Comparison of antibody titres between intradermal and intramuscular rabies vaccination using inactivated vaccine in cattle in Bhutan

Karma Wangmo; Richard Laven; Florence Cliquet; Marine Wasniewski; Aaron Yang

doi:10.1371/journal.pone.0209946

Abstract

In developing countries, the cost of vaccination limits the use of prophylactic rabies vaccination, especially in cattle. Intradermal vaccination delivers antigen directly to an area with higher number of antigen-presenting cells. Therefore, it could produce equivalent or higher antibody titres than conventional intramuscular vaccination even when a lower dose is given. This study aimed to compare the antibody response in cattle vaccinated intramuscularly with 1mL of inactivated rabies vaccine (Raksharab, Indian Immunologicals) against intradermally vaccinated cattle with 0.2mL of the same vaccine. The study was conducted in Haa province of Bhutan where rabies is not endemic. One hundred cattle from 27 farms were selected for the study. Virus neutralising antibody (VNA) response was measured using the fluorescent antibody virus neutralisation test on the day of vaccination (day 0) and 14, 30, 60 and 90 days later. Overall, 71% of intradermally vaccinated cattle and 89% of the intramuscularly vaccinated cattle produced an adequate response (≥0.5IU/mL). On days 14 and 30 post vaccination fewer cattle (P<0.02) in the intradermal group had adequate titres with 36% and 58%, respectively, having titres ≥0.5 IU/mL compared to the equivalent figures of 78% and 77% in the intramuscular group. The mean VNA titres were lower for the intradermal group than intramuscular group (p<0.001) with the mean difference being > 0.6 IU/mL. Although low dose intradermal vaccination did produce a detectable antibody response, it was inferior to intramuscular vaccination. Thus, although intradermal vaccination has the potential to reduce the cost of vaccination by reducing the dose required, this study showed that a single dose of 0.2 mL intradermally was inferior to an intramuscular dose of 1 mL. Further research evaluating dose and dose regimen is needed before intradermal vaccination using the Raksharab rabies vaccine can be recommended in cattle.

Citation: Wangmo K, Laven R, Cliquet F, Wasniewski M, Yang A (2019) Comparison of antibody titres between intradermal and intramuscular rabies vaccination using inactivated vaccine in cattle in Bhutan. PLoS ONE 14(6): e0209946. https://doi.org/10.1371/journal.pone.0209946

Editor: Didier Raboisson, National Veterinary School of Toulouse, FRANCE

Received: December 10, 2018; Accepted: May 10, 2019; Published: June 10, 2019

Copyright: © 2019 Wangmo et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Data Availability: All relevant data are within the paper and its Supporting Information files.

Funding: The study was funded by New Zealand development scholarship, Massey University and ANSES, Nancy Laboratory for Rabies and Wildlife, France. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests: The authors have declared that there are no competing interest.

Introduction

Rabies is a fatal zoonotic viral disease inducing an acute disease of the central nervous system in almost all mammals [1]. In cattle, rabies causes significant economic losses to livestock farmers [2–5] principally through increased mortality. Furthermore, costs of euthanasia, diagnosis, replacement and vaccination of at risk herds add to the loss [6, 7].

In Bhutan, rabies is endemic in 29% of the country [8] with the dog being the principle vector and reservoir host. Cattle contract the disease as a spill-over infection from dogs; nevertheless over 80% of the economic loss due to rabies in Bhutan is due to cattle deaths [9]. A total of 1070 rabies cases were reported in animals in Bhutan between 1996 and 2016 of which 58 were recorded in 2016 [10]. The extensive grazing system (>60% of farms [11], free movement of cattle around their compound (>70%) and limited restrictions on access even when they are housed increase the risk of cattle contracting rabies from rabid dogs [12]. This risk is exacerbated by the large number of dogs in Bhutan (dog population estimated at 120 000 dogs, equivalent to one dog for every 2.5 cattle) of which 40% are stray dogs, and 31% owned but free roaming [13]. Mass vaccination against rabies and sterilization of dogs have been carried out throughout the country on an annual basis to reduce the risk of rabies. However, preventive control measures in cattle, especially vaccination, have been a lower priority, because the risks of virus transmission from cattle to humans are low [14]. Nevertheless, in endemic areas of Bhutan, rabies remains common in cattle and causes considerable economic losses to smallholder cattle farmers. Until rabies is eliminated in the reservoir hosts (principally dogs), free movement of reservoir hosts across the India/Bhutan border and from rabies endemic to non-endemic areas combined with limited and accessible housing of cattle means that rabies will continue to be a major concern in Bhutanese cattle with a significant economic issue for the individual affected smallholders.

As economic constraints at both national and local levels are key drivers for the lack of prophylactic vaccination in Bhutanese cattle, finding a way to reduce the costs of bovine vaccination could be useful for initiating stakeholders to improve vaccination coverage of cattle against rabies without markedly increasing the overall cost of rabies control. One potential method of reducing the cost of vaccination is to use intradermal vaccination. Compared to subcutaneous or intramuscular vaccination, intradermal vaccination results in the direct stimulation of a large population of active antigen-presenting cells [15]. This could, potentially, increase the magnitude of the immune response even if vaccination is used at a lower dose than that required for the intramuscular route.

Lower dose intradermal vaccination against rabies has been shown to be effective in humans, laboratory animals and dogs [16–18]. Data on use of intradermal rabies vaccination in cattle are sparse. A study by Koprowski et al. [19] reported that cattle (11 animals) vaccinated intradermally in the neck region with an attenuated rabies vaccine (1mL) produced protective antibody titres 30 days after vaccination. Asokkumar et al. [20] reported that intradermal vaccination of cattle with an inactivated vaccine (at 1/10 dose) was as effective at stimulating virus neutralising antibody (VNA) titres as the standard intramuscular dose. However, this was a post-exposure study and each animal received multiple doses of vaccine, so it has limited utility in determining the value of preventive intradermal vaccination. Bharti et al. [21] have also reported on experimentation with intradermal rabies vaccination as part of post‐ and pre‐exposure treatments. Benisek et al. [22] compared intradermal and intramuscular vaccination in a pre-exposure prophylaxis in cattle and found that intradermal vaccination (at 1/5 dose) produced higher mean VNA titres than the intramuscular route. This study had a small sample size (only 10 animals per group) and a limited statistical analysis. In addition, the variance in VNA titres of cattle treated using intradermal vaccination was much higher than that of cattle vaccinated intramuscularly. Thus, although these studies support the hypothesis that intradermal rabies vaccination could be effective in cattle, they do not provide sufficient proof to recommend it for preventive vaccination of cattle during mass parenteral vaccination programmes.

