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Efficacy of passively transferred antibodies in cats with acute viral upper respiratory tract infection
Accepted Manuscript Title: Efficacy of passively-transferred antibodies in cats with acute viral upper respiratory tract infection Author: Yvonne Friedl, Bianka Schulz, Anne Knebl, Chris Helps, Uwe Truyen, Katrin Hartmann PII: DOI: Reference:
S1090-0233(14)00185-3 http://dx.doi.org/doi:10.1016/j.tvjl.2014.05.002 YTVJL 4136
To appear in:
The Veterinary Journal
Accepted date:
2-5-2014
Please cite this article as: Yvonne Friedl, Bianka Schulz, Anne Knebl, Chris Helps, Uwe Truyen, Katrin Hartmann, Efficacy of passively-transferred antibodies in cats with acute viral upper respiratory tract infection, The Veterinary Journal (2014), http://dx.doi.org/doi:10.1016/j.tvjl.2014.05.002. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Efficacy of passively-transferred antibodies in cats with acute viral upper respiratory tract infection Yvonne Friedl a,*, Bianka Schulz a, Anne Knebl a, Chris Helps b, Uwe Truyen c, Katrin Hartmann a
a
Clinic of Small Animal Medicine, Ludwig-Maximilians-Universitaet Veterinaerstrasse 13, 80539 Munich, Germany
Munich,
b
Molecular Diagnostic Unit, Langford Veterinary Services, University of Bristol, Langford House, Bristol BS40 5DU, United Kingdom c
Institute of Animal Hygiene and Public Veterinary Services, University of Leipzig, An den Tierkliniken 1, 04103 Leipzig, Germany
* Corresponding author. Tel.: +49 094 315 5371. E-mail address: [email protected] (Y. Friedl).
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Abstract
26
A commercial hyperimmune serum, containing antibodies against feline calicivirus
27
(FCV), feline herpesvirus 1 (FHV-1), and feline panleukopenia virus is available for
28
treatment of cats with feline upper respiratory tract disease (FURTD), but its efficacy has not
29
been rigorously evaluated in scientific studies. The aim of this randomised, placebo-
30
controlled, double-blind clinical trial was to evaluate the efficacy of passive immunisation in
31
cats with acute viral FURTD caused by FCV and/or FHV-1 infection. All cats received
32
symptomatic treatment during the study period. Hyperimmune serum was administered to
33
one group (n = 22) and an equivalent amount of saline was administered to the control group
34
(n = 20) as placebo, for 3 consecutive days. In the treatment group, cats ≤ 12 weeks old
35
received 2 mL, cats > 12 weeks old received 4 mL, subcutaneously once daily and topically
36
into eyes, nostrils, and mouth every 8 h. Clinical signs, including a ‘FURTD score’ and
37
general health status were recorded daily for 8 days and again on day 21. FCV shedding was
38
determined by quantitative PCR on days 0 and 21.
39 40
Clinical signs and health status in both groups improved significantly over time (P <
41
0.001). Cats receiving hyperimmune serum significantly improved in terms of ‘FURTD
42
score’ (P = 0.046) and general health status (P = 0.032) by day 3, while cats in the placebo
43
group only improved significantly by day 7. There was no significant difference in the
44
number of cats shedding FCV between the two groups. Thus, administration of hyperimmune
45
serum led to a more rapid improvement of clinical signs in cats with acute viral FURTD, but
46
by day 7, clinical signs had improved equally in both groups.
47 48
Keywords: Feline calicivirus; Feline herpesvirus 1; Hyperimmune serum; Upper respiratory
49
tract infection
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Introduction
51
Feline upper respiratory tract disease (FURTD) can be caused by several pathogens,
52
leading to similar clinical signs of disease. Two viruses in particular, feline calicivirus (FCV)
53
and feline herpesvirus 1 (FHV-1), are responsible for at least 80% of FURTD cases (Helps et
54
al., 2005; Di Martino et al., 2007; Gould, 2011). In addition, bacteria, including
55
Chlamydophila felis, Bordetella bronchiseptica, and Mycoplasma spp., can act as primary
56
infectious agents (Jacobs et al., 1993; Hartmann and Hartmann, 2010; Hartmann et al., 2010).
57
After initial infection, FHV-1 develops a state of latency, residing mainly in the trigeminal
58
and vestibular ganglions and cats become carriers, shedding virus during period of
59
recrudescence (Townsend et al., 2004; Gaskell et al., 2007; Parzefall et al., 2010). FCV-
60
infected cats can also become asymptomatic carriers, but shed virus persistently (Wardley
61
and Povey, 1977; Radford et al., 2007, 2009).
62 63
Several antiviral drugs are effective in vitro against FHV-1 (Nasisse et al., 1989;
64
Maggs et al., 2000; Maggs and Clarke, 2004; Williams et al., 2004; Siebeck et al., 2006; Van
65
der Meulen et al., 2006) and FCV (Povey, 1978a). Some treatment options are available
66
clinically for treating cats affected with FHV-1, with most of these drugs being administered
67
topically, e.g., cidofovir, idoxuridine, vidarabine, and trifluridine (Stiles, 1995; Fontenelle et
68
al., 2008). Adverse effects can occur when anti-viral drugs are used systemically (Weiss et
69
al., 1993; Nasisse et al., 1997), although oral administration of famciclovir (Famvir, Novartis)
70
has recently been shown to improve clinical signs in FHV-1-infected cats without causing
71
adverse effects (Malik et al., 2009). In contrast, there is no specific treatment option available
72
for FCV that has proven efficacy and tolerable adverse effects (Povey, 1978b; Hennet et al.,
73
2011).
74
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The commercially-available product, Feliserin (IDT Biologika), is reported to contain
76
antibodies against FCV, FHV-1, and feline panleukopenia virus and is marketed in Germany
77
(Product License number 35a/92) for the treatment of acute viral FURTD and feline
78
panleukopenia. Its efficacy, however, has not been rigorously assessed in scientific studies.
79
Therefore, the aim of the present study was to evaluate whether administration of Feliserin
80
was beneficial in cats affected with acute viral FURTD.
81 82
Materials and methods
83
Study design
84
The study was performed as a randomised, placebo-controlled, double-blind clinical
85
trial in cats affected with acute viral FURTD. Thirty-two cats (22 receiving Feliserin and 10
86
placebo) were prospectively recruited into the study and randomised to either treatment or
87
placebo group. Data from a previous study from an additional 10 cats that had received the
88
same symptomatic treatment protocol were also included as controls. When a cat was
89
recruited, medications were drawn up and injected by a veterinarian not involved in the study
90
to ensure that neither owner nor clinician were aware which group the cat had been assigned
91
to. Decoding occurred after the study was completed and data entered for statistical
92
evaluation.
93 94
The study fulfilled the general German guidelines for prospective studies with
95
informed owner consent and was carried out with permission from the responsible German
96
veterinary authority (Government of Upper Bavaria, Maximilianstrasse 39, 80538 Munich,
97
reference number 55.2-1-54-2532-05-12).
98 99
Study population
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Included in the study were 42 cats that presented with clinical signs of FURTD of less
101
than 7 days duration and in which FCV and/or FHV-1 had been detected by quantitative
102
polymerase chain reaction (qPCR) from oropharyngeal and/or conjunctival swabs. Cats were
103
excluded if demonstrated to be infected with feline immunodeficiency virus (FIV) or feline
104
leukaemia virus (FeLV), using a commercial immunoassay (FeLV Antigen/FIV Antibody
105
Test Kit, IDEXX Laboratories). Cats that were affected with corneal ulceration, requiring
106
surgical treatment, and those that were pregnant or lactating were excluded. Cats were also
107
excluded if they showed evidence of systemic disease, as determined by clinical examination,
108
complete blood count (Cell-Dyn 3500, Abbott) and serum biochemistry (Hitachi 911, Roche).
109
Cats with a prior history of FURTD episodes, had received antimicrobial drugs within the
110
previous 3 days, or those that had been vaccinated or received any type of passive
111
immunisation, paramunity inducer, antiviral treatment, or glucocorticoid within the previous
112
4 weeks were also excluded.
113 114
The study population consisted of 38 European Shorthair cats, three longhair
115
crossbreeds, and one Persian cat. Of the 22 females, one was neutered; all 20 males were
116
intact. The youngest kitten was 3 weeks old, the oldest cat 13 years (median, 0.15 years; 35
117
out of 42 cats (83%) were 12 weeks or younger).
118 119
Treatment protocol
120
Twenty-two cats received Feliserin and 20 cats received placebo (physiological
121
saline) subcutaneously once daily for 3 consecutive days. Cats younger than 12 weeks
122
received 2 mL per injection, older cats received 4 mL per injection. This protocol was
123
recommended by the manufacturer and has been used in Europe since 1992. Additionally,
124
two drops (~ 0.1 mL) of antiserum were administered into eyes, nostrils, and on the oral
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mucosa every 8 h for 3 consecutive days. All cats received symptomatic treatment with 12.5
126
mg/kg amoxicillin-clavulanate (Synulox, Zoetis) orally twice daily for 10 days; bromhexine
127
(Bisolvon, Boehringer Ingelheim) 0.5 mg/kg orally every 8 h for 8 days; inhalation with
128
physiological saline and camomile (Kamillosan, MEDA) once daily for 8 days; cleaning of
129
eyes and nostrils; nasal flushing with physiological saline once daily, as well as fluid and
130
nutritional support where necessary.
131 132
Clinical examination
133
A clinical examination was performed each day from days 0 to 7, as well as on day
134
21. A ‘FURTD score’ was calculated, based on a bespoke scoring system that includes 13
135
parameters, graded from 0 to 3, according to their severity (Appendix A: Supplementary
136
Table 1). These clinical signs and the total ‘FURTD score’ were judged at each examination.
137
In addition, quality of life and well-being of each cat were evaluated daily using the modified
138
Karnofsky’s score ranging from 100% (no signs of disease) to 0% (death) (Hartmann and
139
Kuffer, 1998).
