Efficacy of DAPPi2-LR Canine Vaccine against Rabies and Leptospira Induced Clinical Signs, Mortality and Renal Carriage

Efficacy of DAPPi2-LR Canine Vaccine against Rabies and Leptospira Induced Clinical Signs, Mortality and Renal Carriage

Corresponding author: Dr. Lionel Cupillard, Lyon, France, Tel: +33 (0)4 72 72 55 27; Fax: +33 (0)4 72 72 29 63; Email: Lionel.Cupillard@merial com

Abstract

Efficacy of the Leptospiral and rabies components of EURICAN DAPPi2-LR, a canine combined vaccine, was demonstrated after a primary course of vaccination using one dose of EURICAN® DAPPi2-L followed by one dose of EURICAN® DAPPi2-LR. Challenges for leptospirosis (serovars Canicola and Icterohaemorrhagiae) were carried out in puppies 14 days after vaccina- tion for the onset of immunity studies (OOI), in adults 13/14 months after vaccination for the duration of immunity studies (DOI), and for rabies in adults 13 months after vaccination. For OOIs, vaccination provided full clinical protection against severe Leptospira challenges and prevented renal carriage and detectable excretion of Leptospira in urine. For DOIs, clini- cal signs of leptospirosis were less severe in controls but were absent or mild and transient in vaccinated dogs, and urinary shedding was significantly reduced (frequency and duration). In addition, a single dose of EURICAN® DAPPi2-LR induced rabies virus neutralizing antibodies in all dogs as early as 2 weeks post-vaccination and provided full protection in a rabies challenge performed after 13 months. Vaccine has a high efficacy profile with a quick onset of immunity as early as 2 weeks post vaccination, providing full protection against fatal leptospirosis caused by L. Canicola and L. Icterohaemorrhagiae and strong seroconversion against rabies. Long-term protection was also demonstrated by challenges performed 13/14 months post vaccination with reduction of mortality, clinical signs, infection, renal carriage, and bacterial excretion for Leptospira and protection against mortality and clinical signs for rabies.

Keywords: Leptospira Canicola; Icterohaemorrhagiae; Rabies; Vaccine; Onset and Duration Of Immunity; Challenge

Introduction

Leptospirosis is the most widespread zoonotic disease in the world, affecting a broad range of mammals. It is caused by in- fection with pathogenic Leptospira species [1]. Clinical signs associated with Leptospira infection in dogs range from sub- clinical infection to acute disease. Common features include anorexia, vomiting, lethargy, muscle pain, dehydration, jaun- dice, abdominal pain, diarrhoea, bloody urine, and death [1- 6]. Acute renal failure is the predominant finding in symp- tomatic dogs, alone or associated with signs of hepatic injury [2, 3 6, 7]. Animals recovering from leptospirosis may be- come asymptomatic carriers harbouring virulent leptospires in the renal tubules for extended periods and shedding infec-

tious leptospires into the environment, thus being a possible source of human leptospirosis [1,7]. Excretion in urine may be intermittent or continuous and the urinary concentra- tion of bacteria may be high [1,4,5]. Infection of naïv� e ani- mals results from contact of intact mucous membranes, such as ocular conjunctival, or abraded skin with infected urine or urine-contaminated soil, water, food, or bedding [1,4]. Humans are incidental hosts and usually become infected through occupational, recreational or domestic contact with the urine of carrier animals (such as the dog), either directly or via contaminated water or soil.

EURICAN® L is a whole cell, non-adjuvanted vaccine pre- pared from inactivated cultures of Leptospira interrogans,

serovars Canicola and Icterohaemorrhagiae. Historically, ca- nine leptospirosis has mainly been associated with serovars Canicola and Icterohaemorrhagiae. Recently, the epidemio- logical situation in Europe was reviewed and the options for changes to the Leptospira components of dog vaccines in Europe were examined. It was concluded that the inclu- sion of serovars Icterohaemorrhagiae and Canicola is still recommended and that the spectrum should be extended [8]. It is unlikely that these two historical serovars will ever be eradicated given the number of unvaccinated dogs and the extensive rat population [3]. Therefore vaccination of the canine population to protect against clinical signs induced by serovars Canicola and Icterohaemorrhagiae should not be discontinued and should even more provide protection against renal shedding [9].

Rabies is a zoonotic disease, caused by the rabies virus, of the Lyssavirus genus, within the family Rhabdoviridae. Domestic dogs are the most common reservoir of the vi- rus, with more than 95% of human deaths caused by dog- transmitted rabies. In a recent study, it is estimated that canine rabies causes worldwide approximately 59,000 hu- man deaths annually [10], many of whom are children bitten by rabies-infected dogs (http://www.who.int/rabies/en). Rabies is a 100% vaccine-preventable disease. Elimination programs often revolve around mass dog vaccination cam- paigns, where at least 70% of the dog population should be covered in order to break the cycle of transmission in dogs, and to humans.

On one hand, in primo vaccinated dogs, it is generally accept- ed that monovalent rabies vaccines offer a better serologi- cal conversion rate than multivalents ones [11], increasing the success of passing the serological test with an antibody threshold at 0.5 IU/ ml. On the other hand, combined vac- cines, in particular the LR combinations are likely to dis- play negative interferences between components. This is a matter of concern in many countries where rabies remains prevalent or perceived as a potential risk, and where veteri- narians, for practical reasons, are still using extensively such combination vaccine. A way to get around this concern is to optimize the antigen ratio between leptospirosis and rabies antigens, which is critical to maintain highest efficacy pro- file of the vaccine and reduce interferences. Unfortunately, very few data, if any, have been published to support this approach and confirm the efficacy of each antigen of such combined vaccines.

Here, we describe level of protection conferred by a biva- lent LR formulation which was optimized and tested on dog that underwent a primary course of one dose of EURICAN® DAPPi2-L followed by one dose of EURICAN® DAPPi2-LR. Leptospiroses challenges (serovars Canicola and Ictero- haemorrhagiae) were carried out according to European Pharmacopoeia 14 days and 13/14 months after vaccination and rabies challenge 13 months after vaccination. Results of these five clinical studies for short-term and long-term pro- tection against leptospirosis and rabies are presented and discussed.

