Calicivirus Felino PDF

Preview

Summary

Download Calicivirus Felino PDF

Description

Properties of a calicivirus isolated from cats dying in an agitated state Y. Sato, K. Ohe, M. Fukuyama, K. Furuhata, S. Kishikawa, S. Sakai, A. Kiuchi, M. Hara, T. Watanabe, Y. Ishikawa, A. Taneno In June 1993, two of five pet cats kept in Yokohama city in Japan suddenly became agitated and died. Feline calicivirus (FCV) was isolated from them. One strain (FCV-S) was isolated from the spinal cord, lung and tonsil of cat 1, another (FCV-B) from the ileum, medulla oblongata and cervical spinal cord of cat 2, and a third (FCVSAKURA ) from the oral cavity of one of the three surviving cats which showed no clinical signs. These three strains were equally resistant to pH 3·0 and serologically similar to each other, but distinct from strain F9. A genetic analysis, using a 208 base pair fragment from region E of the capsid, showed that FCV-Ari had a 70·4 per cent nucleotide and 77·3 per cent amino acid homology and FCV-F9 had a 68·6 per cent nucleotide and 73·9 per cent amino acid homology with the three strains, indicating that these two strains were genetically distinct from the three new isolates. Unvaccinated cats and cats which had been vaccinated against FCV-F9 developed watery diarrhoea but did not become agitated after the administration of FCV-S. The FCV-S strain did not induce signs of excitability after it was administered orally to specific pathogen-free cats.

FELINE calicivirus (FCV) is a common viral pathogen of cats (Fastier 1957, Umehashi and others 2000) which can induce mild rhinitis, conjunctivitis, palatine and lingual ulcers and occasionally bronchopneumonia (Holzinger and Kahn 1970). Experimentally infected cats develop relatively mild transient signs (Povey and Hale 1974), frequently recover but occasionally die with interstitial pneumonia (Holzinger and Kahn 1970, Love 1975). Cats infected with some strains develop a systemic infection and viraemia and have a mortality rate of 30 per cent (Kahn and Gillespie 1971). Bittle and others (1961) showed that the mortality rate was low in cats infected with a calicivirus alone, but increased to 82 per cent in cats also infected with a panleukopenia virus. The source of infection is healthy cats that have recovered but continue to excrete the virus for long periods. In some cases virus is excreted from the oropharynx for more than two years (Povey and others 1973, Wardley 1976, Wardley and Povey 1977a, b). Acute or chronic FCV infections affect breeding colonies, animal shelters, boarding catteries, pet shops and households. In practice preventive vaccination is used, but rare cases of vaccinerelated disease have been reported in which some cats become lame after an initial vaccination; the virus isolated from these cases was serologically similar to the vaccine virus (Dawson and others 1993). In addition, DNA sequence analysis has suggested that the parenteral vaccine F9 may be the causative agent in some cases which developed signs of flu (Radford and others 1997). On the other hand, field strains that are serologically distinct from the F9 strain have been isolated from diseased cats (Hohdatsu and others 1999). The virus is usually associated with respiratory disease but it can also result in lameness (Pedersen and others 1983, Dawson and others 1994), chronic gastritis and gingivitis (Thompson and others 1984, Knowles and others 1990), abortion (Ellis 1981, Van Vuuren and others 1999) and urinary tract infections (Fabricant and others 1971, Hara and others 1976, Kruger and others 1996). It has been reported that a calicivirus of oceanic origin, San Miguel sea lion virus serotype 5 is a human pathogen which causes a vesicular disease of the hands and feet (Smith and others 1977, 1978, 1998), but there is no report that FCV causes human disease. Recently, a virulent strain of FCV, FCV-Ari, has been reported which caused a severe disease with a high mortality rate; the condition in cats was similar to the disease caused in rabbits by the calicivirus of rabbit haemorrhagic disease (Pedersen and others 2000). The disease appeared suddenly and disap-

