Department of Veterinary ..... would become infected quite quickly, either through wounds or flea or tick bites. ... veterinary surgeons (18.6 per cent) and veterinary technicians. (8-9 per cent) ..... the box at the top right corner of the online article.
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Evidence of Bartonella henselae infection in cats and dogs in the United Kingdom A. BARNES, S. C. BELL, D. R. ISHERWOOD, M. BENNETT, S. D. CARTER Sera from cats and dogs in the UK were tested by ELISA for antibodies to Bartonelia henselae. Seropositivity confirmed in 28 of 69 pet cats (40-6 per cent), 33 of 79 feral cats (41-8 per cent) and three of 100 pet dogs. Reactivity to specific B henselae antigens was confirmed by Western blotting and demonstrated that consistent antigenic bands were bound by sera from the cats and dogs.
was
Veterinary Record (2000) 147, 673-677 A. Barnes, S. C. Bell, PhD, D. R. Isherwood, FIMLS, S. D. Carter, BSc, FIMLS, PhD, MRCPath, Department of Veterinary Clinical Science and
Animal Husbandry, M. Bennett, BVSc, PhD, MRCVS, Department of Veterinary Pathology, University of Liverpool,
Liverpool L69 3BX
Correspondence to Dr Carter
INFECTION with Bartonella species has potential clinical consequences in man. In the Bartonella genus there are 11 species, several of which were previously classified in the Rochalimaea and Grahamella genera. In terms of human health, one of the most important Bartonella species is Bartonella henselae, which has a broad host range including domesticated species. A link between cats infected with B henselae and human diseases has been identified (Regnery and others 1992, Schwartzman 1992, 1996, Caniza and others 1995, Chomel and others 1995, Yoshida and others 1996, Bergmans and others 1997). The two main human diseases caused by B henselae, cat-scratch disease and bacillary angiomatosis, are particularly important in immunocompromised individuals (Childs and others 1995, Flexman and others 1995, Hamilton and others 1995, Regnery and Tappero 1995, Drancourt and others 1996). As its name suggests, catscratch disease follows a cat scratch or bite, and it causes acute signs and symptoms. Lymphadenopathy, skin lesions, fever and vomiting are the commonest, but they can include encephalopathy, osteomyelitis, pulmonary disease and optic neuritis. It is usually a self-limiting disease and is often missed owing to the multiplicity of symptoms and because it is not often included in differential diagnoses. Bacillary angiomatosis also primarily affects immunocompromised patients, usually those infected with the human immunodeficiency virus, and it causes vascular lesions which may resemble Kaposi's sarcoma (Koehler and others 1988). Because children and immunocompromised individuals are particularly susceptible to these two diseases, their significance is increasing. The association with cat infections has led to studies of the prevalence of B henselae infection in populations of cats in many parts of the world (Table 1), but no data have been published about the infection in cats in the UK and thus their potential to transmit the diseases to man. Furthermore, there is little information about the route of transmission or of any possible detrimental effect of B henselae infection on cats themselves. Although B henselae infection has been reported in dogs, with the subsequent transmission of disease to man (Keret and others 1998, Tsukahara and others 1998, Kusaba and others 1999), there has been no survey to determine whether they are important reservoirs of the organism.
MATERIALS AND METHODS Serum samples taken from 69 cats and 100 dogs for diagnostic purposes, and from 79 feral cats in colonies throughout the UK were stored at -200C. The domestic cats were grouped into four categories. In addition to these cats, a colony of 10, 10- to 12-week-old specific pathogen-free (SPF) cats was selected as a negative control group. The domestic cats were divided into groups which had either known flea infections (four cats) or central nervous system disorders (CNS) (23 cats), or miscellaneous non-infec-
The Veterinary Record, December 9, 2000
tious conditions such as constipation, or had been referred for surgical procedures, for example, for urethral obstruction, spaying, or a defective palate (42 cats). Some of the cat sera had already been tested for antibodies to Borrelia burgdorferi and Leptospira species (May and others 1994) and for feline immunodeficiency virus (FIV) and feline leukaemia virus (FeLV). The cats' age and breed were recorded where known. The dog sera were selected from samples submitted for autoimmune screening. All the samples were tested for antibodies to B henselae by ELISA and Western blotting.
Antigen preparation B henselae was grown on blood-agar plates. The organisms were harvested from the plates with sterile phosphate buffered saline (PBS). The suspension was centrifuged at 1000 g and washed three times with PBS, resuspended in 2 per cent Nonidet P40 (Sigma) and stored at -20°C overnight. After thawing, the suspension was sonicated with a MSE (UK) sonicator by four, 10-second bursts, at maximum current, on ice. The suspension was then centrifuged at 10,000 g for 45 minutes at 20°C; the supernatant was retained and dialysed against PBS, with three changes, for 72 hours.