140 141
Nucleic acid preparation
142
Oropharyngeal and/or conjunctival swabs were stored at -80 °C immediately after
143
sampling until analysis. Total nucleic acid (DNA and RNA) was extracted using the
144
Nucleospin Blood Kit (Macherey Nagel). Cotton swabs were placed in a solution consisting
145
of 200 L phosphate-buffered saline (PBS), 200 L of buffer BQ1, and 20 L of proteinase
146
K. Swabs were incubated at 70 °C for 15 min with shaking at 700 rpm, then the
147
manufacturer’s protocol was followed. Total nucleic acid was eluted with 100 L of buffer
148
BE and stored at -80 °C.
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149 150
Quantitative PCR
151
On day 0, qPCR for FCV and FHV-1 was performed for inclusion purposes and
152
repeated for FCV on day 21 to detect viral shedding. Primers used in the study are detailed in
153
Appendix A: Supplementary Table 2. Quantitative PCR was used to detect FHV-1 and feline
154
28S rDNA (endogenous internal control) as described by Helps et al. (2005), using an Agilent
155
MX3005P thermocycler. Each reaction contained 5 L of genomic DNA, 12.5 L of 2×
156
GoTaq PCR Master mix (Promega), 200 nM each of 28S rDNA primers, 100 nM each of
157
FHV-1 primers, 50 nM 28S rDNA Texas Red-BHQ2 probe, 50 nM FHV-1 CY5-BHQ3
158
probe, 4.5 mM MgCl2 (final concentration), and water to a final volume of 25 L. Reactions
159
were incubated at 95 °C for 2 min followed by 45 cycles of 15 s at 95 °C and 30 s at 60 °C.
160
Fluorescence was measured at 610 nm and 665 nm after each annealing/elongation step.
161 162
Two separate real-time quantitative reverse-transcription (qRT) PCR assays were
163
performed for FCV, due to genetic variability. PCR primers for the two FCV assays were
164
designed to anneal to conserved regions of the FCV genome, which were determined by
165
multiple sequence alignment. Ten microlitres of total nucleic acid were reverse transcribed by
166
adding 4 L of 5× RT buffer, 2.4 L of 25 mM MgCl2, 1 L of 10 mM dNTP, 1 L of
167
random hexamer primer (0.5 g/L), 0.6 L of water, and 1 L of Improm II reverse
168
transcriptase (Promega), and incubated at 20 °C for 5 min, 42 °C for 30 min then 70 °C for 15
169
min in a MJ PTC 200 thermocycler. Thirty microlitres of RNase-free water were added to
170
each 20 L cDNA sample and stored at -20 °C prior to analysis.
171
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For qPCR, an Agilent MX3005P thermocycler was used with reactions consisting of 5
173
L cDNA, 12.5 L GoTaq 2× PCR Master mix, 200 nM primers (either FCV1, FCV2, or 28S
174
rDNA; Appendix A: Supplementary Table 2), 0.5 L 1:2000 SYBR green I (Sigma–Aldrich)
175
and water to a final volume of 25 L. Samples were incubated at 95 °C for 2 min, followed
176
by 40 cycles of 15 s at 95 °C and 30 s at either 60 °C (28S rDNA and FCV1) or 64 °C
177
(FCV2). Fluorescence was measured at 516 nm after each annealing/elongation step.
178
Following completion of the PCR, melting curve analysis was performed by incubating
179
reactions at 70 °C for 10 s and taking fluorescence readings as the temperature increased
180
incrementally by 1 °C for 10 s. Melting temperatures of FCV1 amplicons were between 82.5
181
and 86.0 °C and those of FCV2 amplicons were between 83.5 and 86.0 °C.
182 183
The 28S rDNA cycle threshold (Ct) value of each sample was used to normalise the
184
FCV Ct values, to take into account different swabbing efficiencies on days 0 and 21. The
185
normalised viral Ct values were converted into relative copy numbers by assuming one copy
186
had a Ct value of 40, with an assay efficiency approximating 100%.
187 188
Detection of antibodies
189
Antibody titres were determined in Feliserin (Batch number: 0170612) and in sera
190
from 13 cats at days 0, 3, 7, and 21, using a protocol modified from Dawson et al. (1998).
191
Clotted blood samples were centrifuged at 1500 g for 10 min and serum heat-inactivated at
192
56 °C for 30 min. Serial dilutions (1:4) of Feliserin or serum were prepared with PBS in
193
sterile 96-well culture plates (Dynatech Laboratories), with each well containing a final
194
volume of 60 L. For detection of FCV antibodies, 100 TCID50 (tissue culture infective dose)
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of FCV 255 was added and for detection of FHV-1 antibodies, 100 TCID50 of FHV-1 605
196
was added (both isolates used by the manufacturer to produce Feliserin). Plates were
197
incubated at 37 °C for 1–1.5 h, then 100 L transferred onto monolayers of Crandell feline
198
kidney cells (CrFK, CCL-94, ATCC) that had been maintained in complete medium
199
consisting of Minimum Essential Medium (MEM, Biochrom) supplemented with 10% fetal
200
calf serum (FCS, Biochrom). The neutralisation test was evaluated by direct microscopy for
201
evidence of viral cytopathic effect, with the greatest dilution of serum capable of completely
202
neutralising the virus (i.e. no cytopathic effect) indicating the antibody titre.
203 204
Statistical methods
205
The software program Graphpad Prism1 was used for statistical analysis of data. The
206
number of animals required was calculated by the Power Analysis and Sample Size Software
207
(PASS, NCSS Statistical Software)2. It was assumed clinically relevant if cats treated with
208
Feliserin improved in terms of their ‘FURTD score’ by three points or more and their general
209
health status by 15% or more, compared to cats in the placebo group, within the first 3 days.
210
Assuming a power of 80% and a confidence interval of 95%, 17 cats per group were required
211
to detect a difference between groups.
212 213
Fisher’s exact test was used for the inter-group comparison of virus distribution on
214
day 0. One-way analysis of variance (ANOVA) with Dunn’s-post test (Kruskal-Wallis test
215
for not normally distributed data) was used to analyse mean values of the two scores of each
216
group and between the groups at days 0, 3, 7, and 21 and relative FCV copy numbers on days
1
See: www.graphpad.com/scientific-software/prism/
2
See: www.statistical-solutions-software.com/ncss-home
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0 and 21. Changes over time (days 3, 7, and 21 vs. day 0) of the groups were compared to
218
each other using the Mann-Whitney U test (not normally distributed data) or unpaired
219
Student’s t test (normally distributed data). FCV shedding between beginning and end of the
220
study was analysed using Fisher’s exact test. The correlation between clinical signs and virus
221
load was assessed by performing Spearman analysis. In all analyses, P < 0.05 was considered
222
significant.
223 224
Results
225
Clinical signs
226
FCV was isolated from 19/42 cats, FHV-1 was isolated from 8/42 cats, and there was
227
co-infection with both viruses in 15/42 cases. There were no significant differences in the
228
distribution of FCV and FHV-1 infection or clinical parameters on day 0, comparing cats
229
receiving Feliserin or placebo. The general health status improved significantly in Feliserin-
230
treated cats from day 0 to day 3 (P < 0.010; Table 1), while placebo-treated cats failed to
231
show significant improvement during this period. This improvement in health status from
232
onset of therapy was significant, comparing the groups at day 3 (P = 0.032). On days 7 and
233
21, however, both groups showed significant improvements in their general health status (P <
234
0.001). Likewise, the ‘FURTD score’ improved significantly in cats receiving Feliserin by
235
day 3 (P < 0.010), but not in cats receiving placebo; consequently, a significant difference (P
236
= 0.046) was seen between responses in the two groups (Fig. 1). On days 7 and 21, both
237
groups showed significant improvement in their ‘FURTD scores’ (P < 0.001, Table 1; Fig.
238
1).
239 240
Feliserin-treated cats also demonstrated significant improvement in their eye
241
discharge by day 3 (P < 0.001; Table 1), which was not seen in the placebo group. This
Page 10 of 23
242
resulted in a significant inter-group difference (P < 0.001), although by days 7 and 21, both
243
groups had improved significantly (P < 0.001). Other parameters (such as nasal discharge and
244
sneezing) improved in both Feliserin- and placebo-treated cats during the treatment period,
245
without any significant inter-group differences. Other clinical signs (such as corneal changes,
246
gingivostomatitis, salivation, or ulcers) did not change significantly during the treatment
247
period.
248 249
FCV shedding
250
There was no significant difference comparing treatment groups in the number of cats
251
shedding FCV (Table 2). Relative FCV copy numbers did not change significantly from day
252
0 to 21 nor between treatment groups. There was no significant correlation between changes
253
(day 21 vs. day 0) in the FCV virus load and changes in the ‘FURTD score’ in cats of the
254
Feliserin group (n = 19, r = -0.046, P = 0.852) nor in cats of the placebo group (n = 15, r =
255
0.450, P = 0.093) when evaluated by Spearman analysis.
256 257
Serological analysis
258
Feliserin contained an FCV antibody titre of 1:1024 and an FHV-1 antibody titre of
259
1:128. On day 0, before start of treatment, none of the cats demonstrated serum antibodies
260
against FCV or FHV-1 (antibody titres all <1:10), except for one cat, which had an FCV
261
antibody titre of 1:10. FCV antibodies could be detected from day 3 to day 7 in all Feliserin-
262
treated cats tested (7/7; Table 3). In contrast, 5/6 cats in the placebo group remained antibody
263
negative, with one control cat showing a rise in FCV antibody titre from days 3 to 7. None of
264
the 13 cats tested showed any evidence of antibodies against FHV-1 (<1:10 at all time-
265
points).
266
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Discussion
268
This study was performed to evaluate the efficacy of Feliserin in cats affected with
269
FURTD caused by FCV and/or FHV-1-infection. There are various treatment options
270
available for treating cats affected with FHV-1 infection, but no drugs have yet been
271
demonstrated to be effective and safe for treatment of FCV infection (Povey, 1978b; Hennet
272
et al., 2011). In the present study, it was shown that Feliserin, administered systemically and
273
topically, resulted in a more rapid improvement of the ‘FURTD score’ and general health
274
status in comparison to cats treated with a more conventional approach. Cats receiving
275
Feliserin demonstrated significantly improved clinical signs by day 3, while cats receiving
276
symptomatic therapy showed significant improvement by day 7.