Materials and Methods

Experimental design

Institutional Animal Care and Use Committee approvals were obtained before conducting the studies. Once clinical signs appeared in control group, clinical examination was performed twice a day and any animals displaying serious and irreversible clinical signs that could lead to suffering were humanely euthanized based on predefined endpoints.

For leptospirosis, four separate vaccination-challenge stud- ies were carried out to investigate the onset (OOI) and du- ration (DOI) of immunity provided by EURICAN® DAPPi2-LR against L. interrogans serovar Canicola and L.interrogans se- rovar Icterohaemorrhagiae (referred to as studies 1-2 and 3-4 for OOI and DOI respectively, Table 1). Onset of immu- nity studies were validated according to European Pharma- copeia. In all studies, puppies received subcutaneously two injections of primary vaccination at the age of 7-9 weeks with EURICAN® DAPPi2-L and at the age of 11-13 weeks with EURICAN® DAPPi2-LR. Dogs from studies 1 and 3 were chal- lenged with Leptospira Canicola at 2 weeks or 14 months post-vaccination, respectively. Similarly, dogs from studies 2 and 4 were challenged with Leptospira Icterohaemorrha- giae at 2 weeks or 13 months post-vaccination, respectively. Dogs were examined for clinical signs consistent with lepto- spirosis. Blood and urine samples were collected at regular intervals for Leptospira isolation, and a kidney sample was aseptically taken from each dog at necropsy.

Designation Study Group # dogs Challenge
Time after

V2

Infecting

agent

Onset of immunity 1 : OOI Lc V 7 2 weeks Lc
C 6
2 : OOI Li V 7 2 weeks Li
C 7
Duration of immunity 3 : DOI Lc V 8 14 months Lc
C 8
4 : DOI Li V 12 13 months Li
C 8
5 : DOI

rabies

V 25 13 months Rabies
C 10

V=Vaccinated, C=Control, V2 = second vaccination Lc = L. Interro- gans serovar Canicola, Li = L. Interrogans serovar Icterohaemor- rhagiae

Table 1. Experimental design

Blood was also collected for serological (studies 1 and 2), haematological (studies 1, 2 and 3) and biochemical analy-

sis (studies 1, 2 and 3). At the end of the observation period or at euthanasia, dogs were necropsied and organs were re- moved for histological examination.

For rabies (referred to as study 5, Table 1), a vaccination- challenge experiment was carried out according to Europe- an Pharmacopeia to study the duration of immunity provid- ed by EURICAN® DAPPi2-LR. Puppies were vaccinated twice subcutaneously at the age of 8-9 weeks with EURICAN® DAPPi2-L and at the age of 12-13 weeks with EURICAN® DAPPi2-LR (vaccinated group, n = 25), or again with EU- RICAN® DAPPi2-L for puppies (control group, n = 10). Dogs were challenged with rabies 13 months after the second vac- cination. All animals were confirmed to be seronegative for rabies and healthy at the beginning of the study. After chal- lenge, dogs were examined for clinical signs consistent with rabies.

Vaccines

For the leptospirosis studies, production batches of EURI- CAN® L and EURICAN® LR (Merial, Lyon, France) vaccines were used as diluent to reconstitute a non-adjuvanted freeze-dried pellet, EURICAN® DAPPi2, which is a vaccine containing live attenuated canine distemper virus, canine adenovirus type 2, canine parvovirus and canine parainflu- enza type 2 virus. The experimental LR vaccine was used for the rabies study. EURICAN L® is a whole cell, non-adjuvanted vaccine prepared from inactivated cultures of Leptospira interrogans, serovars Canicola and Icterohaemorrhagiae. EURICAN® LR is an alumine hydroxyde adjuvanted vaccine containing inactivated whole cells of Leptospira interrogans, serovars Canicola and Icterohaemorrhagiae and inactivated rabies antigen. All batches of EURICAN® L or EURICAN® LR complied with the potency requirements of monograph 0447 (Leptospira) of the European Pharmacopoiea (2002). EURICAN® LR complied with the potency requirements of monograph 0451 (rabies) of the European Pharmacopoiea (1998).

Leptospirosis Challenges Studies Animals

Beagle puppies aged from 7 to 9 weeks without detectable agglutinating antibodies against the principal serovars of pathogenic Leptospira were purchased from commercial suppliers and enrolled into each study (table 1).

Challenge strains

Leptospira interrogans serogroup and serovar Canicola, strain Moulton (National Veterinary Services Laboratory (NVSL), Ames, Iowa, USA) was used as challenge inoculum in studies 1 and 3. Leptospira interrogans serogroup and serovar Icterohaemorrhagiae, strain 193 (Pasteur Institute, Paris, France) was used as challenge inoculum in studies 2 and 4, respectively. The identity of all serovars was con- firmed by the Pasteur Institute, Paris, France, using restriction fragment analysis.

Challenge Protocol

After an initial culture in Ellinghausen-McCullough-John- son-Harris (EMJH) medium, the strains were back-passaged twice in hamsters to prevent loss of virulence through ad- aptation to culture conditions. Moribund hamsters were hu- manely euthanised and their livers and kidneys or spleens were aseptically removed and homogenated in sterile saline. After harvest, the challenge strains were passaged once in vitro (EMJH medium) to allow a more precise quantification of the bacterial suspension. Each dog received 11 ml (studies 1, 2 and 3) or 13 ml (study 4) of challenge suspension with

0.5 ml instilled in the ventral conjunctival sac of each eye and the remainder administered intra-peritoneally. In stud- ies 1 and 2, the total challenge dose per dog was around 3 x 109 organisms for L. Canicola and L. Icterohaemorrhagiae, respectively. In studies 3 and 4, each dog received around 6 x 109 organisms of L. Canicola and L. Icterohaemorrhagiae, respectively.

Clinical Examination

All animals were observed daily for 28 (studies 1 and 2) or 35 days (studies 3 and 4) after challenge for signs consis- tent with leptospirosis, including, apathy/prostration, de- hydration, conjunctivitis, vomiting, diarrhoea, and cutaneo- mucosal signs. Signs were scored by use of a standardized protocol (Table 2). Rectal temperatures were taken on day 3 (study 1) or 5 (study 2) before challenge and recorded daily for 7 days (studies 1 and 2) or 14 days (studies 3 and 4) af- ter challenge. Temperatures above 39.5°C were considered as hyperthermia. Dogs were weighed once a week until the end of the study. During the post-challenge clinical examina- tion, any animals displaying serious and irreversible clini- cal signs that lead to suffering were humanely euthanized. For post-mortem examination, animals were necropsied and subjected to a macroscopic examination.

