The seroprevalence of the canine respiratory coronavirus in the Netherlands
Selina Norbart
The canine respiratory coronavirus (CRCoV) is one of many coronaviruses circulating among animals and was discovered in the UK in 2003 by Erles et all 1 . Like every coronavirus, CRCoV contains a single-stranded RNA molecule, a capsid and projecting spikes that give the virus its specific appearance in electron micrographs.
Coronaviruses are classified into three different antigenic groups and can cause respiratory, enteric, hepatic and even neurological symptoms in humans and animals. The CRCoV belongs to group 2a and is genetically very similar to the bovine coronavirus. Group 2 coronaviruses have an additional protein, the hemagglutin-esterase protein (HE-protein) 2, 3 .
CRCov itself causes no or only mild respiratory symptoms like coughing, sneezing and rhinitis, but in combination with other pathogens, severe clinical signs can be seen. It is thought that CRCoV plays a role in the canine infectious respiratory disease (CIRD). The most important pathogens causing CIRD are canine parainfluenza virus (CPIV), canine adenovirus type 2 (CAV-2), canine influenza virus (CIV) and Bordetella bronchiseptica. These pathogens can cause (severe) clinical signs on their own, but can invade the body much more easy when the respiratory tract is made vulnerable by, for example, the CRCoV 4 .
In a couple of countries, the seroprevalence of infections with the CRCoV was already studied 5-9 . The seroprevalences range from 17,8% in Japan to 59,1% in Canada. In the
Netherlands there are no data on the seroprevalence and during this study this seroprevalence was determined using Virus Neutralization tests. The aim of the current study was also to develop an ELISA based on the HE protein of the Bovine coronavirus to determine if these results correlate with the results of the VN tests. Also it was tried to develop an ELISA based on the HE protein of the CRCoV itself and these results were also compared to the results of the BCoV-HE + ELISA and the VN tests.
2

The seroprevalence of the canine respiratory coronavirus in the Netherlands
Selina Norbart
Serum samples
The sera used during this study were collected from all over the country in 2005.
Veterinarians were asked to send in blood samples with some information about the dog from which the blood was taken. 47 veterinary practices send in blood from dogs of different ages. The blood was centrifuged and serum was stored at -20 °C. Information about the dogs was stored in a database (vaccinations, age, housing conditions etc).
Cell Culture
HRT-18 cells
The cells used for most of the experiments during this study were human rectal adenocarcinoma (HRT-18) cells. These cells were cultured in T75 and T225 flasks using
Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% inactivated fetal calf serum (fcs) and penicillin/streptomycin (p/s) and incubated at 37 °C, 5% CO
2
. Prior to use, the fcs was inactivated for 30 minutes at 56 °C and it was stored at 4 °C. The p/s was stored at -20 °C.
Every time a confluent monolayer was formed, the cells were passaged to prevent the cells from clotting. Dulbecco’s Phosphate Buffered Saline (DPBS) without Ca 2+ and Mg 2+ (PBS0) was used for washing the cells and trypsin EDTA for loosening the cells. During this study, the cells from passage 25 to 44 were used.
HEK 293T cells
The cells used for transfection were human embryonic kidney (HEK 293T) cells. These cells were cultured in T75 and T225 flasks using DMEM also supplemented with 10% inactivated fcs and p/s and incubated at 37 °C, 5% CO
2
. When passaging these cells, they were treated the same way as the HRT-18 cells, but in contrast to the HRT-18 cells, the HEK 293T cells did not need to be washed for a long time with trypsin to detach from the bottom of the culture flask.
Virus stocks
Bovine Coronavirus Mebus
HRT-18 cells were cultured in three T225 flasks using DMEM supplemented with 10% fcs and p/s. When a confluency of 80% was reached, these cells were infected with BCoV passage 2, kindly provided by Raoul de Groot and Arno van Vliet. After removing the culture medium, the cells were washed with DPBS diethyl-aminoethyl Dextran (PBS DEAE). Two T225 were then inoculated with DMEM + 2% fcs + p/s containing an amount of BCoV with a MOI of 0,01 and one T225 was inoculated with just DMEM + 2% fsc + p/s (mock).
The three flasks were incubated for one hour at 37 °C, 5% CO
2
, washed with DPBS with Ca 2+ and Mg 2+ and fresh medium, again DMEM + 2% + p/s, was added to the flasks. The flasks were incubated until cytopathogenic effect (cpe) was visible.
After five days, the supernatant was removed and stored in volumes of 0,5 ml at -80 °C.
3