277 278
During viral infection, production of protective levels of neutralising antibodies can
279
take 6–14 days (Planz et al., 1996). Such antibodies specifically bind to surface antigens of
280
viruses, interfering with attachment and infection of host target cells (Verdaguer et al., 1997).
281
The incubation period of FCV is 2–10 days (Radford et al., 2009) and that for FHV-1 is 3–7
282
days (Lindt et al., 1965). Thus, clinical signs are usually present during the lag phase of the
283
immune response, before circulating antibodies are evident. This was seen in the present
284
study, since none of the study cats whose blood samples were available for antibody
285
detection demonstrated protective antibody levels at presentation, with all but one cat being
286
FCV and FHV-1 antibody-negative and the latter having a low FCV titre of 1:10.
287
Furthermore, only one cat in the placebo group seroconverted during the observation period.
288 289
In one experimental study, administration of antibodies against FCV and FHV-1
290
showed a promising effect (Umehashi et al., 2002). Feline-adapted murine antibodies
291
specifically directed against FCV (F1D7) and FHV-1 (FJH2) were administered to 16 week
Page 12 of 23
292
old, specific pathogen-free cats that had been experimentally infected with FCV or FHV-1.
293
Antibody-treated cats remained relatively free of clinical signs of FURTD, compared to
294
controls. In contrast, the present study was undertaken using naturally-infected cats and
295
therefore, infectious dose, infection status, and time between onset of clinical signs and
296
initiation of treatment were not standardised. Despite these confounding factors, this study
297
was still able to show some benefit of post-exposure treatment with specific anti-serum.
298
Thus, cats treated with Feliserin showed improvement in clinical signs within a shorter time-
299
frame (i.e. by day 3 compared to day 7 for controls). However, it might be difficult to justify
300
such antiviral treatment, when symptomatic therapy alone also leads to resolution of most
301
clinical signs within a week. No adverse effects were observed with Feliserin treatment, but
302
the cost implications and the additional amount of stress in administering the therapy to the
303
animal should be considered.
304 305
Three Feliserin-treated cats ceased shedding FCV by day 21, while all placebo-treated
306
cats continued to shed FCV up to the end of the trial (Table 2). This difference, however, was
307
not significant. Quantification of viral load in oropharyngeal swabs demonstrated that there
308
was a wide range of values in the cats infected with FCV on day 0. Cats showing
309
improvement in clinical signs still showed high virus loads (Tables 1 and 2) and there was no
310
correlation between viral shedding and clinical manifestation, as demonstrated by Spearman
311
analysis. In contrast to FHV-1-infected cats, whose shedding of virus is generally restricted to
312
the acute phase (around three weeks) and during episodes of viral recrudescence (Thiry et al.,
313
2009), FCV-infected cats can continuously shed virus for a prolonged period after recovery
314
(Wardley, 1976; Radford et al., 2009).
315
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None of the cats tested had protective levels of antibodies against FCV or FHV-1 at
317
presentation. Since the majority of cats enrolled into the trial were < 12 weeks of age, it is
318
likely that they were born from mothers without specific antibodies or they did not receive
319
adequate amounts of colostrum. FCV antibodies were detected in all Feliserin-treated cats
320
post-treatment, which reduced in titre from days 7 to 21, attributable to the relatively short-
321
duration of protection with passive immunisation (Shibata et al., 1983; Young, 1984).
322
Interestingly, no antibodies against FHV-1 were detected in any of the cats tested. The
323
antibody titre against FHV-1 was lower than that for FCV in the Feliserin product (1:128 vs.
324
1:1024, respectively), which is likely associated with this observation. It is not clear from the
325
results whether the relatively low concentration of FHV-1 antibodies in Feliserin were
326
effective, although the numbers were too low to compare Feliserin responses between FCV
327
and FHV-1 infected cats. It is possible, however, that topical application (rather than
328
parenteral administration) plays a major role in reducing viral pathology.
329 330
Inclusion of 10 cats from a previous study, although treated in a similar manner as the
331
controls prospectively recruited, is a confounding factor. Another limitation was that
332
antibody detection was not undertaken in all cats because some of the cats were relatively
333
young and the amount of blood that could reasonably be taken was limited.
334 335
Conclusions
336
The present study has shown that use of Feliserin, administered systemically and
337
topically on three consecutive days, resulted in significant improvement in clinical signs of
338
FURTD and of the general health status of FCV- and/or FHV-1-infected cats within the first
339
3 days when compared to placebo. However, this beneficial effect was transient, with no
Page 14 of 23
340
differences seen comparing Feliserin- and placebo-treated cats by day 7. Administration of
341
Feliserin did not decrease shedding of FVC in infected cats.
342 343 344 345
Conflict of interest statement None of the authors of this paper has a financial or personal relationship with other people or organisations that could inappropriately influence or bias the content of the paper.
346 347
Acknowledgements
348
We would like to thank Professor Dr. Ralf S. Mueller, Clinic of Small Animal
349
Medicine, for his assistance in the statistical examination, the technicians in the Molecular
350
Diagnostic Unit, Langford Veterinary Services, for performing the qPCR assays, as well as
351
the technicians of the Institute of Animal Hygiene and Public Veterinary Services, University
352
of Leipzig, for performing the virus neutralisation tests.
353 354
Parts of the results were presented as an abstract and oral presentation at the 21th
355
annual conference of the German Society of Internal Medicine and Clinical Pathology of the
356
German Veterinary Association (DVG) in Munich, 1-2 February 2013.
357 358 359 360
Appendix A: Supplementary material Supplementary data associated with this article can be found in the online version at doi: setters please insert doi number
361 362 363 364 365 366 367
References Dawson, D.A., Carman, J., Collins, J., Hill, S., Lappin, M.R., 1998. Enzyme-linked immunosorbent assay for detection of feline herpesvirus 1 IgG in serum, aqueous humor, and cerebrospinal fluid. Journal of Veterinary Diagnostic Investigation 10, 315-319.
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Di Martino, B., Di Francesco, C.E., Meridiani, I., Marsilio, F., 2007. Etiological investigation of multiple respiratory infections in cats. The New Microbiologica 30, 455-461. Fontenelle, J.P., Powell, C.C., Veir, J.K., Radecki, S.V., Lappin, M.R., 2008. Effect of topical ophthalmic application of cidofovir on experimentally induced primary ocular feline herpesvirus-1 infection in cats. American Journal of Veterinary Research 69, 289-293. Gaskell, R., Dawson, S., Radford, A., Thiry, E., 2007. Feline herpesvirus. Veterinary Research 38, 337-354. Gould, D., 2011. Feline herpesvirus-1: ocular manifestations, diagnosis and treatment options. Journal of Feline Medicine and Surgery 13, 333-346. Hartmann, A., Hartmann, K., 2010. Treatment and management of Chlamydophila felis infections in cats. Tieraerztliche Praxis 38, 217-226. Hartmann, A.D., Hawley, J., Werckenthin, C., Lappin, M.R., Hartmann, K., 2010. Detection of bacterial and viral organisms from the conjunctiva of cats with conjunctivitis and upper respiratory tract disease. Journal of Feline Medicine and Surgery 12, 775-782. Hartmann, K., Kuffer, M., 1998. Karnofsky's score modified for cats. European Journal of Medical Research 3, 95-98. Helps, C.R., Lait, P., Damhuis, A., Bjornehammar, U., Bolta, D., Brovida, C., Chabanne, L., Egberink, H., Ferrand, G., Fontbonne, A., et al., 2005. Factors associated with upper respiratory tract disease caused by feline herpesvirus, feline calicivirus, Chlamydophila felis, and Bordetella bronchiseptica in cats: experience from 218 European catteries. Veterinary Record 156, 669-673. Hennet, P.R., Camy, G.A., McGahie, D.M., Albouy, M.V., 2011. Comparative efficacy of a recombinant feline interferon omega in refractory cases of calicivirus-positive cats with caudal stomatitis: a randomized, multi-centre, controlled, double-blind study in 39 cats. Journal of Feline Medicine and Surgery 13, 577-587. Jacobs, A.A., Chalmers, W.S., Pasman, J., van Vugt, F., Cuenen, L.H., 1993. Feline bordetellosis: challenge and vaccine studies. Veterinary Record 133, 260-263. Lindt, S., Mühlethaler, E., Bürki, F., 1965. Enzootic virus-induced cat flu at an animal shelter: clinical features, histopathology, aetiology and epizootiology. Schweizer Archiv fur Tierheilkunde 107, 91-101. Maggs, D.J., Clarke, H.E., 2004. In vitro efficacy of ganciclovir, cidofovir, penciclovir, foscarnet, idoxuridine, and acyclovir against feline herpesvirus type-1. American Journal of Veterinary Research 65, 399-403. Maggs, D.J., Collins, B.K., Thorne, J.G., Nasisse, M.P., 2000. Effects of L-lysine and Larginine on in vitro replication of feline herpesvirus type-1. American Journal of Veterinary Research 61, 1474-1478.
