-
Journal of Feline Medicine and Surgery (2008) 10,
529e541doi:10.1016/j.jfms.2008.02.006
Pathogenesis of feline enteric coronavirus infection
Niels C Pedersen DVM, PhD1,2*, Claire E Allen BS2, Leslie A
Lyons PhD3
1Department of Medicine andEpidemiology, School of
VeterinaryMedicine, University of California,Davis, CA 95694,
USA2Center for Companion AnimalHealth, School of
VeterinaryMedicine, University of California,Davis, CA 95694,
USA3Department of Population Healthand Reproduction, School
ofVeterinary Medicine, University ofCalifornia, Davis, CA 95694,
USA
*Corresponding author. Present addressAnimal Health, Room 213,
CCAH BldMedicine, University of California, Davþ1 - 5 3 0 - 7 5 2 -
7 4 0 2 , F a x : þ1 - [email protected]
1098-612X/08/060529+13 $34.00/0
Fifty-one specific pathogen-free (SPF) cats 10 weeks to 13 years
of age wereinfected with a cat-to-cat fecaleoral passed strain of
feline enteric coronavirus(FECV). Clinical signs ranged from
unapparent to a mild and self-limitingdiarrhea. Twenty-nine of
these cats were FECV na€ıve before infection andfollowed
sequentially for fecal virus shedding and antibody responses overa
period of 8e48 months. Fecal shedding, as determined by
real-timepolymerase chain reaction (RT-PCR) from rectal swabs,
appeared within a weekand was significantly higher in kittens than
older cats. FECV shedding remainedat high levels for 2e10 months
before eventually evolving into one of threeexcretion patterns.
Eleven cats shed the virus persistently at varying levels overan
observation period of 9e24 months. Eleven cats appeared to have
periods ofvirus shedding interlaced with periods of non-shedding
(intermittent orrecurrent shedders), and seven cats ceased shedding
after 5e19 months (average12 months). There was no change in the
patterns of virus shedding among catsthat were excreting FECV at
the time of a secondary challenge exposure. Fourcats, which had
ceased shedding, re-manifested a primary type infection
whensecondarily infected. Cats with higher feline coronavirus
(FCoV) antibody titerswere significantly more likely to shed virus,
while cats with lower titers weresignificantly less likely to be
shedding. Twenty-two kittens born toexperimentally infected project
queens began shedding virus spontaneously, butnever before 9e10
weeks of age. Natural kittenhood infections appeared to below grade
and abortive. However, a characteristic primary type
infectionoccurred following experimental infection with FECV at
12e15 weeks of age.Pregnancy, parturition and lactation had no
influence on fecal shedding byqueens. Methylprednisolone acetate
treatment did not induce non-shedders toshed and shedders to
increase shedding.
Date accepted: 29 February 2008 � 2008 ESFM and AAFP. Published
by Elsevier Ltd. All rights reserved.
Feline enteric coronavirus (FECV) is a ubiq-uitous, worldwide,
intestinal virus of cats(Pedersen et al 1981a, 2004). The name
feline coronavirus (FCoV) has been appliedsomewhat
interchangeably to FECV. Technically,FCoV includes all strains
(numerous), serotypes(types I and II) and biotypes (enteric or
infectiousperitonitis viruses) of the genus. Several strainsof FECV
have been studied by experimentalfecal-oral infection with
cat-to-cat passed virus.The original FECV strain was designated
FECV-University of California, Davis (UCD) (Pedersenet al 1981a)
and a second isolate FECV-Rogers
: Center for Companiong., School of Veterinaryis, CA 95694, USA.
Tel:5 2 - 7 7 0 1 . E - m a i l :
� 2008 ESFM a
and Morris (RM) (Hickman et al 1995). Both ofthese strains
belong to serotype I, possessing a fe-line- rather than
canine-coronavirus spike pro-tein. Several additional FECV strains
have beenstudied in the field using polymerase chain reac-tion
(PCR) (Foley et al 1997b, Benetka et al 2006).FECV is tropic for
the mature apical epitheliumof the intestinal villi (Pedersen et al
1981a) andboth type I and II serotypes use species- andprobably
type-specific (Dye et al 2007) variantsof aminopeptidase-N as a
receptor (Tresnanet al 1996, Tusell et al 2007). FECV infection
isusually unapparent or manifested by a transientgastroenteritis
(Hayashi et al 1982, Pedersen et al1981a, Mochizuki et al 1999); it
is rarely fatalwhen in its native biotype (Kipar et al 1998).