Laboratory Analyses

For serology, whole blood was collected before vaccination (day 0) then 3-5 days before and 28 days after challenge. Selected sera were tested for antibodies using microscopic agglutination titres (MAT) by the National Reference Centre for Leptospira (CNRL), Institut Pasteur, Paris, France, against

L. interrogans serovar Canicola (study 1) or L. interrogans serovar Icterohaemorrhagiae (study 2). Since serology has limited value for evaluating the efficacy of vaccines against leptospirosis, sera from studies 3 and 4 were not tested. Antibody titers were expressed as the highest serum dilu- tion that induced agglutination. The threshold of positivity was set at 100 (1/dil).

For haematology, blood samples were collected in heparin from dogs of studies 1 and 2 on day 3 or 5 before challenge and then on days 2, 3, 4, 5, 8 and 11 after challenge. In study 3, blood was collected on the day of challenge, then daily for

7 days after challenge. In studies 1 and 2, counts of plate- lets were performed by ORBIO Laboratory, Bron, France. In study 3, counts of white blood cells and platelets were per- formed using a MS-9 cell counter analyser (Melet Schloesing, France). In studies 1 and 2, platelet counts were compared to normal values provided by ORBIO laboratory. Haematology analyses were not performed for study4.

Clinical sign Degree Score
General appearance Good 0
Apathy 1
Prostration 2
Death 5
Conjunctivitis Absence of sign 0
Presence of conjonctivitis 1
Dehydration Absent 0
Slight 1
Severe 2
Digestive signs, vomiting Absence of signs 0
Vomiting without abdominal pain 1
Severe vomiting alone or severe vomiting with blood or severe vomiting with abdominal pain or severe vomiting with blood and abdominal pain or abdominal pain alone 2
Digestive signs, diarrhoea Absence of signs 0
Non bloody diarrhoea 1
Bloody diarrhoea 2
Cutaneo-mucosal signs Absence of signs 0
Slight icterus visible only on ocular mucosa 1
Obvious icterus (of all non-pigmented body surfaces) alone or obvious icterus with mucosa congestion or obvious icterus with haemorrhagic disorders (petechiae, suffusions, haemorrhages) or obvious icterus with mucosa congestion and haemorrhagic disorders 2

Table 2. Clinical scoring protocol for canine leptospirosis

For blood chemistry, whole blood samples were collected on heparin from dogs of studies 1 and 2 before challenge and then on days 2, 3, 4, 5, 8 and 11 after challenge. Sera were analyzed for urea, creatinine, alanine transaminase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP) by ORBIO Laboratory, Bron, France. Urea, creatinine,

10 after challenge. Blood samples were immediately inocu- lated in liquid EMJH medium (1 ml of blood in 9 ml medium) and transferred to the laboratory. Serial 10-fold dilutions (up to 10-3) were made in the same media and incubated at 30°C. All the cultures were incubated for a maximum of 6 weeks and observed weekly for the presence of leptospires using dark field microscopy. For the detection of leptospires in urine and kidney, urine samples were collected before challenge (day -5/day -3) and on days 3, 5, 8, 11, 14, 21 and 28 after challenge for studies 1 and 2, or before challenge (day -11/day 0) and at 2, 3 and 5 weeks after challenge for studies 3 and 4. Urine samples were collected either spon- taneously after subcutaneous injection of the diuretic furo- semide (DIMAZON® , Intervet, France) (0.5 to 1 ml/kg body- weight) (females) via urinary catheterisation (males) or by cystocentesis (males and females), or by direct bladder tap at the time of euthanasia. Samples were immediately inocu- lated in liquid EMJH medium added with 5 Fluorouracil at a concentration of 100 µg/ml (1 ml of blood in 9 ml medium) and transferred to the laboratory. Serial 10-fold dilutions (up to 10-3) were made in the same media. Samples from kid- neys were collected aseptically. Approximately 5-8 grams of organ tissue were macerated into 10 ml of liquid EMJH me- dium added with 5 Fluorouracil at a concentration of 100µg/ ml. Tissue debris was allowed to settle, and serial 10-fold dilutions were made as for urine samples. All the cultures (urine and kidney) were incubated at 30°C for a maximum of 6 weeks and observed weekly for the presence of leptospires using dark field microscopy. Samples of kidneys and livers were fixed with 10% buffered formalin and processed for microscopic examination following Hemalun-Eosine-Safran staining.

 

Result

Statistical analyses were carried out using SAS® software.

The level of significance was set at P 0.005

Clinical Scores

Global Clinical Score (GCS) was derived from the assess- ments of each Individual Clinical Sign (ICS) measured on each puppy throughout the monitoring period (number of days of monitoring = ndm) by taking into account the effec- tive length of the monitoring period in the case of the death of the puppy (effective number of days of monitoring = ndme).

For each puppy (i), GCS was defined as the sum of the ICS

(k) over the day of measurement (j) and was computed as follows:

ndm

j k i

ALT, AST and ALP were compared to normal values provided by the same laboratory. Biochemistry analyses were not per-

GCSwi =

 ICS j,k ndme

formed for studies 3 and 4.

For the detection of leptospires in blood, in studies 1 and 2, blood samples were collected on heparin tubes before challenge (day -5/day -3) and on days 2, 3, 4, 5, 8, 11 after challenge. In studies 3 and 4, blood was collected before challenge (day 0/day -11), then daily for 7 days and on day

The comparison of the vaccinated group to the control group was performed using the Mann-Whitney (Wilcoxon) test.

Leptospiremia and Leptospiruria

A dog was considered to have Leptospiraemia or leptospiru- ria if at any time point at least one blood culture or urine

culture was positive. The frequency of dogs with Leptospi- raemia, leptospiruria or positive kidneys was compared be- tween vaccinated and control dogs using Fisher exact test. The number of days with Leptospiraemia or leptospiruria for each dog was compared between vaccinated and control dogs using Mann-Whitney test.

