Prevention of renal infection and urinary shedding in dogs

by a Leptospira vaccination

Paul Schreiber *, Virginie Martin, Wojciech Najbar, Annaelle Sanquer,Sylvie Gueguen, Bernard Lebreux

Biological R&D, VIRBAC Laboratories, B.P. 27, 06511 Carros Cedex, France

Received 17 November 2004; received in revised form 15 March 2005; accepted 18 March 2005

www.elsevier.com/locate/vetmic

Veterinary Microbiology 108 (2005) 113–118

Abstract

Prevention of urinary shedding of Leptospira interrogans spp. by chronically infected dogs remains a key objective of the

vaccination in dogs against leptospirosis which is a zoonotic disease. An inactivated bivalent vaccine composed of Leptospira

interrogans serovars icterohaemorrhagiae [L. icterohaemorrhagiae] and canicola [L. canicola] bacterins was tested for its ability

to protect puppies against a challenge exposure with L. icterohaemorrhagiae. The vaccine was administered twice at a 3-week

interval to six puppies aged from 8 to 9 weeks. Six other puppies were used as unvaccinated controls. All puppies were

challenged 2 weeks after the second vaccine injection by intraperitoneal (IP) administration of L. icterohaemorrhagiae (day 0).

Clinical signs, haematological and biochemical changes and evidence of Leptospira in blood, urine and kidney were monitored

for 4 weeks after the challenge exposure (days 0–28). Puppies were euthanised on day 28 for post-mortem and histological

examinations of liver and kidney. Control group presented clinical pictures of severe or subclinical infection. One dog developed

severe clinical signs (hypothermia, depression, anorexia, abdominal pain, dehydration, icterus, weight loss) and died on post-

infection day (PID) 7 due to an acute renal failure. Gross and microscopic lesions were in accordance with this clinical pattern. In

the five remaining control dogs, the challenge exposure induced mainly a systemic infection including leptospiraemia,

leptospiruria and renal carriage. The vaccinated group remained healthy throughout the study period. In conclusion,

immunisation with a Leptospira vaccine was shown to protect dogs against symptomatology and leptospiraemia, urine

shedding and renal infection.

# 2005 Elsevier B.V. All rights reserved.

Keywords: Leptospirosis vaccination; Dog; Challenge; Renal infection

* Corresponding author. Tel.: +33 492087316;

fax: +33 492087199.

E-mail address: pschreib@virbac.fr (P. Schreiber).

0378-1135/$ – see front matter # 2005 Elsevier B.V. All rights reserved

doi:10.1016/j.vetmic.2005.03.007

1. Introduction

In Europe and North America, vaccination against

leptospirosis has been carried out in dogs since the

70s. Inactivated vaccines used for dog immunisation

contain L. canicola and L. icterohaemorrhagiae

.

P. Schreiber et al. / Veterinary Microbiology 108 (2005) 113–118114

bacterins because leptospirosis in dog has been his-

torically associated with these serovars (McDonough,

2001). Serological analysis carried out in France

during the last years demonstrated that the serovar

L. icterohaemorrhagiae was the most prevalent in

dogs (Andre-Fontaine, 2002). Likewise this serogroup

showed the highest prevalence in humans in France

during the last years (Leptospira Molecular Genetics

Server, 2004). L. icterohaemorrhagiae was also shown

to be the most prevalent serogroup in both humans and

dogs in other European countries (Benito Calavia

et al., 1997; Cerri et al., 2003; Christova et al., 2003;

Ciceroni et al., 2000; Holk et al., 2000). In order to

prevent this zoonotic disease, immunisation of dogs

with Leptospira vaccine (including L. icterohaemor-

rhagiae and L. canicola) provides the most of efficient

way considering that the human infection is usually

linked to a direct or indirect contact with the urine of

an infected animal (Levett, 2001). We have assessed a

commercially available vaccine for its ability to

prevent systemic infection and symptomatology in

dog after a challenge with L. icterohaemorrhagiae.

