RESULTS
A total of 35 dogs were evaluated, with 19 in the treatment group receiving
oseltamivir and 16 in the control group receiving a placebo. There were 3 shelter dogs
included in the study, all of which were in the control group. Most dogs (30/35) 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. No statistically significant
differences were found between groups in the baseline characteristics of age, sex,
vaccination status or duration of clinical signs prior to presentation. The median age of
dogs in the control group was 14 (8, 36) weeks, while that of the treatment group was 12 (8, 44) weeks (p=0.50). There were 21 (60%) female dogs, with 10 (48%) randomized to
the control group and 11 (52%) randomized to the treatment group. Alternatively, there
were 14 (40%) male dogs, with 6 (43%) randomized to the control group and 8 (57%) to
the treatment group (p=1.0). All dogs were sexually intact. Vaccination status was
known for 10/16 (62%) dogs in the control group and 13/19 (68%) dogs in the treatment
group. Of these, 5 (50%) dogs in the control group had received at least one vaccination
against CPV, whereas 6 (46%) in the treatment group had been vaccinated at least once
against CPV (p=1.0). The duration of clinical signs prior to presentation was known for
15/16 (94%) control dogs and 16/19 (84%) treatment dogs. Mean days sick for the
control group was 1.4 (+/-0.9) days, while that for the treatment group was 1.8 (+/-1.0)
days (p=0.31).
There was no significant difference in the degree of estimated dehydration at
entry of dogs between groups. The control dogs were estimated to have a mean
dehydration deficit of 6.3% (+/-1.54%), while the treatment dogs were estimated at 6.9%
(+/-1.7%) (p=0.35). In addition, there was no statistical difference amongst the weights
at entry or the weights at discharge between the two groups. Dogs in the control group
had a median entry weight of 6.7kg (14.7lb) (1.8, 28.2kg [3.96, 62lb]), and those of the
treatment group 4kg (8.8lb) (1.6, 25kg [3.5, 55lb]) (p=0.21). At discharge, the median
weight of control dogs was 6.5kg (14.3lb) (1.8, 27.3kg [3.96, 60lb]) and that of the
treatment group was 4.4kg (9.7lb) (1.6, 28.6kg [3.5, 63lb]) (p=0.42). However, a
significant difference was found in the weight change from entry until discharge.
Dogs
in the control group experienced a median change of -0.21kg (-0.46lb) (-2.8, 0.5kg [-6.2,
1.1lb]), while those in the treatment group had a median gain of 0.07kg (0.15lb) (-1, percentage of change in body weight ([discharge weight ñ entry weight / entry weight] *
100%) (Figure 1). Dogs in the control group had a mean change of -4.5% (+/- 6.9%), and
those in the treatment group a mean change of +2.6% (+/- 7.1%) (p=0.006). When this
analysis was repeated for survivors only, the results were still found to be significant.
The control dogs contained all 3 non-survivors, and the new calculation without these
dogs resulted in a median percent weight change of -3.9% (-2.8, 0.5), compared to the
median percent weight change of 1.3% (-1, 3.6) for the treatment dogs (p=0.012).
The percentage of days in the hospital that SIRS criteria were met was calculated
for each dog. There was no significant difference between groups, with control dogs
meeting SIRS criteria a mean of 52% of days, versus 54% for treatment dogs (p=0.91).
No difference was found in the duration of hospitalization between the two
groups. Dogs in the control group had a mean stay of 5.9 (+/- 2.6) days, and those of the
treatment group 6.0 (+/- 2.3) days (p=1.0). Colloid therapy was not required often, as the
median number of days on colloids for the control group was 0 (0, 3) days and also 0 (0,
5) days for the treatment group (p=0.5). None of the 16 dogs in the control group
received a blood transfusion, while 2/19 (10%) dogs in the treatment group did (p=0.5).
The addition of chlorpromazine was necessary in 10/19 (53%) dogs in the treatment
group, and 9/16 (56%) dogs in the control group. Dogs in the treatment group that did
receive chlorpromazine did so an average of 21% of their days in the hospital, while the
dogs requiring it in the control group were given chlorpromazine an average of 30% of
their days in the hospital. The overall survival rate was 91% (32/35). Three dogs died,
all from the control group, giving this group a survival rate of 81% (13/16). One animal was euthanized after severe progression of clinical signs despite treatment, and it was
deemed that the dog was suffering and would not recover, while the other 2 suffered
natural deaths. Survival in the treatment group was 100% (19/19). However, this
difference was not found to be significant (p=0.09).
Post mortem examinations were performed on all non-surviving dogs. Findings
were consistent with a diagnosis of CPV enteritis.
All dogs had diffuse, severe,
necrotizing enteritis. Bone marrow was examined in 2/3 dogs, both showing sections of
moderate hypocellularity. While 2 dogs were noted to have diffuse congestion and
edema in their lungs, the third dog was found to have a mild interstitial pneumonia.
The white blood cell values that were evaluated (initial counts for WBC,
neutrophils and lymphocytes, the nadir values and day of nadir, and significant decreases
in counts for WBC, neutrophils and lymphocytes), were compared between groups. No
significant differences were found for any of these values between groups. Although it
was not found to be significant, it was noted that the treatment dogs had a very slightly
lower average WBC (7,500 cells/ul) and neutrophil count (6,220 cells/ul) at presentation
than the control dogs (8,540 cells/ul WBC and 6,830 cells/ul neutrophils). Both groups
experienced a decline in numbers with an average nadir of WBC at 2,680 and 2,810
cells/ul for the treatment and control groups, respectively, and 1,160 and 1,240 cells/ul
for the neutrophils for the treatment and control groups, respectively. While by Day 5 the
control dogs had rebounded their WBC (4,580 cells/ul) and neutrophil counts (2,270
cells/ul), the treatment dogs showed a slightly higher value for each at the same time
point (6,920 cells/ul WBC and 4,310 cells/ul neutrophils). Seven of the 16 (44%) control
dogs had a total WBC nadir occur at less than 100 cells/ul. Two of these 7 (29%) dogs did not survive (1 died, 1 euthanized). In the treatment group, 8/19 (42%) dogs had their
total WBC nadir occur at less than 100 cells/ul. None of these dogs died.
Comparison of significant decreases in white blood cells, neutrophils and
lymphocytes on initial presentation and on Day 4 of hospitalization is shown in Figure 2.
Based on the expected timeline of disease progression as previously described,11 Day 4
was chosen for this comparison to represent a period in time that should have
encompassed neutrophil and clinical recovery for most dogs. In general, a higher
percentage of treatment dogs versus control dogs fulfilled the criteria for significant
decreases in initial white blood cells and neutrophil counts. However, by Day 4, more
control dogs were significantly affected.
When the clinical scores were compared day by day for each category as well as
the cumulative total for that day, no significant differences were found between groups
(Figures 3) with the exception of Day 6 for the appetite score (p=0.02). There was also a
difference for attitude on Day 6, but this was not significant (p=0.086). Looking at the
scores for dogs in each group as compared to their white blood cell counts on the same
day (Table 2), it can be seen that the treatment dogs did have mildly improved scores
across the board on Days 4 and 5 while also having a quicker rebound of WBC and
neutrophil counts. Days 4 and 5 were chosen for examination for the same reason as
stated above.
DISCUSSION
Findings
While the use of oseltamivir in addition to standard therapy for naturally occurring CPV
enteritis did not exhibit a significant effect to help decrease hospitalization time,
additional treatments needed or disease morbidity as determined by a clinical scoring
system, it was shown to be associated with a significant increase in weight change versus
control dogs from entry until discharge. The control dogs tended to lose weight, while
the treatment dogs gained. This finding was not affected by the degree of dehydration at
presentation, as it was found that there was no significant difference between the two
groups in this variable.
The importance and implications of this finding are unknown at this time. Other
studies have shown that a significant change in weight in study subjects is also associated
with an improved outcome. In one study using a mouse model of human influenza
infection with secondary bacterial pneumonia, it was found that prophylactic treatment
(i.e. dosage begun 4 hours prior to viral infection) with oseltamivir resulted in an average
loss of only 5% of body weight in the mice, compared to an average of 25% loss in the
placebo mice.18 This study also showed that all mice in the prophylactic oseltamivir
group survived the viral and bacterial challenge, versus none of the mice in the placebo
group.
A second study was investigating the effect of early enteral nutrition (EEN) on
dogs with parvoviral enteritis.10 In this study, it was found that dogs in the EEN group
had a significant increase in weight gain from entry on all days of the study, while the
conventional group had no significant change in weight. In addition, dogs in the EEN
group showed a more rapid clinical improvement, based on normalization of clinical scores, than did the conventional group. While the difference was not significant, it was
also found that the 2 dogs that did not survive were both from the conventional group,
giving it a survival rate of 87% compared to 100% for the EEN group. These survival
statistics are very similar to those found in the present study. These results suggest that
the significant change in weight associated with oseltamivir use in this study could imply
that more significant beneficial effects on survival and/or disease recovery could also be
a plausible effect of the oseltamivir that was not brought out here.
The use of oseltamivir did not appear to be associated with any significant
adverse effects. The main side effects reported in humans are gastrointestinal effects
apparently due to direct local irritation of the gastric mucosa.15 In the experience of the
authors, dogs will also often react to the taste of the oseltamivir suspension and nausea
and vomiting can be encountered. Dilution with water just prior to administration
appears to minimize these effects. This practice was utilized in this study in an effort to
not only avoid uncovering group assignment and thereby instituting a bias, but also in an
effort to keep the clinical scores an accurate representation of the disease process in the
animal and not obscure these scores with drug reaction.
Analysis of the clinical scores, specifically vomiting and appetite, did not show
any difference between the treatment and control groups. This would tend to support the
lack of any significant adverse effects of oseltamivir administration versus placebo in this
patient population. The possibility that oseltamivir did have an effect to improve the
clinical effects of CPV enteritis in the treatment dogs, but itself caused increased
vomiting and nausea as a side effect of the drug and therefore concealed any benefit
evident by analysis of the clinical scores does exist. However, subjective observation was that administration of the oral medications (oseltamivir or placebo) was not
associated with initiating increased nausea or vomiting directly afterwards. This supports
the interpretation of results indicating that side effects of oseltamivir were minimal, as
were beneficial effects on decreasing disease morbidity.
The intensity of treatment required and the expected cost of treatment were
inferred based on additional therapy, such as colloid infusion or blood transfusions, as
well as prolonged hospitalization times, which were needed. Colloids were not
frequently used, in contrast to a previous report.26 Specifications for the institution of
colloid therapy were not elucidated in the previous study.
Protocol in this study required
significant decrease in total solids to 3.5g/dl. Much more modest decreases, often around
4.0 to 4.5g/dl, are frequently used in the clinic setting as indication for treatment with
colloids. A higher cutoff value as a trigger for colloid infusion likely would have
increased the incidence of its use in this study. Whether this increased use would
translate into a difference in usage between groups is unknown, but seems unlikely based
on the findings of a previous study showing no difference in albumin concentrations
between an EEN group and a conventional therapy group.10 Blood transfusions were also
rarely indicated. The 2 cases that did require a transfusion received fresh whole blood,
rather than packed red blood cells. Packed red blood cell availability was very limited at
the time of the first required transfusion. Fresh whole blood was collected from a donor
dog and administered in its place. In order to minimize differences in treatment between
dogs, when the second transfusion became necessary, although packed red blood cells
were available, fresh whole blood from the previous donor was again administered. The
total volume of crystalloids as well as colloids administered between the two groups may have been helpful to evaluate. Dogs experiencing greater fluid loss from vomiting and/or
diarrhea as well as having less voluntary intake per os would have a greater intravenous
fluid need. This would correlate with an increased severity of clinical signs and
manifestation of the disease process. Unfortunately, the collection of this data had
significant gaps or was outright missing from certain dogs due to recording error or
technical difficulties. Therefore this value was not analyzed.
The duration of clinical signs prior to presentation was not different between the
groups. It has been reported that for human influenza viral infections, oseltamivir is most
effective if started within the first 12 hours of clinical signs, with efficacy decreasing up
to 48 hours.24 For every 6 hour delay in starting oseltamivir, the duration of illness is
predicted to increase by approximately 8%.25 Whether this also holds true for its use in
CPV enteritis is unknown. Because replication of CPV does not depend on
neuraminidase, administration of oseltamivir in the early stages of infection is unlikely to
diminish viral replication and dissemination as with the influenza virus, and thus a timeefficacy
response is not expected. Rather, with the proposed mechanism against bacterial
translocation, oseltamivir may have a greater impact when administered during the period
of leukopenia and severe clinical signs. Further investigation is needed to expand on
these speculations.