The aim of this study was to compare rabies VNA titres produced by intradermal vaccination at 1/5 of the recommended intramuscular dose (i.e. 0.2 mL) against 1 mL of intramuscular vaccination using the same vaccine in cattle in Bhutan.

Materials and methods

All animal use was approved by the Livestock Research Ethical Committee of the Ministry of Agriculture and Forest, Royal Government of Bhutan dated 10^th March 2017 and in accordance with the requirements of the Research Code of Practice for the Care and Use of Animals for Scientific Purposes.

Study design

The study was a multi-site non-inferiority trial with animals randomly allocated to either intradermal rabies vaccination using 1/5 of the recommended dose or standard intramuscular rabies vaccination.

Study area

The study was conducted in Haa district, which is located in north-western part of Bhutan. As of 2016, the district had approximately 9119 cattle and 1031 farms or household with cattle. Herd size ranged from one to hundred cattle [11]. As of March 2017, there had been no record of rabies in either dogs or cattle in Haa for five consecutive years (personal communication, Veterinary Officer, Haa). However, there are risks of future outbreaks as this district shares borders with other rabies endemic districts.

Sample size calculation

Sample size was calculated using the power calculation for a continuous outcome non-inferiority trial (https://www.sealedenvelope.com/power/continuous-noninferior/). At 5% significance level with 95% power, 0.63 as the standard deviation of the outcome [22] and a non-inferiority limit of 0.5, 35 animals were required in each group to detect if there was truly no difference between the intramuscular and intradermal route of vaccination in eliciting adequate rabies virus neutralising antibody titres in the vaccinated cattle. An additional 10 cattle were included in each group to account for losses during the course of the study. The non-inferiority limit of 0.5 was chosen considering the threshold limit for adequate rabies antibody titres of 0.5 IU/mL [23], and an expectation that peak titres produced by intramuscular vaccination would be at least 1.0 IU/mL [23].

Farm selection

The district annual livestock statistics records were used to select the animals for this study. All the data were recorded in an Excel sheet (Microsoft, USA). Of the six sub districts, three were excluded as they practiced the transhumant system of rearing, which meant that animals would not be available for follow up. The remaining three sub-districts, which as of 2016, had 523 farms and 3312 cattle were included in the study. All farms recorded as having less than four cattle in the herd were excluded by manual selection, leaving 271 farms. Twenty-five farms were selected randomly from this list using a random allocation table. Based on the census, these 25 farms had 260 cattle with maximum herd size of 50.

Animal selection

A minimum of four cattle per farm was required to get a final sample of 100 animals from 25 farms. However, on the day of vaccination, 12 of the pre-selected farms had less than four cattle in their herd, so extra farms that were located near to the pre-selected farms were used in addition to the original 25 farms to reach the target of at least 90 vaccinated cattle. In addition, on four farms more than four cattle were selected as four of the pre-selected farms practised the transhumant system of rearing. Only animals of age six months and above were eligible for selection in order to avoid any interference from maternal antibodies. On each farm, cattle were randomly assigned to treatment using a random allocation table. Ninety animals were designated for treatment ‘A’ or treatment ‘B with 45 cattle in each treatment group. Ten cattle were kept as controls.

The age, body condition score (BCS) (0–5 scale) [24] and breed of the selected cattle were recorded. Age ranged from 6 months to 15 years, BCS from 2–3, and the selected animals included a mixture of local and Jersey cross breeds.

Vaccination

Selected cattle were given either 1 mL of rabies vaccine [25] intramuscularly into the middle third of the neck, or 0.2 mL of the same vaccine intradermally at the same site based on Benisek et al. study [22]. All animals were vaccinated by the same veterinarian who also recorded the accuracy of placement of the intradermal vaccine by observing whether a bleb was formed at the injection site. An excellent bleb was formed in 43 of the 45 (>90%) intradermally vaccinated cattle.

The vaccine used was an inactivated cell culture vaccine authorised for intramuscular or subcutaneous use in cattle, dogs and cats (Raksharab company data). Potency testing at the OIE/EU/WHO reference laboratory on Rabies, Nancy, France showed that the vaccine used in the study had a potency of 8.5 IU/mL (minimum and maximum potency of 2.2 IU/mL and 37.4 IU/mL, respectively), greater than the recommended minimum of 1 IU/dose [23, 26].

On farms that had more than four cattle a control cow was selected for blood sampling but not vaccination. These control animals were equally distributed across the three selected sub districts.

All animals, irrespective of treatment group were managed as normal by the owners who were blinded to treatment allocation. Cattle on 17 of the 27 farms were housed permanently during the winter and stall fed; on 10 farms cattle were sent for grazing during the daytime in the winter. On 26/27 farms, during the summer, all adult cattle were released for grazing during the daytime and housed only at night. One farm housed cattle throughout the year, using two different sheds, one for winter and one for summer. The study started in late winter (March) and continued through to summer (June). Day 0 sample collection and vaccination was undertaken on 9^th to 11^th of March. On most farms, animals were housed for the first three sample collections (days 0, 14 and 30), but on days 60 and 90 all farms had started summer grazing except for the farm which housed their cows throughout the year.

Sample collection

Blood samples were collected on day 0 before vaccination and on days 14, 30, 60 and 90 after vaccination. To avoid grazing times, samples were collected from 6 am to 11 am and from 4 pm to 8 pm; approximately 10 mL of blood was collected via jugular venepuncture into plain vacutainers (BD, India). After collection, blood samples were allowed to settle for 10 to 15 minutes before moving on to the next farm.