The importance of FECV as a primary intestinalpathogen is
minimal. However, FECV commonly
nd AAFP. Published by Elsevier Ltd. All rights reserved.
mailto:[email protected]
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530 NC Pedersen et al
mutates in vivo and at least one mutant form(ie, biotype) causes
a highly fatal disease knownas feline infectious peritonitis (FIP)
(Poland et al1996, Vennema et al 1998). The precise nature ofthe
mutation that causes this change in virulencehas been variably
ascribed to differences in thespike protein (Rottier et al 2005) or
to non-synon-ymous or deletion mutations in the 3c (small
en-velope) gene (Vennema et al 1998). The incidenceof the enteric /
FIP biotype mutation followingFECV infection is unknown but may be
as highas 20% (Poland et al 1996) and is more likely tomanifest
clinically in kittens (Foley et al 1997a)or immunocompromised cats
(Poland et al1996). FIP virus (FIPV) differs from its strictly
in-testinal tropic FECV parent in its affinity for mac-rophages
(Pedersen 1987, Stoddart and Scott1989). This altered tropism
allows the virus to be-come a systemic pathogen of macrophages,
andthe resultant disease involves a complex interac-tion between
host cellular and humoral immu-nity and infected macrophages
(Pedersen andBoyle 1980 Pedersen 1987).
Although there have been numerous studies ofFIPVs, studies of
FECVs have been surprisinglyfew. Experimentation with FECV has been
ham-pered by its lack of growth in tissue culture.Therefore,
infection studies have often relied onextracts of feces from cats
infected with cat-to-cat passed virus (Pedersen et al 1981a,
Polandet al 1996). Although one report suggests thata cultured
strain of FCoV, WSU-79-1683, is a pro-totypic FECV (Pedersen et al
1984b), this authornow believes it to be a tissue culture
attenuatedrecombinant of canine and feline coronavirus.This is
given support by the complex patternsof recombination that have
been described forWSU-79-1146 (a highly virulent FIPV)
andWSU-79-1683, which were both isolated fromthe same laboratory at
the same time (Herre-wegh et al 1998). WSU-79-1683 also lacks the7b
gene, which is intact in cat passed FECVs(Herrewegh et al 1995).
Therefore, studies ofFECV should use biotype confirmed fecal
pas-saged virus until a proper FECV is adapted totissue
culture.
The present study was designed initially toprove that resistance
and susceptibility to FECVinfection were under genetic control,
just as ge-netics appears to play an important role inFIPV
resistance (Foley et al 1997a). Young catswere infected with the RM
strain of FECV andtheir patterns of fecal virus shedding
quantifiedover extended periods of time by periodic sam-pling. Cats
that stopped shedding the virus after
8e12 months were than bred to cats with a simi-lar profile, and
cats that appeared to be long-term shedders were bred to chronic
shedders.Their kittens were then infected with FECVat 10e23 weeks
of age and the cycle continued.The goal was to create two
bloodlines, one resis-tant and one susceptible. Once this was
accom-plished, the genetic basis for resistance/susceptibility was
to be determined. After morethan 3 years, it became apparent that
FECV resis-tance and susceptibility may not be definable bysimple
Mendelian genetics. Therefore, a decisionwas made to concentrate on
what was learnedabout FECV pathogenesis.
Methods
Experimental animals
Twenty-nine FECV na€ıve cats, ranging from kit-tens to aged
animals, were obtained from thespecific pathogen-free (SPF)
breeding colony ofthe Feline Nutrition Laboratory, UCDavis.
Catswere housed in the feline research facilities ofthe Center for
Companion Animal Health(CCAH), UCDavis. Care was provided by
staffof the CCAH under the supervision of the Centerfor Laboratory
Animal Services, UCDavis. Stud-ies were done under United States
Departmentof Agriculture required Institutional AnimalCare and Use
Committee approved protocols.Males and females were not neutered
for thisstudy. Select animals were chosen for breedingduring the
course of the study and 22 kittensproduced from these mating’s
added to thestudy over time.
Experimental infection
Cats were infected with 0.5 ml orally of a fecalextract (Poland
et al 1996) of the RM strain ofFECV (Hickman et al 1995). The
initial groupof cats was infected several days after acquisi-tion,
while kittens reared during the studywere infected at 12e15 weeks
of age and ob-served for signs of acute or chronic disease.Cats
were housed in open rooms, with nomore than five animals per room.
These groupsremained relatively stable, except when tomsor queens
were transferred for breeding orqueens isolated for birthing and
kitten rearing.Reasonable precautions were taken to limitspread of
contaminated litter by caretakers; dis-posable coveralls, boots,
foot baths, hand wash-ing, gloves were used.
