Abstract
A new inactivated and adjuvanted Chlamydophila felis vaccine was developed and its efficacy in cats was compared with that of commercially available inactivated and live vaccines. Two commercial vaccines conferred insufficient immunity on inoculated cats, as evaluated by antibody production and a challenge experiment, whereas cats administered the newly generated vaccine produced high-titre antibodies and acquired sufficient immunity. The cats immunised with the new vaccine revealed no or only mild clinical signs, and no chlamydiae were recovered from their tissue samples after exposure to a virulent C felis. However, they shed chlamydiae in their nasal and conjunctival secretions after challenge, as did those immunised with the commercial vaccines and the non-vaccinated controls. The newly developed vaccine caused no adverse reaction in the inoculated cats. These findings suggest that the new vaccine prepared here may be promising for practical use in controlling C felis infection in cats.
Chlamydophila felis (C felis) is an obligate intracellular microorganism that causes acute and chronic conjunctivitis and pneumonia in cats. 1 C felis was first isolated from the lung of a cat with naturally occurring pneumonia in the United States in 1942. 2 Since then, the presence of C felis infection in cats with ocular or upper respiratory signs has been reported in many countries worldwide, including Great Britain, 3 Australia, 4 Switzerland, 5 Germany, 6 Italy, 7 Sweden, 8 Canada, 9 New Zealand 10 and Japan. 11,12
The use of an effective C felis vaccine for cats would seem to be desirable and should be recommended to prevent infections in cat populations. Several inactivated and modified live vaccines have been developed and used to protect cats against C felis infection. However, only a few reports have addressed the efficacy of C felis vaccines in cats. 13–15
In this study, we generated a new inactivated and adjuvanted C felis vaccine and compared its efficacy in cats with that of commercially available inactivated and modified live vaccines.
Materials and Methods
Vaccines
The experimental vaccine evaluated here was produced using L cells, derived from mouse fibroblasts (provided by Gifu University, Japan) and the Fe/C-P8 strain of C felis, isolated from a cat with sneezing and conjunctivitis in Japan in 1999. 16 Briefly, L-cell cultures in suspension form were inoculated with the Fe/C-P8 strain and incubated at 37°C for 5 days. The infected L-cell cultures were freeze–thawed once and centrifuged (1000×g, 10 min, 4°C). The supernatants were recovered and centrifuged again (9000×g, 1 h, 4°C). The resulting pellets were resuspended in 1/100 volume phosphate-buffered saline (PBS), mixed with formalin at a final concentration of 0.1%, and incubated at 37°C for 2 days to inactivate the living chlamydiae. Then, the inactivated preparation and oil adjuvant were mixed at a ratio of 3:7 to prepare the final product. The oil adjuvant used was ISA-70 (Seppic SA, France). The amount of chlamydiae contained in the final vaccine product was estimated to be 108.2 ELD50 (50% embryo lethal dose)/dose by the yolk-sac inoculation test.
The commercial vaccines used for the comparison with our experimental vaccine were two feline quintuple (feline herpesvirus-1, feline calicivirus, feline panleukopenia virus, feline leukemia virus (FeLV) and C felis) combined vaccines licensed in the United States. One was an inactivated and adjuvanted vaccine, Fel-O-Vax Lv-K IV (Fort Dodge Laboratories, USA). The other was a modified live vaccine, Eclipse 4+FeLV (Intervet Schering–Plough Animal Health, USA). Unfortunately, no information about the amount of C felis antigen or the formulation of the adjuvant of the commercial inactivated vaccine was available. However, the latter commercial live vaccine contained 103.5 ELD50/dose of C felis, as determined by our yolk-sac inoculation test.