For the blood samples collected in the morning, serum separation was undertaken during the afternoon (1 pm-3 pm), while for those collected in the evening, serum separation was done at night (9 pm-11 pm). Blood samples were centrifuged at 1000g for 15 to 20 minutes, and serum transferred to screw-capped cryovials for storage at -20°C [27]. Duplicate aliquots were collected for each sample. Each sample from a cow was assigned a sample identification number (order of sampling) so that the laboratory was blinded to the treatment group.

At the end of the sample collection period (i.e. late June), one serum aliquot per cow was transported by air to the OIE/EU/WHO reference laboratory on Rabies, Nancy in France for analysis.

Sero-neutralization test

The serum samples were analysed for VNA titres using the standard fluorescent antibody virus neutralisation test (FAVN) protocol as described by Cliquet et al. [28]. Briefly, the test was performed on 96 wells microtiter plate with serial threefold dilutions of the positive, negative controls and the test sera. The Challenge Virus Standard-11 ATCC number: VR 959, rabies virus produced in BHK-21 cell lines (ATCC number: CCL-10) was used as the challenge virus and Dulbecco’s modified Eagle’s medium with 10% fetal calf serum and antibiotics as a diluent.

50 μL of diluted challenge virus containing around 100 TCID₅₀ was added into the wells containing diluted samples and diluted positive and negative controls. A back titration of the diluted challenge virus was also performed. The microplates were then incubated at 36°C+/-2°C in a 5% carbon dioxide (CO2) humidified incubator for 1 hour. Following incubation, 50 μL of diluted BHK-21 cell suspension with concentration of 4 X 10⁵ cells/mL was added to each well and further incubated at 36°C±2°C in a 5% CO2 humidified incubator for 48 hours. The cell culture medium was then discarded and the microplates rinsed once in phosphate buffer solution and then in 80% acetone. The microplates were then fixed in 80% acetone at room temperature for 30 minutes which was drained off and air dried at room temperature.

Fluorescein isothiocyanate conjugate was used for staining microplate wells. The wells were read using a fluorescent microscope (between 100X and 125X magnifications) and examined for the presence or absence of fluorescent cells. It is an “all or nothing” reading therefore the well was considered positive if at least one fluorescent cell was detected.

Data analysis

In order to indicate response to vaccination, three antibody titre thresholds were used. These were the OIE minimum titre for seropositivity (≥0.5 IU/mL) [23], a lower minimum seropositive titre (≥0.24 IU/mL) recommended by several authors [29–31], and a titre indicating simply a vaccine response (≥0.17 IU/ml). The proportion of cattle meeting these thresholds at each time point were compared for the two treatment groups using a generalised linear model with a binomial output and a logit link, with cow as a random effect and time since vaccination and treatment group (and their interaction) as fixed effects.

The effects of time and treatment group on antibody titre were then analysed. The titre data were significantly right skewed so were log transformed before analysis. A repeat measures mixed model was used with log VNA titres as the outcome variable, cow as a random effect and time since vaccination and treatment group (and their interaction) as fixed effects. A heterogeneous first-order autoregressive covariance structure was used based on minimising the Akaike information criterion. These analyses were undertaken using SPSS statistics 24 (IBM, USA).

Further analysis was then undertaken to include factors other than treatment and time, and to establish; 1) variables that affected the probability of response to vaccination (titre ≥0.17 IU/mL), and 2) variables that were associated with strength of protection given a response.

An intercept-only repeated measures multilevel (including village, farm and cow levels) linear mixed model was fitted using log VNA titre as an outcome variable to determine whether the village and farm level random effects needed to be included. Once it was confirmed that they did not, a generalised estimating equation (GEE) model [32] with a binomial error distribution was then used to model the probability of response (titre ≥0.17 IU/mL). Initially several univariable models were created, with age, BCS, sex, breed and herd size. For this analysis BCS was recorded as 2, 2.5 and ≥3, herd size as 1–6, 7–8 and 9–11, and age as <2-years old, 2–5 years old and >5-years old to create categories with even group sizes. Independent variables that were related (p ≤ 0.20) were then included in a multivariable GEE model. Backward elimination was then used until all the independent variables in the model had p ≤0.05, except that a confounder that changed coefficients or standard errors of independent variables by ≥20% was forced into the model, regardless of its p-value. Independent variables that had been removed were then added back into the model one by one, and retained if p ≤0.05. After building the main effect model, two-way interactions were created using all pairs of independent variables, interactions were included in the final model if p ≤0.05. This model building strategy was then used to build a GEE model of the strength of protection (log transformed titre). This model had first order auto-regressive work correlation matrix and normal error distribution. These analyses were carried out using STATA 13.1 (StataCorp, USA).

Results

Descriptive data

The characteristics of the cattle included under each treatment group; intramuscular and intradermal are summarised in Table 1. Although all the cattle were randomly allocated to treatment, the proportion of cattle in the intradermal group which were <2 years of age was higher than in the intramuscular group (chi-square test, P-value <0.001). However, according to the chi‐square test, there was no significant heterogeneity in any of the other factors (sex, breed, herd size, BCS, chi-square test, P-values 0.157, 1, 0.967, 0.338 respectively).

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Table 1. Characteristics of cattle in each treatment group.

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Proportion of animals that responded to the vaccination

On day 0, all tested cattle had titres <0.17 IU/mL. Based on this cut-off point, the overall response rate for the intramuscular group was 98% and for the intradermal group 96% (Table 2). According to the p values calculated using generalised linear model, the overall proportion of the cattle with VNA titre ≥ 0.17 IU/mL was not affected by route of vaccination (p = 0.35) or the interaction between vaccination route and time after vaccination (p = 0.31). However, compared to Day 0, the proportion of vaccinated cattle with a titre ≥ 0.17 IU/mL was greater on all other sample days (p<0.001); this was also the case for days 14 and 30 compared to day 90 (p≤0.02).

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Table 2. Proportion of cattle that responded to rabies vaccination (based on VNA titre ≥0.17 IU/mL).

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Proportion of animals with an adequate VNA titre

The proportion of vaccinated cattle with VNA titre ≥0.24 IU/mL) is summarised in Table 3.