The treatment group may have been further along in their course of disease,
despite the lack of difference in reported duration of clinical signs. This would be
supported by the fact that this group, on average, had a slightly lower white blood cell
and neutrophil counts at entry, as well as a somewhat quicker rebound after these
numbers dropped. The clinical scores also showed the trend of quicker improvements in the treatment group. With the inherent inaccuracies of estimation of duration of clinical
signs by the owner or caretaker, it is plausible that the times reported at entry were not
correct, and that the treatment group did include dogs at a more advanced stage of
disease. Conversely, if the reported duration of signs was accurate, the seemingly
quicker recovery of the treatment dogs could be attributed to a beneficial effect of
oseltamivir.
A clinical scoring system was utilized in order to evaluate the subjective criteria
of attitude and appetite, as well as to quantify the severity of vomiting and characterize
the feces to allow for comparison across dogs. One investigator (MRS) had the
responsibility of assigning scores to all dogs to minimize inter-observer variability. This
investigator was also blinded to group assignment in order to minimize bias.
Values for each category and cumulative scores were compared between the 2 groups for
each day, and although mild trends could be seen for lower scores in the treatment group,
there were no significant differences. This could be attributed to the small sample size in
this study, as well as the variability in the timeline of illness among dogs. Since dogs
presented in all stages of their disease (i.e. clinical symptoms for less than 12 hours to up
to 4 days), a straight out comparison of scores per day may not illustrate true differences
and a larger group would be needed to further examine this effect. In addition, the
clinical scoring system utilized is a very simple system, and as such, was relatively
insensitive in its ability to differentiate between various stages of aberrancy in each of the
clinical attributes. There was not much room to allow for representation of subtle yet
clinically significant differences. A scoring system with a greater degree of stratification between assigned values may allow for a greater sensitivity and a more accurate
representation of the clinical status of the patient.
CPV is not reliant on neuraminidase for replication. However, anecdotal reports
of the use of oseltamivir in dogs with CPV enteritis have claimed decreased morbidity
and shortened recovery time in the treated dogs. It is speculated that the drug may inhibit
bacterial translocation that subsequently leads to endotoxemia, sepsis, SIRS and death.
Bacterial adherence and colonization of respiratory epithelial cells is potentiated in the
presence of viral NA, and inhibited with NA blocking agents.17-19 It is believed that the
bacteria that commonly invade the lower respiratory tract express their own NA, thus
enabling them to penetrate the protective mucin layer and infect the epithelial cells.17
Although unproven, a similar mechanism may exist in the gastrointestinal tract.
Oseltamivir may exert a beneficial effect by inhibiting NA on enteric bacteria, preventing
their translocation across the gastrointestinal mucosal barrier. In CPV enteritis, the
mucosal barrier is already impaired, allowing easier passage of bacteria. If bacterial NA
plays a role similar to that in the lungs, the NA would cleave sialic acid residues on the
gut epithelium, exposing receptor sites for bacterial adherence and further encouraging
translocation. In addition, CPV suppresses the dogís immune system, both humoral and
cell-mediated factors, allowing for systemic spread of bacteria and the resultant
deleterious effects. Further studies are needed to accurately define the actual mechanism
behind the observed anecdotal benefits of the use of oseltamivir to treat CPV enteritis.
Limitations
Limitations of this study do involve the concern of administration of an oral
medication to a vomiting patient, and its variable systemic absorption in the face of a diseased gastrointestinal tract. Gastric emptying times are quicker for liquid substances
as compared to solids, with gastric emptying starting as soon as 10-30 minutes after
administration. The oseltamivir suspension thus has the probability of starting to move
through the gastrointestinal system and being absorbed before an episode of vomiting
occurs. In addition, the act of vomiting is reported to only empty 40-50% of stomach
contents. This would suggest that even with the presence of vomiting, it is reasonable to
expect that at least a portion of the drug will remain in the system. Early safety studies of
oseltamivir show that it has a bioavailability of 73% in healthy dogs, with detectable
levels of the drug in plasma within approximately 30 minutes after oral administration.27
Oseltamivir does require transformation to its active metabolite by esterases located
within the liver, and to a certain degree, within the intestinal system. 27, 28 The
importance of the intestinal system esterases is unknown. Due to this need for
transformation, it is believed that oseltamivir effects are not due to a purely local action,
but do require systemic distribution. The effect of a diseased gastrointestinal system such
as that seen in CPV enteritis on the absorption, systemic distribution and transformation
is unknown at this time and future pharmacokinetic studies, in this situation especially,
are needed.
The reasoning behind any beneficial effect of oseltamivir in the treatment of CPV
enteritis suggests that it helps decrease bacterial translocation and therefore the ensuing
endotoxemia, SIRS and MODS that can develop. In this study, although certain physical
and clinic pathologic parameters were monitored, there was no direct test for the presence
of bacterial translocation or sepsis. SIRS criteria were evaluated, but this is a fairly crude
assessment, especially considering the confounding factors. As the dogs were starting to feel better, they would oftentimes become very excitable when being handled. This
frequently resulted in an elevation of their heart rate to over 140 bpm, or their respiratory
rate to greater than 40/min. The presence of these two variables would classify these
healthy, excitable puppies as being positive for SIRS. In addition, the effect of the virus
itself on the white blood cell count confounds the definition slightly. A WBC of less than
6,000 cells/ul can simply reflect destruction of progenitor cells in the bone marrow and is
not necessarily associated with systemic inflammation. Other methods to evaluate for the
presence of bacterial translocation, endotoxemia or SIRS would be more fruitful. Culture
of mesenteric lymph nodes is considered the gold standard in human medicine and
animal models for evaluation of bacterial translocation.29 The feasibility of this
procedure in this patient population (client-owned, live animals) and setting is
questionable. Other methods, such as blood cultures or measurement of serum endotoxin
levels or other inflammatory mediators may provide a more accessible method for
differentiating animals in which bacterial translocation is present from those in which it is
not. Further investigation would be needed before any true conclusions can be made.
CONCLUSIONS
CPV enteritis can be a devastating disease process. The financial constraints
often encountered with treatment can be very frustrating given the treatable nature of this
disease. Despite the anecdotal reports touting the success of oseltamivir to decrease the
disease morbidity and mortality of CPV enteritis, scientific evidence of this was not
found in this study. However, a significant difference in the change in body weight
during hospitalization stay was established, as was the apparent safety of the drug in this
31
patient population. It is believed that, given the paucity of adverse side effeccts and the
findings presented in this study, further investigation is warranted not only for its effects
in CPV enteritis, but possibly any disease state in which bacterial translocation is a
concern.
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6. Otto CM, Drobatz KJ, Soter C. Endotoxemia and tumor necrosis factor activity in
dogs with naturally occurring parvoviral enteritis. J Vet Intern Med 1997; 11:65-
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7. Mann FA, Boon GD, Wagner-Mann CC, et al. Ionized and total magnesium
concentrations in blood from dogs with naturally acquired parvoviral enteritis. J
Am Vet Med Assoc 1998; 212:1398-1401. 8. Rewerts JM, McCaw DL, Cohn LA, et al. Recombinant human granulocyte
colony-stimulating factor for treatment of puppies with neutropenia secondary to
canine parvovirus infection. J Am Vet Med Assoc 1998; 213:991-992.
9. Mischke R, Barth T, Wohlsein P, et al. Effect of recombinant human granulocyte
colony-stimulating factor on leukocyte count and survival rate of dogs with
parvoviral enteritis. Res Vet Sci 2001; 70:221-225.
10. Mohr AJ, Leisewitz AL, Jacobson LS, et al. Effect of early enteral nutrition on
intestinal permeability, intestinal protein loss, and outcome in dogs with severe
parvoviral enteritis. J Vet Intern Med 2003; 17:791-798.
11. Cohn LA, Rewerts JM, McCaw D, et al. Plasma granulocyte colony stimulating
factor concentrations in neutropenic, parvoviral enteritis-infected puppies. J Vet
Intern Med 1999; 13:581-586.
12. Dimmitt R. Clinical experience with cross-protective antiendotoxin antiserum in
dogs with parvoviral enteritis. Canine Pract 1991; 16:23-26.
13. De Mari K, Maynard L, Eun HM, et al. Treatment of canine parvoviral enteritis
with interferon-omega in a placebo-controlled field trial. Vet Rec 2003; 152:105-
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14. Martin V, Najbar W, Gueguen S, et al. Treatment of canine parvoviral enteritis
with interferon-omega in a placebo-controlled challenge trial. Vet Micro 2002;
89:115-127.
15. Gubareva LV, Kaiser L, Hayden FG. Influenza virus neuraminidase inhibitors.
Lancet 2000; 335:827-835. 16. Kaiser L, Wat C, Mills T, Mahoney R, Ward P, Hayden F. Impact of oseltamivir
treatment on influenza-related lower respiratory tract complications and
hospitalizations. Arch Intern Med 2003; 163:1667-1672.
17. McCuller JA, Bartmess KC. Role of neuraminidase in lethal synergism between
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18. McCullers JA. Effect of antiviral treatment on the outcome of secondary bacterial
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19. Peltola VT, Murti KG, McCullers JA. Influenza virus neuraminidase contributes
to secondary bacterial pneumonia. J Infect Dis 2005; 192:249-257.
20. Bhatia A, Kast RE. How influenzaís neuraminidase promotes virulence and
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21. Matheson NJ, Harden AR, Perera R, Sheikh A, Symmonds-Abrahams M.
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viernes, 27 de marzo de 2015
USE OF OSELTAMIVIR IN THE TREATMENT OF CANINE PARVOVIRAL ENTERITIS. Part 1. Michelle R. Savigny 2008
THESIS ABSTRACT
USE OF OSELTAMIVIR IN THE TREATMENT OF CANINE PARVOVIRAL
ENTERITIS
Michelle R. Savigny
Master of Science, May 10, 2008
(DVM, Texas A&M University, 2004)
(B.A., State University of New York at Buffalo, 2000)
Directed by Douglass K. Macintire
Despite the availability of an effective vaccine, canine parvovirus (CPV) enteritis remains a significant cause of disease in veterinary medicine. Appropriate treatment of the disease can result in a favorable survival rate; however, lack of treatment is almost universally fatal. Unfortunately, the treatment tends to be costly to the client, often times deterring medical care. There exists a need for an effective treatment option that will ameliorate disease morbidity in addition to hospitalization time, thereby decreasing client cost. Oseltamivir, a neuraminidase inhibitor antiviral drug, has resounding anecdotal support for achieving this goal. However, scientific validation at this point is lacking.
This study, using a prospective, blinded, randomized, placebo controlled clinical trial, aimed to investigate the effects of oseltamivir as an adjunct to standard treatment of CPV enteritis. Results show the only significant difference between groups to be in weight change during hospitalization in the oseltamivir group versus placebo.
TABLE OF CONTENTS
LIST OF TABLES .......................................................................................................viii
LIST OF FIGURES........................................................................................................ix
CHAPTER I. INTRODUCTION....................................................................................1
CHAPTER II. LITERATURE REVIEW........................................................................3
STATEMENT OF RESEARCH OBJECTIVE ..............................................................12
CHAPTER III. MATERIALS AND METHODS..........................................................12
Study Design......................................................................................................12
Standard Treatment ............................................................................................13
Oseltamivir Treatment .......................................................................................15
Monitoring/Data Acquisition..............................................................................16
Clinical Scoring System.....................................................................................17
Statistical Analysis.............................................................................................18
CHAPTER IV. RESULTS............................................................................................18
CHAPTER V. DISCUSSION AND CONCLUSIONS..................................................23
Findings .............................................................................................................23
Limitations.........................................................................................................28
Conclusions .......................................................................................................30
FOOTNOTES ...............................................................................................................32
REFERENCES..............................................................................................................33
vi ii
LIST OF TABLES
Table 1. Clinical Scoring System .................................................................................37
Table 2. White Blood Cell Counts ................................................................................38
TLIST OF FIGURES
Figure 1. Weight change over time for individual dogs .................................................39
Figure 2. Averaged white blood cell count over time ....................................................40
Figure 3. Box plot of cumulative total scores by day.....................................................41
INTRODUCTION
Canine parvovirus (CPV) is a single-stranded DNA virus that was first discovered in 1978.1 It is a hardy, highly contagious virus that remains a cause of significant disease process in young dogs. It is estimated that over one million dogs are affected each year in the United States2 despite the availability of an effective vaccine.