The seroprevalence of the canine respiratory coronavirus in the Netherlands
Selina Norbart
Recombinant HE protein
The BCoV-HE + protein that was used during this study, was kindly provided by Raoul de
Groot and Arno van Vliet. The CRCoV-HE 0 protein was made during this study via transformation, purification and transfection.
Transformation
Day 1
The plasmid pCD5-CRCoV-HE 0 -Tx-cIg was diluted to 1 ng/μl and an E-coli suspension
(MC10611 P3) was added to this plasmid mix in an eppendorf tube. The eppendorf tube was put on ice for 20 minutes and after that it was incubated for 1,5 min at 42 °C in a heatblock
(heatshock). After the heatshock, the eppendorf tube was put back on ice again. 1 ml warm
Lysogeny Broth (LB) medium was added to the eppendorf tube and the tube was incubated for one hour at 37 °C.
One LB-agar/amp/tet plate was incubated to RT and 100 μl of the mix in the eppendorf tube was divided over the plate with a little L-shaped stick. The plate was incubated o/n at 37 °C.
Day 2
The plate was incubated at 4 °C for storage. Two colonies were picked and each one was resuspended in LB/amp/tet medium in an Erlenmeyer flask. The two flasks were incubated
20-22 hours at 37 °C while shaking.
Plasmid purification
The plasmid was purified with the QIAGEN® plasmid purification Maxi Kit according to the manufacturer’s instructions (QIAGEN Benelux B.V.).
Transfection
Day 0
Ten T225 flasks were filled with 1 x 10 7 HEK 293T cells and 50 ml DMEM + 10% FCS + p/s each and incubated at 37 °C, 5% CO 2 .
Day 1
10 ml out of each T225 was aspirated. The plasmid, DMEM0 and Polyethylenimine (PEI) were mixed and incubated at RT for 30 minutes. The mix was added dropwise to each T225 flask and the flasks are incubated o/n.
Day 2
The medium was totally aspirated and replaced by 40 ml expression medium. The medium was mixed for 15 minutes and sterilized through a 45 μm filter unit.
Day 7
The medium was collected in 50 ml tubes and the tubes were centrifuged for 10 minutes at
1000 rpm (or 5 minutes at 4500 rpm). The supernatant was put in new 50 ml tubes and put on ice.
Protein-A Sepharose beads were washed three times using PBS0 and then suspended in PBS
(50% V/V). The suspension was divided over the tubes with supernatant and the tubes were incubated o/n rotating at 4 °C.
4