-
531Feline enteric coronavirus infection
Quantitation of FECV shedding
FCoV RNAwas quantified using purification pro-cedures and
specific primers reported by Gut et al(1999). Feces were collected
by inserting standardcotton tipped swabs into the rectum prior to
infec-tion and at 1 week intervals for at least 2 months,and then
at 1e2 month intervals thereafter. RNAwas isolated from the swabs
(van der Hoek et al1995). Five microliters of the purified RNA
wasadded to 7 ml of PCR mixture containing 6 ml ofTaqMan One Step
RT-Master Mix (Applied Bio-systems, Foster City, CA), 0.31 ml of
MuLV/RNaseInhibitor, 0.24 ml each of forward and reverseprimers,
and 0.10 ml of RNase-free water. The12-ml reaction went through a
reverse transcrip-tase step for 30 min at 48�C and AmpliTaq
Gold(Applied Biosystems, Foster City, CA) activationfor 10 min at
95�C. The samples were put through40 cycles of 95�C for 15 s and
60�C for 60 s for RNAamplification. PCR was performed using
AppliedBiosystems (Foster City, CA) 7300 Real-time poly-merase
chain reaction (RT-PCR) System and 7300System Software. The
positive/negative cutoff ofthe assay was around 75e100 RNA
transcripts/swab. Therefore, swabs that were negative at1� log 10
were considered negative. The numberof RNA transcripts per swab was
considered equalto the number of viral particle (Gut et al
1999),given that each FECV particle contains only oneRNA
transcript. There was no evidence for fecalinhibitors of the RT-PCR
assay used in this study;SPF cat fecal samples were always
negative, butbecame rapidly and progressively positive
afterexperimental infection. Therefore, internal DNA(Monteiro et al
1997) or RNA (Escobar-Herreraet al 2006) fragment controls were not
employed.
FCoV antibody tests
Serum antibody titers to FECV were undertakenwith an indirect
fluorescent antibody (IFA)procedure (Pedersen 1976) using
FIPV-UCD1infected Fcwf-4 cells (Pedersen et al 1981b). Cellswere
grown in 12-well Teflon coated microscopicslides and infected with
FIPV-UCD1 tissue culturefluid when three-quarters confluent. Slides
wereharvested after 24e48 h and fixed in absolute ace-tone. Each
serum was tested at 1:5, 1:25, 1:100,1:400 and 1:1600 dilutions in
Hank’s buffered sa-line solution. Serum was allowed to react for 1
h,slides washed, and a 1:50 dilution of rabbit anti-cat IgG
(Antibodies Incorporated, Davis, CA95616) was over layered for 1 h.
Slides were thanwashed, stained with dilute Evan’s blue dye,and
cover slips mounted with 1:1 glycerin:saline.
Slides were read on an indirect fluorescent micro-scope and the
titer listed as the last dilution ofserum that still produced
noticeable fluorescence.
Statistical analysis
Data was recorded on Excel spread sheets (Mi-crosoft Office
2003, Microsoft, Redmond, WA98004), and statistical analyses, when
indicated,undertaken with JMP Statistical Discovery Soft-ware (SAS,
Cary, NC 27513) (www.jmp.com/software/). Significance (P� 0.05) was
deter-mined by the program’s Student t-test.
Results
Outcome of primary infection
Thirty-three cats were infected with FECVand fol-lowed
sequentially for fecal virus shedding overa period of 14e48 months
(Table 1). Twenty-ninecats were FECV na€ıve at the start of the
study.Four of these cats (A01eA04) were born duringthe course of
the study to project queens and,therefore, not FECV na€ıve, but
were virus nega-tive at the time of primary infection. Fecal
shed-ding rose within a week and remained atconsistently high
levels of 1012e1016 particles/swab for 2e10 months (Figs 1e3; Table
1). Peak vi-rus levels tended to drop to levels of 106e109
par-ticles per swab in the secondary stage of infectionthat
followed (Figs 1e3).
Three different patterns of virus shedding werenoted in the
secondary infection stage. Eleven catsshed the virus continuously
at greatly varyinglevels over an observation period of 14e24
months(persistent infection) (Table 1; Fig 1). Twelve catshad brief
periods of recovery, interlaced with pe-riods of virus shedding
(intermittent or recurrentshedders) (Table 1; Fig 2), and 10 cats
ceased shed-ding at 7e18 months (average 12.3 months) (Table1; Fig
3). Three representative cats were graphedfor each of the three
infection outcomes (Figs1e3). None of the cats developed FIP.