CPV infects and replicates in rapidly dividing cells, most notably the lymphoid organs, latter myeloid progenitor cells in the bone marrow, and intestinal epithelial cells.
Replication results in cell destruction, causing a clinical disease characterized by severe vomiting, hemorrhagic diarrhea, dehydration and neutropenia. This disease is almost universally fatal without treatment, with reported survival rates of only 9% in an experimental model.3 Treatment increases this figure significantly, with reported survival rates ranging from 64-95%.2, 4-10 Care given in a university setting may be associated with increased survival rates of 96-100%2, 8, versus 67-75%2, 7 at local day clinics. It is postulated that this increase may be due to the 24 hour care available at these universities, as well as the likelihood for more intensive treatments such as plasma or colloid therapy.2
Therapy for CPV enteritis remains mainly supportive and aimed at controlling the clinical signs of disease. Multiple more directed therapies have been studied, such as human recombinant granulocyte colony stimulating factor8, 9, 11, equine antiendotoxin7, 12, recombinant bactericidal/permeability-increasing protein2, and interferon omega13, 14 with variable or disappointing results. Early enteral nutrition has been the only modality to date to show promise for a shortened recovery time and decreased disease morbidity associated with CPV enteritis.10 However, the need still remains for a therapeutic agent hat will help ameliorate the morbidity and mortality of this disease, and in doing so also decrease the cost of treatment. While the survival rate with aggressive treatment is very good, financial constraints often result in suboptimal treatment with lower survival, or even euthanasia. This fact demonstrates the clear need for an agent to help make the appropriate treatment for CPV enteritis a more financially feasible option for many owners or shelters.
Oseltamivira (Tamiflu) is a neuraminidase inhibitor originally designed to treat human influenza virus.15 It has recently also shown efficacy in the treatment of avian influenza. Oseltamivir inhibits the viral neuraminidase enzyme, and thus prevents the cleavage of sialic acid residues. This cleavage is necessary for liberating newly formed virons from the host cell, as well as for preventing the aggregation of viral particles. Both the viron liberation and decreased aggregation are mechanisms necessary for the spread and dissemination of the virus throughout the host in order to further infection. The neuraminidase enzyme is also needed for the virus to cleave the sialic acid residues
in mucin to allow for penetration of this protective layer and infection of respiratory epithelial tissue. Anecdotal reports of oseltamivir use in veterinary medicine claim that it is associated with a less severe form of disease and a quicker recovery when administered to dogs with CPV enteritis. However, unlike the influenza virus, CPV does not rely on neuraminidase for effective replication. Therefore, any beneficial effects that may be present would not be due to a direct antiviral action. Human studies have shown a ignificant decrease in the development of bacterial infections secondary to influenza when oseltamivir is used.15-21 This effect is believed to be due to a decrease in bacterial
permeation through the mucin layer of the respiratory epithelial cells. An extension of this is that oseltamivir could also have a similar inhibitory effect on bacterial permeation through the mucin layer of the gut epithelial cells. This would decrease bacterial translocation, resulting in a potentially lower incidence of endotoxemia, sepsis, systemic inflammatory response and eventual organ failure that is thought to be the main mechanism behind the mortality of CPV enteritis. Thus, oseltamivir could have an effect on the disease process of CPV enteritis in a non-viral dependent manner. In one report, 90% of dogs that died from CPV enteritis had Escherichia coli cultured from their liver
and lungs.22 ARDS-like changes in the lungs of these dogs on histopathologic exam was also present, demonstrating the possibility for organ failure secondary to SIRS development.22
This study was designed to investigate the influence of oseltamivir when added to standard treatment on the disease process of CPV enteritis, as well as to monitor for significant adverse side effects associated with its use. Our hypothesis was that oseltamivir would help to ameliorate the disease morbidity and mortality associated with naturally occurring CPV enteritis, thereby decreasing hospitalization time, the need for colloid and other adjunctive therapies, and thus the cost associated with treatment.
LITERATURE REVIEW
Canine parvovirus (CPV) is a single-stranded, non-enveloped DNA virus that
infects and destroys rapidly dividing cells of the host. It is a highly contagious and hardy
virus, resistant to environmental conditions and many cleaning agents. The virus is
transmitted via the fecal-oral route. It primarily affects young dogs six weeks to six
months old, with the majority of older dogs showing immunity from either natural
infection or vaccination. CPV has a propensity to infect rapidly dividing cells. These
cells in dogs over eight weeks old include those of lymphoid organs, the latter myeloid
progenitor cells in the bone marrow, and intestinal epithelial cells. Rarely, the lungs,
liver and/or kidney can be infected. Dogs younger than eight weeks or pups in utero can
also have the virus infect myocardial cells, resulting in a myocarditis that can lead to
acute death or chronic heart failure symptoms with death by six months of age.1, 11, 23
Once infected, CPV localizes to the lymphoid tissue of the oropharanyx where it
replicates before entering the bloodstream. Viremia will usually occur two to three days
post-infection, and is manifested by a slight anorexia and fever. Dogs recover from this
stage briefly before progressing into clinical enteritis, typically between days five and six
post-infection.11 Clinical signs of enteritis include anorexia, vomiting, diarrhea and
severe dehydration. Severity of signs is related to age, stress level (other disease
processes, weaning), immune status and presence of intestinal parasites. These signs will
usually pinnacle around day six to seven post-infection.11
CPV infects and destroys the epithelial cells of the intestinal crypts. These cells
are responsible for the regeneration and differentiation into the villous epithelial cells.
Therefore, without the intestinal crypt cells, there is marked villous blunting, atrophy and
even necrosis, as well as malabsorption of nutrients with resultant diarrhea. Intestinal
inflammation accompanies this destruction and can lead to the breakdown of the gutblood
barrier. Translocation of bacteria from the gut lumen into the bloodstream, along
with an impaired immune response, can result in bacteremia. Bacteria are most often
gram negative, releasing endotoxin upon their destruction. Endotoxemia can then lead to
systemic inflammatory response syndrome through the activation of various cytokine
mediators, progressing into multiple organ dysfunction syndrome and death.
Leukopenia is considered a characteristic and often diagnostic quality of CPV
infection. However, this finding is reported to be evident in as few as 33% of cases when
diarrhea first developes.6 Most pronounced of the decreased white blood cells is the
neutrophil portion. This neutropenia is postulated to be due to a combination of
significant intestinal tract inflammation acting as a sink for the cells, rapid depletion of
bone marrow stores, and infection of the granulocyte precursors in the bone marrow.
Interestingly, it appears that the CPV spares the early hematopoietic precursors and
destroys mainly the later stages. Neutrophil nadir was found to coincide with the days
when clinical signs were at their worst at roughly day seven to eight post-infection. In
addition, neutrophil recovery coincides with clinical recovery at approximately days eight
through 12.11 A rebound neutrophilia is often observed as granulopoiesis is stimulated
with a decreasing peripheral consumption of mature neutrophils and a decreasing central
destruction of their progenitor cells. Hypoproteinemia, in particular hypoalbuminemia, is
another common clinicopathologic abnormality associated with CPV enteritis. This is a
result of a combination of factors, including intestinal loss, decreased synthesis as a
negative acute phase protein and decreased nutritional intake. Other laboratory
abnormalities vary, and can include anemia, azotemia, or increased liver enzymes.
Maternal antibodies can offer protection to pups from CPV for the first few weeks
of life. The period of protection varies, depending on maternal antibody level, amount of colostrum ingested, and size of the litter. The titer of the pup will equal approximately
50-60% of the bitch’s titer at whelping, meaning that proper vaccination of the mother is
essential to conferring protection to her pups.23 Maternal antibodies have a half-life of
around 10 days in the pup, and can interfere with proper immune response to
vaccination.23 The primary cause for vaccine failure is interference by maternal
antibodies. The high titer (higher amount of virus per dose), low passage (fewer times
virus grown on tissue culture) vaccines tend to be more effective despite maternal
antibody levels.23 A schedule of vaccination starting at six weeks old with boosters every
three weeks until 12-16 weeks old is the current recommendation to avoid interference
and provide proper protection to the pup. Proper vaccination is highly recommended, as
unvaccinated dogs have been found to be 12.7 times more likely to develop CPV enteritis
when compared to vaccinated dogs.24 Local antibody activity is responsible for binding
intestinal CPV to prevent further virus shedding in the feces. Shedding will typically
occur until between days three and 10 post infection. However, it is the systemic
humoral immune response or the circulating antibodies that confer protection as they
inactivate the virus in the blood before it has a chance to invade intestinal, lymphoid or
bone marrow cells.
Treatment of CPV consists principally of supportive care. Maintaining hydration
is the staple of this supportive care. Balanced electrolyte solutions supplemented as
needed with potassium and dextrose are the primary fluid type of choice. Colloidal
solutions can be used when indicated to support colloidal osmotic pressure. Antibiotic
use is an area where opinions vary, although their use is generally accepted once the
neutrophil count falls below 1500 cells/ul. Antibiotics targeting enteric bacteria are recommended, such as a combination of ampicillin and enrofloxacin. A lower dose of
5mg/kg q12 hours and use for a limited time (i.e. less than 5 days if possible) are
precautions taken to help avoid the side effect of cartilage deformities in these young
dogs. Antiemetics such as a constant rate infusion of metaclopramide and/or
chlorpromazine are frequently used to help control the vomiting associated with the
disease. The concept of starvation to rest the gut is being challenged lately. A recent
study found that dogs fed through a nasoesophageal tube soon after admission and
through vomiting episodes showed a significantly more rapid clinical improvement
without major adverse side effects.10 These dogs were found to have a quicker recovery
of appetite and normal attitude as well as recovery from vomiting and diarrhea when
compared to the food restricted group.
Several other potential treatments have been tested, often with conflicting or
disappointing results. Treatment with equine LPS endotoxin antiserum has shown
varying results. One source reports that its use decreased mortality from 48% to 17%.12
Another study found that its use was associated with an increased risk of death in puppies
less than 16 weeks.7 Recombinant human granulocyte colony stimulating factor (rhGCSF)
has been shown to help reverse neutropenia associated with radiation therapy and
drugs as well as cyclic neutropenia in dogs. However, its use to treat the neutropenia of
CPV infection has not shown significant benefit in recovery time or neutrophil
response.8, 9 Bactericidal/permeability-increasing protein (BPI) is an enzyme located in
the granules of polymorphonuclear cells. It is cytotoxic to gram negative bacteria by
increasing membrane permeability, and will also bind to free LPS to block its effects.
With the finding that 82% of CPV enteritis cases have endotoxemia, and that a relationship exists between increasing detectable tumor necrosis factor activity and
mortality,6 it was postulated that administration of a recombinant BPI (rBPI21) would
help improve survival. Unfortunately, rBPI21 treatment was not found to have a
significant effect on outcome, hospitalization time or plasma endotoxin levels in one
study.2 Interferons have antiviral effects that vary depending on dose, species and virus
type. A recombinant form of feline interferon omega (rFeIFN) has shown promise in
reducing severity of disease and mortality,13, 14 warranting further investigation
It is not necessarily the infection of the rapidly dividing cells and resultant
gastroenteritis that causes the high morbidity and mortality associated with CPV disease.
Gnotobiotic dogs infected with CPV were found to have only mild clinical signs.6 This is
highly suggestive that it is the bacterial translocation and resultant septicemia that is
significantly related to the morbidity and mortality of CPV enteritis. Endotoxemia with
subsequent systemic inflammatory response syndrome (SIRS) and multiple organ
dysfunction syndrome (MODS) are associated with a high mortality in and of themselves.
E. coli has been cultured from the lungs and liver in up to 90% of dogs that have died
from CPV,22 illustrating the high potential for bacteremia to develop as well as its
devastating consequences.
Oseltamivir is a drug that was designed to inhibit the human influenza A and B
viruses through inhibition of neuraminidase. Hemagglutinin (HA) and neuraminidase
(NA) are two viral surface proteins that interact with receptors containing terminal
neuraminic acid residues.15 The purpose of the HA is to mediate viral attachment to the
host cell membranes to allow entry and subsequent replication. NA has multiple
functions that allow for the replication of the virus as well as enhancing its virulence. First, NA cleaves the sialic acid residue (N-acetyl-neuraminic acid is synonomous with
sialic acid) of the cellular receptor binding newly formed virons.20 This frees the virons
from the host cell, and allows their systemic release. Secondly, NA prevents the
aggregation of the virons, allowing more effective systemic distribution with subsequent
cellular infectivity.20 Thirdly, in order to come in contact with and invade respiratory
epithelial cells, the influenza virus must first penetrate through the protective mucin layer
covering these cells. This mucin contains abundant sialic acid residues. NA cleaves
these residues, facilitating the viral infiltration through mucin to the epithelial cells which
the virus then invades and replicates within.20 Inhibition of NA will result in a direct
antiviral effect in viruses, such as the influenza virus, that contain the cell surface protein
NA. These effects include decreased replication, decreased viral release, increased viral
aggregation and decreased dissemination. In viruses that lack NA receptors such as CPV,
NA inhibition may prevent viral migration through mucin and subsequent invasion of
respiratory epithelial or gastrointestinal epithelial cells.