peared quickly, but because FCV mutates rapidly, these outbreaks suggest that strains of higher pathogenicity might reappear. Infections with FCV induce various clinical signs and infected cats are commonly in a depressed or suppressed state, and have a poor appetite. However, an FCV infection inducing agitation has not been observed. This paper describes the isolation and properties of strains of FCV isolated from cats that died after becoming severely agitated, showing signs similar to the signs of Aujeszky’s disease. MATERIALS AND METHODS Outbreak of disease In June 1993, two of the five cats living in a household in Yokohama city, Japan, became irritable and agitated, running about the room and hypersalivating. The following day they were still hypersalivating, were dehydrated and had a fever (above 38·4°C) and a slightly swollen throat; they died the next day (Table 1). Cat 1 was a seven-year-old male Siamese, and cat 2 was a four-year-old male mongrel (Table 2). The visible mucous membranes of cat 1 were pale and its serum creatine phosphokinase (CPK) activity was 1004 U/litre (normal range 7 to 28 U/litre; Kawamura 1997). Cell cultures and isolation of virus Feline kidney (CRFK), monkey kidney (MA-104) and pig kidney (PK-15) cells were used for virus isolation (Crandell and others 1973, Hara and others 1987). The cells were cultured in Eagle’s minimum essential medium (EMEM ) supplemented with 5 per cent bovine fetal serum (Table 2). Small pieces of tissue were collected from the cervical spinal cord, cerebrum, medulla oblongata, tonsil, lungs, jejunum and ileum of the two affected cats, and conjunctival, oral and nasal swabs were collected from the three surviving cats that had showed no clinical signs; oropharyngeal swabs were not collected from the two cats that died. A 10 per cent (w/v) homogenate of each tissue sample was prepared in Earle’s balanced salt solution supplemented with 1000 µg of streptomycin, 1000 units of penicillin and 10 µg of fungizone/ml. The homogenate was centrifuged at 4500 g for 30 minutes and the supernatant was used to isolate virus. A sample of the supernatant (0·2 ml) was inoculated on to four freshly prepared cell cultures. The culture fluids were harvested three days after inoculation and all the samples were passaged twice more and then examined for The Veterinary Record, December 18/25, 2004

Veterinary Record (2004) 155, 800-805 Y. Sato, BVSc, K. Ohe, BVSc, M. Fukuyama, BVSc, PhD, K. Furuhata, BVSc, PhD, S. Kishikawa, BVSc, MSc, PhD, S. Sakai, BVSc, A. Kiuchi, BVSc, MSc, PhD, M. Hara, BVSc, PhD, Department of Microbiology, School of Veterinary Medicine, Azabu University, 1-17-71 Fuchinobe, Sagamihara-shi, Kanagawa-ken, 229-8501, Japan T. Watanabe, University of Agriculture, 1737 Funako, Atugi-shi, Kanagawa-ken, 243-0034, Japan Y. Ishikawa, BVSc, PhD, A. Taneno, BVSc, Central Research Laboratories, Intervet, 1103 Fukaya, Kasumigaura-cho, Niihari-gun, Ibaraki-ken, 300-0134, Japan

Veterinary Record: first published as 10.1136/vr.155.25.800 on 18 December 2004. Downloaded fromhttp://veterinaryrecord.bmj.com/on September 26, 2020 at Universidad Nacional Andres Bello Biblioteca Central. Protected by copyright.

Papers & Articles

TABLE 1: Course of disease in two cats infected with feline calicivirus Date

Course of disease

June 3

After a period of severe excitement, anorexia and depression, no vomiting and diarrhoea, no past disease Excessive salivation, no nasal secretion, dehydration, mucosal anaemia (cat 2), body temperature of over 38·4°C, mild laryngeal swelling Dehydration alleviated partially by transfusion, but both cats died from aspiration of vomit