Outcome of secondary infection
Nineteen cats were used for this study and dividedinto two
groups of four and 15 based on their virusshedding patterns prior
to reinfection. The fourcats that had low or non-measurable virus
shed-ding at the time of secondary exposure were clearlyreinfected.
Fecal shedding for one of these cats is il-lustrated in Fig 4.
Figure 5 shows the mean virus
-
Table 1. Description of 33 cats used to study patterns of fecal
FECV shedding following primary infectionand in the study on the
effect of methylprednisolone acetate induced stress in 18 of these
animals
Catnumber
Gender/age(months)
Observationperiod (months)
Duration of primaryinfection (months)
Outcome ofinfection
Time to recovery(months)
94309 F/151 16 4 Persistent NA*94529 F/154 16 3 Recurrent
NA98462 F/108 13 3 Recurrent NA99402 F/96 12 6 Recurrent NA00417
F/96 14 4 Recurrent NA01282 F/33 13 2 Recurrent NA02136y F/59 48 5
Recurrent NA04096 F/51 10 4 Persistent NA04139 F/52 13 10 Recurrent
NA04140 F/52 13 8 Recurrent NA04141 F/52 10 2 Persistent NA04144
F/52 9 2 Persistent NA04146 F/52 9 3 Persistent NA04161 M/51 9 2
Persistent NA04224 M/50 9 4 Persistent NA04225 M/50 12 5 Persistent
NA98272y M/90 24 4 Persistent NA04099z M/51 36 4 Recurrent NA05243y
F/52 24 3 Recurrent NA05244z M/52 24 3 Recurrent NA05246y F/52 23 5
Recovered 1905249z F/52 23 5 Recovered 1805325z M/52 23 5
Persistent NA05326y M/52 23 5 Persistent NA06028y M/52 14 2
Recovered 1506029z F/53 14 2 Recovered 1506032y F/53 14 4 Recovered
1206033z F/53 14 2 Recovered 1206034y M/53 14 3 Recovered 15A01z
M/2 13 4 Recovered 9A02y M/2 12 2 Recovered 11A03z F/2 16 3
Recurrent NAA04y F/2 8 5 Recovered 5
*NA¼ not applicable.yMethylprednisolone acetate treatment
group.zNon-methylprednisolone acetate treatment group.
532 NC Pedersen et al
shedding levels for all four of the cats that were
re-infectable; the peak levels of virus shedding wereas high as
observed during primary infection andthe duration was similar (4e7
months). No evi-dence for reinfection was observed in cats thathad
been shedding high levels of virus at thetime of secondary
challenge exposure (Fig 6).
Relationship of age to peak virus sheddingduring primary
infection
Cats were divided into three age groups: (1) kit-tens 2e4 months
of age at the time of primary in-fection (n¼ 22), (2) mature cats
2e8 years of age(n¼ 25), and (3) aged cats 8e13 years of age
(n¼ 4). The peak level of virus shedding duringtheir primary
phase of FECV infection was com-pared between groups (Fig 7).
Kittens shed sig-nificantly higher peak levels of virus than
cats2e8 years of age; virus shedding was also higherthan for aged
cats, but this difference was notsignificant. Aged cats (8e13 years
of age) alsotended to shed higher levels than 2e8 yearolds, but the
difference was also not significant.
Relationship of serum antibody titersand virus shedding
status
Antibodies to FCoV were measured sequentiallyby the IFA
procedure in 16 animals over a period
-
1
100
10000
1E+06
1E+08
1E+10
1E+12
1E+14
0 2 2 3 4 5 6 7 8 9 10
0 2 2 3 4 5 6 7 8 9 10
Months following primary infection
FE
CV
p
articles p
er sw
ab
1
100
10000
1E+06
1E+08
1E+10
1E+12
1E+14
Months following primary infection
FE
CV
p
articles p
er sw
ab
1
100
10000
1E+06
1E+08
1E+10
1E+12
1E+14
1E+16
1E+18
0 1 2 3 3 5 6 7 8 9 10 12 13 14 15 16Months following primary
infection
FE
CV
p
articles p
er sw
ab
Fig 1. Typical fecal FECV shedding patterns of cats
demonstrating a persistent pattern of infection.
533Feline enteric coronavirus infection
of 12e24 months. These cats were randomly se-lected from among
the 33 animals whose infectioncourse had been established. A total
of 241 timematched serum/feces samples were analyzed(Fig 8). FCoV
antibody titers were significantly(P¼ 0.05) higher among cats that
were virus shed-ders at the time of testing than in the group of
catsthat were non-shedders. Conversely, cats withtiters of 1:25 and
lower, as a group, were signifi-cantly (P¼ 0.05) more likely to be
non-shedders.However, there was considerable overlap in titers
and virus shedding status among individual catsin the two
groups; virus shedders and non-shedders were to be found in
individuals withthe lowest (5e25) and highest (1600) titers.