The above listed functions are accepted and proven effects of NA. In addition,
there currently are hypothesized effects of NA on the immune system itself. Secretory
IgA provides the body’s primary immunologic defense of mucosal surfaces such as the
respiratory or the gut epithelium. Under normal circumstances, sialic acid residues are
covalently bound to the hinge region of the IgA molecule. Removal of these residues
converts IgA into an appropriate ligand for the liver asialoglycoprotein receptor, which
effectively removes IgA from circulation and results in its clearance.20 Working on the
unproven assumption that there is an equilibrium between circulating and mucosal IgA,
the loss of IgA mediated by NA would result in impaired mucosal defenses. The IgA of mucosal surfaces is secreted by B cells under the influence of gamma-delta T cells. Both
of these cells contain sialic acid moieties on their surfaces.20 If cleaved by NA, the
lymphocyte homing would be altered from the mucosa to the bone marrow, resulting in a
localized immunosuppression for that mucosal surface. It is also believed that NA itself
may allow the increased production of certain cytokines,15 resulting in a proinflammatory
effect. Thus, inhibition of neuraminidase has the potential to improve
mucosal immunity by increasing the amount of mucosal IgA, decreasing the clearance of
circulating IgA, allowing normal function of the B and T cells and decreasing the
cytokine release associated with the proinflammatory response. All of these effects could
be beneficial to a dog with severe gastrointestinal inflammation secondary to CPV
infection.
Effects of the neuraminidase inhibitors beyond direct antiviral effects have been
appreciated in multiple studies. Bacterial complications following influenza virus
infection in children and adults are common. The incidence of acute otitis media
complicating influenza infection in children ranges between 21% to over 50%.12 Use of
oseltamivir has been reported to reduce the incidence of otitis media by 44% (children 1-
12 years) and 56% (children 1-5 years). 21 In adults, secondary complications were
reduced from 15% in the placebo group to 7% in the oseltamivir group in one study.25
Secondary bacterial pneumonias are responsible for an estimated 25% of influenzarelated
deaths19 in adults. Streptococcus pneumoniae is a bacteria commonly associated
with pneumonia secondary to influenza infection. A lethal synergism between the
influenza virus and S. pneumonia has been established,17 presumably accounting for the
high morbidity and mortality associated with the combination. It has been shown that the influenza virus promotes adherence of S. pneumonia to respiratory epithelium.17, 18 Viral
NA cleaves the sialic acid residues on the host respiratory epithelial cell, revealing
cryptic binding sites for the bacteria, 17, 18 thus facilitating colonization. Oseltamivir
reverses this effect in vitro.17
In a mouse model of influenza with secondary pneumococcal pneumonia,
oseltamivir use alone increased survival from 0% to 75%.18 This study also found that
oseltamivir use in addition to proper antibiotics resulted in 100% survival, compared to
0% in mice treated with antibiotics alone. Prophylactic use of oseltamivir resulted in
significantly less weight loss than placebo treated mice (prophylactic use, 5%; placebo,
25%).18 Prophylactic as well as delayed treatment use decreased the development of
pneumonia, increased survival and slowed the development and progression of disease in
those animals that did acquire pneumonia.18 In adults, oseltamivir use was found to
decrease the incidence of lower respiratory tract complications (LRTC) in influenza
patients overall by 55%.16 Patients considered at risk for secondary complications
showed a decrease in LRTC and antibiotic use by 34%, and in otherwise healthy patients
by 67%.16 In these studies, the beneficial effect of oseltamivir in preventing secondary
bacterial infections is most likely the result of NA inhibition preventing bacterial and
viral penetration of the mucin layer to allow bacteria to bind to epithelial cells.
Oseltamivir is labeled for use in children over 1 year old; use in children less than
1 year is discouraged based on fatality seen in animal toxicology studies in seven day old
rats with high doses of drug.21 Although the etiology of such adverse effects has not been
specifically identified, there is concern over lack of a fully developed blood brain barrier.
Side effects in children over 1 year are minimal, with vomiting being the only significant effect versus placebo.21 Similarly, nausea (12% vs 4%) and vomiting (10% versus 3%)
were found to be significant side effects over placebo in adults that subsided after the first
few days.27 These effects can be diminished with administration of a small amount of
food along with the drug. Studies in adult humans have shown an 80% bioavailability of
oseltamivir after oral dosing.21
STATEMENT OF RESEARCH OBJECTIVES
This study was designed to investigate the influence of oseltamivir when added to
standard treatment on the disease process of CPV enteritis, as well as to monitor for
significant adverse side effects associated with its use. Our hypothesis was that
oseltamivir would help to ameliorate the disease morbidity and mortality associated with
naturally occurring CPV enteritis, thereby decreasing hospitalization time, the need for
colloid and other adjunctive therapies, and thus the cost associated with treatment.
MATERIALS AND METHODS
Study Design
The present study was a prospective, randomized, placebo-controlled, blinded
clinical trial of the effects of oseltamivir in dogs with naturally occurring parvoviral
enteritis. Cases of CPV enteritis were recruited from the local area and animal shelter
between April 2005 and August 2006. A financial incentive was offered to the owners or
agent to allow their dogs to participate in the study. Inclusion criteria was simply a
positive CPV fecal antigen test, presence of appropriate clinical signs (vomiting, diarrhea,
lethargy, anorexia), and lack of any treatment initiated prior to presentation. Informed consent was received from all owners or agents prior to any treatment initiation at the
Auburn University Veterinary Teaching Hospital. In addition, all procedures were
reviewed and approved by the Animal Care and Use Committee of Auburn University.
Dogs were excluded if they lacked a positive fecal antigen test for CPV, if treatment had
been administered prior to presentation, or if consent was not granted. Based on these
criteria, only one dog that was eligible during the trial period was not included due to
lack of owner consent. All dogs that entered the study completed the trial.
Randomization was achieved via assignment of numbers with predetermined
treatment designation in blocks of 5. Assignment was allocated by hospital personnel not
directly involved in the trial, and these assignments were uncovered to the investigators
only at the conclusion of the study. Power analysis was not performed as part of the
design of the study, but post-hoc analysis revealed that 47 dogs would be needed in each
group to give the study a power of 0.80 at a significance level of 0.05.
Standard Treatment
A treatment protocol was designed to standardize therapy between the two groups
with the exception of oseltamivir administration to the treatment group and a placebo to
the control group. No deviations were made from this protocol for any of the patients.
An intravenous catheter was placed in all dogs, either a peripheral cephalic catheter or
central venous catheter in a jugular vein. Intravenous fluids consisting of a balanced
electrolyte solution b, c were administered at an initial rate to replace an estimated
dehydration deficit over a minimum of 2 hours and a maximum of 4 hours. In the case
were it was determined that shock was present, the same intravenous fluids were bolused
at a dose of 10-30ml/kg over approximately 20 minutes. Boluses were repeated until it was assessed that the state of shock had been resolved, and the remaining fluid deficit
estimated to replace dehydration was administered as above. No patient required the
administration of colloidal therapy to treat a state of shock. Assessment of hydration and
perfusion status, as well as fluid rates were decided upon by a single investigator (MRS)
for all dogs. After rehydration, the hydration status was reassessed via physical
parameters and the fluid rate adjusted as deemed appropriate. Fluids were supplemented
with potassium chlorided and/or dextrosee as needed based on laboratory results.
All dogs received intravenous (IV) antibiotics. Ampicillinf was administered at
22mg/kg (11mg/lb) IV every 8 hours and enrofloxacing at 5mg/kg (2.5mg/lb) IV every 12
hours. A metoclopromideh constant rate infusion was also initiated in all patients at a rate
of 1-2mg/kg/day (0.5-1mg/lb/day). If vomiting persisted at a rate of more than twice per
12 hour period despite a maximum rate of metoclopramide, chlorpromazinei was added at
0.5mg/kg (0.25mg/lb) subcutaneously every 8 hours. Pyrantel pamoatej (10mg/kg
[5mg/lb] per os) was administered to all dogs within the first 3 days of arrival to eradicate
intestinal parasites. If the total solids by refractometery fell below 3.5g/dl, Hetastarch
6%k was infused at a rate of 10-20ml/kg/day (5-10ml/lb). If anemia, defined as a PCV of
less than or equal to 20%, was present, a fresh whole blood transfusion from an available
donor was given at 10-20ml/kg (5-10ml/lb) over 4 hours. Packed red blood cell
availability was very limited at the time of the first required transfusion. Fresh whole
blood was collected from a donor dog and administered in its place. In order to minimize
differences in treatment between dogs, when the second transfusion became necessary,
although packed red cells were available, fresh whole blood from the previous donor was
again administered. If vomiting episodes were less than 4 per 12 hour period, water and a bland dietl were offered. If either appeared to induce nausea, they were immediately
removed from the animal. Voluntary eating was allowed through mild vomiting but food
was not forced upon any dog. True client cost for the treatment administered to the dogs
was not calculated; this figure can only be inferred from the intensity of treatments
(additional antiemetics, colloid or transfusion therapy, increased length of
hospitalization) required for each dog between the groups.
Discontinuation of intravenous fluids was at the discretion of a single investigator
(MRS). This was accomplished primarily when the animal was no longer vomiting and
consistently eating and drinking sufficiently to maintain hydration. Once fluids were
discontinued, all patients were monitored for at least 12 hours before discharge from the
hospital in order to ensure no relapse of clinical signs. The day of discharge was
assigned as the day when it was deemed the dog was healthy enough to be discharged. If
the dog stayed in the hospital for non-medical reasons, these extra days were not included
when figuring length of hospitalization. In those animals that did not survive, the day of
death was considered an endpoint equivalent to the day of discharge for the purpose of
data analysis. Post-mortem exams were performed on all non-surviving dogs.
Oseltamivir Treatment
Dogs assigned to the treatment group received oseltamivir at 2mg/kg (1mg/lb) per
os every 12 hours. An equivalent volume of a placebo, consisting of a standard
suspension agent with color additive, was administered per os every 12 hours to dogs in
the control group. Prior experience with oseltamivir by the authors has shown that dogs
often react to its taste and frequently vomit shortly after administration. Dilution of the
oseltamivir with water (1:1) prior to administration seemed to decrease these reactions; therefore, in this study, both oseltamivir and placebo were diluted out as above to lessen
the risk of adverse reaction to the oseltamivir. In addition, in dogs receiving
chlorpromazine for protracted vomiting, it was attempted to dose this medication 30-60
minutes before administration of the oseltamivir or placebo to minimize the risk of
vomiting either suspension as often as coincidental timing of the drugs allowed (typically
for the morning treatment). Given its propensity for gastric side effects (vomiting,
nausea),15 if significant adverse effects were associated with the administration of
oseltamivir to these patients despite these precautions, it should have been reflected in the
clinical scoring system employed.
Monitoring/Data Acquisition
Historical data, including previous vaccination against CPV and the duration of
clinical signs as noted by the owner or caretaker (rounded to the closest 12 hour period of
time), was obtained when this information was available at study entry. Signalment for
each dog was also recorded, as was its estimated percent dehydration on entry. Baseline
bloodwork was evaluated for all dogs on entry, consisting of a complete blood count
(CBC), a spun packed cell volume (PCV), total solids (TS) via refractometery, serum
electrolyte concentrations (sodium, potassium and chloride), blood glucose concentration
and blood lactate concentrationm. These values were monitored daily for all dogs. For
white blood cell values, the following variables were evaluated: initial total white blood
cell (WBC), neutrophil (neut) and lymphocyte (lymph) counts, the absolute nadir value
and the day of hospitalization (with day of presentation being day 1) on which the nadir
occurred for each of the WBC, neutrophil and lymphocyte counts, and the number of
days for which the counts for each value were significantly decreased, as defined by a WBC less than 3,000 cells/ul, neutrophils less than 2,000 cells/ul and lymphocytes less
than 1,000 cells/ul. Body weight was recorded twice daily, with the same scale used to
monitor each dog during their stay for consistency. In addition, vital parameters (heart
rate, respiratory rate, rectal temperature, mucous membrane color and capillary refill
time) were assessed a minimum of twice daily, as were hydration status and mentation.