June 4 June 5

changes by staining with a May-Giemsa solution. Portions of the tissues were placed in 10 per cent phosphate-buffered formalin for histological examinations. Paraffin sections of the fixed tissues were prepared and stained with haematoxylin and eosin. Viruses The FCVs isolated from the cervical spinal cord of cat 1, the medulla oblongata of cat 2 and the oral cavity of the unaffected cat 3 were designated FCV-S, FCV-B and FCV-SAKURA, respectively. All three viruses were cloned, by limiting dilution in CRFK cells, and their physicochemical and serological properties were examined. Genetic analyses were made of these three strains and of the previously isolated strains in the laboratory; FCV-2 (1989), FCV-11 (1990) and FCV-15 (1990) from cats with respiratory disease; FCV-43 (1994) from cats with respiratory disease and diarrhoea; FCV-6 (1990) from a clinically normal cat and the ATCC VR-782 (F9) strain (Bittle and Rubic 1975). For experimental infection, FCV-S from the third subculture was used without cloning. The 208 base pair (bp) nucleotide sequence of capsid region E of FCV strain F9 was obtained from Genebank (M86379) and the sequence of FCV-Ari from the publication by Pedersen and others (2000). Biochemical tests To determine the heat stability of the strains they were heated at 56°C for 50 minutes. The effects of various pH values and buffer anions on virus infectivity were tested by mixing 1 ml of virus stock in 9 ml of 0·1M citric acid-sodium citrate buffer adjusted to pH 3·0 or 0·1M phosphorous acid acid-sodium

TABLE 2: Isolation of strains of feline calicivirus ( FCV) from the tissues of the two cats which became agitated and the three cats which showed no clinical signs Cat

Specimen

1

Cervical spinal cord† Lungs Tonsil Cerebrum Small intestine Ileum Medullar oblongata‡ Cervical spinal cord Ocular Oral§ Nasal Ocular Nasal Ocular Nasal

2

3 4 5

Viral isolation* 4 4 4 0 0 3

Age Bodyweight Clinical (years) (kg) sign

Breed

Sex

Siamese

M

7

4·0

Death two Unknown days after becoming agitated

Mongrel

M

4

3·8

Death two days after becoming agitated

Yes

1 1 0 2 0 0 0 0 0

Vaccination

M

6

2·4

No sign

Yes

Chinchilla

M

2

2·6

No sign

No

No sign

Unknown

* Number of positive cultures of the four cultures tested † FCV-S ‡ FCV-B

Genetic analysis To prepare cDNA from the RNA extracted from the partially purified virus, 0·1 mM oligo(dT) was added, and the cDNA was synthesised by reaction with Molony murine leukaemia virus reverse transcriptase (M-MLV RT) (Gibco BRL ). The cDNA prepared from the isolates was amplified by reverse transcriptase PCR (RT-PCR) by using the primers of the sense 5 -TTCGGCCTTTTGTGTTCC-3 and the anti-sense 5 -TTGAGAAT TGAACACAT-3 to obtain a fragment of 671 nucleotides from positions 1087 to 1757 in the capsid region (B-F), by the method described by Seal (1994). The PCR-amplified DNA product was cloned by using TA cloning kits (Invitrogen) as described by Mead and others (1991). The sequences of the cloning plasmids were established by determining the plasmid nucleotides with an ABI autosequencer (Applied Biosystems) (Prober and others 1977, Smith and others 1986). The nucleotide and deduced amino acid sequences were determined and the homology comparisons and phylogenetic analyses were made by using Genetyx-MAC Version 10 and Genetyx-MAC/ATSQ3 software (Software Development). Immune serum Strain FCV-F9 was inoculated orally (20 ml at 107·0 TCID50/ml) into a specific pathogen-free (SPF) cat of about six months of age and five weeks later blood was collected; the serum collected constituted the polyclonal F9 antiserum. Antiserum to FCV-S was prepared in a similar manner. Neutralising test Virus (200 TCID50) and an equal volume of a serum dilution were incubated at room temperature for one hour before being inoculated on to cultured CRFK cells in 96-well microtitre plates. The presence or absence of a cytopathic effect (cpe) was judged 48 hours later, and the neutralising titre of the serum was determined on the basis of the results. Experimental infection of cats Six SPF cats were used, two unvaccinated and four vaccinated. The vaccinated cats had been given a single dose of F9 vaccine six months before they were challenged. The challenge strain was FCV-S and 40 ml (107·0 TCID50/ml) was administered orally. A second identical challenge was given three weeks after the first. White blood cells were counted with a PC 608 particle counter (Erma). RESULTS