Experimental animals
Specific pathogen-free (SPF) cats, 3–4 months old, 12 males and 12 females, were used. The experiment was performed in accordance with the institutional guidelines for animal experimentation. The SPF status of the cats was verified, as described previously.17
Experimental designs
The cats were randomly divided into four groups: A, B, C and D. Groups A and D contained seven cats each, and groups B and C had five cats each. Cats in groups A, B, and C were inoculated subcutaneously twice (first over the right shoulder and then, 3 weeks later, over the left) with 1 ml of the vaccine developed in this study, Fel-O-Vax Lv-K IV, and Eclipse 4+FeLV, respectively. The remaining seven cats, group D, served as non-vaccinated controls and were given 1 ml of PBS twice instead of vaccine. All cats were subjected to daily palpation at the inoculation site and the surrounding area for 21 days after the first and the second vaccinations. At 21 days after the second vaccination, all cats, including the non-vaccinated controls, were challenge-exposed to 25 μl each of droplets containing 103.0 ELD50 of the virulent and egg-grown B166 strain of C felis, 18 via both nasal and ocular routes.
Clinical eye signs were observed daily and scored for 21 days after challenge, as described previously, 17 and the means of clinical eye scores in each group were analysed by the Mann–Whitney test. P-values<0.05 were deemed to indicate statistical significance. Rectal temperatures were also recorded daily for 21 days after challenge.
Conjunctival and nasal swabs were collected from all cats on post-challenge days (PCD) 1, 3, 5, 7, 14, and 21, and blood samples were also obtained on PCD 7, 14, and 21. These samples were tested for chlamydiae by the yolk-sac inoculation method, as described previously. 17 Serum samples were collected periodically from all cats and tested for anti-chlamydial antibodies by an indirect micro-immunofluorescence (MIF) test according to a method reported by Pudjiatmoko. 19 All cats were euthanased and autopsied on PCD 21, and selected tissues (lung, tonsil, liver, spleen, and kidney) were collected, homogenised to make 20% suspension with chlamydia transport medium, 20 and stored at −80°C. The homogenates were tested for chlamydiae by the yolk-sac inoculation method, as described previously.17
Results
Reaction of vaccinated cats
No cat exhibited any unexpected or untoward response generally, and no adverse local reaction at the injection site occurred after the first or second vaccination with the experimental or commercial vaccines.
Antibody responses
Antibody responses of the cats are shown in Table 1. Although a single inoculation with the experimental vaccine elicited low-titre anti-chlamydial antibodies in only 5/7 cats, they all produced antibodies, of titre 64–128, after the second inoculation. On the other hand, no anti-chlamydial antibodies were detected in cats after the first inoculation with Fel-O-Vax Lv-K IV, and low-titre antibodies were barely found in 4/5 cats after the second booster inoculation. Additionally, Eclipse 4+FeLV did not trigger the production of detectable anti-chlamydial antibodies, even after the second vaccination. Challenge exposure to the virulent C felis induced antibody production or led to an increase in antibody titres in cats inoculated with the commercial vaccines and in the non-vaccinated controls, whereas antibody titres in cats immunised with the experimental vaccine were unchanged after the challenge.
Table 1.
MIF antibody responses of cats after vaccination and challenge exposure to Chlamydophila felis.
| Group | Vaccine inoculated | MIF antibody | |||||||
|---|---|---|---|---|---|---|---|---|---|
| 1st Vaccination | 2nd Vaccination | Challenge | |||||||
| Pre* | 21† | 7 | 14 | 21 | 7‡ | 14 | 21 | ||
| A | New inactivated vaccine | <8.0§ | 7.2 | 29.0 | 95.1 | 115.9 | 156.0 | 156.0 | 156.0 |
| B | Fel-O-Vax Lv-K IV | <8.0 | <8.0 | 5.3 | 4.6 | 7.0 | 10.6 | 84.4 | 97.0 |
| C | Eclipse 4+FeLV | <8.0 | <8.0 | <8.0 | <8.0 | <8.0 | <8.0 | 6.1 | 12.1 |
| D | Non-vaccinated control | <8.0 | <8.0 | <8.0 | <8.0 | <8.0 | <8.0 | 4.9 | 14.5 |
*Pre-vaccination.
†Post-vaccination day.
‡PCD.
§Geometric means of antibody titres.