2. Material and methods

2.1. Animals

Twelve specific pathogen-free (SPF) Beagle dogs

were obtained from a commercial supplier (Harlan

Table 1

Results of blood, urine and kidney cultures after challenge of dogs with

Group Dog no. Days after challenge

Blood

0 2 3 4 5 6

Vaccinated 77891 � � � � � �78666 � � � � � �80254 � � � � � �83897 � � � � � �85511 � � � � � �90365 � � � � � �

Control 81032 � + + � � �81763 � + + � � �84083 � + � � � �84233 � + + � � �85252 � + + + � �87851 � + � � � �

(�) Negative cultures; (+) positive cultures; (ND) not done (euthanised o

Sprague Dawley, USA). They were randomly allo-

cated to two groups (vaccinated and control) and

housed in two separate and isolated rooms. The pups

were fed once a day with a commercial diet and water

was available ad libitum. All procedures were

approved by internal Ethics Committee and were

performed in compliance with the requirements of the

European Convention for the protection of vertebrate

animals used for experimental purpose.

2.2. Vaccines

CANIGEN1 L (VIRBAC S.A., Carros, France) is a

non-adjuvanted liquid vaccine containing a suspen-

sion of inactivated whole organisms of L. canicola and

L. icterohaemorrhagiae. The vaccine is mixed before

use with CANIGEN1 DHPPi, a lyophilised vaccine

containing live canine distemper virus, canine

adenovirus type 2, canine parvovirus and canine

parainfluenza virus. The combination vaccine is

denoted CANIGEN1 DHPPi/L.

2.3. Experimental design

Six 8- to 9-week-old pups were immunised

subcutaneously twice at a 3-week interval with

CANIGEN1 DHPPi/L (vaccinated group) and the

six remaining pups were not vaccinated (control

group) (Table 1). Two weeks after the second vaccine

injection (day 0), each dog was inoculated via the IP

Leptospira interrogans serovar icterohaemorrhagiae

Urine Kidney

7 11 0 7 14 21 28 28

� � � � � � � �� � � � � � � �� � � � � � � �� � � � � � � �� � � � � � � �� � � � � � � �

� � � � + + + �� � � + + + � +

� � � � � + � +

� � � � + + + +

� ND � + ND ND ND ND

� � � + + + + �n day 7 post-challenge).

P. Schreiber et al. / Veterinary Microbiology 108 (2005) 113–118 115

route with 2.5 � 109 to 5 � 109 organisms of L.

icterohaemorrhagiae strain I 109/90A (identified by

Institut Pasteur, Paris). A clinical examination was

carried out once daily for 4 weeks. For Leptospira

isolation, blood samples were taken eight times (days

0, 2, 3, 4, 5, 6, 7 and 11), urine samples were collected

weekly (days 0, 7, 14, 21 and 28) and the right kidney

was taken at the end of the study (day 28). Blood was

also sampled weekly for serological, haematological

and biochemical analyses. On day 28, an autopsy was

performed to collect tissue samples from kidney (left)

and liver for histological examination.

2.4. Serology

Sera were tested by microscopic agglutination

(MA) for antibodies against L. icterohaemorrhagiae.

The MA titre is expressed as the reciprocal of the

highest serum dilution that agglutinates 50% or more

of the Leptospira relative to the control (Levett, 2001).

2.5. Haematology

The white blood cell (WBC), red blood cell (RBC)

and platelet counts and haematocrit were obtained by

use of Coulter Act Diff haematology analyser (Beck-

man Coulter). For each haematological parameter,

individual values were obtained from all the dogs

sampled prior to the inoculation (day 0) and are

expressed as a range about the mean including two

standard deviations. These baseline values were used

in order to check that each individual value was

normal during the PI period (Tvedten, 1981).

2.6. Biochemistry

Urea, total bilirubin, creatinine, alkaline phospha-

tase (AP), aspartate aminotransferase (ASAT) and

alanine aminotransferase (ALAT) were determined

using OLYMPUS AU640 chemistry analyser. Like

haematology, biochemical results were assessed

according to the baseline values.