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Table 3. Proportion of cattle in each vaccination group with VNA titre ≥ 0.24 IU/mL.

https://doi.org/10.1371/journal.pone.0209946.t003

As with the proportion of cattle with a titre ≥0.17 IU/mL, according to the p values calculated using a generalised linear model, there was no effect of vaccination route or interaction between vaccination route and time on the odds of a vaccinated animal having a titre ≥0.24 IU/mL (p = 0.67 and 0.38, respectively), but there was an effect of time since vaccination (p<0.001) S1 Fig.

Proportion of animals with a VNA titre above the OIE minimum for sero‐positivity (0.5 IU/mL).

According to the p values calculated using a generalised linear model, no effect of vaccination route on proportion of titres ≥0.5 IU/mL was found (p = 0.538). However, both the interaction between vaccination route and time and time alone were significant at the 4% level (p = 0.039 and <0.001, respectively). On both day 14 and day 30 the proportion of cattle with a VNA titre ≥ 0.5 IU/mL was lower in the intradermally vaccinated group than in the intramuscularly vaccinated group (p<0.001 and 0.02, respectively) (Table 4).

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Table 4. Proportion of cattle with VNA titre ≥ 0.5 IU/mL at 0,14,30,60 and 90 days.

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Effect of vaccination route and time on VNA titres

Time, vaccination route and their interaction were found to have an effect on VNA titres (p<0.001) (see Fig 1). In both groups, VNA titres were higher after vaccination throughout the study period than before vaccination (p<0.001 for all comparisons), with the mean VNA titres of both groups peaking on day 30. The decline in titres after the peak was more marked in the intramuscularly vaccinated group than the intradermal group with titres being significantly lower on day 60 than on day 30 in the former group (p<0.001), but only by day 90 in the intradermally vaccinated group.

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Fig 1. Geometric mean VNA titres of intramuscularly (im) and intradermally (id) vaccinated cattle on 0, 14, 30, 60 and 90 days post vaccination.

The error bars indicate 95% confidence interval.

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Mean antibody titres were significantly lower on days 14 and 30 in the intradermally vaccinated group than in the intramuscularly vaccinated group. On day 14 the back transformed mean difference between the VNA titres of intramuscularly and intradermally vaccinated animals was 0.62 (95% CI 0.02 to 1.3) IU/mL, whereas on day 30 it was 0.66 (95% CI 0.22 to 1.39) IU/mL. Thus, in terms of the antibody response, the intradermal vaccination was inferior to the intramuscular vaccination.

Effect of cow and farm level factors on response to vaccination

The GEE modelling process found that there was a significant interaction between route and age on the likelihood of a sample having a titre ≥0.17 IU/mL. For cattle <2 years old the odds of a sample titre being ≥0.17 IU/mL; was 4.8 (95%CI: 1.3–17.7) times greater for intradermal than intramuscular vaccinated animals group, whereas for animals between 2 and 5 years of age the equivalent odds ratio was 0.24 (95%CI: 0.06–0.97), and for cattle >5 years old 0.34 (95%CI: 0.04–2.73). Apart from time the only factor which influenced the odds of an antibody response ≥0.17 IU/mL, was herd size, with samples from herds with more than six cattle having lower odds of titres ≥0.17 IU/mL.

For the actual titres, apart from time, the only significant effect found was an effect of age and its interaction with route of vaccination. Geometric mean VNA titres from GEE model were 0.53IU/mL for cattle <2 years, 0.38 IU/mL for 2–5 years and 0.33 IU/mL for cattle >5 years. For intramuscularly vaccinated cattle, mean titre increased with age whereas for intradermally vaccinated cattle it decreased. Titres of <2-year-old cattle vaccinated intradermally were higher than those of <2-year-old cattle vaccinated intramuscularly; this was reversed in the other two age categories.

Discussion

This is the first study to compare the efficacy of intramuscular and intradermal routes of rabies vaccination in cattle in Bhutan under field conditions. It is also, as far as the authors are aware, the first study of intradermal vaccination against rabies in cattle to use more than 10 cattle per treatment group. The study was designed as a non-inferiority trial with the aim of confirming whether the mean VNA titres produced by intradermal vaccination were no more than 0.5 IU/mL lower than the titres produced by the standard intramuscular vaccination. In addition, three thresholds of vaccination response were used in order to further compare the response of the two vaccination routes.

The geometric mean VNA production by intradermal vaccination using 1/5 (0.2mL) of the dose used in standard intramuscular (1mL) route was significantly lower than the standard intramuscular route on days 14 and 30 post vaccination. The back transformed mean difference between intramuscular and intradermal groups was >0.6 IU/mL, indicating that based on the criteria of the study, intradermal vaccination was inferior to intramuscular vaccination. Furthermore, the geometric mean titre in the intradermally vaccinated cattle did not achieve the WHO and OIE recommended threshold titre of ≥0.5 IU/mL on any day post vaccination. Furthermore, the proportion of intradermally vaccinated cattle with titres ≥0.5 IU/mL were significantly lower (P≤0.02) than in the intramuscular group on days 14 and 30 post vaccination—with 36 and 58% having titres ≥0.5 IU/mL on day 14 and 30, respectively compared to the equivalent figures of 78 and 77% in the intramuscular group. Nevertheless, overall 71% (32/45) of the intradermally vaccinated cattle had a titre ≥0.5 IU/mL on at least one day (in comparison to 89% (41/45) of the intramuscularly vaccinated cattle).

This finding is in contrast to the findings of Asokkumar et al. [20] that used the same vaccine brand as used in this study. They measured VNA titres using Rapid Fluorescent Focus Inhibition Test (RFFIT) and reported intradermal vaccination produced titres equivalent to those produced by the intramuscular route despite using 1/5 of the dose (VNA titre in IU/mL; Zero day- 0.07 for IM and ID, 14^th day 0.89 for IM and 0.6 for ID; 28^th day 2.81 for IM and 2.54 for ID). However, in addition to being a small study (8 cattle per treatment group), this was a post-exposure prophylaxis study, so cattle were vaccinated on days 0, 3, 7, 14 and 28, significantly increasing the chance of a response. Furthermore, as a post-exposure study with no untreated controls, it is not clear whether any of the response was due to exposure to wild-type virus.