Natural transmission to kittensborn to project queens
Twenty-two kittens were born to eight differentqueens, and data
was available for 12 of themfor the first 24 weeks of their lives.
None of
-
110
1001000
100001000001E+061E+071E+081E+091E+101E+111E+121E+131E+14
0 1 1 2 3 4 5 6 7 8 9 10 11 12 13 14Months following primary
infection
FE
CV
p
articles p
er sw
ab
110
1001000
100001000001E+061E+071E+081E+091E+101E+111E+121E+131E+141E+15
0 1 2 2 3 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17Months following
primary infection
FE
CV
p
articles p
er sw
ab
110
1001000
100001000001E+061E+071E+081E+091E+101E+111E+12
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 17 18 19 20 21 22 23
24 27 30 32 35 37 38Months following primary infection
FE
CV
p
articles p
er sw
ab
Fig 2. Typical fecal FECV shedding patterns of cats
demonstrating an intermittant pattern of infection.
534 NC Pedersen et al
these 12 kittens shed FECV before 9 weeks ofage, while all
kittens tested at 9e11 weeksof age were shedding as a result of
natural ex-posure (Fig 9). However, the level of virusshedding was
relatively low, from 103 to 108
particles/swab, and declined to very low ornon-detectable levels
by 13e15 weeks (Fig 9).All of the kittens were infected with
FECV-RM at 10e17 weeks of age (average 13 weeks)regardless of their
prior FECV shedding status.The subsequent pattern of fecal virus
sheddingresembled that observed during primary
FECV exposure in coronavirus na€ıve cats (Figs1e3, 9).
Effects of pregnancy, parturition andlactation on FECV
shedding
Fecal virus shedding was measured for a period of12 weeks before
and 12 weeks after parturition inseven queens and nine litters (Fig
10). There wasno significant difference in the levels of
FECVshedding as a result of pregnancy, parturition orlactation.
-
110
1001000
100001000001E+061E+071E+081E+091E+101E+111E+121E+131E+14
Months following primary infection
FE
CV
articles p
er sw
ab
110
1001000
100001000001E+061E+071E+081E+091E+101E+111E+121E+13
0 12Months following primary infection
FE
CV
p
articles p
er sw
ab
110
1001000
100001000001E+061E+071E+081E+091E+101E+111E+121E+131E+14
Months following primary infection
FE
CV
p
articles p
er sw
ab
1 2 3 4 5 6 7 8 9 10 11 13 14 15 16
0 121 2 3 4 5 6 7 8 9 10 11 13 14 15 16
0 121 2 3 4 5 6 7 8 9 10 11 13 14 15 16
17
17
Fig 3. Typical fecal FECV shedding patterns of cats
demonstrating a self-limiting (recovery) pattern of infection.
535Feline enteric coronavirus infection
Effects of methylprednisolone acetatetreatment on fecal FECV
shedding
Methylprednisolone acetate (5 mg/kg) was ad-ministered twice
intramuscularly at a 3-week in-terval to 10 randomly selected cats
from theproject; eight cohort cats were given saline(Table 1).
There was no statistical change in the
levels of virus shedding post-treatment in catsgiven
methylprednisolone acetate (Fig 11) or sa-line (data not shown).
Furthermore, cats in eithergroup that were shedding at the time of
treat-ment were not induced to shed more virus andcats that were
non-shedders did not start shed-ding (data not shown).
-
1
100
10000
1E+06
1E+08
1E+10
1E+12
1E+14
1E+16
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23
24Months following primary infection
FE
CV
p
articles p
er sw
ab
Fig 4. Levels of virus shedding prior to and after reinfection
(arrow).