The seroprevalence of the canine respiratory coronavirus in the Netherlands
Selina Norbart
Day 8
The tubes were centrifuged for 3 minutes at 2000 rpm and the medium was aspirated. The beads were resuspended in and washed 3 times with cold Thrombin Cleaving Buffer (TCB).
The beads were resuspended as a 50% suspension in TCB. Thrombin was added and the tubes with beads, TCB and Thrombin were incubated o/n rotating at RT.
Day 9
The tubes were centrifuged for 2 minutes at 3000 rpm and the supernatant was put in 12 ml tubes on ice. The beads were washed in TCB, the tubes were centrifuged again and the supernatant was added to the supernatant first taken.
Benzamidine Sepharose beads were washed 3 times in TCB and suspended in TCB (50% V/V).
This suspension was added to the thrombin cleaved CRCoV-HE 0 and the tubes were incubated for 1 hour rotating at 4 °C. Again the tubes were centrifuged and the supernatant was used for concentration using Pierce Concentrator 20K. CRCoV-HE 0 was concentrated at
4000 rpm at 4 °C.
Between these steps, the concentration of protein present in the supernatant was measured a couple of times and at one point, no protein was measured anymore. The most likely cause for this phenomenon was that no thrombin cleavage site was present. In this case, the protein was still attached to the Protein-A Sepharose beads, so the next couple of steps were made to elute the protein from the beads.
0,1 M Citric Acid was added the tubes still containing the Protein-A Sepharose beads and the total was incubated for 3 minutes at RT
1 M TrisHCl was added to the tubes and after mixing the tubes were centrifuged and the supernatant was taken and measured again.
The supernatant was stored in small amounts of 100 μl at -20 °C
Gel electrophoresis
To confine the presence of the CRCoV-HE in the supernatant, a gel electrophoresis was performed. A 12,5% running gel with a total volume of 10 ml and a stacking gel with a total volume of 4,5 ml were used.
The first time a gel electrophoresis was performed was because no protein was measured anymore at one point of the purifying of the CRCoV-HE 0 . It was necessary to determine if the
CRCoV-HE 0 was present somewhere in de different samples. The next samples were running through the gel for 1,5 hour:
Thrombin
Ladder
What was bound to the Protein-A Sepharose beads
Unconcentrated supernatant after using Benzamidine Sepharose beads
Concentrated supernatant after using Benzamidine Sepharose beads
What was bound to the Benzamidine Sepharose beads
The second time a gel electrophoresis was performed was to determine if (only) one or more proteins were present in the stored supernatant. Only two samples were running through the gel for 1,5 hour:
Ladder
Supernatant
After running, the gel was washed 3 times 5 minutes with MilliQ while shaking. Coomassie
Brilliant Blue was added to the gel and the gel was incubated for 1 hour at RT while shaking.
The gel was washed again every 30 minutes while shaking. At the end of the day fresh MilliQ was added to the gel and the gel was left shaking o/n. The next day the gel was dried.
5

The seroprevalence of the canine respiratory coronavirus in the Netherlands
Selina Norbart
Determination of TCID
50
Harvested virus
Before starting the virus neutralization tests, it was necessary to perform a titration of the harvested BCoV on HRT-18 cells and to calculate the TCID
50
.
On day one, six 96-wells flat-bottomed plates were coated with HRT-18 cells. The same method was used as when passaging the cells, but when the cells were loosened by trypsin, they were resuspended in 50 ml DMEM + 10% fcs + p/s. A counting chamber was used to calculate the average amount of cells present. This should be 1 x 10 5 cells/ml.
Each well of a 96-wells plate was filled with 100 μl of the resuspended cells and incubated overnight.
On day two 10-fold serial dilutions (10 -1 to 10 -12 ) of the harvested virus were made in DMEM
+ 2% fcs + p/s. The dilutions were made in a micronic block and the dilutions were added in eightfold to the plate containing the HRT-18 cells (eight rows of the same dilution, 100 μl in each well) after it was washed with PBS DEAE Dextran. The last three wells of the twelfth column of the coated plate were filled with the negative control DMEM + 2% + p/s.
The plate was incubated at 37 °C, 5% CO
2
and CPE was checked and scored after five and six days.
The TCID
50
/ml was calculated with the help of the Spearmann-Kärber formula:
Log ID
50
/ Volume = x
0
– d/2 + d/n * x
1 x
0
= the negative logarithm of the highest dilution of which all wells were positive d = the dose distance in logarithms (usually log10 = 1) n = the number of wells per dilution x
1
= the sum of all positive wells from (and inclusively) the dilution at x
0
The TCID
50 of the made virus stock was 10 6,5 TCID
50
/ml.
Virus Neutralization
HRT-18 cells
The day before performing VN-tests, 96-wells flat-bottomed plates were coated with 1 x 10 5 cells/ml as previously described and incubated overnight at 37 °C, 5% CO
2
.
Sera
A total of 208 dog sera and a pathogen free dog serum (SPF) were used in the VN tests.
Prior to use the sera were inactivated at a temperature of 56 °C for 30 minutes.
Three-fold serial dilutions of the sera were made in micronic blocks in DMEM + 2% fcs + p/s
+ gentamycine, starting at dilution 1:5, and every serum was put three times on a 96-wells plate. This means that a total of four sera could be put on one plate. Every time a virus neutralization test was started, a positive control (H043), a SPF serum, a TCID
50
control and cell controls were present.
The first dilution was made with 240 μl DMEM + 2% fcs + p/s + gentamycine and 60 μl serum. The second dilutions was made of 200 μl DMEM + 2% fcs + p/s + gentamycine and
100 μl of the first dilution, etc. After making the last dilution, 100 μl out of that dilution was discarded so every micronic block tube contained an equal volume of DMEM + serum.
6