Haematological and Biochemical Data

In studies 1 and 2, for each dog and at each time point, a scoring system based on a 3-point scale was used: 0 if no parameter was outside the normal values range, 1 if 1 or 2 parameters were outside the normal values range and 2 if 2 or more parameters were outside the normal values range. A global biochemical and haematological score (GBS) was calculated for each dog corresponding to the sum of daily scores throughout the monitoring period. GBS were com- pared between vaccinated and control groups using Mann- Whitney test.

In study 3, platelet and WBC counts of vaccinated and con- trol groups were compared using a mixed model with re- peated measurements.

Rabies Challenge Study Animals

Eight to 9-week-old beagle puppies were purchased from a commercial supplier.

Challenge strains

Rabies, strain New York (CDC, Laurenceville, USA) was used as challenge inocula in study 5.

Challenge protocol

All dogs were confirmed to be in good health and injected sub-cutaneously with ROBINUL-V ND (0.25 ml/5kg), before the anesthesia. Dogs were anesthesied with Ketamine (Im- algene 1000ND) and Xylazine (ROMPUNND) (0.05 ml/kg) of each) before being injected 13 months after the second vac- cination with 0.5 ml per muscle crotaphyte of a dilution of the rabies challenge virus in order to obtain a titre of 103.8 CCID50/ml. The rabies challenge virus titer was confirmed by backtitration after challenge at 103.4 CCID50/ml.

Clinical examination

Dogs were monitored and scored daily during 95 days after challenge for rabies clinical signs (changes in behavior, sen- sitivity disorders, rabid cry, swallowing difficulties, saliva- tion, loss of appetite, furious behavior, paresis, paralysis and death). Dogs were euthanised on day 95 (D95). Brains of all dogs were collected and tested for rabies virus detection by immunofluorescence [12] at the ANSES (French Agency for Food, Environmental and Occupational Health Safety, Nancy Laboratory for Rabies and Wildlife, France).

Laboratory analyses

Serum anti-rabies neutralizing antibody titers were mea- sured on day 0 (injection with L2R combo), days 14, 28, 56,

84, 147, 175, 203, and day-2 before challenge, and on the day of euthanasia. Rapid fluorescent focus inhibition test (RFFIT) was used according to the technique described by [13], with a positive threshold of 0.5 IU/ml. In this assay, a 1.41 log10 titer is equivalent to 0.5 IU/ml of neutralizing antibodies.

 

Result 2

Leptospirosis

Humoral Responses to Vaccination and Challenge

At the start of studies 1 and 2 (OOI, Lc and Li respectively), all dogs were seronegative for serovars Canicola or Icterohaem- orrhagiae, respectively. None of the serum of the vaccinated dogs had MAT detectable antibody titres against serovars Canicola or Icterohaemorrhagiae after vaccination. In study 1, antibody titres against serovar Canicola were detected 28 days after challenge in all 7 vaccinated dogs (range: 50-800) and in 5 out of 6 controls (range 100-200). In study 2, an- tibody titres against serovar Icterohaemorrhagiae were de- tected 28 days after challenge in 3 out of 7 vaccinated dogs (range: 100-400) and in 4 out of 7 controls (range: 50-400).

Clinical Signs

In studies 1 and 2 (OOI, Lc and Li respectively), all controls developed clinical signs (apathy/prostration, dehydration, vomiting, bloody diarrhoea and/or icterus), except one pup- py which died suddently on day 4 after Canicola challenge, without any clinical signs in the preceding observations. All other controls were humanely euthanised between day 4 and day 11 post-challenge (DPC). In contrast, all vaccinates remained healthy and did not show clinical signs, except for four vaccinates which presented transient conjunctivitis probably due to the inoculation route of the challenge sus- pension, and one vaccinate which presented with mild dehy- dration from days 14 to 28 after Canicola challenge (Table 3). GCS were significantly higher in controls than vaccinates in studies 1 (p = 0.001) and 2 (p <0.001).

As expected, clinical signs were less severe in control adults. In study 3 (DOI, Lc), dehydration, diarrhoea and/or prostra- tion were observed in 5 out of 8 controls leading to the eutha- nasia of one animal on DPC7 after having shown bloody di- arrhoea, severe dehydration and prostration. Conjonctivitis was observed in all controls and in 3 of 8 vaccinates. In study 4 (DOI, Li), one control was humanely euthanised on DPC5 after having shown bloody diarrhoea, vomiting, icterus and prostration. Conjonctivitis was observed in 7 out 8 controls and in 4 of 12 vaccinates. In contrast, 7 out of 8 vaccinated dogs in study 3 and 10 out of 12 vaccinated dogs in study 4 did not show any clinical signs during the observation pe- riod. One vaccinate in study 3 had petechiae on DPC13 and DPC15 and two vaccinates in study 4 were apathetic for 1 day only (DPC 1 and DPC8) (Table 3). GCS were significantly higher in controls than vaccinates in study 3 (p = 0.002) and in study 4 (p <0.001).

Onset of immunity Duration of immunity
Canicola Icterohaemorrhagiae Canicola Icterohaemorrhagiae
Clinical signs V C V C V C V C
Apathy/prostration 0/7 5/6 0/7 6/7 0/8 1/8 2/12 1/8
Conjunctivitis 4/7 3/6 4/7 6/7 3/8 8/8 4/12 7/8
Dehydration 1/7 4/6 0/7 6/7 0/8 4/8 0/12 1/8
Vomiting 0/7 4/6 0/7 1/7 0/8 1/8 0/12 1/8
Diarrhoea 0/7 5/6 0/7 7/7 0/8 3/8 0/12 1/8
Icterus/petechiae 0/7 5/6 0/7 7/7 1/8 0/8 0/12 1/8
Global Clinical Score 8.1 61.4 2.7 70.6 2.1 23.7 5.7 34.6
p-value* 0.001 <0.001 0.002 <0.001

Abbreviations: V= vaccinated; C= control.

