group is a beneficial effect of the disease process. Unfortunately, the collection of this oseltamivir administration. data was not possible from certain dogs due to record- The main adverse effects of oseltamivir reported in ing errors or technical difficulties. Therefore this vari- humans are gastrointestinal effects apparently due to di- able was not analyzed. rect local irritation of the gastric mucosa.15 In the expe- The duration of clinical signs before presentation was rience of the authors, dogs will also often react to the taste not different between the groups. It has been reported of the oseltamivir suspension and nausea and vomiting that for human influenza viral infections, oseltamivir is can be encountered. Dilution with water just before ad- most effective if started within the first 12 hours of ministration appears to minimize these effects. This prac- clinical signs, with efficacy decreasing up to 48 hours.26 tice was utilized in this study in an effort to not only For every 6-hour delay in starting oseltamivir, the du- avoid uncovering group assignment and thereby institut- ration of illness is predicted to increase by approxi- ing a bias, but also in an effort to keep the clinical scores mately 8%.26 Whether this also holds true for its use in an accurate representation of the disease process in the CPV enteritis is unknown. Because replication of CPV animal and not obscure these scores with drug reaction. does not depend on NA, administration of oseltamivir However, it is still possible that oseltamivir caused in- in the early stages of infection is unlikely to diminish creased vomiting and nausea as a side effect of the drug, viral replication and dissemination as with the influ- thereby concealing any benefit evident by analysis of the enza virus, and thus a time-efficacy response is not ex- clinical scores. There was a lack of any significant differ- pected. Rather, with the proposed mechanism against ence in clinical scores between groups, specifically vom- bacterial translocation, oseltamivir may have a greater iting and appetite scores, as well as the subjective impact when administered during the period of leu- observation that administration of the oral medications kopenia and severe clinical signs. Further investigation (oseltamivir or placebo) was not associated with increased is needed to expand on these speculations. nausea or vomiting directly afterwards. Both of these ob- A clinical scoring system was utilized in order to servations support the interpretation of results indicating evaluate the subjective criteria of attitude and appetite, & Veterinary Emergency and Critical Care Society 2009, doi: 10.1111/j.1476-4431.2009.00404.x 139
M.R. Savigny & D.K. Macintire as well as to quantify the severity of vomiting and ensuing endotoxemia, SIRS, and organ dysfunction that characterize the feces to allow for comparison across can develop. In this study, although certain physical and dogs. One investigator (M.R.S.) had the responsibility clinic pathologic parameters were monitored, there was of assigning scores to all dogs to minimize interobserv- no direct test for the presence of bacterial translocation er variability. This investigator was also blinded to or sepsis. SIRS criteria were evaluated, but this is a group assignment in order to minimize bias. crude assessment, especially considering the confound- Values for each category and cumulative scores were ing factors. As the dogs were starting to feel better, they compared between the 2 groups for each day, and would oftentimes become very excitable when being although mild trends could be seen for lower scores in handled. This frequently resulted in an elevation of their the treatment group, there were no significant differ- heart rate to over 140/min, or their respiratory rate to ences. This could be attributed to the small sample size 440/min. The presence of these 2 variables would in this study, as well as the variability in the timeline of classify these healthy, excitable puppies as illness among dogs. Because dogs presented in all fulfilling the criteria SIRS. In addition, the effect of the stages of their disease (ie, clinical symptoms for o12 h virus itself on the WBC count confounds the definition to up to 4 d), a comparison of scores per day may not slightly. A WBC of o6.0 109/L can simply reflect illustrate true differences and a larger group would be destruction of progenitor cells in the bone marrow and needed to further examine this effect. In addition, the is not necessarily associated with systemic inflam- clinical scoring system utilized is a very simple system, mation. Other methods to evaluate for the presence of and as such, was relatively insensitive in its ability to bacterial translocation, endotoxemia, or SIRS may be differentiate between various stages of aberrancy in more fruitful. Culture of mesenteric lymph nodes is each of the clinical attributes. This scoring