The seroprevalence of the canine respiratory coronavirus in the Netherlands
Selina Norbart
Virus
A total of 200 μl of 100TCID
50
/50 μl was added to the 200 μl of serum dilution in each micronic block tube. After the virus was added, the micronic blocks were incubated for one hour at 37 °C, 5% CO
2
.
96-wells plates with HRT-18 cells
After an hour the 96-wells plates containing HRT-18 cells were taken from the incubator and washed with PBS DEAE Dextran. The dilutions were then added to the cells (according to layout at table 1 , 100 μl per well) and the plates were incubated at 37 °C, 5% CO
2
again.
After five and six days the CPE was checked and scored.
Table 1. Plate layout VN tests
1:15
1:45
1:135
1:405
1:1215
1:3645
1:10935
1:32805
Cell controls
Ipox
Ipox was performed to detect viral antigen on some fixated plates, just to double check if the visually scored CPE was correct and sometimes because of the toxicity of some sera which made it difficult to score the CPE visually.
Fixation of HRT-18 cells
Six days after the start of the VN tests or a titration, the cells were fixated prior to performing Ipox. The supernatant was stored in new 96-wells plates at -20 °C and the cells were washed with PBS + Ca 2+ and Mg 2+ . Methanol/acidic acid was added to the wells and the plates were incubated at -20 °C for ten minutes. The cells were then washed with PBS0 and stored at 4 °C with some PBS0 still on the cells to prevent the cells from drying.
The stored supernatant was used in a pNPA assay.
Ipox on 96-wells plates used for titration
The blockbuffer (BB) used during ipox was PBS0/0,05%Tween-20/5%NormalGoatSerum
(NGS). Ipox consists of many incubation steps and these steps are mentioned here:
The cells were washed three times with PBS0, BB/0,02%H2O2 was added to each well and the plates were incubated at room temperature (RT) for five minutes.
7

The seroprevalence of the canine respiratory coronavirus in the Netherlands
Selina Norbart
The cells were washed three times for five minutes with PBS0, BB was added to each well and the plates were incubated at RT for fifteen minutes.
The BB was removed, BB with cow127-αBCoV serum (1:150) was added to the first six rows of the plates and to two of the three mock wells, BB with fcs (1:150) was added (as a negative control) to the last two rows of the plates and to the last mock well and the plates were incubated at RT for 45 minutes. See table 2 for the plate layout.
The cells were washed three times for five minutes with PBS0/0,05%Tween-20, BB with Rabbit-αCow-HRPO (1:200) was added to each well and the plates were incubated at RT for 45 minutes.
The cells were washed three times for five minutes with PBS0/0,05%Tween-20, two times short with PBS0, AEC-substrate was added (using gloves!) to each well and the plates were incubated at RT for 15-30 minutes.
The cells were washed three times with PBS0, PBS0 was added to each well to prevent the cells from drying, staining of the cells was checked and CPE scored and the plates were stored at 4 °C.
Table 2. Plate layout ipox on plates used for titration
1
10 -1 10 -2 10 -3 10 -4 10 -5 10 -6 10 -7 10 -8 10 -9 10 -10 10 -11 10 -12
2
3
4
5
6
7
8
BB with cow127-αBCoV serum
BB with fcs
Mock
Ipox on 96-wells plates used in virus neutralization tests
The same procedure as described above was used, but when BB with cow127-αBCoV serum of BB with fcs was added to the wells, another plate layout was used. See table 3 for this plate layout.
8