  • Mann-Whitney (Wilcoxon) test

Table 3. Clinical signs after challenge with L. Canicola and L. Icterohaemorrhagiae in vaccinated and control groups performed 14 days or 14/13 months after primary course of two doses of vaccine (Studies 1- 4)

Haematology and Biochemistry

Studies 1 and 2 (OOI, Lc and Li respectively): All controls be- came thrombocytopenic (number of platelets < 150 x 103/ mm3) for several days after challenge. In contrast, a transient thrombocytopenia was recorded in 4 out of 7 vaccinates in study 1 (Lc) from one to three days and in 2 out of 7 vacci- nates in study 2 (Li) for a single day. All controls showed an increase in urea, creatinine, AST, ALT and/or ALP values. In contrast, the values of the vaccinates remained normal. The GBS was then significantly lower in vaccinates than in con- trols (p < 0.001 in both studies).

In study 3 (DOI, Lc), the platelet count was significantly low- er in the controls than in the vaccinates over the 1 to 7 days post-challenge period (p < 0.001) and the WBC count was significantly higher in the controls than in the vaccinates over the 3 to 7 days post-challenge period (p = 0.006).

Isolation from the Blood

All blood samples were negative for leptospires before challenge.

Studies 1 and 2 (OOI, Lc and Li respectively): Leptospires could be isolated from the blood of all controls for at least 3 days following challenge. In contrast, none of the vacci- nated dogs developed Leptospiraemia, except one dog on a single day (day 3) after L. canicola challenge, indicating that infection was not established in any of the vaccinated dogs (Tables 4A and 4B). The frequency and duration of Leptospi- raemia were significantly lower in vaccinates than in con- trols in study 1 (p = 0.005 and p = 0.001, respectively) and in study 2 (p < 0.001 and p < 0.001, respectively).

Studies 3 and 4 (DOI, Lc and Li respectively): all animals (controls and vaccinates) developed Leptospiraemia (Tables 4C and 4D). The duration of Leptospiraemia was significantly shorter in vaccinates than in controls in studies 3 (p=0.003) and 4 (p=0.009).

Isolation of Leptospires from Urine and Kidney

Studies 1 and 2 (OOI, Lc and Li respectively): Leptospires were isolated from the kidneys of all control dogs. In study 1 (OOI, Lc), no urine sample could be collected from one control dog, and on the day of euthanasia (DPC4 or DPC5) from 2 other dogs, but leptospires were recovered from the urine samples from the 3 other control dogs. In study 2 (OOI, Li), all controls, except 1 dog euthanised on DPC5 (no urine sample was collected at that time), had a least one positive urine sample between DPC3 and DPC11. In contrast, none of the vaccinated dogs had positive urine or kidney cultures (Tables 4A and 4B). The frequency of dogs with at least one positive urine sample and the duration of leptospiruria were significantly lower in vaccinates than in controls in studies 1 (p = 0.046) and 2 (p = 0.005). The frequency of dogs with a positive kidney was significantly lower in vaccinates than in controls in both studies (p < 0.001).

Studies 3 and 4 (DOI, Lc and Li respectively): All controls from studies 3 and 4 shed leptospires in the urine, and the kidneys of 3 out of 8 controls in study 3 (DOI, Lc) and of all controls in study 4 (DOI, Li) were cultured positive for Lep- tospira. Leptospires were isolated from the urine of 1 out of 8 vaccinates in study 3 and from 3 out of 12 vaccinates in

study 4. No vaccinates in study 3, and 4 vaccinates out of 12 in study 4 had positive kidneys (Tables 4C and 4D). The fre- quency of dogs with at least one positive urine sample and the duration of leptospiruria were significantly lower in vac- cinates than in controls in study 3 (p = 0.010 and p = 0.009, respectively) and in study 4 (p = 0.001 and p < 0.001, respec- tively). The frequency of dogs with a positive kidney was sig- nificantly lower in vaccinates than in controls in study 4 (p

= 0.005), but was not significant in study 3, due to the low

number of control dogs with positive kidneys.

Necropsy and Histopathology

In studies 1 and 2 (OOI, Lc and Li respectively), renal lesions

Group Dog No. Days after challenge (L. Canicola)

Blood Urine Kidney

-3 2 3 4 5 8 11 -3 3 5 8 11 14 21 28 28
1
2
3
Vaccinated 4
5
6
7 +
positive 0/7 0/7 1/7 0/7 0/7 0/7 0/7 0/7 0/7 0/7 0/7 0/7 0/7 0/7 0/7 0/7
8 + + + d d d NS NS d d d d d +
9 + + + + + d NS + d d d d +
Control 10 + + + d d d NS d d d d d +
11 + + + + d d NS d d d d d +
12 + + + + + d + + + d d d d +
13 + + + + + d + + d d d d +
positive 0/6 6/6 6/6 6/6 4/4 3/3 NA 0/6 1/5 2/2 3/3 NA NA NA NA 6/6

+ culture positive

– culture negative

d : euthanasia/death NA: not applicable

NS: no sample (empty bladder)

Table 4A. Leptospira isolation from blood, urine and kidney after L. interrogans serovar Canicola challenge (Study 1, OOI Lc). Dogs were challenged 14 days after a primary course of two doses of vaccine

Group Dog No. Days after challenge (L. Icterohaemorrhagiae)

Blood Urine Kidney
-5 2 3 4 5 8 11 -5 3 5 8 11 14 21 28 28
1 – –
2 – –
3 – –
Vaccinated 4 – –
5 – –
6 – –
7 – –
positive 0/7 0/7 0/7 0/7 0/7 0/7 0/7 0/7 0/7 0/7 0/7 0/7 0/7 0/7 0/7 0/7
8 + + + + d – – + + d d d d +
9 + + + + d d – – + d d d d d +
Control 10 + + + + d – – + + d d d d +
11 + + + + d d – – NS d d d d d +
12 + + + + d – – + d d d d +
13 + + + – + + + + d d d +
14 + + + + d d – + NS d d d d d +
positive 0/7 6/7 7/7 7/7 6/7 1/4 0/1 0/7 2/7 5/5 3/4 1/1 NA NA NA 7/7

+ culture positive

– culture negative

d : euthanasia/death NA: not applicable

NS: no sample (empty bladder)

Table 4B. Leptospira isolation from blood, urine and kidney after L. interrogans serovar Icterohaemorrhagiae challenge (Study 2, OOI Li). Dogs were challenged 14 days after a primary course of two doses of vaccine

Group Dog No. Days after challenge (L. Canicola)