Days on which dogs demonstrated SIRS criteria were also calculated as a percentage of
days of their total stay (days with positive SIRS criteria/total days in hospital x 100%).
SIRS was defined as to presence of at least 2 of the following 4 criteria: 1) temperature
greater than 102.5°F (39.2°C) or less than 100.0°F (37.8°C), 2)heart rate greater than 140
beats per minute (bpm), 3) respiratory rate greater than 40/minute or 4) total white blood
cell count greater than 19,000 cells/ul or less than 6,000 cells /ul.
Clinical Scoring System
A previously published clinical scoring system10 was used to evaluate 4 clinical
attributes of each patient: attitude, appetite, vomiting and feces. A score of 0 represented
a clinically normal parameter, with increasing severity of signs as the score increased up
to a maximum of 3 for each variable (Table 1). Scores were assigned twice daily, to
encompass the previous 12 hour period, and were assigned by the same investigator for
all dogs (MRS). The clinical scores were totaled for each dog per day for each of the 4
categories (attitude, appetite, vomit, feces), with a possible score ranging from 0 (normal)
to 6 (most severely affected) per 24 hour period. This total consisted of the combined
scores (ranging from 0 to 3) for each 12 hour periods for that day, assigned to each
category. In addition, a cumulative score consisting of a total across all 4 categories was calculated for each dog per day, with a possible score ranging from 0 (normal) to 24
(most severely affected).
STATISTICAL ANALYSIS
The Shapiro-Wilks test was used to evaluate the distribution of continuous
variables. Median (minimum, maximum) was used to describe continuous variables not
normally distributed and mean (+/- standard deviation [SD]) was used to describe
normally distributed variables. The Wilcoxon ranksum test was used to compare not
normally distributed continuous variables while the t test was used for normally
distributed variables. Categorical variables were described using percent and the Fisher’s
exact test was used to compare these variables between the treatment and control groups.
For all comparisons, a p-value <0 .05="" all="" analyses="" considered="" p="" significant.="" statistical="" was="">were performed using a statistical software programn.
USE OF OSELTAMIVIR IN THE TREATMENT OF CANINE PARVOVIRAL
ENTERITIS
Michelle R. Savigny
Master of Science, May 10, 2008
(DVM, Texas A&M University, 2004)
(B.A., State University of New York at Buffalo, 2000)
Directed by Douglass K. Macintire
Despite the availability of an effective vaccine, canine parvovirus (CPV) enteritis remains a significant cause of disease in veterinary medicine. Appropriate treatment of the disease can result in a favorable survival rate; however, lack of treatment is almost universally fatal. Unfortunately, the treatment tends to be costly to the client, often times deterring medical care. There exists a need for an effective treatment option that will ameliorate disease morbidity in addition to hospitalization time, thereby decreasing client cost. Oseltamivir, a neuraminidase inhibitor antiviral drug, has resounding anecdotal support for achieving this goal. However, scientific validation at this point is lacking.
This study, using a prospective, blinded, randomized, placebo controlled clinical trial, aimed to investigate the effects of oseltamivir as an adjunct to standard treatment of CPV enteritis. Results show the only significant difference between groups to be in weight change during hospitalization in the oseltamivir group versus placebo.
TABLE OF CONTENTS
LIST OF TABLES .......................................................................................................viii
LIST OF FIGURES........................................................................................................ix
CHAPTER I. INTRODUCTION....................................................................................1
CHAPTER II. LITERATURE REVIEW........................................................................3
STATEMENT OF RESEARCH OBJECTIVE ..............................................................12
CHAPTER III. MATERIALS AND METHODS..........................................................12
Study Design......................................................................................................12
Standard Treatment ............................................................................................13
Oseltamivir Treatment .......................................................................................15
Monitoring/Data Acquisition..............................................................................16
Clinical Scoring System.....................................................................................17
Statistical Analysis.............................................................................................18
CHAPTER IV. RESULTS............................................................................................18
CHAPTER V. DISCUSSION AND CONCLUSIONS..................................................23
Findings .............................................................................................................23
Limitations.........................................................................................................28
Conclusions .......................................................................................................30
FOOTNOTES ...............................................................................................................32
REFERENCES..............................................................................................................33
vi ii
LIST OF TABLES
Table 1. Clinical Scoring System .................................................................................37
Table 2. White Blood Cell Counts ................................................................................38
TLIST OF FIGURES
Figure 1. Weight change over time for individual dogs .................................................39
Figure 2. Averaged white blood cell count over time ....................................................40
Figure 3. Box plot of cumulative total scores by day.....................................................41
INTRODUCTION
Canine parvovirus (CPV) is a single-stranded DNA virus that was first discovered in 1978.1 It is a hardy, highly contagious virus that remains a cause of significant disease process in young dogs. It is estimated that over one million dogs are affected each year in the United States2 despite the availability of an effective vaccine.
CPV infects and replicates in rapidly dividing cells, most notably the lymphoid organs, latter myeloid progenitor cells in the bone marrow, and intestinal epithelial cells.
Replication results in cell destruction, causing a clinical disease characterized by severe vomiting, hemorrhagic diarrhea, dehydration and neutropenia. This disease is almost universally fatal without treatment, with reported survival rates of only 9% in an experimental model.3 Treatment increases this figure significantly, with reported survival rates ranging from 64-95%.2, 4-10 Care given in a university setting may be associated with increased survival rates of 96-100%2, 8, versus 67-75%2, 7 at local day clinics. It is postulated that this increase may be due to the 24 hour care available at these universities, as well as the likelihood for more intensive treatments such as plasma or colloid therapy.2
Therapy for CPV enteritis remains mainly supportive and aimed at controlling the clinical signs of disease. Multiple more directed therapies have been studied, such as human recombinant granulocyte colony stimulating factor8, 9, 11, equine antiendotoxin7, 12, recombinant bactericidal/permeability-increasing protein2, and interferon omega13, 14 with variable or disappointing results. Early enteral nutrition has been the only modality to date to show promise for a shortened recovery time and decreased disease morbidity associated with CPV enteritis.10 However, the need still remains for a therapeutic agent hat will help ameliorate the morbidity and mortality of this disease, and in doing so also decrease the cost of treatment. While the survival rate with aggressive treatment is very good, financial constraints often result in suboptimal treatment with lower survival, or even euthanasia. This fact demonstrates the clear need for an agent to help make the appropriate treatment for CPV enteritis a more financially feasible option for many owners or shelters.
Oseltamivira (Tamiflu) is a neuraminidase inhibitor originally designed to treat human influenza virus.15 It has recently also shown efficacy in the treatment of avian influenza. Oseltamivir inhibits the viral neuraminidase enzyme, and thus prevents the cleavage of sialic acid residues. This cleavage is necessary for liberating newly formed virons from the host cell, as well as for preventing the aggregation of viral particles. Both the viron liberation and decreased aggregation are mechanisms necessary for the spread and dissemination of the virus throughout the host in order to further infection. The neuraminidase enzyme is also needed for the virus to cleave the sialic acid residues
in mucin to allow for penetration of this protective layer and infection of respiratory epithelial tissue. Anecdotal reports of oseltamivir use in veterinary medicine claim that it is associated with a less severe form of disease and a quicker recovery when administered to dogs with CPV enteritis. However, unlike the influenza virus, CPV does not rely on neuraminidase for effective replication. Therefore, any beneficial effects that may be present would not be due to a direct antiviral action. Human studies have shown a ignificant decrease in the development of bacterial infections secondary to influenza when oseltamivir is used.15-21 This effect is believed to be due to a decrease in bacterial
permeation through the mucin layer of the respiratory epithelial cells. An extension of this is that oseltamivir could also have a similar inhibitory effect on bacterial permeation through the mucin layer of the gut epithelial cells. This would decrease bacterial translocation, resulting in a potentially lower incidence of endotoxemia, sepsis, systemic inflammatory response and eventual organ failure that is thought to be the main mechanism behind the mortality of CPV enteritis. Thus, oseltamivir could have an effect on the disease process of CPV enteritis in a non-viral dependent manner. In one report, 90% of dogs that died from CPV enteritis had Escherichia coli cultured from their liver
and lungs.22 ARDS-like changes in the lungs of these dogs on histopathologic exam was also present, demonstrating the possibility for organ failure secondary to SIRS development.22
This study was designed to investigate the influence of oseltamivir when added to standard treatment on the disease process of CPV enteritis, as well as to monitor for significant adverse side effects associated with its use. Our hypothesis was that oseltamivir would help to ameliorate the disease morbidity and mortality associated with naturally occurring CPV enteritis, thereby decreasing hospitalization time, the need for colloid and other adjunctive therapies, and thus the cost associated with treatment.
LITERATURE REVIEW
Canine parvovirus (CPV) is a single-stranded, non-enveloped DNA virus that
infects and destroys rapidly dividing cells of the host. It is a highly contagious and hardy
virus, resistant to environmental conditions and many cleaning agents. The virus is
transmitted via the fecal-oral route. It primarily affects young dogs six weeks to six
months old, with the majority of older dogs showing immunity from either natural
infection or vaccination. CPV has a propensity to infect rapidly dividing cells. These
cells in dogs over eight weeks old include those of lymphoid organs, the latter myeloid
progenitor cells in the bone marrow, and intestinal epithelial cells. Rarely, the lungs,
liver and/or kidney can be infected. Dogs younger than eight weeks or pups in utero can
also have the virus infect myocardial cells, resulting in a myocarditis that can lead to
acute death or chronic heart failure symptoms with death by six months of age.1, 11, 23
Once infected, CPV localizes to the lymphoid tissue of the oropharanyx where it
replicates before entering the bloodstream. Viremia will usually occur two to three days
post-infection, and is manifested by a slight anorexia and fever. Dogs recover from this
stage briefly before progressing into clinical enteritis, typically between days five and six
post-infection.11 Clinical signs of enteritis include anorexia, vomiting, diarrhea and
severe dehydration. Severity of signs is related to age, stress level (other disease
processes, weaning), immune status and presence of intestinal parasites. These signs will
usually pinnacle around day six to seven post-infection.11
CPV infects and destroys the epithelial cells of the intestinal crypts. These cells
are responsible for the regeneration and differentiation into the villous epithelial cells.
Therefore, without the intestinal crypt cells, there is marked villous blunting, atrophy and
even necrosis, as well as malabsorption of nutrients with resultant diarrhea. Intestinal
inflammation accompanies this destruction and can lead to the breakdown of the gutblood
barrier. Translocation of bacteria from the gut lumen into the bloodstream, along
with an impaired immune response, can result in bacteremia. Bacteria are most often
gram negative, releasing endotoxin upon their destruction. Endotoxemia can then lead to
systemic inflammatory response syndrome through the activation of various cytokine
mediators, progressing into multiple organ dysfunction syndrome and death.
Leukopenia is considered a characteristic and often diagnostic quality of CPV
infection. However, this finding is reported to be evident in as few as 33% of cases when
diarrhea first developes.6 Most pronounced of the decreased white blood cells is the
neutrophil portion. This neutropenia is postulated to be due to a combination of
significant intestinal tract inflammation acting as a sink for the cells, rapid depletion of
bone marrow stores, and infection of the granulocyte precursors in the bone marrow.
Interestingly, it appears that the CPV spares the early hematopoietic precursors and
destroys mainly the later stages. Neutrophil nadir was found to coincide with the days
when clinical signs were at their worst at roughly day seven to eight post-infection. In
addition, neutrophil recovery coincides with clinical recovery at approximately days eight
through 12.11 A rebound neutrophilia is often observed as granulopoiesis is stimulated
with a decreasing peripheral consumption of mature neutrophils and a decreasing central
destruction of their progenitor cells. Hypoproteinemia, in particular hypoalbuminemia, is
another common clinicopathologic abnormality associated with CPV enteritis. This is a
result of a combination of factors, including intestinal loss, decreased synthesis as a
negative acute phase protein and decreased nutritional intake. Other laboratory
abnormalities vary, and can include anemia, azotemia, or increased liver enzymes.