The seroprevalence of the canine respiratory coronavirus in the Netherlands
Selina Norbart
Table 3. Plate layout ipox on plates used in VN tests
1:15
1:45
1:135
1:405
1:1215
1:3645
1:10935
1:32805
Cell controls
BB with cow127-αBCoV serum
BB with fcs
Enzyme-linked immunosorbent assay (ELISA)
High binding flat-bottomed 96-wells plates were coated with antigens at a concentration of
1,5 μg/ml in PBS0 and incubated overnight at 4 °C.
BCoV-HE + antigens
An amount of BCoV-HE + at a concentration of 2 mg/ml was kindly provided by Raoul de
Groot and Arno van Vliet. These antigens were also derived from the BCoV strain Mebus.
CRCoV-HE 0 (S 40 A)-cIg antigens
The first plate coated with these antigens was coated with three different dilutions of this antigen to determine the dilution at which the results were optimal. The concentration of
0,79 mg/ml was diluted to 7,5 μg/ml, 1,5 μg/ml and 0,3 μg/ml. Based on the results of the different antigen concentrations, next plates were coated with a concentration of 1,5 μg/ml.
Sera
The blockbuffer (BB) used in these experiments was PBS0/1%BSA (bovine albumin serum).
This BB was added to each well and incubated at 37 °C, 5% CO
2
for 45 minutes.
In the meanwhile, three-fold serum dilutions were made in BB. ELISA using BCoV-HE + was performed with 75 different sera. Sera that were tested negative in the neutralization tests were diluted from 1:15 to 1:405 and sera that were tested positive in the neutralization tests were diluted from 1:15 to 1:32805. This was done because of the small amount of BCoV-HE + available; more sera could be tested on one plate in this way. ELISA using CRCoV-HE 0 was performed on 11 different sera that were also tested on BCoV-HE + . The plates were washed three times with PBS0 and the serum dilutions were added to the plates. See table 4 for the plate layout of the plates coated with CRCoV-HE 0 and table 5 for the plate layout of most of the plates coated with BCoV-HE + . Two plates coated with BCoV-HE + had the same plate layout as the plates coated with CRCoV-HE 0 . The plates were incubated for one hour at 37
°C, 5% CO
2
.
9

The seroprevalence of the canine respiratory coronavirus in the Netherlands
Selina Norbart
Table 4. Layout of the plates coated with CRCoV-HE 0 .
Serum 1 Serum 2 Serum 3
1:15
1:45
1:135
1:135
1:405
Serum 2
1:15
1:45
1:135
1:405
1:1215
1:3645
1:10935
1:32805
Table 5. Layout of the plates coated with BCoV-HE + .
Serum 1 Serum 3 Serum 5
1:15
1:45
Serum 4 Serum 6
Serum 4
1:15
Serum 7
1:45
1:135
1:405
1:1215
1:3645
1:10935
Serum 5
Serum 8
Serum 6
Serum 9
1:405 1:32805
Conjugate
An ELISA was performed on two different sera (one SPF and one positive serum) to determine the optimal dilution of the conjugate that was going to be used. Three dilutions of the conjugate goat-αdog IgG HRPO were used in this ELISA: 1:2000, 1:4000 and 1:8000. Also the known optimal dilution of (previously in ELISA used) rabbit-αdog was used on both the
SPF and the positive serum as a control. Too little of this rabbit-αdog was available to use in this study.
After washing the plates three times with PBS0/0,0,5%Tween-20 and three times with PBS0, the conjugate was added to the wells and the plates were incubated again for one hour at 37
°C, 5% CO
2
.
Substrate
Tetramethylbenzidine was used as the substrate in all of the ELISA. After washing the plates three times with PBS0/0,05%Tween-20 and three times with PBS0 again, the substrate was added to the plates. The reaction was stopped when the blue color was clearly visible with
2M H
2
SO
4
. The optical density (OD) was measured at 450 nm.
The dilution of the conjugate of 1:4000 seemed to be the optimal dilution, so in all of the performed ELISA this dilution was used.
10

The seroprevalence of the canine respiratory coronavirus in the Netherlands
Selina Norbart
P-nitrophenyl acetate (pNPA) assay
Six days after starting VN tests, the cells in the 96-wells plates were fixated and the supernatant was stored at -20 °C. This supernatant was used in a pNPA assay to determine the enzymatic activity in the supernatant (HE-protein). The more enzymatic activity the supernatant shows, the more virus is present and so replication of the virus can be measured quantitatively. To read the plates, an ELISA reader was used. On the computer
(that was in contact with the ELISA reader) the program GEN5 was opened. The protocol
‘pNPA assay’ was selected and the plate layout, time and intervals of reading was set
(reading the OD every minute for one hour).
A 96-wells plate containing the supernatant of one of the plates on which titration was performed was taken out of the -20 °C freezer. 5 μl out of (almost) each well was put into a new 96-wells plate (maintaining the same plate layout, see table 2 ). The wells A12, B12 and
C12 contained 5 μl fresh DMEM + 10% FCS + p/s as a control instead of the supernatant. 100
μl 100 mM pNPA was added to 10 ml PBS0 and mixed well. 95 μl of this mix was added to each well and the plate was put in the ELISA reader immediately and the reading was started. The OD was measured at 405 nm.
11