536 NC Pedersen et al
DiscussionThe present study added to our understandingof the
course of FECV infection in domesticcats. There was a distinct
primary stage of infec-tion that lasted from 7 to 18 months; the
highestlevel of virus shedding occurred during thisstage. This
primary stage was resolved in oneof three manners: (1) recovery,
(2) persistentshedding, and (3) recurrent or intermittent
shed-ding. These findings corroborated earlier studiesof naturally
occurring FECV infection. In a com-prehensive study of 275 purebred
cats from sixcatteries, fecal samples were collected every1e3
months for a year and virus shedding quan-tified by RT-PCR (Foley
et al 1997b). A large pro-portion of these cattery cats shed virus
at anygiven time, but most manifested cycles of infec-tion and
shedding. Similarly, Harpold et al(1999) found that all adult cats
in an Abyssinian
110
1001000
10000100000
100000010000000
1000000001000000000
1e+101e+11
FE
CV
p
articles p
er sw
ab
-15 -12 -10 -4 -1 0 2 4 6 9 11Weeks post reinfection
Fig 5. One-way analysis of levels of fecal FECV sheddingin a
group of four cats that were shedding very low ornon-detectable
levels of virus prior to infection. Virus levelsfollowing
reinfection were higher at all time points than theywere prior to
infection, but because of the small group size,only weeks 3 and 4
were significantly different.
cattery shed virus in their feces at least once dur-ing the year
and 4/15 cats were shedding greaterthan 75% of the time. Rohner
(1999) reported thatFECV shedding dramatically decreased over
2years in a group of naturally infected cats. Herre-wegh et al
(1997) studied the persistence andevolution of endemic FECV
infection in a closedcat-breeding facility. Viral RNA was detected
byRT-PCR in the feces and/or plasma of 36 of 42cats (86%) tested.
Four of five infected catswere still shedding when tested 111 days
later.Two cats were then placed in strict isolationand virus
shedding was found to last up to 7months in one animal.
Persistent and recovered infections might beexplained by
relative differences in the strengthof local gut immunity. However,
an immunologicexplanation for the recurrent pattern of sheddingwas
not so apparent. It would be tempting toblame periods of recurrent
shedding on frequentreinfections. Reinfection is a common
occurrence
1
100
10000
1000000
100000000
1e+10
1e+12
1e+14
1e+16
FE
CV
p
articles p
er sw
ab
-15 -12 -10 -4 -1 0 2 4 6 9 11Weeks post reinfection
Fig 6. One-way analysis of levels of fecal FECV sheddingin a
group of 15 cats that were shedding virus at the timeof their
secondary challenge exposure. There was no signif-icant change in
virus shedding following reinfection.
-
Fig 7. One-way analysis of the mean peak levels of FECVfecal
shedding during primary infection in cats infected at2e4 months of
age, >2< 8 years of age, and >8 years of age.
537Feline enteric coronavirus infection
for many gut pathogens, because local immunityoften requires
persistence of the organism anddoes not possess strong memory
(Brandtzaeg2007). However, successful reinfection in fourcats
resembled a primary infection in magnitudeand duration, which was
not true for recurrentbouts of shedding. It is possible that
recurrentshedding was an artifact of the assay procedure.If the
assay failed to delineate low level sheddingfrom non-shedding,
recurrent and persistentshedders would be basically the same
acceptfor amplitude. The alternative possibility wasthat these
periods of reshedding were due to re-activation of a latent or
sub-detectable infection.However, this was not supported by studies
ofnatural or artificial stress (see below).
FECV was shed at very high levels followingprimary infection and
the levels were signifi-cantly higher in kittens than in adult
cats. Rohner(1999) also found that the levels of FECV were
Shedding status
010203040506070
5-25 100 400FECV antibody titer
1600
PositiveNegative
Nu
mb
er o
f sam
ples
Fig 8. FCoV indirect IFA antibody titers in serum collectedfrom
cats over a 12e24 month period. Their fecal FECVshedding status was
measured at the same time.
many log10 higher during early than late infec-tion. Foley et al
(1997b) were the first to showhigher levels of FECV shedding in
kittens thanolder cats in shelters. These findings have an
im-portant collective implication for FIP. FIP is muchmore common
among younger cats (Pedersen1976, Foley et al 1997a). If kittens
are infected be-fore their immune systems are fully matured,levels
of FECV replication would be higher, andgreater levels of virus
replication would favorFECV / FIPV mutations. Relative
age-relatedimmunodeficiency could also prevent kittensfrom
containing the FIP mutant virus. This sce-nario is supported by
research with FECV-RMinfection in cats that were
immunocompromisedby long-standing FIV infection (Poland et al1996).
Chronic FIV infected cats shed 10e100times more FECV than
age-matched non-FIV in-fected cats, just like FECV in kittens, and
2/19 ofthem developed FIP vs none of the 20 FIV freecohorts. It was
concluded that immunosuppres-sion caused by chronic FIV infection
enhancedthe creation and selection of FIPV mutants by in-creasing
the rate of FECV replication in thebowel, as well as by inhibiting
the host’s abilityto combat the mutant viruses once they
occurred.
The study also followed kittens born to FECVinfected queens.