Maternal antibodies can offer protection to pups from CPV for the first few weeks
of life. The period of protection varies, depending on maternal antibody level, amount of colostrum ingested, and size of the litter. The titer of the pup will equal approximately
50-60% of the bitch’s titer at whelping, meaning that proper vaccination of the mother is
essential to conferring protection to her pups.23 Maternal antibodies have a half-life of
around 10 days in the pup, and can interfere with proper immune response to
vaccination.23 The primary cause for vaccine failure is interference by maternal
antibodies. The high titer (higher amount of virus per dose), low passage (fewer times
virus grown on tissue culture) vaccines tend to be more effective despite maternal
antibody levels.23 A schedule of vaccination starting at six weeks old with boosters every
three weeks until 12-16 weeks old is the current recommendation to avoid interference
and provide proper protection to the pup. Proper vaccination is highly recommended, as
unvaccinated dogs have been found to be 12.7 times more likely to develop CPV enteritis
when compared to vaccinated dogs.24 Local antibody activity is responsible for binding
intestinal CPV to prevent further virus shedding in the feces. Shedding will typically
occur until between days three and 10 post infection. However, it is the systemic
humoral immune response or the circulating antibodies that confer protection as they
inactivate the virus in the blood before it has a chance to invade intestinal, lymphoid or
bone marrow cells.
Treatment of CPV consists principally of supportive care. Maintaining hydration
is the staple of this supportive care. Balanced electrolyte solutions supplemented as
needed with potassium and dextrose are the primary fluid type of choice. Colloidal
solutions can be used when indicated to support colloidal osmotic pressure. Antibiotic
use is an area where opinions vary, although their use is generally accepted once the
neutrophil count falls below 1500 cells/ul. Antibiotics targeting enteric bacteria are recommended, such as a combination of ampicillin and enrofloxacin. A lower dose of
5mg/kg q12 hours and use for a limited time (i.e. less than 5 days if possible) are
precautions taken to help avoid the side effect of cartilage deformities in these young
dogs. Antiemetics such as a constant rate infusion of metaclopramide and/or
chlorpromazine are frequently used to help control the vomiting associated with the
disease. The concept of starvation to rest the gut is being challenged lately. A recent
study found that dogs fed through a nasoesophageal tube soon after admission and
through vomiting episodes showed a significantly more rapid clinical improvement
without major adverse side effects.10 These dogs were found to have a quicker recovery
of appetite and normal attitude as well as recovery from vomiting and diarrhea when
compared to the food restricted group.
Several other potential treatments have been tested, often with conflicting or
disappointing results. Treatment with equine LPS endotoxin antiserum has shown
varying results. One source reports that its use decreased mortality from 48% to 17%.12
Another study found that its use was associated with an increased risk of death in puppies
less than 16 weeks.7 Recombinant human granulocyte colony stimulating factor (rhGCSF)
has been shown to help reverse neutropenia associated with radiation therapy and
drugs as well as cyclic neutropenia in dogs. However, its use to treat the neutropenia of
CPV infection has not shown significant benefit in recovery time or neutrophil
response.8, 9 Bactericidal/permeability-increasing protein (BPI) is an enzyme located in
the granules of polymorphonuclear cells. It is cytotoxic to gram negative bacteria by
increasing membrane permeability, and will also bind to free LPS to block its effects.
With the finding that 82% of CPV enteritis cases have endotoxemia, and that a relationship exists between increasing detectable tumor necrosis factor activity and
mortality,6 it was postulated that administration of a recombinant BPI (rBPI21) would
help improve survival. Unfortunately, rBPI21 treatment was not found to have a
significant effect on outcome, hospitalization time or plasma endotoxin levels in one
study.2 Interferons have antiviral effects that vary depending on dose, species and virus
type. A recombinant form of feline interferon omega (rFeIFN) has shown promise in
reducing severity of disease and mortality,13, 14 warranting further investigation
It is not necessarily the infection of the rapidly dividing cells and resultant
gastroenteritis that causes the high morbidity and mortality associated with CPV disease.
Gnotobiotic dogs infected with CPV were found to have only mild clinical signs.6 This is
highly suggestive that it is the bacterial translocation and resultant septicemia that is
significantly related to the morbidity and mortality of CPV enteritis. Endotoxemia with
subsequent systemic inflammatory response syndrome (SIRS) and multiple organ
dysfunction syndrome (MODS) are associated with a high mortality in and of themselves.
E. coli has been cultured from the lungs and liver in up to 90% of dogs that have died
from CPV,22 illustrating the high potential for bacteremia to develop as well as its
devastating consequences.
Oseltamivir is a drug that was designed to inhibit the human influenza A and B
viruses through inhibition of neuraminidase. Hemagglutinin (HA) and neuraminidase
(NA) are two viral surface proteins that interact with receptors containing terminal
neuraminic acid residues.15 The purpose of the HA is to mediate viral attachment to the
host cell membranes to allow entry and subsequent replication. NA has multiple
functions that allow for the replication of the virus as well as enhancing its virulence. First, NA cleaves the sialic acid residue (N-acetyl-neuraminic acid is synonomous with
sialic acid) of the cellular receptor binding newly formed virons.20 This frees the virons
from the host cell, and allows their systemic release. Secondly, NA prevents the
aggregation of the virons, allowing more effective systemic distribution with subsequent
cellular infectivity.20 Thirdly, in order to come in contact with and invade respiratory
epithelial cells, the influenza virus must first penetrate through the protective mucin layer
covering these cells. This mucin contains abundant sialic acid residues. NA cleaves
these residues, facilitating the viral infiltration through mucin to the epithelial cells which
the virus then invades and replicates within.20 Inhibition of NA will result in a direct
antiviral effect in viruses, such as the influenza virus, that contain the cell surface protein
NA. These effects include decreased replication, decreased viral release, increased viral
aggregation and decreased dissemination. In viruses that lack NA receptors such as CPV,
NA inhibition may prevent viral migration through mucin and subsequent invasion of
respiratory epithelial or gastrointestinal epithelial cells.
The above listed functions are accepted and proven effects of NA. In addition,
there currently are hypothesized effects of NA on the immune system itself. Secretory
IgA provides the body’s primary immunologic defense of mucosal surfaces such as the
respiratory or the gut epithelium. Under normal circumstances, sialic acid residues are
covalently bound to the hinge region of the IgA molecule. Removal of these residues
converts IgA into an appropriate ligand for the liver asialoglycoprotein receptor, which
effectively removes IgA from circulation and results in its clearance.20 Working on the
unproven assumption that there is an equilibrium between circulating and mucosal IgA,
the loss of IgA mediated by NA would result in impaired mucosal defenses. The IgA of mucosal surfaces is secreted by B cells under the influence of gamma-delta T cells. Both
of these cells contain sialic acid moieties on their surfaces.20 If cleaved by NA, the
lymphocyte homing would be altered from the mucosa to the bone marrow, resulting in a
localized immunosuppression for that mucosal surface. It is also believed that NA itself
may allow the increased production of certain cytokines,15 resulting in a proinflammatory
effect. Thus, inhibition of neuraminidase has the potential to improve
mucosal immunity by increasing the amount of mucosal IgA, decreasing the clearance of
circulating IgA, allowing normal function of the B and T cells and decreasing the
cytokine release associated with the proinflammatory response. All of these effects could
be beneficial to a dog with severe gastrointestinal inflammation secondary to CPV
infection.
Effects of the neuraminidase inhibitors beyond direct antiviral effects have been
appreciated in multiple studies. Bacterial complications following influenza virus
infection in children and adults are common. The incidence of acute otitis media
complicating influenza infection in children ranges between 21% to over 50%.12 Use of
oseltamivir has been reported to reduce the incidence of otitis media by 44% (children 1-
12 years) and 56% (children 1-5 years). 21 In adults, secondary complications were
reduced from 15% in the placebo group to 7% in the oseltamivir group in one study.25
Secondary bacterial pneumonias are responsible for an estimated 25% of influenzarelated
deaths19 in adults. Streptococcus pneumoniae is a bacteria commonly associated
with pneumonia secondary to influenza infection. A lethal synergism between the
influenza virus and S. pneumonia has been established,17 presumably accounting for the
high morbidity and mortality associated with the combination. It has been shown that the influenza virus promotes adherence of S. pneumonia to respiratory epithelium.17, 18 Viral
NA cleaves the sialic acid residues on the host respiratory epithelial cell, revealing
cryptic binding sites for the bacteria, 17, 18 thus facilitating colonization. Oseltamivir
reverses this effect in vitro.17
In a mouse model of influenza with secondary pneumococcal pneumonia,
oseltamivir use alone increased survival from 0% to 75%.18 This study also found that
oseltamivir use in addition to proper antibiotics resulted in 100% survival, compared to
0% in mice treated with antibiotics alone. Prophylactic use of oseltamivir resulted in
significantly less weight loss than placebo treated mice (prophylactic use, 5%; placebo,
25%).18 Prophylactic as well as delayed treatment use decreased the development of
pneumonia, increased survival and slowed the development and progression of disease in
those animals that did acquire pneumonia.18 In adults, oseltamivir use was found to
decrease the incidence of lower respiratory tract complications (LRTC) in influenza
patients overall by 55%.16 Patients considered at risk for secondary complications
showed a decrease in LRTC and antibiotic use by 34%, and in otherwise healthy patients
by 67%.16 In these studies, the beneficial effect of oseltamivir in preventing secondary
bacterial infections is most likely the result of NA inhibition preventing bacterial and
viral penetration of the mucin layer to allow bacteria to bind to epithelial cells.
Oseltamivir is labeled for use in children over 1 year old; use in children less than
1 year is discouraged based on fatality seen in animal toxicology studies in seven day old
rats with high doses of drug.21 Although the etiology of such adverse effects has not been
specifically identified, there is concern over lack of a fully developed blood brain barrier.
Side effects in children over 1 year are minimal, with vomiting being the only significant effect versus placebo.21 Similarly, nausea (12% vs 4%) and vomiting (10% versus 3%)
were found to be significant side effects over placebo in adults that subsided after the first
few days.27 These effects can be diminished with administration of a small amount of
food along with the drug. Studies in adult humans have shown an 80% bioavailability of
oseltamivir after oral dosing.21
STATEMENT OF RESEARCH OBJECTIVES
This study was designed to investigate the influence of oseltamivir when added to
standard treatment on the disease process of CPV enteritis, as well as to monitor for
significant adverse side effects associated with its use. Our hypothesis was that
oseltamivir would help to ameliorate the disease morbidity and mortality associated with
naturally occurring CPV enteritis, thereby decreasing hospitalization time, the need for
colloid and other adjunctive therapies, and thus the cost associated with treatment.
MATERIALS AND METHODS
Study Design
The present study was a prospective, randomized, placebo-controlled, blinded
clinical trial of the effects of oseltamivir in dogs with naturally occurring parvoviral
enteritis. Cases of CPV enteritis were recruited from the local area and animal shelter
between April 2005 and August 2006. A financial incentive was offered to the owners or
agent to allow their dogs to participate in the study. Inclusion criteria was simply a
positive CPV fecal antigen test, presence of appropriate clinical signs (vomiting, diarrhea,
lethargy, anorexia), and lack of any treatment initiated prior to presentation. Informed consent was received from all owners or agents prior to any treatment initiation at the
Auburn University Veterinary Teaching Hospital. In addition, all procedures were
reviewed and approved by the Animal Care and Use Committee of Auburn University.
Dogs were excluded if they lacked a positive fecal antigen test for CPV, if treatment had
been administered prior to presentation, or if consent was not granted. Based on these
criteria, only one dog that was eligible during the trial period was not included due to
lack of owner consent. All dogs that entered the study completed the trial.
Randomization was achieved via assignment of numbers with predetermined
treatment designation in blocks of 5. Assignment was allocated by hospital personnel not
directly involved in the trial, and these assignments were uncovered to the investigators
only at the conclusion of the study. Power analysis was not performed as part of the
design of the study, but post-hoc analysis revealed that 47 dogs would be needed in each
group to give the study a power of 0.80 at a significance level of 0.05.
Standard Treatment
A treatment protocol was designed to standardize therapy between the two groups
with the exception of oseltamivir administration to the treatment group and a placebo to
the control group. No deviations were made from this protocol for any of the patients.
An intravenous catheter was placed in all dogs, either a peripheral cephalic catheter or
central venous catheter in a jugular vein. Intravenous fluids consisting of a balanced
electrolyte solution b, c were administered at an initial rate to replace an estimated
dehydration deficit over a minimum of 2 hours and a maximum of 4 hours. In the case
were it was determined that shock was present, the same intravenous fluids were bolused
at a dose of 10-30ml/kg over approximately 20 minutes. Boluses were repeated until it was assessed that the state of shock had been resolved, and the remaining fluid deficit
estimated to replace dehydration was administered as above. No patient required the
administration of colloidal therapy to treat a state of shock. Assessment of hydration and
perfusion status, as well as fluid rates were decided upon by a single investigator (MRS)
for all dogs. After rehydration, the hydration status was reassessed via physical
parameters and the fluid rate adjusted as deemed appropriate. Fluids were supplemented
with potassium chlorided and/or dextrosee as needed based on laboratory results.