Clinical responses after challenge
As shown in Fig 1, non-vaccinated control cats showed severe febrile responses and developed conjunctivitis accompanied by conjunctival hyperemia, serous and mucopurulent ocular discharges, and swelling of the eyelids on PCD 5–7, which persisted for more than 10 days. Similarly, cats in groups B and C manifested severe ocular signs after challenge, although their febrile responses were milder than those of non-vaccinated controls.
Fig 1.
Mean clinical eye scores and rectal temperatures of cats after challenge exposure to C felis. Upper and lower columns show clinical eye scores and rectal temperature, respectively. Side axis indicates PCD, and left axis indicates clinical eye score (points) or rectal temperature (°C). Groups A, B, and C were inoculated with a new inactivated vaccine developed in this study, Fel-O-Vax Lv-K IV, and Eclipse 4+FeLV, respectively. Group D was the non-vaccinated control.
Clinical responses of cats in group A were milder than those of cats in groups B, C and D. Six of seven cats in group A exhibited only mild conjunctivitis on PCD 6–11, which persisted for several days, and the others developed no observable clinical signs after challenge. A significant difference in the mean clinical eye scores was found between groups A and B, A and C, and group A and the non-vaccinated group D (P<0.01). Although the difference in the mean clinical eye scores between groups B and D was statistically significant (P<0.01), no significant difference was found between groups C and D (P>0.05).
Chlamydial isolation
The results of chlamydial isolation from clinical samples are shown in Tables 2 and 3. Chlamydiae were recovered consistently from the conjunctival and nasal swabs of all cats, regardless of vaccination, between PCD 3 or 5 and the end of the experiment (PCD 21). No chlamydiae were recovered from the blood of any cat in the vaccinated groups A, B and C, whereas four cats in the non-vaccinated group D were positive for chlamydial isolation from blood on PCD 21 (Table 2). At autopsy, no chlamydiae were isolated from any tissue tested in group A, whereas the livers of six cats, the spleens of five cats, and the lungs, tonsils, and kidneys of four cats of the control group D were positive for chlamydiae. In group B, chlamydiae were detected in lungs and tonsils collected from two cats, and in the liver and spleen of one cat. Chlamydiae were also recovered from the tonsils and spleens of two cats, and from the lungs and liver of one cat in group C (Table 3).
Table 2.
Isolation of chlamydiae from conjunctival and nasal swabs and blood samples of cats challenged with Chlamydophila felis.
| Group | Vaccine inoculated | Conjunctival swab | Nasal swab | Blood | ||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| 1* | 3 | 5 | 7 | 14 | 21 | 1 | 3 | 5 | 7 | 14 | 21 | 7 | 14 | 21 | ||
| A | New inactivated vaccine | 0/7† | 4/7 | 7/7 | 7/7 | 7/7 | 7/7 | 0/7 | 2/7 | 7/7 | 7/7 | 7/7 | 7/7 | 0/7 | 0/7 | 0/7 |
| B | Fel-O-Vax Lv-K IV | 0/5 | 3/5 | 5/5 | 5/5 | 5/5 | 5/5 | 0/5 | 3/5 | 5/5 | 5/5 | 5/5 | 5/5 | 0/5 | 0/5 | 0/5 |
| C | Eclipse 4+FeLV | 0/5 | 3/5 | 5/5 | 5/5 | 5/5 | 5/5 | 0/5 | 3/5 | 5/5 | 5/5 | 5/5 | 5/5 | 0/5 | 0/5 | 0/5 |
| D | Non-vaccinated control | 0/7 | 7/7 | 7/7 | 7/7 | 7/7 | 7/7 | 0/7 | 6/7 | 7/7 | 7/7 | 7/7 | 7/7 | 0/7 | 0/7 | 4/7 |
*PCD.
†Number of cats positive for chlamydiae isolation/number of cats tested.
Table 3.