A more directly comparable study is that by Benisek et al. [22] who in unexposed cattle reported that the VNA response in their intradermally vaccinated group was significantly greater than intramuscular group despite using only 1/5^th of the dose in the former group. The VNA titres, measured by RFFIT, for day 14 were 0.51 IU/mL and 0.73 IU/mL for intramuscular and intradermal routes respectively. Similarly, the day 35 and 90 were also greater for the intradermal group than the intramuscular (1.64 IU/mL vs 1.07 IU/mL and 1.40 IU/mL vs 0.94 IU/mL respectively).

It is unclear why Benisek et al. [22] found different results from this study. Although they used a different brand of vaccine (Rabicell) it was also an inactivated rabies vaccine with an aluminium hydroxide adjuvant. The response to the intramuscular vaccination reported by Benisek et al. [22] was different from that seen in this study. The mean VNA titres in their study were still >0.5 IU/mL 180 days after intramuscular vaccination compared to this study in which mean VNA titres after intramuscular vaccination were <0.5 IU/mL within 90 days. Furthermore, the proportion of cattle with a titre ≥0.5 IU/mL on days 14 and 30 in the intramuscularly vaccinated group in this study were lower than the WHO targets for tissue culture rabies vaccine of almost 100% [33].

The results of this study seem to be in contrast to the undoubted efficacy of intradermal rabies vaccination in humans [18]. However, in humans pre-exposure vaccination is a multidose regimen that requires three to four doses of vaccine [34, 35]. Furthermore, the results of the current study are consistent with the statement made by WHO [36] that ‘antibody titres are higher and more sustained after intramuscular injection’.

One potential issue with intradermal vaccination is that it is more difficult than subcutaneous vaccination, so some vaccines could have been incorrectly administered into adipose or subcutaneous tissue. However, >90% of vaccinations were recorded as definitively going intradermally. Another potential issue was that as there was no licensed rabies vaccine for intradermal use in cattle, a 10 mL vaccine vial was used for this study. Repeated drawing of vaccine from this multidose vial could have resulted in some animals receiving a dose less than 0.2 mL. Finally, cattle were released for grazing after vaccination and were not monitored afterwards. As intradermal administration can cause irritation at the vaccination site [37], rubbing induced by irritation at the injection site could have led to leakage of vaccine before being absorbed into the system. Thus, it is plausible that despite a sufficient dose being given intradermally, the vaccine was not retained long enough in the dermal tissue to be absorbed.

Nonetheless, all of these issues would affect individual cows, thereby increasing variability in VNA response between animals not vaccinated correctly and those which were. However, there was no evidence of variability in this study, in contrast to Benisek et al. [22] study in which intradermal vaccination was associated with an increase in variability of VNA titres. Thus, the most plausible rationale for the difference between the results of this study and that of Benisek et al. [22] is differences between vaccines, different tests for VNA measurements (RFFIT vs FAVN test) or between the cattle in each study. One hypothesis is the age of the animals used in the two studies. Whereas this study used animals of all ages, Benisek et al. [22] reported that they used ‘young bulls’ in their study, The data from this study suggest that young cattle (<2 years of age) vaccinated intradermally responded better than adult cattle, so this difference in age range could be partly responsible for the difference between the two studies. Furthermore, a classical antibody response curve was produced in this study with a rise on day 14^th after vaccination, peak at day 30 and fall on day 60 and 90. However, this titre curve was not observed by Benisek et al. [22].

The finding of this study that young cattle (< 2years old) seem to be better at responding to intradermal vaccination than older cattle could be potentially valuable in developing prophylactic regimes against rabies in cattle. However, it needs further confirmation as there were only five young cattle in the intramuscular group (Table 1). Therefore, the results are only suggestive of a possible effect that requires further research with age group factored into the study design.

Even disregarding the impact of age the effect of route on VNA titres may not have been as great as suggested in Fig 1. When the cut-off for a protective titre was reduced from ≥0.5 to ≥0.24 IU/mL, there was no difference in response between the two routes and geometric mean VNA titres in both groups were ≥0.24 IU/mL throughout the duration of the study following vaccination. This cut-off point was chosen based on the vaccination and challenge studies conducted in cattle, dogs, cats [29, 30, 38, 39] and foxes [31]. Côrtes et al. [30] reported a protection rate of 80% in cattle with titre ≥0.24 IU/mL when challenged with a virulent strain of rabies virus. However, there was only a 5% increase in the proportion protected with titre ≥ 0.5 IU/mL.

Thus, although a higher VNA titre is preferred, any seroconversion following vaccination indicates some degree of protection [29, 39], particularly against natural infection, which is usually less severe than the experimental infection used to set thresholds [29, 40]. However, much of this is based on data from dogs, which are reservoir hosts and there may therefore be a certain degree of host-virus adaptation that reduces the risk of infection in dogs compared to dead end hosts such as cattle [29].

Thus, the results of this study do not support the routine use of intradermal vaccination of cattle using the Raksharab vaccine at a dose rate of 0.2 mL, except, perhaps, in cattle <2 years old. Further studies are needed, in particular on the use of a booster vaccination 60 days after primary vaccination, or on the use of a higher dose than the 0.2 IU/mL used in this study. The latter is likely to have lower costs and be more feasible, and increasing the intradermal dose is easier in cattle than humans as cattle skin is relatively thick and it is therefore easier to administer larger quantities of vaccine at one site [41].

Conclusion

Intradermal vaccination induced protective titres (≥ 0.5 IU/mL) in 71% of cattle, despite using 1/5 of the recommended dose. However, the proportion of cattle with a protective titre was significantly lower than for cattle given the standard dose (1mL) intramuscularly (71 vs 89%). In addition, mean antibody titres in the intradermally vaccinated cattle were significantly lower than the intramuscularly vaccinated cattle on days 14 and 30 post vaccination. Intradermal vaccination using 1/5 dose was inferior to intramuscular vaccination using the standard dose. However, the antibody responses obtained in this study were good enough to support further testing of intradermal vaccination with an increased dose.

Supporting information

S1 Dataset. Vaccination groups and description of study cattle.