Blood Urine Kidney

0 1 2 3 4 5 6 7 10 0 16 21 35 35
1 + + + +
2 + + +
3 + + + + C C
4 + + + +
Vaccinated 5 + + + + +
6 + + + +
7 + + + C
8 + + + + + + +
positive 0/12 8/8 8/8 8/8 6/8 2/8 0/8 0/8 0/8 0/6 1/8 1/7 0/8 0/8
9 – + + + + – C + +
10 – + + + + + + + – – + +
11 – + + + + + + – C + + + +
12 – + + + + + + – – + +
Control 13 – + + + + + + + – – + C +
14 – + + + + + + – C + C + +
15 – + + + + + – C C
16 – + + + + + + + – – +* d d +
positive 0/8 8/8 8/8 8/8 8/8 6/8 4/8 6/8 0/8 0/4 7/8 4/4 3/7 3/8

+ culture positive

– culture negative

d : euthanasia/death NA= not applicable

*urine sample collected on DCP7 (day of death)

Table 4C. Leptospira isolation from blood, urine and kidney after L. interrogans serovar Canicola challenge (Study 3, DOI Lc). Dogs were challenged 14 months after a primary course of two doses of vaccine

Group Dog No. Days after challenge (L. Icterohaemorrhagiae)

Blood Urine Kidney

-11 1 2 3 4 5 6 7 10 -11 14 21 35 35
1 + + + + + +
2 + + + + +
3 + + + + C +
4 + + + + +
5 C C C + C + + +
6 + + + + C +
Vaccinated 7 + + + + + +
8 + + + + + + + +
9 + + + + +
10 + + + + + + +
11 + + + + + + + + +
12 + + + + + +
positive 0/12 6/11 8/11 8/11 10/12 7/11 6/10 11/12 8/12 0/12 0/12 3/12 0/12 4/12
13 – + + + + + d d d – +* d d +*
14 – + + + + + + + + – + + +
15 – + + + + + + + + – + + + +
Control 16 – + + + + + + C + – + + + +
17 – + + + + + C + – + + + +
18 – + + + + + + + + – + + + +
19 – + + + + + + + – + +
20 – + + + + + + C + – + + + +
positive 0/8 8/8 8/8 8/8 7/8 8/8 6/7 4/4 7/7 0/8 8/8 6/7 5/7 8/8

+culture positive

– culture negative

  • urine sample collected on DPC5 (day of euthanasia)

C=contaminated

d : euthanasia/death

Table 4D. Leptospira isolation from blood, urine and kidney after L. interrogans serovar Icterohaemorrhagiae challenge (Study 4, DOI Li). Dogs were challenged 13 months after a primary course of two doses of vaccine

Cite this article: Lionel Cupillard. Efficacy of DAPPi2-LR Canine Vaccine against Rabies and Leptospira Induced Clinical Signs, Mortality and Renal Carriage.

J J Vaccine Vaccination. 2016, 2(1): 015.

were observed in all controls, ranging from rare and discrete clusters of inflammatory cells (lymphocytes, plasma cells) within cortical interstitium (study 2) to extensive mono- nuclear cell inflammatory infiltrates throughout the cortex, protein within tubules, tubular degeneration, glomerular senescence or cortical fibrosis (study 1). Hepatic lesions including discrete margination of neutrophils and lympho- cytes into sinusoids, mild periportal, intraparenchymatous and centrolobular infiltration of lymphocytes and neutro- phils, images of hepatocytic necrosis, dissociation of hepa- tocyte plates, or acute centrilobular degeneration were ob- served in all controls. No specific lesions were found in the vaccinated dogs. In studies 3 and 4 (DOI, Lc and Li respec- tively), macroscopic lesions were detected at necropsy in control dogs that were humanely euthanised. Gross findings included dehydration, icteric mucosa, liquid faecal material tinged with blood, petechiae or haemorrhagic lungs, jeju- num, ileum, caecum, colon, liver and kidneys, and enlarged kidneys. When urine was present from these animals, it was tinged with blood up to severely haematuric. Apart from some reactive mesenteric lymph nodes and some enlarged spleens, control dogs that survived the experimental infec- tion appeared normal on gross visual examination, as well as all vaccinated dogs. In study 4, kidney or liver lesions such as severe diffuse hemorrhagic nephritis, moderate to severe interstitial nephritis associated or not with tubular lesion of atrophy or pigmentary overload and an excess of bile pig- ment compatible with an hepatic cholestasis were observed in all controls. Vaccinated dogs had no lesions.

All results from the four studies are summarized in Table 5.

Rabies

Humoral Responses to Vaccination and Challenge

All controls animals remained negative for rabies (Figure 1), until the day of challenge with mean titers of 0.12 IU/ml (range: 0.06-0.18 IU/ml) at the time of vaccination and 0.07 IU/ml (range: 0.04-0.11 IU/ml) at the time of challenge. Vac- cinated animals had a mean titer of 0.12 IU/ml at the time of vaccination (identical to control group, but in a range of 0.03-0.32 IU/ml, indicating that some dogs had traces of ma- ternal antibodies). All vaccinated dogs seroconverted with antibody titres above the WHO positivity threshold value of

0.5 UI/ml, with a peak of antibody on day 28 post-vaccina- tion (mean titer of 1.97 IU/ml, range 0.24-6.59 IU/ml). The mean antibody titres then decreased on days 84 (0.44, range 0.06-1.66 IU/ml), 175 (0.43 IU/ml, range 0.04-1.66 IU/ml), and became negative before challenge (0.18 IU/ml, range: 0.05-0.55). Mean specific antibody titers of the vaccinated group were higher than the mean values of the control group during the whole pre-challenge period, from 2 weeks post vaccination (1.16 IU/ml versus 0.10 IU/ml) and until the day of challenge (0.18 IU/ml versus 0.07 IU/ml).