system failed considered the gold standard in human medicine and to identify subtle yet clinically significant differences. A animal models for evaluation of bacterial transloca- scoring system with a greater degree of stratification tion.30 The feasibility of this procedure in this patient between assigned values may allow for a greater sen- population (client-owned, live animals) and setting is sitivity and a more accurate representation of the clin- questionable. Other methods, such as blood cultures or ical status of the patient. measurement of serum endotoxin levels or other in- Limitations of this study do involve the concern of flammatory mediators could be used to differentiate administration of an oral medication to a vomiting animals in which bacterial translocation is present from patient, and its potential for variable systemic absorption those in which it is not. Further investigation would be in the face of a diseased gastrointestinal tract. The specific needed before any true conclusions can be made. site of oseltamivir absorption from the gastrointestinal Oseltamivir administration to animals is not an tract has not yet been fully elucidated. It has been shown approved use of the drug. However, this would not to be absorbed equally from the stomach, proximal, and be the first instance of off-label use of a medication in distal small intestinal segments in an experimental veterinary medicine. Yet concern exists over the accept- study.27 Colonic absorption does appear to occur, but to ability of using this drug in animals. This concern a lesser extent and with a greater delay than other sites. revolves around the potential that use of the drug Early safety studies of oseltamivir show that it has a may enhance resistance to this medication, specifically bioavailability of 73% in healthy dogs, with detectable by the avian influenza virus. Oseltamivir is consi- levels of the drug in plasma within approximately 30 dered the first line agent in the prevention of a viral minutes after oral administration.27 Oseltamivir does re- epidemic if the avian influenza virus appears in the quire transformation to its active metabolite by esterases United States. There are a very limited number of drugs located within the liver, and to a certain degree, within currently available to treat or prevent avian influenza in the intestinal system.28,29 The importance of the intestinal humans; oseltamivir is one of these. If resistance to system esterases is unknown. Because of this need for oseltamivir develops, the ability to treat and prevent a transformation, it is believed that oseltamivir effects are viral epidemic could be severely hampered. Recently, not due to a purely local action, but do require the World Health Organization has found that the systemic distribution. The effect of a diseased gastroin- number of strains of the avian influenza virus exhib- testinal system such as that seen in CPV enteritis on the iting resistance to oseltamivir has increased.31 How- absorption, systemic distribution, and transformation ever, the significance, etiology, and impact of this is unknown at this time and future pharmacokinetic increased resistance are unknown at this time. While studies, in this situation especially, are needed. oseltamivir was used in a companion pet population The reasoning behind any beneficial effect of oseltam- in this study, the appropriateness of its continued use in ivir in the treatment of CPV enteritis suggests that it this setting in light of this increased resistance has yet helps decrease bacterial translocation and therefore the to be determined. 140 & Veterinary Emergency and Critical Care Society 2009, doi: 10.1111/j.1476-4431.2009.00404.x
Oseltamivir in parvoviral enteritis
Conclusions 5. Macintire DK, Smith-Carr S. Canine parvovirus part II. Clinical
signs, diagnosis, and treatment. Compend Contin Educ Pract Vet
CPV enteritis can be a devasting disease process. The 1997; 19(3):291–302.
6. Otto CM, Drobatz KJ, Soter C. Endotoxemia and tumor necrosis
financial constraints often encountered with treatment factor activity in dogs with naturally occurring parvoviral enteri-
can be very frustrating given the treatable nature of this tis. J Vet Intern Med 1997; 11:65–70.
disease. Despite the anecdotal reports touting the success 7. Mann FA, Boon GD, Wagner-Mann CC, et al. Ionized and total
magnesium concentrations in blood from dogs with naturally ac-
of oseltamivir to decrease the disease morbidity and quired parvoviral enteritis. J Am Vet Med Assoc 1998; 212:1398–1401.
mortality of CPV enteritis, concrete scientific evidence of 8. Rewerts JM, McCaw DL, Cohn LA, et al. Recombinant human
this was not found in this study. However, a significant granulocyte colony-stimulating factor for treatment of puppies
with neutropenia secondary to canine parvovirus infection. J Am
difference in the change in body weight during hospi- Vet Med Assoc 1998; 213:991–992.