Chinchilla

Unknown Unknown Unknown Unknown

phosphate buffer adjusted to pH 7·0. Infectivity titrations were carried out after incubation for three hours at room temperature. Bile sensitivity was tested by using deoxycholic acid sodium salt (DOC) (Nakarai Tesque); the virus was mixed with an equal volume of 0·2 per cent DOC in EMEM, incubated at 37°C for one hour and then titrated in cell cultures. The protease sensitivity of the strains was tested by using trypsin (1:250; Nakarai Tesque); the virus fluid was mixed with an equal volume of 1 per cent trypsin in EMEM, incubated at 37°C for one hour and then titrated.

Virus isolation No viruses were isolated on PK-15 and MA104 cells, but FCVs were isolated on CRFK cells; they were isolated from the cervical spinal cord (FCV-S), lung and tonsil of cat 1, from the ileum, medulla oblongata (FCV-B) and cervical spinal cord of cat 2, and from the oral cavity (FCV-SAKURA) of cat 3, one of the three surviving cats. Histopathological examination No marked inflammation, such as perivascular cell infiltration or chromatolysis, was observed in any of the specimens,

§ FCV-SAKURA

M Male

The Veterinary Record, December 18/25, 2004

Veterinary Record: first published as 10.1136/vr.155.25.800 on 18 December 2004. Downloaded fromhttp://veterinaryrecord.bmj.com/on September 26, 2020 at Universidad Nacional Andres Bello Biblioteca Central. Protected by copyright.

Papers & Articles

nor were there nuclear inclusion bodies in the nerve cells of the medulla oblongata, the cervical region of the spinal cord or the brain, unlike Aujeszky’s disease. No cytoplasmic inclusion bodies, like the Negri bodies characteristic of rabies, were observed. Physicochemical properties The sensitivities of the three isolates, of other field isolates and of F9 to heat, pH, treatment for an hour with 0·1 per cent bile acid, and treatment for three hours with 0·05 per cent trypsin were tested (Table 3). All the strains showed a marked decrease in their infectivity titre after heating. No difference between the effects of pH 3 and pH 7 on the isolates FCV-S, FCV-B and FCV-SAKURA was observed, and the diarrhoeacausing FCV-43 was also pH-resistant. The other strains were pH-sensitive, and only the diarrhoea-causing FCV-43 was resistant to bile acid.

TABLE 3: Physicochemical characteristics of the strains of feline calicivirus ( FCV) isolated from the five cats and of some field strains of the virus Strain