Immunoglobulin G (IgG) anti-B henselae ELISA A checkerboard range of dilutions was used to establish the correct dilutions of reagents. The ELISA was optimised and validated by using feline sera from the USA supplied by the Center for Disease Control (CDC), Atlanta, which were either positive or negative for B henselae antibodies. For the ELISA, 96-well microtitre plates (Dynex) were coated with the B henselae antigen preparation (1 ag/ml, 50 pl per well) in PBS and incubated in a humidified chamber for one hour at 37°C and then at 4°C overnight. The wells were then washed three times with PBS and 50 p1 of cat or dog serum diluted 1:100 in PBS/0-05 per cent Tween (PBS/T) were added to each well and incubated at 37°C in a humidified chamber for two hours and then at 4'C overnight. The wells were then washed three times with PBS/T and 50 pl of either monoclonal mouse anti-cat IgG (Clone KIM2-1A; Serotec) diluted 1:2000 in PBS/T or anti-dog IgG (alkaline phosphatase-conjugated; Sigma) diluted 1:2000 in PBS/T were added. The plates were again incubated in a humid chamber at 37°C for one hour and then at 4°C for 30 minutes. The cat serum plates were washed with PBS/T and the bound mouse anti-cat IgG antibodies were detected by the addition of alkaline phosphatase-conjugated goat anti-mouse IgG (1:20,000 dilution in PBS/T) for one hour at 37°C followed by an incubation at 4°C for 30 minutes. Both the cat and dog plates were washed three times with PBS/T and 50 p1 of substrate S-104 (Sigma) diluted in glycine buffer (pH 10.1) to 1 mg/ml were added to the wells. The absorbance was read at 405 nm on a Titertec Plus Multiscan (Life Sciences) and for the feline ELISA, expressed on a 0 to 9 linear matrix scale (a standard high positive cat serum was always set on 9 to obviate any interplate variation). OD405 data above this range were 673
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PAPERS & ARTICLES
20
Detection method Positive (%) Reference
19
Country
18 17
0
16
0
15
0
14
0
13
.rx
S
5-55 53 8-3 15-1 23 35 15 62.6 54
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0
Glaus and others (1997) Ueno and others (1995) Kelly and others (1996) Branley and others (1996) Haimerl and others (1999) Chomel and others (1999) Marston and others (1999)
Domestic Domestic Domestic
Domestic/feral Domestic Domestic Domestic/feral
incubated for one hour at room temperature with the previously described monoclonal antibody to cat IgG at 1:2000 in PBS/T. The dog membranes were incubated with horseradish peroxidase-conjugated anti-dog IgG at 1:2000 in PBS/T, again at room temperature. The cat blots were washed as before and then incubated at room temperature with horseradish peroxidase-conjugated anti-mouse IgG antiserum at 1:10,000 (Sigma). Both sets of blots were washed with PBS/T and the colour developed with 3,3'-diaminobenzidine (Sigma). The staining patterns on the blots were then scanned on to a computer, the images recorded, and the molecular weights estimated by interpolation from known molecular weight standards.
10 9
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mm
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RESULTS
0 SPF
Fleas
NI
CNS
Feral
FIG 1: Levels of immunoglobulin G antibodies to Bartonella henselae in 10 specific pathogen-free (SPF) cats, 42 cats with non-infectious diseases (NI), four cats with fleas, 23 cats with central nervous system (CNS) disorders, and 79 feral cats
extrapolated. For analysis of canine serological data, the OD405 values were used as, due to the absence of a known seropositive dog, it was not possible to calculate a linear matrix scale.
Sodium dodecylsulphate polyacrylamide gel electrophoresis (SDS-PAGE) and Westem blotting The B henselae antigen preparation was separated on 10 per cent SDS-PAGE gels (Laemmli 1970) and stained with Coomassie blue or transferred electrophoretically to nitrocellulose membranes in a wet blotter (Bio-Rad) using CAPS buffer, 1OmM, pH 11 (Sigma). After blocking the nitrocellulose for one hour with 1 per cent gelatin (Sigma) at room temperature and washing three times for 10 minutes with PBS/T, the membranes were probed with cat or dog sera, diluted 1:100 in PBS/T, that were negative or positive by the ELISA for IgG B henselae antibodies and also with known negative or positive cat sera from CDC. The blots were washed three times for 10 minutes in PBS/T and then the cat membranes were
IgG anti-B henselae ELISA Cats The SPF cats were considered the least likely to be infected with B henselae and were used as a negative control group. The mean (matrix value) of the SPF cats was 1.9 and the mean +3sd was 4-44. Serum samples with a matrix value of 4-5 and above were therefore considered positive for B henselae antibodies. Twenty-eight of the 69 domestic cats (40.6 per cent) were seropositive, and 20 (47-6 per cent) of the 42 cats with noninfectious conditions, one of the four cats with fleas, and seven (30.4 per cent) of the 23 cats with CNS disorders were 1.1 0
1*0 00
0.9 Mean +3sd LC
0t
~0
0-7
.0
0-6
0
a IMEWIF
Dorset
Unknown Ifton Manor (Wales) Cardiff Nofwich
0-5
00"00 *-0009****
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Number of cats Tested Seropositive (O) 5
Newport
4 16 6 16 7
Chepstow
14
Blackpool
so *00
5 MMM.
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Area of colony
0.8
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11
5 (100) 4 (100) 11 (68.8) 4 (66 7) 2 (12-5) 6 (85-7) 0 (0) 3 (21-4)
...............
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Mean
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FIG 2: Levels of
0.1
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Dogs 674
Population
Jameson and others (1995) Domestic Urban stray Heller and others (1997)
12
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Antibody Culture Switzerland Antibody Japan Antibody South Africa and Zimbabwe Antibody Australia Culture Germany Antibody Philippines Antibody Indonesia Antibody USA France
The Veterinary Record, December 9, 2000
immunoglobulin G antibodies to Bartonella henselae (ELISA) in 100 dogs. OD Optical density
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cats which tested positive for FeLV were seropositive to B henselae. Thirty-three (41.8 per cent) ofthe feral cats were seropositive (Fig 1) but the seropositivity rate was very different in different regions (Table 2). By the Mann-Whitney U test there were no significant differences between the groups of domestic cats. The only significant differences were between the SPF cats and the domestic cats as a whole (P