talization stay was established, as was the apparent 9. Mischke R, Barth T, Wohlsein P, et al. Effect of recombinant human
safety of the drug in this patient population. It was also granulocyte colony-stimulating factor on leukocyte count and
survival rate of dogs with parvoviral enteritis. Res Vet Sci 2001;
shown that the control dogs had a significant decline in 70:221–225.
their WBC counts from Day 1 to Days 2 through 4 of 10. Mohr AJ, Leisewitz AL, Jacobson LS, et al. Effect of early enteral
hospitalization, while treatment dogs seemed to be pro- nutrition on intestinal permeability, intestinal protein loss, and
outcome in dogs with severe parvoviral enteritis. J Vet Intern Med
tected from this effect, and had no significant change 2003; 17:791–798.
during this time period. Whether this represents a true 11. Cohn LA, Rewerts JM, McCaw D, et al. Plasma granulocyte colony
benefit of oseltamivir administration or rather a differ- stimulating factor concentrations in neutropenic, parvoviral en-
teritis-infected puppies. J Vet Intern Med 1999; 13:581–586.
ence in timelines of disease between groups is open to 12. Dimmitt R. Clinical experience with cross-protective antiendotox-
debate, although the latter appears to be unlikely in this in antiserum in dogs with parvoviral enteritis. Canine Pract 1991;
study. It is believed that, given the paucity of adverse 16:23–26.
13. De Mari K, Maynard L, Eun HM, et al. Treatment of canine par-
effects and the findings presented in this study, further voviral enteritis with interferon-omega in a placebo-controlled
investigation is warranted for its effects in CPV enteritis. field trial. Vet Rec 2003; 152:105–108.
14. Martin V, Najbar W, Gueguen S, et al. Treatment of canine par-
voviral enteritis with interferon-omega in a placebo-controlled
Acknowledgement challenge trial. Vet Microbiol 2002; 89:115–127.
15. Gubareva LV, Kaiser L, Hayden FG. Influenza virus ne-
The authors would like to acknowledge Dr. Kenneth uraminidase inhibitors. Lancet 2000; 335:827–835.
16. Ward P, Small I, Smith J, et al. Oseltamivir (Tamiflu) and its po-
Drobatz for his assistance with the statistical analysis. tential for use in the event of an influenza pandemic. J Antimicrob
Chemother 2005; 55(suppl 1):i5–i21.
17. Hayden FG, Pavia AT. Antiviral management of seasonal and
Footnotes pandemic influenza. J Infect Dis 2006; 194(suppl 2):S119–S126.
a
18. Kaiser L, Wat C, Mills T, et al. Impact of oseltamivir treatment on
SNAP Parvo antigen test, IDEXX Laboratories Inc, Westbrook, ME. influenza-related lower respiratory tract complications and hos-
b
Lactated Ringer’s solution, Abbott Laboratories, North Chicago, IL.
c pitalizations. Arch Intern Med 2003; 163:1667–1672.
Normosol-R, Abbott Laboratories.
d 19. McCuller JA, Bartmess KC. Role of neuraminidase in lethal syn-
Potassium chloride, Phoenix Pharmaceutical Inc, St Joseph, MO.
e ergism between influenza virus and Streptococcus pneumoniae.
Dextrose 50% solution, Veterinary Laboratories Inc, Lenexa, KS.
f
Ampicillin, Abraxis Pharmaceutical Products, Schaumburg, IL. J Infect Dis 2003; 187:1000–1009.
g
Enrofloxacin injectable solution, 2.27%, Bayer HealthCare LLC, Animal 20. McCullers JA. Effect of antiviral treatment on the outcome of sec-
Health Division, Shawnee Mission, KS. ondary bacterial pneumonia after influenza. J Infect Dis 2004;
h
Metoclopramide injection USP, SICOR Pharmaceuticals Inc, Irvine, CA. 190:519–526.