The seroprevalence of the canine respiratory coronavirus in the Netherlands
Selina Norbart
Virus Neutralization tests
A total of 208 sera were tested and the CRCoV titers ranged from < 5 (negative) to 405. Of these sera, 46 (present on 12 plates earlier used in VN tests) were also tested with Ipox. Of seven tested sera, the visually scored CPE was only slightly different from the CPE scored after Ipox, indicating that the visual scoring of CPE works for BCoV infected HRT-18 cells.
As a second control, a pNPA assay was performed, but this assay did not gave good results.
The OD of the supernatant was only slightly higher than the OD of the negative control
(uninfected supernatant). Besides that, something in the sera and even in freshly added medium (A12, B12 and C12 on the titration plates) was also reacting in the assay, resulting in the same or even a higher OD than the OD of the supernatant containing the virus that should be measured (its enzymatic activity).
For example, the supernatant containing a serum that showed a high titer in both the VN tests and the ELISA tests, showed a very high OD in the pNPA assay in the first couple of dilutions. This should not be the case, because the assay should measure active virus and the virus was captured by antibodies (the virus was made inactive) in these first couple of dilutions, concluding that there are other reacting factors present in the supernatant. The pNPA assay was therefore not further used for screening of the supernatant.
Table 6 gives an overview of how many sera showed which titers.
Table 6. Overview VN tests.
<5 (negative) 55
5
15
15
26
45
135
52
51
26,44%
7,21%
12,50%
25,00%
24,52%
405
1215
3645
9
-
-
4,33%
-
-
10935
-
-
The most frequent found titers were 1:45 and 1:135. In Japan the most frequent found titer using VN tests was 1:160 8 , so neutralization titers of CRCoV seem to be low in general.
The percentage of negative sera was found to be (as is seen in the table) 26,44% and the seroprevalence of infection with the CRCoV in the Netherlands was found to be 73,56%.
The next table and graphic ( table 7 and graphic 1 ) show the seroprevalence of infection with CRCoV in its relation to the ages of the dogs.
12

The seroprevalence of the canine respiratory coronavirus in the Netherlands
Selina Norbart
6
7
8
9
10
11
12
13
14
Table 7. Seroprevalence of infection in its relation to the ages
Age
Percentage of
CRCoV positive dogs
Number of
CRCoV positive dogs
0
1
33,33%
72,22%
27
37
2
3
4
5
50,00%
50,00%
77,78%
100%
20
8
18
10
100%
100%
100%
78,57%
100%
77,78%
92,86%
100%
100%
9
18
7
14
7
9
14
3
2
Graphic 1. Seroprevalence of infection in its relations to the ages
100,00%
90,00%
80,00%
70,00%
60,00%
50,00%
40,00%
30,00%
20,00%
10,00%
0,00%
0 2 4
CRCoV positive dogs
6 8
Age in years
10 12
15 50% 2
The highest seroprevalences of infection with CRCoV can be seen among older dogs (5-8, 10,
12-14 years). This was also the case in other studies; the highest seroprevalences measured in North America, the UK/ROI and southern Italy lay around the ages of 7-9, 11-12 and 9 years respectively 5, 7 .
Ipox
A total of 46 sera (on 12 plates earlier used in VN tests) and two plates on which titration was performed were tested with Ipox. Of 39 of these sera the results of the visually scored
CPE coincided with the staining of the cells in an Ipox. Of seven tested sera, the visually scored CPE was only slightly different from the CPE scored after Ipox, indicating that the visual scoring of CPE works for BCoV infected HRT-18 cells.
ELISA
BCoV-HE + ELISA
A total of 75 sera, previously tested in VN tests, were also tested in a BCoV-HE + ELISA. Wells were considered positive if the OD was higher than two times the OD of the negative (SPF) serum. Every time ELISA was performed, a SPF serum was also present on one of the 96wells plates. The CRCoV titers ranged from 1:15 to 1:10935, so the titers found in BCoV-HE +
ELISA turned out to be much higher than in the VN tests. Despite this fact, a good correlation could be seen between the titers found in VN tests and the titers found in BCoV-HE + ELISA
( graphic 2 ).
14 16
13