None of these kittens shed virusprior to 9 weeks of age, while all
kittens thatwere tested between 10 and 15 weeks were pos-itive.
These findings were in concordance withthose of Foley et al
(1997a,b), who were notable to detect virus in feces before 10
weeks ofage in cattery kittens. However, Harpold et al(1999) found
that kittens in an Abyssinian catterystarted shedding virus at
33e78 days (5e11weeks) of age (mean 9.6 weeks). Gut et al(1999)
studied 77 kittens from 12 catteries andfound a progressive rise in
fecal shedding fromaround 2e4 weeks onwards, with a peak at 9weeks
of age. Therefore, the period of 9 weeksof age is probably when
most kittens are in-fected, although it may occur at an earlier
ageunder certain conditions. These chronologicalfindings support
FCoV control programs that ad-vocate isolation of pregnant queens
and earlyweaning of their kittens (Addie et al 2004).
The-oretically, if queens are strictly isolated and theirkittens
separated at the earliest possible time(
-
0
2
4
6
8
10
12
14
16
8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24Weeks of age
FE
CV
sh
ed
din
g p
er sw
ab
(lo
g10)
C01C02D01E01E02E03
A01A02A03A04B01B02
Fig 9. Fecal virus shedding levels in kittens born to project
queens. Kittens were infected naturally at 9e10 weeks of age,but
this infection appeared transient. Kittens were experimentally
infected at 10e17 weeks of age (average 13 weeks).
538 NC Pedersen et al
transmitted from room to room by caregiverseven with very good
containment facilities andprocedures (Pedersen et al 1981a). The
ease of fo-mite transmission and ubiquitous nature of thevirus
makes it extremely difficult to keep catsfree of the virus.
However, delaying FECV infec-tion until the kittens are older
(>16 weeks), byisolation and early weaning, may have another
0
2
4
6
8
10
12
-12 -10 -8 -6 -4 -2 0Weeks after par
FE
CV
fecal sh
ed
din
g p
er sw
ab
(lo
g10)
Fig 10. Average levels of FECV fecal shedding before and a
depressing effect on FIP regardless of whetheror not they are
infected later in life. The levelsof virus replication might be
lower in older kit-tens, thus decreasing the likelihood of
mutants,and the immune system would be more compe-tent in
containing any FIPV that might arise.
Kittens born into the study, and infected natu-rally,
demonstrated a peculiar form of infection.
2 4 6 8 10 12turition
5243
4140(1)
4140(2)
5246
5249(1)
5249(2)
4139
2136
6032
fter parturition in seven queens during nine pregnancies.
-
1
100
10000
1000000
100000000
1e+10
1e+12
1e+14
Viru
s p
artic
le
s p
er s
wa
b
-11 -9 -6 -1 0 1 2 3 4 5 6 7 8Weeks post injection of
depoMedrol
Fig 11. Levels of fecal FECV in cats that were positive
shed-ders and treated with two injections of
methylprednisoloneacetate at week 0 and 4. There was no significant
differencein levels of virus shed in the feces after treatment.
539Feline enteric coronavirus infection
The levels of virus replication in naturally in-fected kittens
were much lower than in olderkittens and cats that had been
experimentallyinfected. The virus shedding also seemed to bemuch
more transient. Unlike cats with longerterm infections, which did
not respond to su-per-infection, kittens responded to an
experi-mental challenge exposure just like na€ıve orrecovered cats.
This suggests that maternal im-munity may have played some role in
alteringthe course of natural infection, although notleading to
either strong premonition or adaptiveimmunity.
The role of stress in reactivating possible latentor subclinical
infections, or increasing virusshedding among shedders, was studied
in twoways: (1) by studying a natural stress, ie, preg-nancy,
parturition and lactation, and (2) inducingan artificial stress
with corticosteroid treatment.The stress of parturition and
lactation is knownto activate latent feline herpesvirus infection
inabout 40% of queens, and this re-excretion of vi-rus is an
important route for infection of kittens(Gaskell and Povey 1977).
This stress can bemimicked by the administration of
corticoste-roids (Gaskell and Povey 1977, Hickman et al1994); a
single dose of methylprednisolone ace-tate is particularly
effective (Reubel et al 1993).Methylprednisolone is also a potent
activator oflatent feline leukemia virus infection (Rojkoet al
1982, Pedersen et al 1984a,b) and willabolish age-related
resistance to FeLV (Pedersenand Johnson, 1991). It will even
reactivatesubclinical dermatophyte infections in kittens inthe
post-recovery phase (Pedersen 1991). Unlikefeline herpesvirus,
neither parturition/lactationnor methylprednisolone treatment
affectedFECV shedding in this laboratory setting.