i
Chlorpromazine HCl injection USP, Baxter, Deerfield, IL. 21. Peltola VT, Murti KG, McCullers JA. Influenza virus neu-
j raminidase contributes to secondary bacterial pneumonia. J Infect
Pyrantel pamoate 50 mg/mL, Pfizer Animal Health, New York, NY.
k
6% Hetastarch, Hospira, Lake Forest, IL. Dis 2005; 192:249–257.
l
Science Diet Canine I/D, Hill’s Pet Nutrition Inc, Topeka, KS. 22. Bhatia A, Kast RE. How influenza’s neuraminidase promotes vir-
m
Oseltamivir phosphate, Roche Laboratories Inc, Nutley, NJ. ulence and creates localized lung mucosa immunodeficiency. Cell
n
StataCorp. 2003, Stata Statistical Software: Release 8, College Station, TX. Mol Biol Lett 2007; 12:111–119.
23. Matheson NJ, Harden AR, Perera R, et al. Neuraminidase inhib-
itors for preventing and treating influenza in children (review).
References Cochrane Lib 2007; 1:1–40.
24. Turk J, Miller M, Brown T, et al. Coliform septicemia and pulmo-
1. Hoskins JD. Canine viral enteritis, In: Greene CE. ed. Infectious nary disease associated with canine parvoviral enteritis: 88 cases
Disease of the Dog and Cat, 2nd ed. Philadelphia: WB Saunders; (1987–1988). J Am Vet Med Assoc 1990; 196:771–773.
1998, pp. 40–44. 25. Mantione NL, Otto CM. Characterization of the use of antiemetic
2. Otto CM, Jackson CB, Rogell EJ, et al. Recombinant bactericidal/ agents in dogs with parvoviral enteritis treated at a veterinary
permeability-increasing protein for treatment of parvovirus en- teaching hospital: 77 cases (1997–2000). J Am Vet Med Assoc 2005;
teritis: a randomized, double-blinded, placebo-controlled trial. 227:1787–1793.
J Vet Intern Med 2001; 15:355–360. 26. Gillissen A, Hoffken G. Early therapy with the neuraminidase in-
3. Kariuki Njenga M, Nyaga PN, Buoro IBJ, et al. Effectiveness of hibitor oseltamivir maximizes its efficacy in influenza treatment.
fluids and antibiotics as supportive therapy of canine parvovirus-2 Med Microbiol Immunol 2002; 191:165–168.
enteritis in puppies. Bull Anim Health Prod Afr 1990; 38:379–389. 27. Oo C, Snell P, Barrett J, et al. Pharmacokinetics and delivery of
4. Glickman LT, Domanski LM, Patronek GJ, et al. Breed-related risk the anti-influenza prodrug oseltamivir to the small intestine and
factors for canine parvovirus enteritis. J Am Vet Med Assoc 1985; colon using site-specific delivery capsules. Int J Pharm 2003; 257:
187:589–594. 297–299.
& Veterinary Emergency and Critical Care Society 2009, doi: 10.1111/j.1476-4431.2009.00404.x 141M.R. Savigny & D.K. Macintire
28. Li W, Escarpe PA, Eisenberg EJ, et al. Identification of GS 4104 as 30. Gatt M, Reddy BS, Macfie J. Review article: bacterial translocation
an orally bioavailable prodrug of the influenza virus ne- in the critically ill – evidence and methods of prevention. Aliment
uraminidase inhibitor GS 4071. Antimicrob Agents Chemother Pharmacol Ther 2007; 25:741–757.
1998; 42:647–653. 31. World Health Organization. WHO/ECDC frequently asked ques-
29. He G, Massarella J, Ward P. Clinical pharmacokinetics of the pro- tions for oseltamivir resistance [document on the internet]. WHO;
drug oseltamivir and its active metabolite Ro 64-0802. Clin Feb 15, 2008. Available at: http://www.who.int/csr/disease/in-
Pharmacokinet 1999; 37:471–484. fluenza/oseltamivir_faqs/en/index.html. Accessed Feb 15, 2009.
142 & Veterinary Emergency and Critical Care Society 2009, doi: 10.1111/j.1476-4431.2009.00404.xYou can also read