Page 16 of 23
417 418 419 420 421 422 423 424 425 426 427 428 429 430 431 432 433 434 435 436 437 438 439 440 441 442 443 444 445 446 447 448 449 450 451 452 453 454 455 456 457 458 459 460 461 462 463 464 465
Malik, R., Lessels, N.S., Webb, S., Meek, M., Graham, P.G., Vitale, C., Norris, J.M., Power, H., 2009. Treatment of feline herpesvirus-1 associated disease in cats with famciclovir and related drugs. Journal of Feline Medicine and Surgery 11, 40-48. Nasisse, M.P., Dorman, D.C., Jamison, K.C., Weigler, B.J., Hawkins, E.C., Stevens, J.B., 1997. Effects of valacyclovir in cats infected with feline herpesvirus 1. American Journal of Veterinary Research 58, 1141-1144. Nasisse, M.P., Guy, J.S., Davidson, M.G., Sussman, W., De Clercq, E., 1989. In vitro susceptibility of feline herpesvirus-1 to vidarabine, idoxuridine, trifluridine, acyclovir, or bromovinyldeoxyuridine. American Journal of Veterinary Research 50, 158-160. Parzefall, B., Schmahl, W., Fischer, A., Blutke, A., Truyen, U., Matiasek, K., 2010. Evidence of feline herpesvirus-1 DNA in the vestibular ganglion of domestic cats. The Veterinary Journal 184, 371-372. Planz, O., Seiler, P., Hengartner, H., Zinkernagel, R.M., 1996. Specific cytotoxic T cells eliminate B cells producing virus-neutralizing antibodies. Nature 382, 726-729. Povey, R.C., 1978a. In vitro antiviral efficacy of ribavirin against feline calicivirus, feline viral rhinotracheitis virus, and canine parainfluenza virus. American Journal of Veterinary Research 39, 175-178. Povey, R.C., 1978b. Effect of orally administered ribavirin on experimental feline calicivirus infection in cats. American Journal of Veterinary Research 39, 1337-1341. Radford, A.D., Addie, D., Belak, S., Boucraut-Baralon, C., Egberink, H., Frymus, T., Gruffydd-Jones, T., Hartmann, K., Hosie, M.J., Lloret, A. et al., 2009. Feline calicivirus infection. ABCD guidelines on prevention and management. Journal of Feline Medicine and Surgery 11, 556-564. Radford, A.D., Coyne, K.P., Dawson, S., Porter, C.J., Gaskell, R.M., 2007. Feline calicivirus. Veterinary Research 38, 319-335. Shibata, Y., Baba, M., Kuniyuki, M., 1983. Studies on the retention of passively transferred antibodies in man. II. Antibody activity in the blood after intravenous or intramuscular administration of anti-HBs human immunoglobulin. Vox Sanguinis 45, 77-82. Siebeck, N., Hurley, D.J., Garcia, M., Greene, C.E., Kostlin, R.G., Moore, P.A., Dietrich, U.M., 2006. Effects of human recombinant alpha-2b interferon and feline recombinant omega interferon on in vitro replication of feline herpesvirus-1. American Journal of Veterinary Research 67, 1406-1411. Stiles, J., 1995. Treatment of cats with ocular disease attributable to herpesvirus infection: 17 cases (1983-1993). Journal of the American Veterinary Medical Association 207, 599603. Thiry, E., Addie, D., Belak, S., Boucraut-Baralon, C., Egberink, H., Frymus, T., GruffyddJones, T., Hartmann, K., Hosie, M.J., Lloret, A. et al., 2009. Feline herpesvirus infection.
Page 17 of 23
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ABCD guidelines on prevention and management. Journal of Feline Medicine and Surgery 11, 547-555. Townsend, W.M., Stiles, J., Guptill-Yoran, L., Krohne, S.G., 2004. Development of a reverse transcriptase-polymerase chain reaction assay to detect feline herpesvirus-1 latencyassociated transcripts in the trigeminal ganglia and corneas of cats that did not have clinical signs of ocular disease. American Journal of Veterinary Research 65, 314- 319. Umehashi, M., Imamura, T., Akiyama, S., Kimachi, K., Tokiyoshi, S., Mikami, T., 2002. Post-exposure treatment of cats with mouse-cat chimeric antibodies against feline herpesvirus type 1 and feline calicivirus. The Journal of Veterinary Medical Science 64, 1017-1021. Van der Meulen, K., Garre, B., Croubels, S., Nauwynck, H., 2006. In vitro comparison of antiviral drugs against feline herpesvirus 1. BMC Veterinary Research 2, 13. Verdaguer, N., Fita, I., Domingo, E., Mateu, M.G., 1997. Efficient neutralization of foot-andmouth disease virus by monovalent antibody binding. Journal of Virology 71, 98139816. Wardley, R.C., 1976. Feline calicivirus carrier state. A study of the host/virus relationship. Archives of Virology 52, 243-249. Wardley, R.C., Povey, R.C., 1977. The clinical disease and patterns of excretion associated with three different strains of feline caliciviruses. Research in Veterinary Science 23, 714. Weiss, R.C., Cox, N.R., Boudreaux, M.K., 1993. Toxicologic effects of ribavirin in cats. Journal of Veterinary Pharmacology and Therapeutics 16, 301-316. Williams, D.L., Fitzmaurice, T., Lay, L., Forster, K., Hefford, J., Budge, C., Blackmore, K., Robinson, J.C., Field, H.F., 2004. Efficacy of antiviral agents in feline herpetic keratitis: results of an in vitro study. Current Eye Research 29, 215-218. Young, L.S., 1984. Symposium on infectious complications of neoplastic disease (Part II). Immunoprophylaxis and serotherapy of bacterial infections. The American Journal of Medicine 76, 664-671.
Page 18 of 23
505
Table 1
506
General health status, ‘FURTD score’, and clinical signs of cats receiving either Feliserin or
507
placebo.
General status (%)
health
Day
Feliserin mean SD
0 3 7 21
57.2712.02 85.2310.96 95.457.85
P-value a
<0.01 <0.001 <0.001
98.643.16 ‘FURTD score’
0 3 7 21
14.234.45 4.593.38 1.732.19
0 3 7 21
0.860.94 0.360.73 0.140.47
<0.010 <0.001 <0.001
0 3 7 21
2.090.61 0.410.59 0.140.35
ns <0.050 <0.010
0 3 7 21
2.050.79 1.140.77 0.410.67
<0.001 <0.001 <0.001
0 3 7 21
1.591.01 0.680.57 0.270.55
ns <0.001 <0.001
0 3 7 21
1.500.80 0.410.67 0.180.39
ns <0.001 <0.001
Thoracic
0 3 7 21
0
2.180.91 0.450.67 0.180.50
6.653.70
ns <0.001 <0.001
0.046 ns ns
3.602.74
1.100.97 0.600.60
ns ns ns
ns ns ns
0.450.60
1.700.80 0.900.72
ns <0.001 <0.001
<0.001 ns ns
0.450.60
1.850.75 1.350.81
ns <0.050 <0.001
ns ns ns
0.850.49
1.250.85 0.650.75
ns <0.010 <0.001
ns ns ns
0.250.44
<0.010 <0.001 <0.001
ns ns ns
<0.010 <0.001 <0.001
ns ns ns
0.150.49 <0.001 <0.001 <0.001
0.090.29 Sneezing (grade)
14.203.68
0.500.61
0.000.00 Nasal discharge (grade)
0.032 ns 0.045
0.300.47
0.000.00 Blepharospasm (grade)
94.505.10
ns <0.001 <0.001
0.300.47
0.090.29 Conjunctivitis (grade)
84.009.68
Feliserin vs. Placebo P-value b
1.701.84
0.050.21 Eye discharge (grade)
64.5016.05
P-value a
98.004.10
0.640.85 Lymph node enlargement (grade)
Placebo mean SD
1.650.59 0.650.75 0.350.59 0.050.22
<0.001 <0.001 <0.001
2.001.03 0.550.69 0.200.41
0.360.58
0.250.44
1.550.86
1.200.77
Page 19 of 23
auscultation (grade)
3 7 21
0.730.77 0.320.65
ns <0.001 <0.001
0.050.21 Respiratory effort (grade)
0 3 7 21
0.860.94 0.270.55 0.090.29
0 3 7 21
0.090.29 0.050.21 0.000.00
ns <0.010 <0.001
0 3 7 21
1.410.80 0.140.47 0.050.21
ns ns ns
1.200.52 0.200.52
<0.001 <0.001 <0.001
ns ns ns
0.050.22 0.000.00
ns ns ns
0.000.00 Appetite (grade)
0.250.55
ns <0.010 <0.001
0.100.31
0.000.00 Gingivostomatit is (grade)
0.600.68
0.200.52 0.100.45 0.050.22
ns ns ns
0.000.00 <0.001 <0.001 <0.001
0.000.00
1.450.94 0.300.66 0.050.22
<0.001 <0.001 <0.001
ns ns ns
0.000.00
508 509
a
510
group over time (days 3, 7, and 21 vs. day 0).
511
b
512
test for normally distributed data) comparing treatment groups in terms of change at each
513
time-point relative to day 0.
514
ns, not statistically significant.
P-values (Kruskal-Wallis test with Dunn’s-post test) of differences within each treatment
P-values (Mann-Whitney U test for data not normally distributed or unpaired Student’s t
515
Page 20 of 23
516
Table 2.
517
Number of feline calicivirus (FCV) shedding cats and relative FCV copy numbers (× 104)
518
before and after receiving Feliserin or placebo.
Day 0 Day 21 P-value a
Feliserin Shedding cats Copy numbers (× 104) 19 (100%) 57.81 98.65 16 (84%) 104.30 253.80 0.8
Placebo Shedding cats Copy numbers (× 104) 15 (100%) 1424 3709 15 (100%) 69.34 143
P-value b ns ns
1.0
519 520
a
P-values (Fisher’s exact test) of the difference between groups over time (day 21 vs. day 0).
521
b
P-values (Kruskal-Wallis test with Dunn’s-post test) of changes within each group over
522
time (day 21 vs. day 0).
523
ns, not statistically significant.
524
Page 21 of 23
525
Table 3.
526
Antibody titres
527
sampled during the study period.
a
against feline calicivirus (FCV) in Feliserin- and placebo-treated cats,
Feliserin-treated group
Placebo group
528
a
Cat
Day 0
Day 3
Day 7
Day 21
1 2 3 4 5 6 7 1 2 3 4 5 6
< 1:10 < 1:10 < 1:10 < 1:10 < 1:10 < 1:10 1:10 < 1:10 < 1:10 < 1:10 < 1:10 < 1:10 < 1:10
1:20 1:80 1:80 1:80 1:160 1:160 1:80 < 1:10 < 1:10 < 1:10 < 1:10 < 1:10 < 1:10
1:80 1:40 1:80 1:80 1:80 1:80 1:160 < 1:10 < 1:10 < 1:10 < 1:10 < 1:10 1:10
1:10 1:10 < 1:10 1:10 1:10 1:20 1:20 < 1:10 < 1:10 < 1:10 < 1:10 < 1:10 1:20
Virus neutralisation assay using 100 TCID50 of FCV 255 in Crandell feline kidney cells.