Isolation of chlamydiae from tissues of cats challenged with Chlamydophila felis.
| Group | Vaccine inoculated | Lung | Tonsil | Liver | Spleen | Kidney |
| A | New inactivated vaccine | 0/7* | 0/7 | 0/7 | 0/7 | 0/7 |
| B | Fel-O-Vax Lv-K IV | 2/5 | 2/5 | 1/5 | 1/5 | 0/5 |
| C | Eclipse 4+FeLV | 1/5 | 2/5 | 1/5 | 2/5 | 0/5 |
| D | Non-vaccinated control | 4/7 | 4/7 | 6/7 | 5/7 | 4/7 |
*Number of cats positive for chlamydiae isolation/number of cats tested.
Discussion
A new inactivated C felis vaccine was developed and its efficacy in cats was compared with commercially available vaccines. The results of this study seem to indicate that the new inactivated vaccine developed was more effective than the commercial vaccines investigated. Our experimental vaccine elicited higher titre antibody responses to C felis in inoculated cats, and their clinical manifestations after challenge exposure to the virulent C felis were relatively mild compared with those in non-vaccinated controls and cats vaccinated with the commercial vaccines.
Although cats in group A showed only mild conjunctivitis or developed no clinical signs, they consistently shed chlamydiae in their conjunctival and nasal swabs after challenge, as did those in groups B, C and D. This may suggest that the inactivated vaccine developed in this study has the potential to induce immunity, to reduce the severity of clinical disease, and to prevent systemic infection, but not to elicit local immunity to protect against infection in the ocular and nasal mucous membranes. The fact that no chlamydiae were recovered from the organs of cats of group A, unlike those in groups B, C and D, may support this suggestion.
The commercial inactivated and modified live vaccines evaluated in this study were less effective and scarcely induced antibodies to C felis in vaccinated cats. Additionally, cats immunised with the commercial modified live vaccine manifested severe ocular signs, as did the non-vaccinated controls, after challenge. Although reasons for the poor potency of the commercial vaccines are unknown, one possible explanation for the difference in effectiveness between the commercial inactivated vaccine and the vaccine generated in this study may be due to the amount of C felis antigen and the adjuvant formulation used in the respective vaccines. Shewen et al reported that an inactivated vaccine consisting of 107.5 ELD50/dose of chlamydiae and the L75 or FD19 adjuvant conferred partial immunity in inoculated cats. 14 Unfortunately, no information was available about the amount of chlamydial antigen or the formulation of the adjuvant in the commercial inactivated vaccine tested. Our formulation consisted of 108.2 ELD50/dose of chlamydiae and the effective adjuvant ISA-70; this may explain why the inactivated vaccine tested here induced good immunity in the cats. Further investigations on the amount of chlamydial antigen and the adjuvant formulation are expected to produce useful information to further improve the C felis vaccine. The commercially available modified live vaccine conferred insufficient protection, although it did contain 103.5 ELD50/dose of C felis. Why the commercial modified live vaccine induced little or no immunity remains unclear.
The ISA-70 adjuvant used in this study has been considered to be more effective than other adjuvants in guinea pigs, rats, chickens, and mice, 21–23 and is used in a commercial feline trivalent inactivated vaccine. 24 Vaccine-associated sarcoma in cats has been recognised as a potential consequence of vaccination for over a decade. 25,26 It has been estimated that more than 0.01% of vaccinated cats develop a tumour at the inoculation site of killed-adjuvanted vaccines in the United States. 27,28 Although palpation of the vaccine inoculation sites revealed no swelling or any other side effects in this study, it will be necessary to carry out longer term studies to assess the safety of the vaccine. Furthermore, it should be noted that oil-adjuvanted vaccines present a potential risk to operators; when hands or fingers are accidentally stabbed by a needle, the oil adjuvant may cause swelling and pain due to inflammation.
The conclusion of this study is that the experimental vaccine, consisting of a large amount of killed C felis antigen and the adjuvant ISA-70, was effective for reduction in disease severity when the vaccinated cats were experimentally infected.
Acknowledgements
We thank Dr H Fukushi of Gifu University, Japan, for providing us with the L cells and B166 strain of C felis used in this study.
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