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(XLSX)

S1 Fig. Pairwise comparisons of the effect of time since vaccination on proportion of vaccinated cattle with rabies VNA titres ≥0.24 IU/mL.

https://doi.org/10.1371/journal.pone.0209946.s002

(DOCX)

S1 Table. Generalised estimating equation model outputs.

https://doi.org/10.1371/journal.pone.0209946.s003

(DOCX)

Acknowledgments

We would like to sincerely acknowledge Alexandre Servat, Valère Brogat, Sébastien Kempf, Anouck Labadie, Estelle Litaize, Jonathan Rieder, Laetitia Tribout and Jean Luc Schereffer at ANSES, Nancy Laboratory for Rabies and Wildlife, France for analysing the rabies vaccine and serum samples.

We are grateful to Dr. Sonam Peldon and her team at Haa, Bhutan for helping to collect and process samples.

We are also thankful to all the cattle owners who agreed to participate in this study and the Royal Government of Bhutan and Department of Livestock for giving the authorizations to conduct this study in Bhutan.

Lastly, we thank Kencho Sum (Triple Gem), my family members and friends (Xue Qi Soon, Dr. Mary Gaddam, Dr. Linda Laven) for giving us the moral support and inspiration to pursue and complete this study.

References

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5. Mayen F. Haematophagous bats in Brazil, their role in rabies transmission, impact on public health, livestock industry and alternatives to an indiscriminate reduction of bat population. Journal of Veterinary Medicine Series B-Infectious Diseases and Veterinary Public Health. 2003;50(10):469–72.
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- Google Scholar
9. Tenzin Dhand NK, Ward MP. Anthropogenic and environmental risk factors for rabies occurrence in Bhutan. Preventive Veterinary Medicine. 2012;107(1–2):21–6. pmid:22673581
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- PubMed/NCBI
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10. National Center for Animal Health. National Rabies Prevention and Control Plan. Bhutan: Department of Livestock; 2017.
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- View Article
- Google Scholar
21. Bharti OK, Sharma UK, Kumar A, Phull A. Exploring the Feasibility of a New Low Cost Intra-Dermal Pre & Post Exposure Rabies Prophylaxis Protocol in Domestic Bovine in Jawali Veterinary Hospital, District Kangra, Himachal Pradesh, India. World. 2018;8:8–20.
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- View Article
- Google Scholar
27. Rodrigues da Silva ADC, Caporale GMM, Gonçalves CA, Targueta MC, Comin F, Zanetti CR, et al. Antibody response in cattle after vaccination with inactivated and attenuated rabies vaccines. Revista do Instituto de Medicina Tropical de Sao Paulo. 2000;42(2):95–8. pmid:10810324
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- PubMed/NCBI
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28. Cliquet F, Aubert M, Sagne L. Development of a fluorescent antibody virus neutralisation test (FAVN test) for the quantitation of rabies-neutralising antibody. Journal of Immunological Methods. 1998;212(1):79–87. pmid:9671155
- View Article
- PubMed/NCBI
- Google Scholar
29. Aubert MF. Practical significance of rabies antibodies in cats and dogs. OIE Revue Scientifique et Technique. 1992;11(3):735–60.
- View Article
- Google Scholar
30. Côrtes JA, Rweyemamu MM, Ito FH, Umehara O, Medeiros Neto RR, De Lucca-Neto D, et al. Immune response in cattle induced by inactivated rabies vaccine adjuvanted with aluminium hydroxide either alone or in combination with avridine. OIE Revue Scientifique et Technique. 1993;12(3):941–55.
- View Article
- Google Scholar
31. Cliquet F, Sagne L, Schereffer JL, Aubert MFA. ELISA test for rabies antibody titration in orally vaccinated foxes sampled in the fields. Vaccine. 2000;18(28):3272–9. pmid:10869772
- View Article
- PubMed/NCBI
- Google Scholar
32. Zeger SL, Liang KY, Albert PS. Models for longitudinal data: A generalized estimating equation approach. Biometrics. 1988;44(4):1049–60. pmid:3233245
- View Article
- PubMed/NCBI
- Google Scholar
33. Sudarshan MK, Ravish HS, Narayana DHA. Time interval for booster vaccination following reexposure to rabies in previously vaccinated subjects. Asian Biomedicine. 2011;5(5):589–93.
- View Article
- Google Scholar
34. Khawplod P, Wilde H, Sirikwin S, Benjawongkulchai M, Limusanno S, Jaijaroensab W, et al. Revision of the Thai Red Cross intradermal rabies post-exposure regimen by eliminating the 90-day booster injection. Vaccine. 2006;24(16):3084–6. pmid:16494972
- View Article
- PubMed/NCBI
- Google Scholar
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- View Article
- PubMed/NCBI
- Google Scholar
36. WHO. WHO expert consultation on rabies: second report: World Health Organization; 2013.
37. Vescovo P, Rettby N, Ramaniraka N, Liberman J, Hart K, Cachemaille A, et al. Safety, tolerability and efficacy of intradermal rabies immunization with DebioJect (TM). Vaccine. 2017;35(14):1782–8. pmid:28317660
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- PubMed/NCBI
- Google Scholar
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- PubMed/NCBI
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- View Article
- PubMed/NCBI
- Google Scholar
40. Larghi OP, Nebel AE. Duration of immunity afforded to cattle by a binary‐ethylenimine inactivated rabies vaccine. Zentralblatt für Veterinärmedizin Reihe B. 1985;32(1–10):609–15.
- View Article
- Google Scholar
41. Itzchak S, Jacob B, Avraham R, Benami P. Enhancement of the immune response by intradermal vaccination in cattle with enterotoxigenic Escherichia coli (ETEC) vaccine without adjuvant. Vaccine. 1992;10(4):217–20. pmid:1561828
- View Article
- PubMed/NCBI
- Google Scholar

[ref1] 1. Rupprecht CE, Hanlon CA, Hemachudha T. Rabies re-examined. Lancet Infectious Diseases. 2002;2(6):327–43. pmid:12144896
View Article
PubMed/NCBI
Google Scholar