All dogs received intravenous (IV) antibiotics. Ampicillinf was administered at
22mg/kg (11mg/lb) IV every 8 hours and enrofloxacing at 5mg/kg (2.5mg/lb) IV every 12
hours. A metoclopromideh constant rate infusion was also initiated in all patients at a rate
of 1-2mg/kg/day (0.5-1mg/lb/day). If vomiting persisted at a rate of more than twice per
12 hour period despite a maximum rate of metoclopramide, chlorpromazinei was added at
0.5mg/kg (0.25mg/lb) subcutaneously every 8 hours. Pyrantel pamoatej (10mg/kg
[5mg/lb] per os) was administered to all dogs within the first 3 days of arrival to eradicate
intestinal parasites. If the total solids by refractometery fell below 3.5g/dl, Hetastarch
6%k was infused at a rate of 10-20ml/kg/day (5-10ml/lb). If anemia, defined as a PCV of
less than or equal to 20%, was present, a fresh whole blood transfusion from an available
donor was given at 10-20ml/kg (5-10ml/lb) over 4 hours. Packed red blood cell
availability was very limited at the time of the first required transfusion. Fresh whole
blood was collected from a donor dog and administered in its place. In order to minimize
differences in treatment between dogs, when the second transfusion became necessary,
although packed red cells were available, fresh whole blood from the previous donor was
again administered. If vomiting episodes were less than 4 per 12 hour period, water and a bland dietl were offered. If either appeared to induce nausea, they were immediately
removed from the animal. Voluntary eating was allowed through mild vomiting but food
was not forced upon any dog. True client cost for the treatment administered to the dogs
was not calculated; this figure can only be inferred from the intensity of treatments
(additional antiemetics, colloid or transfusion therapy, increased length of
hospitalization) required for each dog between the groups.
Discontinuation of intravenous fluids was at the discretion of a single investigator
(MRS). This was accomplished primarily when the animal was no longer vomiting and
consistently eating and drinking sufficiently to maintain hydration. Once fluids were
discontinued, all patients were monitored for at least 12 hours before discharge from the
hospital in order to ensure no relapse of clinical signs. The day of discharge was
assigned as the day when it was deemed the dog was healthy enough to be discharged. If
the dog stayed in the hospital for non-medical reasons, these extra days were not included
when figuring length of hospitalization. In those animals that did not survive, the day of
death was considered an endpoint equivalent to the day of discharge for the purpose of
data analysis. Post-mortem exams were performed on all non-surviving dogs.
Oseltamivir Treatment
Dogs assigned to the treatment group received oseltamivir at 2mg/kg (1mg/lb) per
os every 12 hours. An equivalent volume of a placebo, consisting of a standard
suspension agent with color additive, was administered per os every 12 hours to dogs in
the control group. Prior experience with oseltamivir by the authors has shown that dogs
often react to its taste and frequently vomit shortly after administration. Dilution of the
oseltamivir with water (1:1) prior to administration seemed to decrease these reactions; therefore, in this study, both oseltamivir and placebo were diluted out as above to lessen
the risk of adverse reaction to the oseltamivir. In addition, in dogs receiving
chlorpromazine for protracted vomiting, it was attempted to dose this medication 30-60
minutes before administration of the oseltamivir or placebo to minimize the risk of
vomiting either suspension as often as coincidental timing of the drugs allowed (typically
for the morning treatment). Given its propensity for gastric side effects (vomiting,
nausea),15 if significant adverse effects were associated with the administration of
oseltamivir to these patients despite these precautions, it should have been reflected in the
clinical scoring system employed.
Monitoring/Data Acquisition
Historical data, including previous vaccination against CPV and the duration of
clinical signs as noted by the owner or caretaker (rounded to the closest 12 hour period of
time), was obtained when this information was available at study entry. Signalment for
each dog was also recorded, as was its estimated percent dehydration on entry. Baseline
bloodwork was evaluated for all dogs on entry, consisting of a complete blood count
(CBC), a spun packed cell volume (PCV), total solids (TS) via refractometery, serum
electrolyte concentrations (sodium, potassium and chloride), blood glucose concentration
and blood lactate concentrationm. These values were monitored daily for all dogs. For
white blood cell values, the following variables were evaluated: initial total white blood
cell (WBC), neutrophil (neut) and lymphocyte (lymph) counts, the absolute nadir value
and the day of hospitalization (with day of presentation being day 1) on which the nadir
occurred for each of the WBC, neutrophil and lymphocyte counts, and the number of
days for which the counts for each value were significantly decreased, as defined by a WBC less than 3,000 cells/ul, neutrophils less than 2,000 cells/ul and lymphocytes less
than 1,000 cells/ul. Body weight was recorded twice daily, with the same scale used to
monitor each dog during their stay for consistency. In addition, vital parameters (heart
rate, respiratory rate, rectal temperature, mucous membrane color and capillary refill
time) were assessed a minimum of twice daily, as were hydration status and mentation.
Days on which dogs demonstrated SIRS criteria were also calculated as a percentage of
days of their total stay (days with positive SIRS criteria/total days in hospital x 100%).
SIRS was defined as to presence of at least 2 of the following 4 criteria: 1) temperature
greater than 102.5°F (39.2°C) or less than 100.0°F (37.8°C), 2)heart rate greater than 140
beats per minute (bpm), 3) respiratory rate greater than 40/minute or 4) total white blood
cell count greater than 19,000 cells/ul or less than 6,000 cells /ul.
Clinical Scoring System
A previously published clinical scoring system10 was used to evaluate 4 clinical
attributes of each patient: attitude, appetite, vomiting and feces. A score of 0 represented
a clinically normal parameter, with increasing severity of signs as the score increased up
to a maximum of 3 for each variable (Table 1). Scores were assigned twice daily, to
encompass the previous 12 hour period, and were assigned by the same investigator for
all dogs (MRS). The clinical scores were totaled for each dog per day for each of the 4
categories (attitude, appetite, vomit, feces), with a possible score ranging from 0 (normal)
to 6 (most severely affected) per 24 hour period. This total consisted of the combined
scores (ranging from 0 to 3) for each 12 hour periods for that day, assigned to each
category. In addition, a cumulative score consisting of a total across all 4 categories was calculated for each dog per day, with a possible score ranging from 0 (normal) to 24
(most severely affected).
STATISTICAL ANALYSIS
The Shapiro-Wilks test was used to evaluate the distribution of continuous
variables. Median (minimum, maximum) was used to describe continuous variables not
normally distributed and mean (+/- standard deviation [SD]) was used to describe
normally distributed variables. The Wilcoxon ranksum test was used to compare not
normally distributed continuous variables while the t test was used for normally
distributed variables. Categorical variables were described using percent and the Fisher’s
exact test was used to compare these variables between the treatment and control groups.
For all comparisons, a p-value <0 .05="" all="" analyses="" considered="" p="" significant.="" statistical="" was="">were performed using a statistical software programn.
jueves, 19 de marzo de 2015
CONTROL DE IBR EN ESPAÑA Mónica Espada , Juanjo Labajos, Luis Figueras , Marta Ruiz de Arcaute y Miren E. Ortega 2015
La crisis afecta a los programas de control de IBR en España
06/05/2013@15:31:57 GMT+1
 |
| Animal con signos clínicos de enfermedad respiratoria. |
La vacunación es una herramienta muy importante en los programas de control frente a la enfermedad, ya que reduce los signos clínicos y la diseminación del virus.
Mónica Espada [1,2], Juanjo Labajos [1], Luis Figueras [1,2], Marta Ruiz de Arcaute [1,2] y Miren E. Ortega [1]1. Gabinete Técnico Veterinario, S.L.
2. Departamento de Patología Animal. Facultad de Veterinaria de Zaragoza.
Imágenes cedidas por los autores
2. Departamento de Patología Animal. Facultad de Veterinaria de Zaragoza.
Imágenes cedidas por los autores
La rinotraqueitis infecciosa bovina (IBR) es una enfermedad infectocontagiosa producida por Herpesvirus tipo 1 (HBV-1). La sintomatología clínica es diversa en función del órgano que se ve afectado. Puede producir:
- Procesos respiratorios (los más importantes), principalmente en animales jóvenes. El cuadro comienza con fiebre alta y tos, alcanzando una alta morbilidad en los lotes infectados. En algunos animales puede complicarse con infecciones secundarias produciendo una enfermedad respiratoria con signos clínicos de respiración abdominal, dificultad en la respiración y anorexia. La mortalidad no suele ser muy alta en aquellos casos en los que se aplican medidas terapeuticas tempranas.
- Procesos reproductivos. Pueden producir vulvovaginitis pustular infecciosa (IPV) y balanopostitis pustular infecciosa (IPB). En algunos casos se ha relacionado con procesos de reabsorción embrionaria en fase temprana.
La gravedad del signo clínico varía dependiendo de la cepa del virus, de la vía de infección, del estado sanitario del hospedador y de los factores ambientales. La gravedad del cuadro es muy variable en función de si se trata de una enfermedad epidémica o endémica en la granja, de si va ligada a la entrada de animales o de si se aplican medidas profilácticas en la explotación. Sin embargo, el IBR suele aparecer como un cuadro de tipo leve que cursa a modo de brotes con signos de conjuntivitis bilateral, lagrimeo y descarga nasal. En gran parte de los animales afectados la sintomatología es subclínica con presencia intermitente de casos clínicos. Si se evitan infecciones secundarias, las consecuencias no son muy graves. La importancia real de esta enfermedad se plantea como un aspecto comercial más que sanitario, porque son muchos los países que han establecido programas de control (algunos de ellos han erradicado la enfermedad), por lo que existe una directiva europea que regula los movimientos de animales intracomunitarios intentando preservar al máximo el estatus sanitario de las zonas indemnes.
Es una enfermedad cuyas pérdidas económicas son muy difíciles de cuantificar porque tanto en el caso del cuadro respiratorio como en el reproductivo pueden existir etiologías muy diversas, lo que dificulta el diagnóstico, exceptuando cuando va ligado a una entrada de animales. En las explotaciones ganaderas en las que se presenta de forma endémica, se debe a la presencia de animales que permanecen infectados de forma latente de modo que actúan como portadores y diseminadores en aquellos momentos en los que la infección se reactiva.
No obstante, en la mayor parte de las explotaciones se contempla como una enfermedad en control para lo cual se implantaron medidas principalmente de tipo profiláctico. En algunas comunidades autónomas de España hubo implicación por parte de la administración planteando el plan de control de IBR dentro de las gestiones realizadas por las asociaciones de defensa sanitaria ganadera (ADSG).
Situación del IBR en Europa
Los programas de control en Europa comenzaron en los años 70, y son muchos los países en los que actualmente el IBR está erradicado o se encuentra en programas de erradicación.
Los países en los que la enfermedad se encuentra erradicada son Finlandia, Suecia, Dinamarca, Suiza Austria, Noruega y la provincia de Bolzano en Italia. En estos países el IBR se encuentra en campaña de erradicación por lo que está prohibida la vacunación, procediéndose al sacrificio de aquellos animales positivos. Este tipo de programas únicamente pueden ejecutarse cuando los niveles de prevalencia son bajos.
Los países con programas de control son Alemania, Bélgica, Francia, Holanda, Gran Bretaña, Hungría, Italia, Lituania y Polonia.
En la Unión Europea existe legislación vigente (Decisión 2007/584/CE, por la que se aplica la Directiva 64/432/CEE) que limita los intercambios intracomunitarios de ganado vacuno con destino a aquellos países en los que la enfermedad se considera erradicada, lo que supone una barrera comercial de entrada de ganado vacuno para países como España.
Situación del IBR en España
Esta enfermedad presenta una alta prevalencia en España (aunque no existen datos concretos, se estima que aproximadamente el 58% de los rebaños son positivos) con grandes diferencias entre comunidades. En previsión de las limitaciones comerciales que podían plantearse a España en los movimientos intracomunitarios, varias comunidades autónomas españolas comenzaron a trabajar en la implantación de programas de control. Actualmente no existe un plan de control a nivel nacional.
Algunas provincias, como Cantabria, Castilla y León y País Vasco han desarrollado programas voluntarios de control. Debido a que se trata de una infección vírica, las medidas de control van dirigidas a controlar la diseminación y la transmisión del virus.
Las medidas de control implantadas es España son de tipo profiláctico:
- Muestreo serológico de todas las explotaciones y aleatoriamente de los animales.
- Plan profiláctico vacunal con la obligación de aplicar únicamente vacuna marcadora*.