Heat stability (50°C) No heat 50 min

FCV-S FCV-B FCV-SAKURA FCV-2 FCV-11 FCV-15 FCV-43 F9

5·5* 6·5 6·5 5·75 5·0 4·5

0·75 0·5 1·0 2·0 0·5 0·5

NT

NT

6·0

3·0

pH stability 3·0 7·0 3·75 4·25 5·25 2·75 0·5 0·5 5·5 2·5

4·5 5·5 6·0 5·75 5·75 4·5 6·5 5·25

Bile sensitivity No bile Bile 6·0 6·5 6·5 6·0 5·5

1·75 2·0 1·75 3·25 1·5

Trypsin sensitivity No trypsin Trypsin

NT

NT

6·0 6·5 6·5 6·0 6·5 6·5

2·75 2·5 2·5 5·5 6·5 6·5

6·5 6·5

6·5 1·5

NT

NT

5·0

2·5

* Log TCID50/0·1 ml NT Not tested

DISCUSSION Serological comparisons The immune serum with a titre of 1:2560 of the F9-immunised cats developed titres of 1:160 to 1:224 against FCV-S, FCV-B and FCV-SAKURA (Table 4). The antiserum with a titre of 1:1280 of the FCV-S-immunised cats developed a titre of 1:2240 against FCV-B and FCV-SAKURA, and 1:1280 against FCV-S, suggesting that the three viruses belonged to the same strain. However, this antiserum had a relatively low titre of 1:620 against F9. Genetic analysis The PCR product of each isolate was inserted into a TA cloning vector. As shown in Fig 1, the resulting plasmid DNA (1245 to 1453) showed that the sequences of the strains FCV-S, FCV-B and FCV-SAKURA were the same. The 208 bp region E of FCV-S and FCV-Ari had only 70·4 per cent nucleotide homology and 77·3 per cent amino acid homology, and FCV-S and F9 had only 68·6 per cent nucleotide homology and 73·9 per cent amino acid homology, indicating that FCV-Ari and F9 were genetically distant from the three strains (Fig 1). Infection experiment FCV-S culture material (40 ml) was administered orally to each of two unvaccinated and four vaccinated cats.One to two days later all the cats developed watery diarrhoea, but recovered three to seven days later (Figs 2, 3, Table 5). The cats did not develop upper respiratory tract signs. They developed a mild leukocytosis and a slight fever, but did not become agitated. Control cats receiving a virus-free cell culture did not develop diarrhoea, and when the cats were re-inoculated with FCV-S in the same way three weeks later they did not have diarrhoea. The unvaccinated cats produced higher neutralising antibody titres to FCV-S (>1:20,480) after the second challenge with FCV-S than the cats previously vaccinated with F9.

The diseased cats died suddenly, showing signs of agitation and hypersalivation, like cats with Aujeszky’s disease (Hara and others 1987, 1991), but in the absence of significant pathological findings such as intranuclear inclusion bodies and inflammation, another underlying cause was suspected. However, no other disease that produces the same clinical signs has been reported. Since an outbreak of Aujeszky’s disease in dogs and cats was reported by Hara and others (1987, 1991) animal hospitals in Japan have paid more attention to diseases which cause agitation, although because in most cases the agitation is transient and the animals recover, no virological tests are made. Pedersen and others (2000) observed a slight increase in CPK in cats with a rabbit haemorrhagic fever-like disease, and suggested that it was probably due to myonecrosis. However, they did not report that the cats became agitated. A strain of FCV was isolated from the cervical spinal cord of the two cats that died. The virus of Aujeszky’s disease is considered to be transmitted from the tonsil (Narita and others 1984) to the spinal cord, medulla oblongata and trigeminal nerve (Rziha and others 1986), inducing agitation or itching in dogs and cats, depending on the site of the infection. It was therefore suspected that the FCV infecting the cervical spinal cord caused the agitation in these cats. The absence of significant pathological changes at this site is characteristic of the acute course of the disease, as in cases of Aujeszky’s disease (Hara and others 1987). On the other hand, a FCV has been isolated from the spinal cord (Povey and Hale 1974), cerebellum (Kahn and Gillespie 1971), and midbrain (Povey and others 1973) of non-agitated cats, suggesting that

Number of nucleotide 1245

TABLE 4: Neutralisation tests of the strains of feline calicivirus ( FCV) isolated from the five cats and of some field strains of the virus Immunised cat serum Strain FCV-S FCV-B FCV-SAKURA FCV-2 FCV-6 FCV-11 FCV-15 FCV-43 F9

* Reciprocal titre

F9

FCV-S

224* 160 160 640 1792 896 160 112 2560

1280 2240 2240 120 160 40 200 40 620

Veterinary Record: first published as 10.1136/vr.155.25.800 on 18 December 2004. Downloaded fromhttp://veterinaryrecord.bmj.com/on September 26, 2020 at Universidad Nacional Andres Bello Biblioteca Central. Protected by copyright.

Papers & A...