[2] View Article

[3] PubMed/NCBI

[4] Google Scholar

[ref2] 2. Anderson A, Shwiff S, Gebhardt K, Ramirez AJ, Shwiff S, Kohler D, et al. Economic Evaluation of Vampire Bat (Desmodus rotundus) Rabies Prevention in Mexico. Transboundary and Emerging Diseases. 2014;61(2):140–6. pmid:22984914
View Article
PubMed/NCBI
Google Scholar

[6] View Article

[7] PubMed/NCBI

[8] Google Scholar

[ref3] 3. Feng Y, Shi YY, Yu MY, Xu WD, Gong WJ, Tu ZZ, et al. Livestock rabies outbreaks in Shanxi province, China. Archives of Virology. 2016;161(10):2851–4. pmid:27422397
View Article
PubMed/NCBI
Google Scholar

[10] View Article

[11] PubMed/NCBI

[12] Google Scholar

[ref4] 4. Jibat T, Mourits MCM, Hogeveen H. Incidence and economic impact of rabies in the cattle population of Ethiopia. Preventive Veterinary Medicine. 2016;130:67–76. pmid:27435648
View Article
PubMed/NCBI
Google Scholar

[14] View Article

[15] PubMed/NCBI

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[ref5] 5. Mayen F. Haematophagous bats in Brazil, their role in rabies transmission, impact on public health, livestock industry and alternatives to an indiscriminate reduction of bat population. Journal of Veterinary Medicine Series B-Infectious Diseases and Veterinary Public Health. 2003;50(10):469–72.
View Article
Google Scholar

[18] View Article

[19] Google Scholar

[ref6] 6. Thiptara A, Atwill ER, Kongkaew W, Chomel BB. Epidemiologic Trends of Rabies in Domestic Animals in Southern Thailand, 1994–2008. American Journal of Tropical Medicine and Hygiene. 2011;85(1):138–45. pmid:21734139
View Article
PubMed/NCBI
Google Scholar

[21] View Article

[22] PubMed/NCBI

[23] Google Scholar

[ref7] 7. Vos A, Un H, Hampson K, De Balogh K, Aylan O, Freuling CM, et al. Bovine rabies in Turkey: patterns of infection and implications for costs and control. Epidemiology and Infection. 2014;142(9):1925–33. pmid:24280252
View Article
PubMed/NCBI
Google Scholar

[25] View Article

[26] PubMed/NCBI

[27] Google Scholar

[ref8] 8. Tenzin Dhand NK, Ward MP. Patterns of rabies occurrence in bhutan between 1996 and 2009. Zoonoses and Public Health. 2011;58(7):463–71. pmid:21843156
View Article
PubMed/NCBI
Google Scholar

[29] View Article

[30] PubMed/NCBI

[31] Google Scholar

[ref9] 9. Tenzin Dhand NK, Ward MP. Anthropogenic and environmental risk factors for rabies occurrence in Bhutan. Preventive Veterinary Medicine. 2012;107(1–2):21–6. pmid:22673581
View Article
PubMed/NCBI
Google Scholar

[33] View Article

[34] PubMed/NCBI

[35] Google Scholar

[ref10] 10. National Center for Animal Health. National Rabies Prevention and Control Plan. Bhutan: Department of Livestock; 2017.

[ref11] 11. MoAF. Livestock statistics. Ministry of agriculture and forest (MoAF); 2016.

[ref12] 12. Dukpa K, Robertson ID, Edwards JR, Ellis TM, Tshering P, Rinzin K, et al. Risk factors for foot-and-mouth disease in sedentary livestock herds in selected villages in four regions of Bhutan. New Zealand Veterinary Journal. 2011;59(2):51–8. pmid:21409730
View Article
PubMed/NCBI
Google Scholar

[39] View Article

[40] PubMed/NCBI

[41] Google Scholar

[ref13] 13. Rinzin K, Tenzin T, Robertson I. Size and demography pattern of the domestic dog population in Bhutan: Implications for dog population management and disease control. Preventive Veterinary Medicine. 2016;126:39–47. pmid:26873612
View Article
PubMed/NCBI
Google Scholar

[43] View Article

[44] PubMed/NCBI

[45] Google Scholar

[ref14] 14. Tenzin Dhand NK, Dorjee J Ward MP. Re-emergence of rabies in dogs and other domestic animals in eastern Bhutan, 2005–2007. Epidemiology and Infection. 2011;139(2):220–5. pmid:20492745
View Article
PubMed/NCBI
Google Scholar

[47] View Article

[48] PubMed/NCBI

[49] Google Scholar

[ref15] 15. Malissen B, Tamoutounour S, Henri S. The origins and functions of dendritic cells and macrophages in the skin. Nature Reviews Immunology. 2014;14(6):417–28. pmid:24854591
View Article
PubMed/NCBI
Google Scholar

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[52] PubMed/NCBI

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[ref16] 16. Ray NB, Ewalt LC, Lodmell DL. Nanogram quantities of plasmid DNA encoding the rabies virus glycoprotein protect mice against lethal rabies virus infection. Vaccine. 1997;15(8):892–5. pmid:9234541
View Article
PubMed/NCBI
Google Scholar

[55] View Article

[56] PubMed/NCBI

[57] Google Scholar

[ref17] 17. Lodmell DL, Ewalt LC, Parnell MJ, Rupprecht CE, Hanlon CA. One-time intradermal DNA vaccination in ear pinnae one year prior to infection protects dogs against rabies virus. Vaccine. 2006;24(4):412–6. pmid:16153757
View Article
PubMed/NCBI
Google Scholar

[59] View Article

[60] PubMed/NCBI

[61] Google Scholar

[ref18] 18. Madhusudana SN, Mani RS. Intradermal vaccination for rabies prophylaxis: conceptualization, evolution, present status and future. Expert Review of Vaccines. 2014;13(5):641–55. pmid:24655026
View Article
PubMed/NCBI
Google Scholar

[63] View Article

[64] PubMed/NCBI

[65] Google Scholar

[ref19] 19. Koprowski H, Black J, Johnson WP. Rabies in cattle. IV. Vaccination of cattle with high egg-passage. Journal of the American Veterinary Medical Association. 1955;127(943):363–6. pmid:13263233
View Article
PubMed/NCBI
Google Scholar