It might be argued that conditions in nature aremore severe;
however, FECV shedding was alsonot affected by parturition and
lactation incattery cats (Foley et al 1997b).
There was a significant relationship betweenserum IFA titers and
virus shedding in this study.Cats that were shedding virus tended
to have IFAtiters of 1:100 and above. Cats that were no
longershedding virus tended to have titers of 1:25 andlower. This
confirmed an earlier report by Rohner(1999). However, such a
relationship was notnoted by Harpold et al (1999) or insinuated
byFoley et al (1997b). This discrepancy may involvethe manner in
which data is viewed. When fecalshedders and non-shedders are
looked at asgroups, the relationship between higher titersand
shedding and lower titers and non-sheddingwas significant. However,
the overlap between ti-ter and shedding was substantial and greatly
di-minished the value of titers in evaluatingshedding status in
individual cats. The value ofapplying group FECV.
Application of this technique to eradication ofFECV under
experimental conditions was firstreported by Hickman et al (1995).
A FECV wasinadvertently introduced into a very large re-search
cat-breeding colony and not recognizeduntil a few cases of FIP were
confirmed overthe next couple of years. In order to save valu-able
blood lines, FECV was eradicated based onserum antibody titers.
Cats with high or rising ti-ters were culled and cats with low and
decreas-ing titers were taken out of breeding andmaintained in
strict isolation. The process of con-tinually selecting cats with
low titers eventuallyyielded a much smaller group of cats that
wereproven to be free of FECV by following titers intheir kittens.
If the titers in kittens were strictlyof maternal origin, they
would become negativeby 12e16 weeks. If the kittens were
infected,they would fall, and then rise again after 12e16weeks.
Such an eradication regimen requires ex-ceptional quarantine
facilities and infection con-trol practices and accurate titer
determinations.These are difficult to achieve in most
multi-catenvironments in the field. Although the use ofgroup titers
for eradication may not be usefulto many multi-cat households and
catteries,group titers may be helpful in looking at theoverall
coronavirus status in cattery or othermulti-cat environments.
Groups of cats thattend to have high titers are likely to have a
signif-icant proportion of FECV shedders, while theconverse would
be true for groups of cats thathave low and negative titers.
Catteries with
-
540 NC Pedersen et al
many high titer cats are known to have a greaterincidence of FIP
(Foley et al 1997a,b).
Immunity to FECV infection was not studiedper se, but it was
possible to infer several thingsfrom the present observations.
First, there isa definite primary stage of infection that
lastsseveral months, followed by a period when virusshedding either
remains persistent at a lowerlevel, becomes intermittent, or
ceases. The immu-nity generated during this primary stage wasslow
to develop, variable in strength, and tenu-ous in duration.
Reinfection also appeared tomirror primary infection, indicating
that immu-nity is not only tenuous, but that it lacks memory.The
finding that immunity is tenuous and rein-fection common mirrors
the conclusions ofAddie et al (2003), who found that FECV
recov-ered cats can be reinfected with the same or dif-ferent
strains of the virus. This is characteristic ofgut immunity in
general (Brandtzaeg 2007) andto coronavirus immunity in particular
(Saif2004). This pattern of infection and immunity isstrongly
influenced by environmental factors.FECV infection would be
self-limiting if groupsof cats were allowed to grow old without
con-stant re-exposure to other infected cats and tonew cats
(especially kittens). Certain husbandrypractices unique to cats may
also favor cat-to-cat (ie, litterboxes and litter) and
cat-to-human-to-cat (fomite) transmission.
AcknowledgementsThis study was funded by Winn Feline Founda-tion
of the Cat Fanciers Association, Manasquan,NJ 08736-0805, and the
Center for CompanionAnimal Health, School of Veterinary
Medicine,University of California, Davis, CA 95616. Theauthors are
also grateful for the technical supportgiven by Ms Kelly
Bettencourt and Ms ElizabethHolmes.
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Available online at www.sciencedirect.com
Pathogenesis of feline enteric coronavirus
infectionMethodsExperimental animalsExperimental
infectionQuantitation of FECV sheddingFCoV antibody
testsStatistical analysis
ResultsOutcome of primary infectionOutcome of secondary
infectionRelationship of age to peak virus shedding during primary
infectionRelationship of serum antibody titers and virus shedding
statusNatural transmission to kittens born to project queensEffects
of pregnancy, parturition and lactation on FECV sheddingEffects of
methylprednisolone acetate treatment on fecal FECV shedding
DiscussionAcknowledgements