2.7. Blood, urine, kidney cultures

For each dog, 0.5 ml of plasma sample was

inoculated into a 25 cm2 culture flask containing

9.5 ml of BSAT medium plus 40 ml of 5-fluorouracil.

The flask was incubated at 30 8C. Cultures were then

checked at least three times for evidence of growth of

Leptospira by an increase in turbidity and by

examination under dark-field microscopy between 2

and 6 weeks (before being discarded as negative). The

urine sample (0.5 ml urine) was put first into BSAT

medium (4.5 ml) before being inoculated into a

culture flask containing 5 ml of BSAT medium. Urine

cultures were carried out as described above for blood

cultures. At autopsy, the right kidney was taken and

perfused with 20 ml of BSAT medium. The fluid

harvested was put into a flask and cultured as

described above for blood cultures.

2.8. Histological examination

The left kidney and liver were collected post-

mortem and samples were fixed with formalin.

Histological sections were stained with haematox-

ylin–eosin.

2.9. Statistical analysis

Statistical analysis was performed using NCSS1

2000 statistical software. Student’s t-test was used for

the assessment of the quantitative parameters (body-

weight, rectal temperature) on the challenge day. The

time courses of these quantitative parameters were

analysed using ANOVA 2 test on repeated measures.

Analysis of survival probability of dogs after

challenge was made using the Kaplan–Meier method.

The log-rank test was used to compare the survival

curves of vaccinated and control groups. The number

of positive blood or urine cultures per dog was

compared between vaccinated and control groups

using a Mann–Whitney test. The proportion of dogs

with positive kidney culture per group and the

proportion of dogs with gross or microscopic lesions

was made using Fischer’s exact test. A P-value <0.05

was considered significant.

3. Results

3.1. Clinical examination

One control dog (no. 85252) was euthanised on day

7 for ethical reasons after it showed severe clinical

P. Schreiber et al. / Veterinary Microbiology 108 (2005) 113–118116

Fig. 1. Mean microscopic agglutination (MA) antibody titres

(�S.D.) to Leptospira interrogans serovar icterohaemorrhagiae in

dogs after challenge with L. icterohaemorrhagiae.

signs including hypothermia (35.6 8C), depression,

anorexia, abdominal pain, dehydration, icterus and

weight loss. Two other control dogs (nos. 81763 and

84083) became febrile (39.6 8C). All vaccinated

animals remained healthy through the 4-week follow

up. There was no significant difference between the

survival curves of vaccinated and unvaccinated

groups. Before challenge, the mean values of body

weight and rectal temperature were not significantly

different between vaccinated and control groups.

3.2. Serology

Unvaccinated control group remained free of

antibodies to L. icterohaemorrhagiae up to the

challenge day. All vaccinated pups showed a

seroconversion 7 days after the second vaccination

but were seronegative at the time of challenge. By day

7 after challenge, all vaccinated animals showed a

sharp increase of antibody titres to L. icterohaemor-

rhagiae which still remained high 4 weeks PID. The

kinetic of the mean antibody titres was similar in the

control group except for the peak of mean antibody

titres, which was lower by day 7 PID (Fig. 1).

3.3. Haematology and biochemistry

Additional blood samples were made on days 5 and

6 in control dog no. 85252 which died on day 7 after

challenge. Significant haematological changes were

recorded three successive days (5, 6 and 7) in the

shape of severe leukocytosis (23,700, 27,900 and

28,900 cells/mm3) and severe thrombocytopenia

(65,000, 132,000 and 158,000 cells/mm3). A sharp

increase in urea (9.4 g/l), creatinine (62 mg/l) and total

bilirubin (123 mg/l) were observed on day 7 in dog no.

85252. The remaining control dogs and the vaccinated

animals showed no significant changes.

3.4. Blood, urine and kidney cultures

In control group, all dogs showed positive blood

cultures during the first week PI. All vaccinated

animals remained negative (Table 1). The number of

positive blood cultures per dog is statistically different

between vaccinated group and control group

(P = 0.0025). All control dogs shed Leptospira in

their urine at least once between the first and the 4th

week PID. All vaccinates remained negative (Table 1).