529
Page 22 of 23
530
Figure legend
531 532
Fig. 1. ‘FURTD score’ of cats receiving Feliserin or placebo, showing grades (0–39) at days
533
0, 3, 7, and 21. Cats of both groups improved significantly (P < 0.001, Kruskal-Wallis test
534
with Dunn’s-post test) by day 7 and day 21; only cats receiving Feliserin improved
535
significantly (P = 0.046, unpaired Student’s t test) between day 0 and day 3.
536
Page 23 of 23
S1090-0233(14)00185-3 http://dx.doi.org/doi:10.1016/j.tvjl.2014.05.002 YTVJL 4136
To appear in:
The Veterinary Journal
Accepted date:
2-5-2014
Please cite this article as: Yvonne Friedl, Bianka Schulz, Anne Knebl, Chris Helps, Uwe Truyen, Katrin Hartmann, Efficacy of passively-transferred antibodies in cats with acute viral upper respiratory tract infection, The Veterinary Journal (2014), http://dx.doi.org/doi:10.1016/j.tvjl.2014.05.002. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24
Efficacy of passively-transferred antibodies in cats with acute viral upper respiratory tract infection Yvonne Friedl a,*, Bianka Schulz a, Anne Knebl a, Chris Helps b, Uwe Truyen c, Katrin Hartmann a
a
Clinic of Small Animal Medicine, Ludwig-Maximilians-Universitaet Veterinaerstrasse 13, 80539 Munich, Germany
Munich,
b
Molecular Diagnostic Unit, Langford Veterinary Services, University of Bristol, Langford House, Bristol BS40 5DU, United Kingdom c
Institute of Animal Hygiene and Public Veterinary Services, University of Leipzig, An den Tierkliniken 1, 04103 Leipzig, Germany
* Corresponding author. Tel.: +49 094 315 5371. E-mail address: [email protected] (Y. Friedl).
Page 1 of 23
25
Abstract
26
A commercial hyperimmune serum, containing antibodies against feline calicivirus
27
(FCV), feline herpesvirus 1 (FHV-1), and feline panleukopenia virus is available for
28
treatment of cats with feline upper respiratory tract disease (FURTD), but its efficacy has not
29
been rigorously evaluated in scientific studies. The aim of this randomised, placebo-
30
controlled, double-blind clinical trial was to evaluate the efficacy of passive immunisation in
31
cats with acute viral FURTD caused by FCV and/or FHV-1 infection. All cats received
32
symptomatic treatment during the study period. Hyperimmune serum was administered to
33
one group (n = 22) and an equivalent amount of saline was administered to the control group
34
(n = 20) as placebo, for 3 consecutive days. In the treatment group, cats ≤ 12 weeks old
35
received 2 mL, cats > 12 weeks old received 4 mL, subcutaneously once daily and topically
36
into eyes, nostrils, and mouth every 8 h. Clinical signs, including a ‘FURTD score’ and
37
general health status were recorded daily for 8 days and again on day 21. FCV shedding was
38
determined by quantitative PCR on days 0 and 21.
39 40
Clinical signs and health status in both groups improved significantly over time (P <
41
0.001). Cats receiving hyperimmune serum significantly improved in terms of ‘FURTD
42
score’ (P = 0.046) and general health status (P = 0.032) by day 3, while cats in the placebo
43
group only improved significantly by day 7. There was no significant difference in the
44
number of cats shedding FCV between the two groups. Thus, administration of hyperimmune
45
serum led to a more rapid improvement of clinical signs in cats with acute viral FURTD, but
46
by day 7, clinical signs had improved equally in both groups.
47 48
Keywords: Feline calicivirus; Feline herpesvirus 1; Hyperimmune serum; Upper respiratory
49
tract infection
Page 2 of 23
50
Introduction
51
Feline upper respiratory tract disease (FURTD) can be caused by several pathogens,
52
leading to similar clinical signs of disease. Two viruses in particular, feline calicivirus (FCV)
53
and feline herpesvirus 1 (FHV-1), are responsible for at least 80% of FURTD cases (Helps et
54
al., 2005; Di Martino et al., 2007; Gould, 2011). In addition, bacteria, including
55
Chlamydophila felis, Bordetella bronchiseptica, and Mycoplasma spp., can act as primary
56
infectious agents (Jacobs et al., 1993; Hartmann and Hartmann, 2010; Hartmann et al., 2010).
57
After initial infection, FHV-1 develops a state of latency, residing mainly in the trigeminal
58
and vestibular ganglions and cats become carriers, shedding virus during period of
59
recrudescence (Townsend et al., 2004; Gaskell et al., 2007; Parzefall et al., 2010). FCV-
60
infected cats can also become asymptomatic carriers, but shed virus persistently (Wardley
61
and Povey, 1977; Radford et al., 2007, 2009).
62 63
Several antiviral drugs are effective in vitro against FHV-1 (Nasisse et al., 1989;
64
Maggs et al., 2000; Maggs and Clarke, 2004; Williams et al., 2004; Siebeck et al., 2006; Van
65
der Meulen et al., 2006) and FCV (Povey, 1978a). Some treatment options are available
66
clinically for treating cats affected with FHV-1, with most of these drugs being administered
67
topically, e.g., cidofovir, idoxuridine, vidarabine, and trifluridine (Stiles, 1995; Fontenelle et
68
al., 2008). Adverse effects can occur when anti-viral drugs are used systemically (Weiss et
69
al., 1993; Nasisse et al., 1997), although oral administration of famciclovir (Famvir, Novartis)
70
has recently been shown to improve clinical signs in FHV-1-infected cats without causing
71
adverse effects (Malik et al., 2009). In contrast, there is no specific treatment option available
72
for FCV that has proven efficacy and tolerable adverse effects (Povey, 1978b; Hennet et al.,
73
2011).
74
Page 3 of 23
75
The commercially-available product, Feliserin (IDT Biologika), is reported to contain
76
antibodies against FCV, FHV-1, and feline panleukopenia virus and is marketed in Germany
77
(Product License number 35a/92) for the treatment of acute viral FURTD and feline
78
panleukopenia. Its efficacy, however, has not been rigorously assessed in scientific studies.
79
Therefore, the aim of the present study was to evaluate whether administration of Feliserin
80
was beneficial in cats affected with acute viral FURTD.
81 82
Materials and methods
83
Study design
84
The study was performed as a randomised, placebo-controlled, double-blind clinical
85
trial in cats affected with acute viral FURTD. Thirty-two cats (22 receiving Feliserin and 10
86
placebo) were prospectively recruited into the study and randomised to either treatment or
87
placebo group. Data from a previous study from an additional 10 cats that had received the
88
same symptomatic treatment protocol were also included as controls. When a cat was
89
recruited, medications were drawn up and injected by a veterinarian not involved in the study
90
to ensure that neither owner nor clinician were aware which group the cat had been assigned
91
to. Decoding occurred after the study was completed and data entered for statistical
92
evaluation.
93 94
The study fulfilled the general German guidelines for prospective studies with
95
informed owner consent and was carried out with permission from the responsible German
96
veterinary authority (Government of Upper Bavaria, Maximilianstrasse 39, 80538 Munich,
97
reference number 55.2-1-54-2532-05-12).
98 99
Study population
Page 4 of 23
100
Included in the study were 42 cats that presented with clinical signs of FURTD of less
101
than 7 days duration and in which FCV and/or FHV-1 had been detected by quantitative
102
polymerase chain reaction (qPCR) from oropharyngeal and/or conjunctival swabs. Cats were
103
excluded if demonstrated to be infected with feline immunodeficiency virus (FIV) or feline
104
leukaemia virus (FeLV), using a commercial immunoassay (FeLV Antigen/FIV Antibody
105
Test Kit, IDEXX Laboratories). Cats that were affected with corneal ulceration, requiring
106
surgical treatment, and those that were pregnant or lactating were excluded. Cats were also
107
excluded if they showed evidence of systemic disease, as determined by clinical examination,
108
complete blood count (Cell-Dyn 3500, Abbott) and serum biochemistry (Hitachi 911, Roche).
109
Cats with a prior history of FURTD episodes, had received antimicrobial drugs within the
110
previous 3 days, or those that had been vaccinated or received any type of passive
111
immunisation, paramunity inducer, antiviral treatment, or glucocorticoid within the previous
112
4 weeks were also excluded.
113 114
The study population consisted of 38 European Shorthair cats, three longhair
115
crossbreeds, and one Persian cat. Of the 22 females, one was neutered; all 20 males were
116
intact. The youngest kitten was 3 weeks old, the oldest cat 13 years (median, 0.15 years; 35
117
out of 42 cats (83%) were 12 weeks or younger).
118 119
Treatment protocol
120
Twenty-two cats received Feliserin and 20 cats received placebo (physiological
121
saline) subcutaneously once daily for 3 consecutive days. Cats younger than 12 weeks
122
received 2 mL per injection, older cats received 4 mL per injection. This protocol was
123
recommended by the manufacturer and has been used in Europe since 1992. Additionally,
124
two drops (~ 0.1 mL) of antiserum were administered into eyes, nostrils, and on the oral
Page 5 of 23
125
mucosa every 8 h for 3 consecutive days. All cats received symptomatic treatment with 12.5
126
mg/kg amoxicillin-clavulanate (Synulox, Zoetis) orally twice daily for 10 days; bromhexine
127
(Bisolvon, Boehringer Ingelheim) 0.5 mg/kg orally every 8 h for 8 days; inhalation with
128
physiological saline and camomile (Kamillosan, MEDA) once daily for 8 days; cleaning of
129
eyes and nostrils; nasal flushing with physiological saline once daily, as well as fluid and
130
nutritional support where necessary.
131 132
Clinical examination
133
A clinical examination was performed each day from days 0 to 7, as well as on day
134
21. A ‘FURTD score’ was calculated, based on a bespoke scoring system that includes 13
135
parameters, graded from 0 to 3, according to their severity (Appendix A: Supplementary
136
Table 1). These clinical signs and the total ‘FURTD score’ were judged at each examination.
137
In addition, quality of life and well-being of each cat were evaluated daily using the modified
138
Karnofsky’s score ranging from 100% (no signs of disease) to 0% (death) (Hartmann and
139
Kuffer, 1998).