Clinical signs

For rabies (table 6), 10 of 10 control dogs showed most of the signs of rabies. All the 25 vaccinated animals survived the rabies challenge. Some dogs showed non specific signs post-challenge, such as loss of appetite, or slightly modified habitus for 2 dogs, or very transient isolated swallowing

Challenge 14 days after vaccination Challenge 13 to 14 months after vaccination

Parameter V C p value V C p value
clinical signs 2/7 5/6** 0.001 3/8 8/8 0.002
L. interrogans leptospiraemia 1*/7 6/6 0.005 (F) and 0.001 (D) 8/8 8/8 NS (F) and 0.003 (D)
serovar leptospiruria 0/7 3/6*** 0.046 (F) and 0.046 (D) 1/8 7/8 0.010 (F) and 0.009 (D)
Canicola positive kidney 0/7 6/6 < 0.001 0/8 3/8 NS
kidney lesions 0/7 6/6 < 0.001 ND
clinical signs 4/7 7/7 <0.001 6/12 8/8 <0.001
L. interrogans leptospiraemia 0/7 7/7 < 0.001 (F) and < 0.001 (D) 12/12 8/8 NS (F) and 0.009 (D)
serovar leptospiruria 0/7 6/7 0.005 (F) and 0.005 (D) 3/12 8/8 0.001 (F) and < 0.001 (D)
rhagiae positive kidney 0/7 7/7 < 0.001 4/12 8/8 0.005
kidney lesions 0/7 7/7 < 0.001 0/12 8/8 ND

Icterohaemor-

V: vaccinates, C:controls D: duration

ND : not determined

F: frequency

NS: not significant

* positive on DPC3 only

** one control died on DPC4 before any clinical signs

*** no or only one urine sample tested for the 3 controls died on DPC 4 or 5

Table 5. Summary of Leptospiroses studies results (Studies 4-1)

disorders without other clinical signs, which could result from individual housing of the animal.

Immunofluorescence results

Brain tissue samples of all vaccinated dogs were found nega- tive by specific immunofluorescence at the end of the study (table 6). In contrast, brain tissue samples from the ten con- trol dogs were positive by immunofluorescence for rabies virus.

pies and adults remained healthy, without clinical signs, with only few animals developing mild and most often tran- sient symptoms (dehydration, apathy, petechiae). The pro- tective effect of the combined vaccine against severe clini- cal signs was demonstrated as soon as 14 days and lasted up to 13 to 14 months after a primary course of two doses of vaccine. Regarding laboratory data, hematological and biochemical abnormalities were observed only in unvacci- nated control puppies and consisted of thrombocytopenia,

Group Neutralizing rabies virus Antibodies seroconversion Challenge resistance Rabies diagnostics (immunofluorecence)
Group 1

(vaccinated with LR combo vaccine)

25/25 25/25 25/25: negative
Group 2 (vaccinated with L combo vaccine) 0/10 0/10 10/10 : positive

Table 6. Summary of rabies seroconversion, challenge result and virus isolation in test groups of dogs (Study 5)

 

Discussion

Since infection with leptospires can lead to an acute, of- ten fatal disease [14], vaccination is recommended in dogs potentially exposed in order to protect them from signs of disease (and death in the worse case scenario) and to reduce significantly the shedding of virulent Leptospira and the zoo- notic risk. Efficacy of vaccines can only be reliably evaluated through challenge studies with significant clinical signs of an acute disease and significant shedding in unvaccinated controls. Our challenge models were thus developped to in- duce typical clinical signs so as to allow demonstration of a complete protection against clinical disease and lesions associated to it. Only few studies in dogs have evaluated the efficacy of leptospirosis vaccines against a severe disease model following experimental challenge with L. Canicola [15-17] or L. Icterohaemorrhagiae [15,17,18]. In our chal- lenge model, all control puppies of the onset of immunity studies showed symptoms of severe leptospirosis that led to euthanasia after L. Canicola or L. Icterohaemorrhagiae challenge. Symptoms observed in control puppies included dehydration, apathy, depression, vomiting, blood diarrhoea, icterus, and were characteristic of leptospirosis [2,3,5,6]. As expected, less severe clinical signs were observed when performing duration of immunity studies in adult controls. Most challenge models used to assess the duration of im- munity induced by leptospirosis vaccines have failed to pro- duce severe clinical signs in adult controls [19-22]. This dif- ference may be related to the challenge strain, the number of passages of the challenge strain in vivo (hamster) before inoculation to dogs or the challenge dose. It is therefore re- markable that our challenge models were capable to induce in adult dogs clinical signs, sometimes leading to euthanasia (one dog each) after challenges performed with L. Canicola and with L. Icterohaemorrhagiae. This confirms observa- tions from a previous experiment using a similar model [17]. Despite this severe challenge model, most vaccinated pup-

increase in urea, creatinine, transaminases (ALT, AST) and/ or alkaline phosphatase activities, supporting the renal and hepatic dysfunctions. In contrast, none of the vaccinated puppies showed hematological or biochemical disorders. Hematological abnormalities were also recorded in adult controls, with a significant decrease in the platelet count and increase in leucocytes in controls compared to vacci- nates. All these hepatic, renal, and haematological signs sup- ported the polysystemic nature of leptospires infection and have commonly been reported after natural or experimen- tal exposure [2,3,5,7,9,16-19,23]. Macroscopic examination was consistent with clinical signs and microscopic lesions of kidneys and/or livers were observed in all control puppies or adult controls (study 4), whereas no specific lesions were detected in vaccinates. These results support the protective effect of EURICAN® LR against renal and hepatic failures af- ter challenge with Leptospira.

Several studies have documented that vaccine-associated Leptospira MAT titers are generally low and short-lived [5,17,19,20,24] and, they should not be used to predict re- sistance to Leptospira infection. Although immunity against leptospirosis is thought to be primarily humoral [1], we did not find any correlation between post-vaccination antibody titers and protection since all vaccinated dogs were protect- ed after challenge whereas none of them seroconverted after vaccination, thereby confirming previous observations [17].

While leptospires were isolated from the blood of all con- trol puppies of the onset of immunity studies for at least 3 consecutive days after challenge with L. Canicola and L. Icterohaemorrhagiae, none of the vaccinated puppies be- came Leptospiraemic, except one. One vaccinated puppy was detected positive on a single occasion on day 3 after chal- lenge with L. Canicola without showing any clinical signs, urinary shedding, positive kidney, hematological or bio- chemical abnormalities, suggesting that the leptospiremia

was very transient. Due to the virulence of challenge strains, all adult controls of the duration of immunity studies but also vaccinated dogs were detected positive for several days when the challenge was performed 13 or 14 months after primary vaccination. However the duration of Leptospirae- mia was significantly reduced in vaccinates after both L. Ca- nicola and L. Icterohaemorrhagiae challenges. The absence of full protection against Leptospiraemia was also reported by others in duration of immunity studies, with either a chal- lenge dose 67 times lower (9 x 107 organsims/dog) [21] than the one used in our challenge model or in a challenge model where 50% only of adult control dogs became Leptospirae- mic [20].