The seroprevalence of the canine respiratory coronavirus in the Netherlands
Selina Norbart
Graphic 2. VN titers against ELISA titers
The sera that were tested negative in the VN tests were most of the time also tested negative in the BCoV-HE + ELISA. Most of the sera that showed high neutralization titers, also showed higher BCOV-HE + ELISA titers. This is shown again in the next table ( table 8 ).
There are some exceptions, like the serum (colored pink in the table) that was tested negative (< 5) in the VN test and showed a high titer in the ELISA (1215). This serum was tested twice and both times the same results were measured. This can be due to the fact that the VN test measures other antibodies (neutralizing antibodies) than the ELISA does; probably this dog did not have many neutralizing antibodies.
Table 8. VN titers against ELISA titers
< 5
5
15
45
135
405
1215
3624
10935
ELISA titers (BCoV-HE)
<15 15 45 135 405
34 5 2 - 2
4 - 1
1 - -
-
-
1
4
- - -
- - -
- - -
- - -
- - -
- - -
1
-
-
-
-
-
-
-
2
-
1
-
1215
1
-
1
2
2
1
-
-
-
3645
-
-
-
1
6
1
-
-
-
10935
-
-
-
2
-
-
-
-
-
32805
-
-
-
-
-
-
-
-
-
CRCoV-HE 0 (S 40 A)-cIg
A total of 11 sera that were previously tested in VN tests ánd in BCoV-HE + ELISA were also tested in CRCoV-HE 0 ELISA. This CRCoV-HE 0 protein lost its enzymatic activity, because of a change in amino acid sequence: a serine was replaced by an alanine on place 40. Because no thrombin cleavage site turned out to be present in the plasmid pCD5-CRCoV-HE 0 -‘Tx’-cIg,
14

The seroprevalence of the canine respiratory coronavirus in the Netherlands
Selina Norbart the CRCoV-HE 0 protein could not be cleaved and was still attached to the Protein-A
Sepharose beads (determined with gel electrophorese). With Citric acid the protein was released, but it still contained the cIg part. To make sure only one protein was present, gel electrophorese was performed again on the stored protein that was released from the
Protein-A Sepharose beads. Just one protein was present, but the cIg part was expected to give cross reactions during ELISA. In table 9 the results of the 11 sera that were tested in all 3 assays are put together.
Table 9. Overview of 11 sera
< 5
15
45
45
< 5
< 5
< 5
< 5
135
405
< 15
< 15
< 15
< 15
15
405
405
1215
1215
405
< 135
< 135
< 135
< 135
< 135
135
405
1215
1215
405
405 3645 3645
As can be seen in table 9 , there are almost no differences between the titers found in BCoV-
HE + ELISA and CRCoV-HE 0 ELISA. The only problem that was encountered using the CRCoV-
HE 0 protein was the much higher background that was present in the first two dilutions (1:15 and 1:45). Again, wells were considered positive when the OD was two times higher than the
OD of the negative (SPF) serum. In this case however, the OD of the first two dilutions of the
SPF serum were already so very high, that every other well containing serum in these same dilutions (1:15 and 1:45) should be all called negative. In this way the sera that were tested positive in VN tests and in the BCoV-HE + ELISA were also positive in the CRCoV-HE 0 ELISA, but this could only be seen in the higher dilutions (starting at 1:135). An example of these results can be seen in table 10 .
15

The seroprevalence of the canine respiratory coronavirus in the Netherlands
Selina Norbart
Table 10. Averages of the OD’s of in duplicate tested sera (CRCoV-HE 0 ELISA)
CRCoV SPF-L H127 H138 H011 H142 H144
1:15 1,401 1,3435 1,317 1,915 1,872 1,8525
1:45
1:135
1,0095
0,626
0,8635
0,504
0,812
0,466
1:405 0,401 0,335 0,322
1:1215 0,29 0,2695 0,259
1:3645 0,2575 0,2415 0,233
1:10935 0,254 0,2345 0,233
1:32805 0,1965 0,1895 0,1985
1,842
1,7825
1,573
1,102
0,6025
0,3685
0,233
1,777
1,5465
1,153
0,6465
0,365
0,456
0,255
1,7465
1,549
1,1155
0,6585
0,3845
0,278
0,1985
16