140 141
Nucleic acid preparation
142
Oropharyngeal and/or conjunctival swabs were stored at -80 °C immediately after
143
sampling until analysis. Total nucleic acid (DNA and RNA) was extracted using the
144
Nucleospin Blood Kit (Macherey Nagel). Cotton swabs were placed in a solution consisting
145
of 200 L phosphate-buffered saline (PBS), 200 L of buffer BQ1, and 20 L of proteinase
146
K. Swabs were incubated at 70 °C for 15 min with shaking at 700 rpm, then the
147
manufacturer’s protocol was followed. Total nucleic acid was eluted with 100 L of buffer
148
BE and stored at -80 °C.
Page 6 of 23
149 150
Quantitative PCR
151
On day 0, qPCR for FCV and FHV-1 was performed for inclusion purposes and
152
repeated for FCV on day 21 to detect viral shedding. Primers used in the study are detailed in
153
Appendix A: Supplementary Table 2. Quantitative PCR was used to detect FHV-1 and feline
154
28S rDNA (endogenous internal control) as described by Helps et al. (2005), using an Agilent
155
MX3005P thermocycler. Each reaction contained 5 L of genomic DNA, 12.5 L of 2×
156
GoTaq PCR Master mix (Promega), 200 nM each of 28S rDNA primers, 100 nM each of
157
FHV-1 primers, 50 nM 28S rDNA Texas Red-BHQ2 probe, 50 nM FHV-1 CY5-BHQ3
158
probe, 4.5 mM MgCl2 (final concentration), and water to a final volume of 25 L. Reactions
159
were incubated at 95 °C for 2 min followed by 45 cycles of 15 s at 95 °C and 30 s at 60 °C.
160
Fluorescence was measured at 610 nm and 665 nm after each annealing/elongation step.
161 162
Two separate real-time quantitative reverse-transcription (qRT) PCR assays were
163
performed for FCV, due to genetic variability. PCR primers for the two FCV assays were
164
designed to anneal to conserved regions of the FCV genome, which were determined by
165
multiple sequence alignment. Ten microlitres of total nucleic acid were reverse transcribed by
166
adding 4 L of 5× RT buffer, 2.4 L of 25 mM MgCl2, 1 L of 10 mM dNTP, 1 L of
167
random hexamer primer (0.5 g/L), 0.6 L of water, and 1 L of Improm II reverse
168
transcriptase (Promega), and incubated at 20 °C for 5 min, 42 °C for 30 min then 70 °C for 15
169
min in a MJ PTC 200 thermocycler. Thirty microlitres of RNase-free water were added to
170
each 20 L cDNA sample and stored at -20 °C prior to analysis.
171
Page 7 of 23
172
For qPCR, an Agilent MX3005P thermocycler was used with reactions consisting of 5
173
L cDNA, 12.5 L GoTaq 2× PCR Master mix, 200 nM primers (either FCV1, FCV2, or 28S
174
rDNA; Appendix A: Supplementary Table 2), 0.5 L 1:2000 SYBR green I (Sigma–Aldrich)
175
and water to a final volume of 25 L. Samples were incubated at 95 °C for 2 min, followed
176
by 40 cycles of 15 s at 95 °C and 30 s at either 60 °C (28S rDNA and FCV1) or 64 °C
177
(FCV2). Fluorescence was measured at 516 nm after each annealing/elongation step.
178
Following completion of the PCR, melting curve analysis was performed by incubating
179
reactions at 70 °C for 10 s and taking fluorescence readings as the temperature increased
180
incrementally by 1 °C for 10 s. Melting temperatures of FCV1 amplicons were between 82.5
181
and 86.0 °C and those of FCV2 amplicons were between 83.5 and 86.0 °C.
182 183
The 28S rDNA cycle threshold (Ct) value of each sample was used to normalise the
184
FCV Ct values, to take into account different swabbing efficiencies on days 0 and 21. The
185
normalised viral Ct values were converted into relative copy numbers by assuming one copy
186
had a Ct value of 40, with an assay efficiency approximating 100%.
187 188
Detection of antibodies
189
Antibody titres were determined in Feliserin (Batch number: 0170612) and in sera
190
from 13 cats at days 0, 3, 7, and 21, using a protocol modified from Dawson et al. (1998).
191
Clotted blood samples were centrifuged at 1500 g for 10 min and serum heat-inactivated at
192
56 °C for 30 min. Serial dilutions (1:4) of Feliserin or serum were prepared with PBS in
193
sterile 96-well culture plates (Dynatech Laboratories), with each well containing a final
194
volume of 60 L. For detection of FCV antibodies, 100 TCID50 (tissue culture infective dose)
Page 8 of 23
195
of FCV 255 was added and for detection of FHV-1 antibodies, 100 TCID50 of FHV-1 605
196
was added (both isolates used by the manufacturer to produce Feliserin). Plates were
197
incubated at 37 °C for 1–1.5 h, then 100 L transferred onto monolayers of Crandell feline
198
kidney cells (CrFK, CCL-94, ATCC) that had been maintained in complete medium
199
consisting of Minimum Essential Medium (MEM, Biochrom) supplemented with 10% fetal
200
calf serum (FCS, Biochrom). The neutralisation test was evaluated by direct microscopy for
201
evidence of viral cytopathic effect, with the greatest dilution of serum capable of completely
202
neutralising the virus (i.e. no cytopathic effect) indicating the antibody titre.
203 204
Statistical methods
205
The software program Graphpad Prism1 was used for statistical analysis of data. The
206
number of animals required was calculated by the Power Analysis and Sample Size Software
207
(PASS, NCSS Statistical Software)2. It was assumed clinically relevant if cats treated with
208
Feliserin improved in terms of their ‘FURTD score’ by three points or more and their general
209
health status by 15% or more, compared to cats in the placebo group, within the first 3 days.
210
Assuming a power of 80% and a confidence interval of 95%, 17 cats per group were required
211
to detect a difference between groups.
212 213
Fisher’s exact test was used for the inter-group comparison of virus distribution on
214
day 0. One-way analysis of variance (ANOVA) with Dunn’s-post test (Kruskal-Wallis test
215
for not normally distributed data) was used to analyse mean values of the two scores of each
216
group and between the groups at days 0, 3, 7, and 21 and relative FCV copy numbers on days
1
See: www.graphpad.com/scientific-software/prism/
2
See: www.statistical-solutions-software.com/ncss-home
Page 9 of 23
217
0 and 21. Changes over time (days 3, 7, and 21 vs. day 0) of the groups were compared to
218
each other using the Mann-Whitney U test (not normally distributed data) or unpaired
219
Student’s t test (normally distributed data). FCV shedding between beginning and end of the
220
study was analysed using Fisher’s exact test. The correlation between clinical signs and virus
221
load was assessed by performing Spearman analysis. In all analyses, P < 0.05 was considered
222
significant.
223 224
Results
225
Clinical signs
226
FCV was isolated from 19/42 cats, FHV-1 was isolated from 8/42 cats, and there was
227
co-infection with both viruses in 15/42 cases. There were no significant differences in the
228
distribution of FCV and FHV-1 infection or clinical parameters on day 0, comparing cats
229
receiving Feliserin or placebo. The general health status improved significantly in Feliserin-
230
treated cats from day 0 to day 3 (P < 0.010; Table 1), while placebo-treated cats failed to
231
show significant improvement during this period. This improvement in health status from
232
onset of therapy was significant, comparing the groups at day 3 (P = 0.032). On days 7 and
233
21, however, both groups showed significant improvements in their general health status (P <
234
0.001). Likewise, the ‘FURTD score’ improved significantly in cats receiving Feliserin by
235
day 3 (P < 0.010), but not in cats receiving placebo; consequently, a significant difference (P
236
= 0.046) was seen between responses in the two groups (Fig. 1). On days 7 and 21, both
237
groups showed significant improvement in their ‘FURTD scores’ (P < 0.001, Table 1; Fig.
238
1).
239 240
Feliserin-treated cats also demonstrated significant improvement in their eye
241
discharge by day 3 (P < 0.001; Table 1), which was not seen in the placebo group. This
Page 10 of 23
242
resulted in a significant inter-group difference (P < 0.001), although by days 7 and 21, both
243
groups had improved significantly (P < 0.001). Other parameters (such as nasal discharge and
244
sneezing) improved in both Feliserin- and placebo-treated cats during the treatment period,
245
without any significant inter-group differences. Other clinical signs (such as corneal changes,
246
gingivostomatitis, salivation, or ulcers) did not change significantly during the treatment
247
period.
248 249
FCV shedding
250
There was no significant difference comparing treatment groups in the number of cats
251
shedding FCV (Table 2). Relative FCV copy numbers did not change significantly from day
252
0 to 21 nor between treatment groups. There was no significant correlation between changes
253
(day 21 vs. day 0) in the FCV virus load and changes in the ‘FURTD score’ in cats of the
254
Feliserin group (n = 19, r = -0.046, P = 0.852) nor in cats of the placebo group (n = 15, r =
255
0.450, P = 0.093) when evaluated by Spearman analysis.
256 257
Serological analysis
258
Feliserin contained an FCV antibody titre of 1:1024 and an FHV-1 antibody titre of
259
1:128. On day 0, before start of treatment, none of the cats demonstrated serum antibodies
260
against FCV or FHV-1 (antibody titres all <1:10), except for one cat, which had an FCV
261
antibody titre of 1:10. FCV antibodies could be detected from day 3 to day 7 in all Feliserin-
262
treated cats tested (7/7; Table 3). In contrast, 5/6 cats in the placebo group remained antibody
263
negative, with one control cat showing a rise in FCV antibody titre from days 3 to 7. None of
264
the 13 cats tested showed any evidence of antibodies against FHV-1 (<1:10 at all time-
265
points).
266
Page 11 of 23
267
Discussion
268
This study was performed to evaluate the efficacy of Feliserin in cats affected with
269
FURTD caused by FCV and/or FHV-1-infection. There are various treatment options
270
available for treating cats affected with FHV-1 infection, but no drugs have yet been
271
demonstrated to be effective and safe for treatment of FCV infection (Povey, 1978b; Hennet
272
et al., 2011). In the present study, it was shown that Feliserin, administered systemically and
273
topically, resulted in a more rapid improvement of the ‘FURTD score’ and general health
274
status in comparison to cats treated with a more conventional approach. Cats receiving
275
Feliserin demonstrated significantly improved clinical signs by day 3, while cats receiving
276
symptomatic therapy showed significant improvement by day 7.