Besides protecting against clinical signs, vaccination should prevent renal colonization and urine shedding, which is characteristic of a carrier or maintenance animal host. The renal carrier state is central to the persistence and epide- miology of leptospirosis [4]. In the onset of immunity stud- ies, 100% of control puppies but none of vaccinated puppies had urine or kidney samples positive for Leptospira isolation after L. Canicola and L. Icterohaemorrhagiae challenges. A complete protection against renal carriage was more dif- ficult to achieve in long-term studies, probably due to the severe challenge conditions in face of fading immunity. We found some discrepancies between urine and kidney isola- tion, this beeing also reported in previous observations [17]. This may be related to the presence of specific inhibiting enzymes from kidney cells [25], high urine osmolarity and pH [26], and/or the intermittent shedding of leptospires [26]. Nevertheless, the vaccine was able to provide a sig- nificant reduction of carriage/shedding under such condi- tions: urine samples were detected positive after L. Canicola and L. Icterohaemorrhagiae challenges in 87.5% and 100% of adult controls, respectively, whereas urine shedding was detected in only 12.5% and 25% of vaccinated adults, respectively. The urinary shedding, in terms of frequency of dogs with leptospiruria and duration of leptospiruria, was significantly reduced in long-term studies after challenges with both serovars. Kidney samples were positive after L. Canicola and L. Icterohaemorrhagiae challenges in 38% and 100% of adult controls, respectively and in 0% and 33% of vaccinated adults, respectively. The difference between vac- cinated and controls was significant after L. Icterohaemor- rhagiae challenge. The difference was not significant after

L. Canicola challenge due to the too low number of positive kidney samples in adult controls. It should be reminded that the challenge dose that we used was probably much higher than the one observed during a natural exposure, suggesting that the protection against renal carriage might be almost complete in the field. Indeed, contrasting results have been published in the literature about the protection against the establishment of a renal carrier stage, with a lack of protec- tive effect against urinary shedding for some vaccines evalu- ated shortly after primo-vaccination [9]. A significant long- term protection against urinary shedding and renal infection has been reported in less severe challenge models [20-22], with some urine samples detected positive for Leptospira in a few vaccinated dogs [20,22].

Rabies study shows serological data and challenge results 13 months after one dose of primary vaccination with the

EURICAN® DAPPi2-LR vaccine. The rabies challenge was vali- dated, as 100% of control dogs showed most of the expected typical signs of rabies and had to be euthanised. Post mor-

tem examinations of brain tissues confirmed the infection. Rabies antigen was detected by immunofluorescence test from brains of all animals from control group. In contrast, all the 25 vaccinated dogs resisted rabies challenge and were protected. Tissus collected from the vaccinates which sur- vived after challenge were found negative, confirming that they were free of virus and protected. As expected, serologi- cal results confirmed the immunogenicity of the vaccine, as 100% of the dogs developed rabies antibody titers above 0.5 IU/ ml after vaccination. This is not necessarily obvious for combination vaccine, as it was previously shown that Lepto- spiral components may strongly interfere with the raise of antibody titers [11]. In addition, in the field, in primovaccin- ated dogs, monovalent vaccines showed a better serological conversion rate than multivalent ones [11]. Interestingly, the EURICAN® DAPPi2-LR vaccine induced also an early se- roconversion 15 days post-vaccination against rabies with a peak observed 29 days post-vaccination, indicating a quick onset of immunity. A rapid decrease of rabies antibodies was observed and confirms the observation made by Cliquet [11]. Despite the absence or low levels of antibibodies spe- cific of rabies (below the WHO positivity threshold value of

0.5 UI/ml), all dogs resisted to rabies challenge 13 months after a single vaccination. These results suggest that quality of the priming and the initial seroconversion level are crucial for protection. Absence of antibody at the time of challenge in such correctly vaccinated animals does not mean absence of protection, as cell mediated response may also play an important role. In summary, these results demonstrate that a single injection with the EURICAN® DAPPi2-LR vaccine in- duced rabies virus neutralizing antibodies of at least 0.5 IU/ ml in all dogs from 2 weeks post-vaccination and that those dogs were protected more than one year after vaccination against a virulent rabies challenge.

EURICAN® DAPPi2-L was previously shown to provide a rapid onset of immunity and a long-lasting protection of at least 14 months against serovars Icterohaemorrhagiae and Canicola. The vaccine was able to prevent clinical disease and renal carrier state [17]. The antigen ratio between leptospirosis and rabies antigens of EURICAN® LR vaccine has been opti- mized, allowing a reduction of 2/3 of total amount of protein per dose compared to historical LEPTORABISIN®. Here, we show that a primary course of one dose of EURICAN® DAP- Pi2-L followed by one dose of EURICAN® DAPPi2-LR provided a quick onset of immunity as early as 2 weeks post vaccina- tion with full protection against fatal leptospirosis caused by L. Canicola and L. Icterohaemorrhagiae and with strong seroconversion against rabies. Long-term protection against leptospirosis and rabies was also demonstrated by challenge 13/14 months post-vaccination, with reduction of mortality, clinical signs, infection, renal carriage, and bacterial excre- tion for Leptospira and protection against mortality and clin- ical signs for all dogs challenged with rabies. These results

demonstrate that the LR formulation of the tested vaccine is optimal, providing good protection against Leptospira infec- tion without hampering the quality of rabies protection. This combined vaccine should help veterinarians to prevent clini- cal disease in dogs and limit the zoonotic risk and transmis- sion of leptospirosis and rabies between animals.

Acknowledgements

The authors wish to acknowledge Merial R&D Departments with a special thank to Jules Minke for their contribution in this work and critical reading of the manuscript. The authors wish to acknowledge Merial Technical services Department with a special thank to Jean-Christophe Thibault for his criti- cal reading of the manuscript.

Conflict Of Interest

Most authors of this paper are employees of Merial, the man- ufacturer of EURICAN® DAPPi2-L and EURICAN® DAPPi2-LR.

A.L. Guiot is an external scientific writer, she has no competing interest.

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