[67] View Article

[68] PubMed/NCBI

[69] Google Scholar

[ref20] 20. Asokkumar M, Ganesan PI, Sekar M, Anuradha P, Balakrishnan S. Vaccination studies against rabies in farm and pet animals using different immunization routes. Indian Veterinary Journal. 2016;93(10):33–6.
View Article
Google Scholar

[71] View Article

[72] Google Scholar

[ref21] 21. Bharti OK, Sharma UK, Kumar A, Phull A. Exploring the Feasibility of a New Low Cost Intra-Dermal Pre & Post Exposure Rabies Prophylaxis Protocol in Domestic Bovine in Jawali Veterinary Hospital, District Kangra, Himachal Pradesh, India. World. 2018;8:8–20.
View Article
Google Scholar

[74] View Article

[75] Google Scholar

[ref22] 22. Benisek Z, Suli J, Svrcek S, Ondrejkova A, Mojzisova J, Ondrejka R. Intradermal anti-rabies immunization—Possibilities of needleless rabies vaccine administration. Bulletin of the Veterinary Institute in Pulawy. 2006;50(2):137–42.
View Article
Google Scholar

[77] View Article

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View Article
Google Scholar

[80] View Article

[81] Google Scholar

[ref24] 24. Roche JR, Kay JK, Friggens NC, Loor JJ, Berry DP. Assessing and managing body condition score for the prevention of metabolic disease in dairy cows. Veterinary Clinics of North America—Food Animal Practice. 2013;29(2):323–36.
View Article
Google Scholar

[83] View Article

[84] Google Scholar

[ref25] 25. Raksharab. Raksharab Profile 2011 [Available from: http://www.poulvet.com/vetproducts/medicine_detail.php?mediid=706.
View Article
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View Article
Google Scholar

[89] View Article

[90] Google Scholar

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View Article
PubMed/NCBI
Google Scholar

[92] View Article

[93] PubMed/NCBI

[94] Google Scholar

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View Article
PubMed/NCBI
Google Scholar

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[97] PubMed/NCBI

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View Article
Google Scholar

[100] View Article

[101] Google Scholar

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View Article
Google Scholar

[103] View Article

[104] Google Scholar

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View Article
PubMed/NCBI
Google Scholar

[106] View Article

[107] PubMed/NCBI

[108] Google Scholar

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PubMed/NCBI
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[111] PubMed/NCBI

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View Article
Google Scholar

[114] View Article

[115] Google Scholar

[ref34] 34. Khawplod P, Wilde H, Sirikwin S, Benjawongkulchai M, Limusanno S, Jaijaroensab W, et al. Revision of the Thai Red Cross intradermal rabies post-exposure regimen by eliminating the 90-day booster injection. Vaccine. 2006;24(16):3084–6. pmid:16494972
View Article
PubMed/NCBI
Google Scholar

[117] View Article

[118] PubMed/NCBI

[119] Google Scholar

[ref35] 35. Permpalung N, Wongrakpanich S, Korpaisarn S, Tanratana P, Angsanakul J. Trend of human rabies prophylaxis in developing countries: Toward optimal rabies immunization. Vaccine. 2013;31(38):4079–83. pmid:23845809
View Article
PubMed/NCBI
Google Scholar

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[122] PubMed/NCBI

[123] Google Scholar

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[ref37] 37. Vescovo P, Rettby N, Ramaniraka N, Liberman J, Hart K, Cachemaille A, et al. Safety, tolerability and efficacy of intradermal rabies immunization with DebioJect (TM). Vaccine. 2017;35(14):1782–8. pmid:28317660
View Article
PubMed/NCBI
Google Scholar

[126] View Article

[127] PubMed/NCBI

[128] Google Scholar

[ref38] 38. Hammami S, Schumacher C, Cliquet F, Tlatli A, Aubert A, Aubert M. Vaccination of Tunisian dogs with the lyophilised SAG2 oral rabies vaccine incorporated into the DBL2 dog bait. Veterinary Research. 1999;30(6):607–13. pmid:10596408
View Article
PubMed/NCBI
Google Scholar

[130] View Article

[131] PubMed/NCBI

[132] Google Scholar

[ref39] 39. Darkaoui S, Fihri OF, Schereffer JL, Aboulfidaa N, Wasniewski M, Zouine K, et al. Immunogenicity and efficacy of Rabivac vaccine for animal rabies control in Morocco. Clinical and Experimental Vaccine Research. 2016;5(1):60–9. pmid:26866025
View Article
PubMed/NCBI
Google Scholar

[134] View Article

[135] PubMed/NCBI

[136] Google Scholar

[ref40] 40. Larghi OP, Nebel AE. Duration of immunity afforded to cattle by a binary‐ethylenimine inactivated rabies vaccine. Zentralblatt für Veterinärmedizin Reihe B. 1985;32(1–10):609–15.
View Article
Google Scholar

[138] View Article

[139] Google Scholar

[ref41] 41. Itzchak S, Jacob B, Avraham R, Benami P. Enhancement of the immune response by intradermal vaccination in cattle with enterotoxigenic Escherichia coli (ETEC) vaccine without adjuvant. Vaccine. 1992;10(4):217–20. pmid:1561828
View Article
PubMed/NCBI
Google Scholar

[141] View Article

[142] PubMed/NCBI

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Figures

Abstract

Introduction

Materials and methods

Study design

Study area

Sample size calculation

Farm selection

Animal selection

Vaccination

Sample collection

Sero-neutralization test

Data analysis

Results

Descriptive data

Proportion of animals that responded to the vaccination

Proportion of animals with an adequate VNA titre

Proportion of animals with a VNA titre above the OIE minimum for sero‐positivity (0.5 IU/mL).

Effect of vaccination route and time on VNA titres

Effect of cow and farm level factors on response to vaccination

Discussion

Conclusion

Supporting information

S1 Dataset. Vaccination groups and description of study cattle.

S1 Fig. Pairwise comparisons of the effect of time since vaccination on proportion of vaccinated cattle with rabies VNA titres ≥0.24 IU/mL.

S1 Table. Generalised estimating equation model outputs.

Acknowledgments

References