277 278
During viral infection, production of protective levels of neutralising antibodies can
279
take 6–14 days (Planz et al., 1996). Such antibodies specifically bind to surface antigens of
280
viruses, interfering with attachment and infection of host target cells (Verdaguer et al., 1997).
281
The incubation period of FCV is 2–10 days (Radford et al., 2009) and that for FHV-1 is 3–7
282
days (Lindt et al., 1965). Thus, clinical signs are usually present during the lag phase of the
283
immune response, before circulating antibodies are evident. This was seen in the present
284
study, since none of the study cats whose blood samples were available for antibody
285
detection demonstrated protective antibody levels at presentation, with all but one cat being
286
FCV and FHV-1 antibody-negative and the latter having a low FCV titre of 1:10.
287
Furthermore, only one cat in the placebo group seroconverted during the observation period.
288 289
In one experimental study, administration of antibodies against FCV and FHV-1
290
showed a promising effect (Umehashi et al., 2002). Feline-adapted murine antibodies
291
specifically directed against FCV (F1D7) and FHV-1 (FJH2) were administered to 16 week
Page 12 of 23
292
old, specific pathogen-free cats that had been experimentally infected with FCV or FHV-1.
293
Antibody-treated cats remained relatively free of clinical signs of FURTD, compared to
294
controls. In contrast, the present study was undertaken using naturally-infected cats and
295
therefore, infectious dose, infection status, and time between onset of clinical signs and
296
initiation of treatment were not standardised. Despite these confounding factors, this study
297
was still able to show some benefit of post-exposure treatment with specific anti-serum.
298
Thus, cats treated with Feliserin showed improvement in clinical signs within a shorter time-
299
frame (i.e. by day 3 compared to day 7 for controls). However, it might be difficult to justify
300
such antiviral treatment, when symptomatic therapy alone also leads to resolution of most
301
clinical signs within a week. No adverse effects were observed with Feliserin treatment, but
302
the cost implications and the additional amount of stress in administering the therapy to the
303
animal should be considered.
304 305
Three Feliserin-treated cats ceased shedding FCV by day 21, while all placebo-treated
306
cats continued to shed FCV up to the end of the trial (Table 2). This difference, however, was
307
not significant. Quantification of viral load in oropharyngeal swabs demonstrated that there
308
was a wide range of values in the cats infected with FCV on day 0. Cats showing
309
improvement in clinical signs still showed high virus loads (Tables 1 and 2) and there was no
310
correlation between viral shedding and clinical manifestation, as demonstrated by Spearman
311
analysis. In contrast to FHV-1-infected cats, whose shedding of virus is generally restricted to
312
the acute phase (around three weeks) and during episodes of viral recrudescence (Thiry et al.,
313
2009), FCV-infected cats can continuously shed virus for a prolonged period after recovery
314
(Wardley, 1976; Radford et al., 2009).
315
Page 13 of 23
316
None of the cats tested had protective levels of antibodies against FCV or FHV-1 at
317
presentation. Since the majority of cats enrolled into the trial were < 12 weeks of age, it is
318
likely that they were born from mothers without specific antibodies or they did not receive
319
adequate amounts of colostrum. FCV antibodies were detected in all Feliserin-treated cats
320
post-treatment, which reduced in titre from days 7 to 21, attributable to the relatively short-
321
duration of protection with passive immunisation (Shibata et al., 1983; Young, 1984).
322
Interestingly, no antibodies against FHV-1 were detected in any of the cats tested. The
323
antibody titre against FHV-1 was lower than that for FCV in the Feliserin product (1:128 vs.
324
1:1024, respectively), which is likely associated with this observation. It is not clear from the
325
results whether the relatively low concentration of FHV-1 antibodies in Feliserin were
326
effective, although the numbers were too low to compare Feliserin responses between FCV
327
and FHV-1 infected cats. It is possible, however, that topical application (rather than
328
parenteral administration) plays a major role in reducing viral pathology.
329 330
Inclusion of 10 cats from a previous study, although treated in a similar manner as the
331
controls prospectively recruited, is a confounding factor. Another limitation was that
332
antibody detection was not undertaken in all cats because some of the cats were relatively
333
young and the amount of blood that could reasonably be taken was limited.
334 335
Conclusions
336
The present study has shown that use of Feliserin, administered systemically and
337
topically on three consecutive days, resulted in significant improvement in clinical signs of
338
FURTD and of the general health status of FCV- and/or FHV-1-infected cats within the first
339
3 days when compared to placebo. However, this beneficial effect was transient, with no
Page 14 of 23
340
differences seen comparing Feliserin- and placebo-treated cats by day 7. Administration of
341
Feliserin did not decrease shedding of FVC in infected cats.
342 343 344 345
Conflict of interest statement None of the authors of this paper has a financial or personal relationship with other people or organisations that could inappropriately influence or bias the content of the paper.
346 347
Acknowledgements
348
We would like to thank Professor Dr. Ralf S. Mueller, Clinic of Small Animal
349
Medicine, for his assistance in the statistical examination, the technicians in the Molecular
350
Diagnostic Unit, Langford Veterinary Services, for performing the qPCR assays, as well as
351
the technicians of the Institute of Animal Hygiene and Public Veterinary Services, University
352
of Leipzig, for performing the virus neutralisation tests.
353 354
Parts of the results were presented as an abstract and oral presentation at the 21th
355
annual conference of the German Society of Internal Medicine and Clinical Pathology of the
356
German Veterinary Association (DVG) in Munich, 1-2 February 2013.
357 358 359 360
Appendix A: Supplementary material Supplementary data associated with this article can be found in the online version at doi: setters please insert doi number
361 362 363 364 365 366 367
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Di Martino, B., Di Francesco, C.E., Meridiani, I., Marsilio, F., 2007. Etiological investigation of multiple respiratory infections in cats. The New Microbiologica 30, 455-461. Fontenelle, J.P., Powell, C.C., Veir, J.K., Radecki, S.V., Lappin, M.R., 2008. Effect of topical ophthalmic application of cidofovir on experimentally induced primary ocular feline herpesvirus-1 infection in cats. American Journal of Veterinary Research 69, 289-293. Gaskell, R., Dawson, S., Radford, A., Thiry, E., 2007. Feline herpesvirus. Veterinary Research 38, 337-354. Gould, D., 2011. Feline herpesvirus-1: ocular manifestations, diagnosis and treatment options. Journal of Feline Medicine and Surgery 13, 333-346. Hartmann, A., Hartmann, K., 2010. Treatment and management of Chlamydophila felis infections in cats. Tieraerztliche Praxis 38, 217-226. Hartmann, A.D., Hawley, J., Werckenthin, C., Lappin, M.R., Hartmann, K., 2010. Detection of bacterial and viral organisms from the conjunctiva of cats with conjunctivitis and upper respiratory tract disease. Journal of Feline Medicine and Surgery 12, 775-782. Hartmann, K., Kuffer, M., 1998. Karnofsky's score modified for cats. European Journal of Medical Research 3, 95-98. Helps, C.R., Lait, P., Damhuis, A., Bjornehammar, U., Bolta, D., Brovida, C., Chabanne, L., Egberink, H., Ferrand, G., Fontbonne, A., et al., 2005. Factors associated with upper respiratory tract disease caused by feline herpesvirus, feline calicivirus, Chlamydophila felis, and Bordetella bronchiseptica in cats: experience from 218 European catteries. Veterinary Record 156, 669-673. Hennet, P.R., Camy, G.A., McGahie, D.M., Albouy, M.V., 2011. Comparative efficacy of a recombinant feline interferon omega in refractory cases of calicivirus-positive cats with caudal stomatitis: a randomized, multi-centre, controlled, double-blind study in 39 cats. Journal of Feline Medicine and Surgery 13, 577-587. Jacobs, A.A., Chalmers, W.S., Pasman, J., van Vugt, F., Cuenen, L.H., 1993. Feline bordetellosis: challenge and vaccine studies. Veterinary Record 133, 260-263. Lindt, S., Mühlethaler, E., Bürki, F., 1965. Enzootic virus-induced cat flu at an animal shelter: clinical features, histopathology, aetiology and epizootiology. Schweizer Archiv fur Tierheilkunde 107, 91-101. Maggs, D.J., Clarke, H.E., 2004. In vitro efficacy of ganciclovir, cidofovir, penciclovir, foscarnet, idoxuridine, and acyclovir against feline herpesvirus type-1. American Journal of Veterinary Research 65, 399-403. Maggs, D.J., Collins, B.K., Thorne, J.G., Nasisse, M.P., 2000. Effects of L-lysine and Larginine on in vitro replication of feline herpesvirus type-1. American Journal of Veterinary Research 61, 1474-1478.