The seroprevalence of the canine respiratory coronavirus in the Netherlands
Selina Norbart
A very high seroprevalence of infections with CRCoV was found in the Netherlands (73,56%) compared to other countries. Most of the seroprevalences in other countries were studied around the year 2006/2007 5-8 and the sera that were tested in this study were already collected in 2005, so the high seroprevalence is not due to the difference in times of sample taking.
It is expected that the seroprevalence is lower among younger dogs, because they did spent less time in close proximity to other dogs compared to older dogs. When more old than young dogs were tested, a higher seroprevalence was expected, but this was not the case; the highest number of dogs tested were dogs in their first, second and third year of life (see table 7 ). In another study 5 , less dogs of these ages were tested compared to the number of older dogs and still the found seroprevalences were lower than in the Netherlands.
The population density in the Netherlands is pretty high, so dogs are in close contact with each other all the time. This is probably the best explanation for the much higher seroprevalence in the Netherlands compared to other countries.
The titers found in the VN tests are all low compared to the titers found in the BCoV-HE +
ELISA. Different antibodies are measured in these two tests though, as previously mentioned. Despite this fact a good correlation can be seen between the VN titers and the
ELISA titers; most of the sera that showed low VN titers, also showed low ELISA titers and when the VN titers became higher, there was also an increase in ELISA titers. Using this
ELISA, still 10 out of 44 sera that were tested negative in the VN tests were found to be positive in the ELISA, although with low titers. Despite this fact the BCoV-HE + ELISA might be used for large scale sero-epidemiological studies.
The used CRCoV-HE 0 protein, still containing the cIg part, was not the optimal protein to use in an ELISA. Although the results seem to be the same as in BCoV-HE+ ELISA, sera with only low titers will not turn out to be positive in this ELISA, because of the high background. This background seem to be due to the cIg part, but a CRCoV-HE 0 protein not containing that part should be tested to see if that is the true cause. Therefore a plasmid truly containing an thrombin cleavage site should be created. This CRCoV-HE 0 ELISA is not yet ready to be used in large scale sero-epidemiological studies.
17

The seroprevalence of the canine respiratory coronavirus in the Netherlands
Selina Norbart
References
1. Erles, K., Toomey, C., Brooks, H. W. & Brownlie, J. Detection of a group
2 coronavirus in dogs with canine infectious respiratory disease. Virology
310, 216-223 (2003).
2. Lai, M. M. Coronavirus: organization, replication and expression of genome. Annu. Rev. Microbiol. 44, 303-333 (1990).
3. Decaro, N. & Buonavoglia, C. An update on canine coronaviruses: viral evolution and pathobiology. Vet. Microbiol. 132, 221-234 (2008).
4. Buonavoglia, C. & Martella, V. Canine respiratory viruses. Vet. Res. 38,
355-373 (2007).
5. Priestnall, S. L., Brownlie, J., Dubovi, E. J. & Erles, K. Serological prevalence of canine respiratory coronavirus. Vet. Microbiol. 115, 43-53
(2006).
6. Decaro, N. et al. Serological and molecular evidence that canine respiratory coronavirus is circulating in Italy. Vet. Microbiol. 121, 225-
230 (2007).
7. Priestnall, S. L., Pratelli, A., Brownlie, J. & Erles, K. Serological prevalence of canine respiratory coronavirus in southern Italy and epidemiological relationship with canine enteric coronavirus. J. Vet.
Diagn. Invest. 19, 176-180 (2007).
8. Kaneshima, T. et al. The prevalence of a group 2 coronavirus in dogs in
Japan. J. Vet. Med. Sci. 68, 21-25 (2006).
9. Knesl, O., Allan, F. J. & Shields, S. The seroprevalence of canine respiratory coronavirus and canine influenza virus in dogs in New
Zealand. New Zealand Vet. J. 57, 295-298 (2009).
18