Detection of virus-neutralising antibodies and associated factors against rabies in the vaccinated household dogs of Kathmandu Valley, Nepal.

BACKGROUND: Rabies is a vaccine-preventable neglected tropical viral zoonosis. It occurs worldwide, creating a very heavy burden in many developing countries, including Nepal. Dogs are the principle vector for the transmission of this disease in urban areas. Vaccination is the most important preventive measure in areas where dogs are the principle source of infection. This study was conducted with the aim of detecting virus-neutralising antibodies and associated factors against rabies in vaccinated household dogs of Kathmandu valley.
METHODS: Blood samples were collected from 110 vaccinated pet dogs in Kathmandu, Bhaktapur, and Lalitpur districts of Nepal. The samples were taken to the laboratory of the National Zoonosis and Food Hygiene Research Center where serum was separated. An indirect immune-enzymatic assay (PlateliaTM Rabies II kit ad usum Veterinarium, Biorad, China) was used for the detection of rabies virus anti-glycoprotein antibodies in the dog serum samples following the manufacturer's recommendations and instructions. Optical density values for unknown samples were compared with the positive sera titers in quantification tests obtained after a direct reading on the standard curve. Results were expressed as equivalent units per ml (EU/ml).
FINDINGS: Of the total samples, 89.09% exceeded the required seroconversion level (≥ 0.5 EU/ml); another 9.09% did not reach the seroconversion level (0.125-0.5 EU/ml); and 1.81% had undetectable seroconversion levels (<0.125 EU/ml) suggesting that the animal had not seroconverted according to the PLATELIA™ RABIES II test. Only one factor, the condition under which the dog was kept, was significantly associated with the antibody titer level. No association was found for any of the other factors included in the study.
INTERPRETATION: Vaccination is the most effective measure for prevention and control of rabies. The locally manufactured brand of vaccine, which is available in Nepal, is potent enough to generate a sufficient amount of protective antibodies, equal to international brands.

Introduction

Rabies is a vaccine-preventable, viral zoonosis belonging to neglected tropical diseases that can infect all mammals, but over 99% of all human death from rabies are caused by domestic dogs [1]. According to the World Health Organization (WHO), 59,000 people die from this disease yearly, of which 30–50% are below 15 years of age. Over 95% of the human rabies cases are concentrated in Asia and Africa, with Asia having 60% of the total number of cases [2] [3]. Dog bites are the most important cause of human deaths from rabies in those regions [4] [5] [6].

Nepal is a landlocked country between China and India; the latter having the largest rabies burden in the world [3] [7]. Nepal has an open border with India and the socioeconomic status is quite similar in the two countries. China has the second highest incidence of rabies [8] with an annual 2,000 to 3,000 human deaths, of which more than 95% are due to the bite of a rabid dog [5]. With the two neighboring countries having a high rabies burden, the disease has become endemic and a priority zoonotic disease in Nepal as well [9]. There, the disease has been confirmed in cattle, buffaloes, goats, alpacas, dogs, and mongooses. Rabies occurs throughout the year, and dogs are the principle vector for the transmission of disease. Unfortunately, the true status of rabies burden due to vectors in Nepal is unknown.

There are two primary and interrelated epidemiological cycles of rabies in Nepal. One is the urban cycle which involves domesticated dogs, and the other is the sylvatic cycle which involves wildlife [10]. A study conducted between 1991 and 2000 showed that more than 96% of rabies patients shared a history of rabid dog exposure, indicating that the urban cycle is the main source of human rabies [9]. Large numbers of human rabies cases in Nepal are due to stray and community dogs. Host wise prevalence study also suggests that 61.7% rabies cases were detected in stray and community dogs [11]. Thus, this rabies cycle is maintained by stray and community dogs. Sometimes there is spill-over from stray and community dogs to pet dogs, which further adds to the human rabies burden.

Vaccination of dogs is one of the most important preventive measures in areas where dogs are the main source of human infection. For the dog rabies mass vaccination program, achieving a coverage of least 70% appears to be sufficient to prevent transmission rabies to humans for at least 6 years [10,11]. A vaccination rate of 60–70% of pet dogs has been shown to result in a drastic decrease in the prevalence of the disease in humans [12]. As vaccination is the primary control measure, it is important to determine the level of anti-rabies antibodies in animals to evaluate the efficacy of the control measures [13].

So far, no studies have been conducted in Nepal to evaluate the efficacy of the vaccines being used, or to determine if the vaccination programs that have been implemented are an effective strategy for the prevention and control of rabies. Various studies have shown that the type of vaccine used, the number of vaccinations, the interval between vaccinations and blood sample collection, age at vaccination, size and breed of dog can influence the antibody response, [14] but that has not been investigated in Nepal. The present study aimed to detect the antibody titer level and associated factors in vaccinated pet dogs to determine the effectiveness of the Nepali vaccination program as a method of prevention of rabies.