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Malik, R., Lessels, N.S., Webb, S., Meek, M., Graham, P.G., Vitale, C., Norris, J.M., Power, H., 2009. Treatment of feline herpesvirus-1 associated disease in cats with famciclovir and related drugs. Journal of Feline Medicine and Surgery 11, 40-48. Nasisse, M.P., Dorman, D.C., Jamison, K.C., Weigler, B.J., Hawkins, E.C., Stevens, J.B., 1997. Effects of valacyclovir in cats infected with feline herpesvirus 1. American Journal of Veterinary Research 58, 1141-1144. Nasisse, M.P., Guy, J.S., Davidson, M.G., Sussman, W., De Clercq, E., 1989. In vitro susceptibility of feline herpesvirus-1 to vidarabine, idoxuridine, trifluridine, acyclovir, or bromovinyldeoxyuridine. American Journal of Veterinary Research 50, 158-160. Parzefall, B., Schmahl, W., Fischer, A., Blutke, A., Truyen, U., Matiasek, K., 2010. Evidence of feline herpesvirus-1 DNA in the vestibular ganglion of domestic cats. The Veterinary Journal 184, 371-372. Planz, O., Seiler, P., Hengartner, H., Zinkernagel, R.M., 1996. Specific cytotoxic T cells eliminate B cells producing virus-neutralizing antibodies. Nature 382, 726-729. Povey, R.C., 1978a. In vitro antiviral efficacy of ribavirin against feline calicivirus, feline viral rhinotracheitis virus, and canine parainfluenza virus. American Journal of Veterinary Research 39, 175-178. Povey, R.C., 1978b. Effect of orally administered ribavirin on experimental feline calicivirus infection in cats. American Journal of Veterinary Research 39, 1337-1341. Radford, A.D., Addie, D., Belak, S., Boucraut-Baralon, C., Egberink, H., Frymus, T., Gruffydd-Jones, T., Hartmann, K., Hosie, M.J., Lloret, A. et al., 2009. Feline calicivirus infection. ABCD guidelines on prevention and management. Journal of Feline Medicine and Surgery 11, 556-564. Radford, A.D., Coyne, K.P., Dawson, S., Porter, C.J., Gaskell, R.M., 2007. Feline calicivirus. Veterinary Research 38, 319-335. Shibata, Y., Baba, M., Kuniyuki, M., 1983. Studies on the retention of passively transferred antibodies in man. II. Antibody activity in the blood after intravenous or intramuscular administration of anti-HBs human immunoglobulin. Vox Sanguinis 45, 77-82. Siebeck, N., Hurley, D.J., Garcia, M., Greene, C.E., Kostlin, R.G., Moore, P.A., Dietrich, U.M., 2006. Effects of human recombinant alpha-2b interferon and feline recombinant omega interferon on in vitro replication of feline herpesvirus-1. American Journal of Veterinary Research 67, 1406-1411. Stiles, J., 1995. Treatment of cats with ocular disease attributable to herpesvirus infection: 17 cases (1983-1993). Journal of the American Veterinary Medical Association 207, 599603. Thiry, E., Addie, D., Belak, S., Boucraut-Baralon, C., Egberink, H., Frymus, T., GruffyddJones, T., Hartmann, K., Hosie, M.J., Lloret, A. et al., 2009. Feline herpesvirus infection.
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ABCD guidelines on prevention and management. Journal of Feline Medicine and Surgery 11, 547-555. Townsend, W.M., Stiles, J., Guptill-Yoran, L., Krohne, S.G., 2004. Development of a reverse transcriptase-polymerase chain reaction assay to detect feline herpesvirus-1 latencyassociated transcripts in the trigeminal ganglia and corneas of cats that did not have clinical signs of ocular disease. American Journal of Veterinary Research 65, 314- 319. Umehashi, M., Imamura, T., Akiyama, S., Kimachi, K., Tokiyoshi, S., Mikami, T., 2002. Post-exposure treatment of cats with mouse-cat chimeric antibodies against feline herpesvirus type 1 and feline calicivirus. The Journal of Veterinary Medical Science 64, 1017-1021. Van der Meulen, K., Garre, B., Croubels, S., Nauwynck, H., 2006. In vitro comparison of antiviral drugs against feline herpesvirus 1. BMC Veterinary Research 2, 13. Verdaguer, N., Fita, I., Domingo, E., Mateu, M.G., 1997. Efficient neutralization of foot-andmouth disease virus by monovalent antibody binding. Journal of Virology 71, 98139816. Wardley, R.C., 1976. Feline calicivirus carrier state. A study of the host/virus relationship. Archives of Virology 52, 243-249. Wardley, R.C., Povey, R.C., 1977. The clinical disease and patterns of excretion associated with three different strains of feline caliciviruses. Research in Veterinary Science 23, 714. Weiss, R.C., Cox, N.R., Boudreaux, M.K., 1993. Toxicologic effects of ribavirin in cats. Journal of Veterinary Pharmacology and Therapeutics 16, 301-316. Williams, D.L., Fitzmaurice, T., Lay, L., Forster, K., Hefford, J., Budge, C., Blackmore, K., Robinson, J.C., Field, H.F., 2004. Efficacy of antiviral agents in feline herpetic keratitis: results of an in vitro study. Current Eye Research 29, 215-218. Young, L.S., 1984. Symposium on infectious complications of neoplastic disease (Part II). Immunoprophylaxis and serotherapy of bacterial infections. The American Journal of Medicine 76, 664-671.
Page 18 of 23
505
Table 1
506
General health status, ‘FURTD score’, and clinical signs of cats receiving either Feliserin or
507
placebo.
General status (%)
health
Day
Feliserin mean SD
0 3 7 21
57.2712.02 85.2310.96 95.457.85
P-value a
<0.01 <0.001 <0.001
98.643.16 ‘FURTD score’
0 3 7 21
14.234.45 4.593.38 1.732.19
0 3 7 21
0.860.94 0.360.73 0.140.47
<0.010 <0.001 <0.001
0 3 7 21
2.090.61 0.410.59 0.140.35
ns <0.050 <0.010
0 3 7 21
2.050.79 1.140.77 0.410.67
<0.001 <0.001 <0.001
0 3 7 21
1.591.01 0.680.57 0.270.55
ns <0.001 <0.001
0 3 7 21
1.500.80 0.410.67 0.180.39
ns <0.001 <0.001
Thoracic
0 3 7 21
0
2.180.91 0.450.67 0.180.50
6.653.70
ns <0.001 <0.001
0.046 ns ns
3.602.74
1.100.97 0.600.60
ns ns ns
ns ns ns
0.450.60
1.700.80 0.900.72
ns <0.001 <0.001
<0.001 ns ns
0.450.60
1.850.75 1.350.81
ns <0.050 <0.001
ns ns ns
0.850.49
1.250.85 0.650.75
ns <0.010 <0.001
ns ns ns
0.250.44
<0.010 <0.001 <0.001
ns ns ns
<0.010 <0.001 <0.001
ns ns ns
0.150.49 <0.001 <0.001 <0.001
0.090.29 Sneezing (grade)
14.203.68
0.500.61
0.000.00 Nasal discharge (grade)
0.032 ns 0.045
0.300.47
0.000.00 Blepharospasm (grade)
94.505.10
ns <0.001 <0.001
0.300.47
0.090.29 Conjunctivitis (grade)
84.009.68
Feliserin vs. Placebo P-value b
1.701.84
0.050.21 Eye discharge (grade)
64.5016.05
P-value a
98.004.10
0.640.85 Lymph node enlargement (grade)
Placebo mean SD
1.650.59 0.650.75 0.350.59 0.050.22
<0.001 <0.001 <0.001
2.001.03 0.550.69 0.200.41
0.360.58
0.250.44
1.550.86
1.200.77
Page 19 of 23
auscultation (grade)
3 7 21
0.730.77 0.320.65
ns <0.001 <0.001
0.050.21 Respiratory effort (grade)
0 3 7 21
0.860.94 0.270.55 0.090.29
0 3 7 21
0.090.29 0.050.21 0.000.00
ns <0.010 <0.001
0 3 7 21
1.410.80 0.140.47 0.050.21
ns ns ns
1.200.52 0.200.52
<0.001 <0.001 <0.001
ns ns ns
0.050.22 0.000.00
ns ns ns
0.000.00 Appetite (grade)
0.250.55
ns <0.010 <0.001
0.100.31
0.000.00 Gingivostomatit is (grade)
0.600.68
0.200.52 0.100.45 0.050.22
ns ns ns
0.000.00 <0.001 <0.001 <0.001
0.000.00
1.450.94 0.300.66 0.050.22
<0.001 <0.001 <0.001
ns ns ns
0.000.00
508 509
a
510
group over time (days 3, 7, and 21 vs. day 0).
511
b
512
test for normally distributed data) comparing treatment groups in terms of change at each
513
time-point relative to day 0.
514
ns, not statistically significant.
P-values (Kruskal-Wallis test with Dunn’s-post test) of differences within each treatment
P-values (Mann-Whitney U test for data not normally distributed or unpaired Student’s t
515
Page 20 of 23
516
Table 2.
517
Number of feline calicivirus (FCV) shedding cats and relative FCV copy numbers (× 104)
518
before and after receiving Feliserin or placebo.
Day 0 Day 21 P-value a
Feliserin Shedding cats Copy numbers (× 104) 19 (100%) 57.81 98.65 16 (84%) 104.30 253.80 0.8
Placebo Shedding cats Copy numbers (× 104) 15 (100%) 1424 3709 15 (100%) 69.34 143
P-value b ns ns
1.0
519 520
a
P-values (Fisher’s exact test) of the difference between groups over time (day 21 vs. day 0).
521
b
P-values (Kruskal-Wallis test with Dunn’s-post test) of changes within each group over
522
time (day 21 vs. day 0).
523
ns, not statistically significant.
524
Page 21 of 23
525
Table 3.
526
Antibody titres
527
sampled during the study period.
a
against feline calicivirus (FCV) in Feliserin- and placebo-treated cats,
Feliserin-treated group
Placebo group
528
a
Cat
Day 0
Day 3
Day 7
Day 21
1 2 3 4 5 6 7 1 2 3 4 5 6
< 1:10 < 1:10 < 1:10 < 1:10 < 1:10 < 1:10 1:10 < 1:10 < 1:10 < 1:10 < 1:10 < 1:10 < 1:10
1:20 1:80 1:80 1:80 1:160 1:160 1:80 < 1:10 < 1:10 < 1:10 < 1:10 < 1:10 < 1:10
1:80 1:40 1:80 1:80 1:80 1:80 1:160 < 1:10 < 1:10 < 1:10 < 1:10 < 1:10 1:10
1:10 1:10 < 1:10 1:10 1:10 1:20 1:20 < 1:10 < 1:10 < 1:10 < 1:10 < 1:10 1:20
Virus neutralisation assay using 100 TCID50 of FCV 255 in Crandell feline kidney cells.
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Figure legend
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Fig. 1. ‘FURTD score’ of cats receiving Feliserin or placebo, showing grades (0–39) at days
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0, 3, 7, and 21. Cats of both groups improved significantly (P < 0.001, Kruskal-Wallis test
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with Dunn’s-post test) by day 7 and day 21; only cats receiving Feliserin improved
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significantly (P = 0.046, unpaired Student’s t test) between day 0 and day 3.
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