- Implantación de medidas de bioseguridad en la granja.
- Monitorización de estos programas con muestreos serológicos.
La vacunación es una herramienta muy importante en este proceso, reduciendo los signos clínicos de los animales infectados y la diseminación del virus y aumentando la protección de los animales sanos frente a las nuevas infecciones.
Es importante no olvidar que los programas vacunales son una herramienta más en los programa de control, por lo que siempre deben ir acompañados de la implantación de medidas de buenas prácticas y de bioseguridad.
¿De qué depende el éxito del programa de control?
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El control del IBR es un tema importante en Europa desde el aspecto económico. España todavía presenta una prevalencia media-alta aunque sí se ha observado una importante mejora en aquellas provincias en las que se han iniciado planes de control. Es importante mantener los programas sanitarios en las ganaderías desde un aspecto sanitario como económico.
El éxito de un programa de control de IBR dependerá de la asociación de varios factores clave: vacunación con vacunas marcadoras, pruebas de diagnóstico fiables, medidas de bioseguridad y una buena gestión del programa.
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Evolución de los programas de control
Tras la aparición en el mercado de las vacunas marcadoras, junto con la implantación de programas de control en varias provincias, fueron muchas las ganaderías que comenzaron a vacunar con dicha vacuna (en Aragón se alcanzaron niveles del 90% de empleo de vacuna marcadora). Muchos veterinarios concienciados con el problema y con las trabas comerciales que pueden dañar de forma importante la exportación de España, apostamos fuerte por la implantación de programas vacunales y muestreos serológicos periódicos.
Pero desde el 2012 la situación ha cambiado. Las dificultades económicas que está atravesando España, afectan de forma negativa por dos vertientes:
Los ganaderos que comenzaron a vacunar en el 2007 con vacuna marcadora se están replanteando si la inversión está justificada cuando no se observa la implantación de un programa nacional a corto plazo. Económicamente supone un aumento del gasto por dosis (incremento del coste de vacuna de un 31,25% si se vacuna con marcadora frente a la que no).
Las administraciones plantean recortes en los programas sanitarios de las ADSG. Los programas sanitarios estaban cofinanciados por la administración, por lo que al reducirse esa partida, los gastos se verán repercutidos al ganadero.
Estos factores están teniendo un efecto directo muy negativo en la lucha frente a esta enfermedad, dejándonos a los veterinarios como meros observadores del retroceso en el trabajo desarrollado durante cinco años, al dejarse de vacunar con vacuna marcadora volviendo a la convencional. Es importante resaltar que esto supone una limitación también a largo plazo porque serológicamente un animal vacunado con vacuna no marcadora se considera positivo de por vida.
Los programas de control únicamente se pueden llevar a cabo si la vacunación se realiza con vacunas marcadoras porque en caso contrario los controles serológicos no nos aportan información acerca de la diseminación del virus ni de la recirculación, por lo que se limita la monitorización. La única posibilidad viable en este caso es dejar un lote de control de novillas (o en el mejor de los casos, plantear la vacunación con vacuna marcadora únicamente en la reposición) y observar si existe seroconversión pero en este caso se pone en riesgo a esos animales o se dificulta el trabajo de vacunación por lo que al final se abandona esta opción.
También la implantación de medidas de bioseguridad supone un aumento en el coste para el ganadero por lo que en estos años se han visto limitadas. El control del gasto de producción en un momento en el que el margen de gasto/beneficio está tan ajustado hace que aquellas medidas que no aporten un beneficio inminente sean restringidas.
*En el año 2006 comenzaron a aparecer en el mercado las llamadas vacunas marcadoras, en las que se elimina el gen que codifica la glicoproteína E (gE), lo que permite el diagnóstico del origen de los anticuerpos (de origen virus de campo o vacunal). De esta manera se puede conocer el porcentaje de nuevas infecciones en un rebaño, las nuevas infecciones, la respuesta inmunitaria de la vacuna y la recirculación del virus dentro de un rebaño.
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| Las pérdidas económicas son muy difíciles de cuantificar porque pueden existir etiologías muy diversas, lo que además dificulta el diagnóstico. |
LENGUA AZUL EN ESPAÑA Ana Cristina Pérez de Diego, Pedro José Sánchez-Cordón y José Manuel Sánchez-Vizcaíno 2015
Evolución histórica y situación actual de la lengua azul en España
En los primeros años del siglo XXI la enfermedad sufrió una expansión geográfica y se detectaron diferentes serotipos en toda Europa.
12/03/2015@10:04:32 GMT+1
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| Culicoides imicola. (Foto: Alan R Walker - CC Licensed) |
Ana Cristina Pérez de Diego [1]*, Pedro José Sánchez-Cordón [2] y José Manuel Sánchez-Vizcaíno [3][1] Consejo de Colegios Profesiones de Veterinarios de Castilla-La Mancha[2] Instituto Pirbright (Reino Unido)[3] Centro de Vigilancia Sanitaria Veterinaria (VISAVET) (UCM)*anacristina@sanidadanimal.info
El virus responsable de la lengua azul (LA) pertenece al género Orbivirus. Se han descrito hasta la fecha 26 serotipos distintos entre los que no existe inmunidad cruzada, lo que complica la lucha frente a la enfermedad y hace que sea necesario vacunar contra cada serotipo específico frente al que se quiera proteger a los animales. La transmisión del virus de la LA no se produce por contacto directo entre animales susceptibles, ya que es necesaria la presencia de un vector, en concreto insectos hematófagos del género Culicoides, para que se produzca la transmisión de la enfermedad.
La enfermedad fue descrita por primera vez en Sudáfrica en 1902 y hasta hace poco se creía limitada entre los paralelos 40S y 35N, por lo que su presencia en el norte de la cuenca mediterránea resultaba poco probable. Sin embargo, en los primeros años del siglo XXI la enfermedad sufrió una expansión geográfica y se detectaron diferentes serotipos en toda Europa que llegaron a afectar a países nórdicos como Dinamarca o Suecia. Los esfuerzos para lograr su control mediante vacunación fueron efectivos y se logró su erradicación en la mayoría de los países, especialmente en los nórdicos.
La historia de la lengua azul en España
En julio de 1956 se detectó el serotipo 10 del virus de la LA en Portugal, justo un mes antes de que apareciera en el oeste de España. Como se comprobó posteriormente, esta cepa estuvo relacionada con el serotipo 10 presente en África en aquel momento, algo que se ha repetido a lo largo de la historia en varias ocasiones. Este serotipo provocó sintomatología clínica en ganado ovino y bovino. La morbilidad alcanzó el 3,5 %, mientras que la mortalidad fue del 2,6 %. Las medidas adoptadas consistieron en la vacunación con vacuna viva atenuada, la restricción de movimientos y el sacrificio en algunos casos. Estas medidas desembocaron en un descenso muy significativo del número de animales afectados en tan solo un año, lo que llevó a su erradicación en 1958.
En 2004, más de cuatro décadas después del último caso de lengua azul en la Península, se detectó el serotipo 4, probablemente procedente de Marruecos, en bovinos centinelas en la provincia de Cádiz. La enfermedad se extendió rápidamente a diferentes comunidades autónomas y se decidió instaurar un programa de control basado en la vacunación con vacuna viva atenuada que se empleó hasta la primavera de 2006, momento a partir del cual se dispuso de una vacuna inactivada que permitió la inclusión del ganado bovino en los programas de vacunación obligatoria. La epidemia se controló en 2006 tras la detección del último caso en Ávila. Durante 2007 y 2008 se continuó con el programa de lucha frente al serotipo 4, para lo que se emplearon vacunas inactivadas y se mantuvo el programa de vigilancia en centinelas, así como un programa de vigilancia entomológico, lo que conllevó la erradicación de este serotipo a finales del año 2008.
El serotipo 1 de la lengua azul
En julio de 2007 apareció en España un nuevo serotipo procedente del norte de África, el serotipo 1, que se expandió a Portugal y Francia . Este serotipo se mostró más virulento que el serotipo 4, y provocó una mortalidad del 7 %.
La vacunación obligatoria frente a este serotipo comenzó en 2007, para lo que se emplearon vacunas monovalentes inactivadas en ovinos y bovinos, lo que produjo un descenso del número de casos en los años siguientes, hasta el punto de modificar la política de vacunación y permitir que ésta fuera voluntaria desde el 30 de junio de 2011. En 2011, 2012 y 2013 se detectaron casos esporádicos de circulación del virus, lo que llevaría a establecer en el año 2013 áreas de vacunación obligatoria en Extremadura y Castilla-La Mancha.
El serotipo 8 en España
Cuando todavía permanecíamos vigilantes frente al serotipo 4, y en plena onda epidémica del serotipo 1, en enero de 2008 se introdujo en España el serotipo 8 procedente del norte de Europa. No obstante, resulta interesante comentar que en este caso la expansión fue mucho menor, posiblemente debido a que el principal vector de este serotipo (Culicoides obsoletus) es mucho menos abundante en la Península que Culicoides imicola, vector principal del resto de serotipos detectados en España hasta la fecha. La vacunación obligatoria frente a este serotipo permitió controlar rápidamente su expansión. Así, se detectó el último caso positivo a finales de 2010, lo que permitió declarar España libre de este serotipo dos años después.
Y el serotipo 4 volvió para quedarse
Cuando en 2010 la situación parecía controlada sin apenas presencia del serotipo 8 y con tan sólo 80 focos del serotipo 1, se produjo una nueva incursión del serotipo 4 en España, el cual fue detectado en octubre de ese año en la provincia de Cádiz. Entre 2010 y 2013 las notificaciones de este serotipo fueron las representadas en la figura 6, lo que condujo en el año 2013 a establecer un área de restricción y vacunación obligatoria en una zona de Andalucía.
¿Y ahora qué? ¿Cuál es la situación actual?
En la figura 7 se muestran los focos de lengua azul notificados en otoño de 2014, tanto del serotipo 1 como del serotipo 4. Mientras el serotipo 1 se ha controlado en las zonas de Extremadura y Castilla-La Mancha, donde estaba presente en 2013, este serotipo se ha vuelto a detectar en Andalucía procedente del norte de África, lo que conducirá a establecer una zona de vacunación obligatoria con el fin de evitar su dispersión hacia otras regiones.
Durante el 2014, el serotipo 4 se ha expandido sin control desde la zona de restricción y vacunación establecida en 2013 en el sur de Andalucía, lo que llevará a realizar una vacunación preventiva durante 2015 en un área mucho mayor a la que se estableció en 2013. Además, resulta interesante comentar que en ciertas zonas de la Península, en concreto en el sur de Andalucía, se debería establecer un sistema de vigilancia y de vacunación lo más exhaustivo posible, teniendo en cuenta la continua circulación de este serotipo en el norte de África, la vía tradicional de entrada a España, así como la circulación continuada en el tiempo que ha sucedido en estas zonas.
Lecciones aprendidas y su aplicación en nuestras explotaciones
La lucha que durante años se ha mantenido frente a esta enfermedad ha permitido mejorar e implementar eficaces planes de vigilancia, control y erradicación. En este sentido, la vacunación del ganado ovino y bovino se ha mostrado como la medida más efectiva para su control y erradicación. Además, al tratarse de una enfermedad vectorial, el sacrificio de los animales presentes en las explotaciones afectadas carece de sentido. Así mismo, la historia nos muestra que la circulación del virus en el norte de África supone una fuente de infección para España, por lo que debe mantenerse una vigilancia especial y un estado de alerta respecto a los serotipos que estén presentes en esa zona.
Para que la lucha frente a esta enfermedad sea efectiva, se requiere de la colaboración de todos los agentes implicados, especialmente de los ganaderos y veterinarios. A este respecto cabe destacar que la vacunación debe realizarse de acuerdo a la normativa vigente en cada momento, puesto que ésta se establece con el propósito de controlar y erradicar la enfermedad con el fin de minimizar las pérdidas económicas derivadas de la sintomatología y de las restricciones comerciales. Por otro lado, la aplicación de medidas como el confinamiento de los animales durante las horas de mayor actividad del vector (amanecer y atardecer) o el uso de insecticidas favorecen el control de esta enfermedad. Finalmente, cabe recordar la obligatoriedad de notificar cualquier sospecha de esta enfermedad a la autoridad competente en sanidad animal, a través de los veterinarios oficiales de las oficinas comarcales agrarias, pues una detección precoz facilitará la puesta en marcha de medidas para su control y limitará sus consecuencias.
Bibliografía disponible en www.albeitar.grupoasis.com/bibliografias/lenguazul183.doc
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