The number of positive urine cultures per dog is

statistically different between vaccinated group and

control group (P = 0.0035) (Table 1). In control group,

the surviving animals at the study end showed either

positive results (three dogs) or negative results (two

dogs) from kidney culture. All vaccinated dogs

showed negative results (Table 1). The proportion

of dogs with positive kidney culture is not significant

between vaccinated and control groups.

3.5. Post-mortem and histological examinations

Only the dog no. 85252 showed macroscopic

changes i.e., a widespread icterus (icteric coloration of

mucosa, skin, subcutaneous tissues, surface of

stomach, intestines, kidneys and bladder), petechia

(cutaneous tissues, omentum, peritoneum) and icteric

discoloration of liver. This dog also showed histolo-

gical changes in both liver (mild and diffuse

congestion) and kidney (diffuse subacute nephritis).

None of the other dogs (control and vaccinated)

developed gross or microscopic lesions.

4. Discussion

After challenge exposure, the control group showed

varied clinical signs from a subclinical infection in

most animals (n = 5) to a fatal disease in one dog. The

fatal case showed significant abnormalities in symp-

P. Schreiber et al. / Veterinary Microbiology 108 (2005) 113–118 117

tomatology (hypothermia, depression, anorexia,

abdominal pain, dehydration, icterus, weight loss),

in haematology (leukocytosis, thrombocytopenia), in

biochemistry (increase of urea, creatinine, total

bilirubin), in gross lesions (icterus, petechia) and in

microscopic lesions (subacute nephritis) after the

experimental inoculation. The lack of any obvious

clinical signs in most of control dogs was consistent

with the subclinical nature of most of leptospiral

infections in dogs (Brown et al., 1996). None of the

vaccinates showed clinical signs or laboratory

abnormalities suggesting a complete protection from

the challenge exposure with L. icterohaemorrhagiae.

The vaccination provided a complete protection

against leptospiraemia (P = 0.0025), leptospiruria

(P = 0.0035) and the renal carriage. Regarding the

renal infection, no significant difference was observed

between vaccinated and control groups because only

three control animals (out of five) showed a positive

culture. However, the two remaining control dogs

(nos. 81032 and 87851) showed a weekly leptospiruria

at least three times (from days 14–28) while the renal

culture was negative. This leptospiruria can be

explained only by a persistent renal infection as the

leptospiraemia, which lasted at the most for 3 days,

cannot be responsible of the urine shedding (Kerr and

Marshall, 1974). A lack of sensibility in the urine

collection (by renal perfusion with BSAT) could

explain these negative results in the culture. This study

also demonstrated that the renal carrier state is

possible without any microscopic lesions of the

kidney. These results are in accordance with pre-

viously published data after challenge exposure with

L. icterohaemorrhagiae or L. canicola (Klaasen et al.,

2003) or after natural infection with L. sejroe (Ruhl-

Fehlert et al., 2000).

If protection against clinical signs of leptospirosis

is the first goal of the dog vaccination, the prevention

of the carrier state is also essential due to the zoonotic

risk (Broughton and Scarnell, 1985). Unfortunately

the clinical and laboratory diagnosis are difficult

because most of chronic infections are asymptomatic

and antibody titres may fall below the detectable levels

while animals are chronically infected (Brown et al.,

1996; Harkin et al., 2003; World Organisation for

Animal Health, 2004). Therefore, vaccination still

remains useful in order to prevent chronic Leptospira

infections.

5. Conclusion

Experimental infection in dogs with L. icterohae-

morrhagiae induced clinical signs and infection of

blood, urine and kidney. Immunisation with a bivalent

Leptospira vaccine was able to protect dogs against

symptomatology and to prevent leptospiraemia, urine

shedding and the renal infection. The complete

prevention of the renal carriage is important in order

to limit the zoonotic risk.

Acknowledgement

The authors are grateful to A. Le Grand for skilful

technical assistance and thank T. Eun for the critical

reviewing of the manuscript.

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