Materials and methods

Ethical consideration

Ethical approvals were taken from the Nepal Veterinary Council and Nepal Health Research Council in context of animal and human (Ref No: 293-2073/74 and Ref No: 759) (S6 and S7 Files). No human intervention was conducted in this study. Written consent was taken from the dog owners before conducting the research (S4 File). For the blood sample collection, agriculture animal use protocol of Chiang Mai University, Thailand was followed (Ref No: FA001/2560[02/2560-08-10]) (S8 File). No anesthesia or analgesia was given to the pet dogs.

Study design and sample calculation

A cross-sectional study was conducted involving 110 vaccinated pet dogs in Kathmandu, Bhaktapur and Lalitpur districts of Kathmandu valley, shown in Map 1. Inclusion criteria were a dog ages above 4 months, receipt of at least one anti-rabies vaccination, no fever or external injury at the time of blood collection, and blood collection done at least one month after the last booster.

Map 1

Sampling sites and sampling locations in three selected districts of Kathmandu Valley.

Blood samples were collected from either the cephalic veins or saphenous vein for antibody detection. Blood was drawn aseptically following the animal use protocol. Samples were kept in an ice box and transferred to the laboratory of the National Zoonosis and Food Hygiene Research Center. The blood samples were allowed to clot and then centrifuged at 2000 rpm for 15 minutes. The serum sample was separated and stored in a cryovial at -18 to -20o centigrade until further testing.

Antibody titre detection using ELISA–Platelia Rabies II kit ad usum Veterinarium

For detection of rabies virus anti-glycoprotein antibodies in the dog serum samples, an indirect immune-enzymatic assay (Platelia Rabies II kit ad usum Veterinarium, Biorad, China) was used, following the manufacturer’s recommendations and instructions. This test was chosen for rapidity in obtaining results (<3 hours), simplicity in comparison to virus neutralization, the quantitative and qualitative results, and safety for the laboratory personnel. This test had 98.6% specificity and 88.8% sensitivity.

The serum samples were diluted into 1/10 ratio (10 μl of sample in 990 μl of dilution solution). The diluted serum samples, the positive and negative controls, and the quantification standard were distributed into microplates and then incubated at 37ºC for one hour. To remove unbound antibodies and other proteins in the samples after incubation, three washing steps were performed. Then 100 μl conjugate-protein A labeled with peroxidase was added to each well, followed by a second incubation at 37ºC for one hour and an additional five washing steps to remove unbound conjugate. The presence of the immune complexes was highlighted by adding to each well, a peroxidase substrate solution and a chromogen followed by incubation at room temperature for 30 minutes and the addition of 100μl solution of H2SO4 1N to stop the enzymatic reaction. The microplates were read bichromatically at 450 and 620 nm.

For the quantitative determination of anti-rabies antibodies, a standard curve was constructed using the quantification standards (S1 to S6 Files), obtained by serial dilutions of the R4b calibrated positive controls. The optical density values for the unknown samples were compared with the positive sera titers in quantification tests, obtained after a direct reading on the standard curve and expressed as equivalent units per ml (EU/ml), a unit equivalent to the international units defined by seroneutralization. The results were categorized as high seroconversion level (>4 EU/ ml), sufficient seroconversion level (0.5–4 EU/ml), insufficient seroconversion level (0.125–0.5 EU/ ml), and undetectable seroconversion (<0.125 EU/ml).

Data collection

A questionnaire was used to gather information regarding each pet dog (age, sex, breed), vaccination details (boosters given or not, age at booster, health status during vaccination, place of vaccination, person who conducted the vaccination, how many vaccines were given together), and dog management (whether the dog lives in the owner’s house or not, whether the dog is restrained or allowed to roam, food given, if they trained or untrained, and, if trained, by whom). (S3 File)

Data analysis

Data analysis was done using the R Foundation for Statistical Computing Software (R version 3.3.2 (2016-10-31)). For the rabies antibody titer, descriptive statistics was applied and proportions, standard curve, and R2 were derived. For the factors potentially associated with rabies, analytical statistics (chi-square test, odds ratio) were applied and P-values were calculated. Factors with P-values <0.05 were listed as the associated factors.

Results

Qualitative results

The obtained serum antibody titer levels were compared with the WHO recommended level of protection (≥ 0.5 IU/ml). The district-wise prevalence of positive results for dog serum is shown in Table 1.

Table 1

Results of dog serum samples by district in Kathmandu Valley.

District	No. of Samples	Positive/ Negative	Criteria Result Validation
Bhaktapur	2	-	Not Seroconverted
	25 (92.59%)	+	Seroconverted
Kathmandu	8	-	Not Seroconverted
	48 (85.71%)	+	Seroconverted
Lalitpur	2	-	Not Seroconverted
	25 (92.59%)	+	Seroconverted
Total	110	- 12 (10.91%)	-
		+ 98 (89.09%)

- : Negative

+ : Positive

Quantitative results

To determine the quantity of anti-rabies antibodies in each sample, the optical density compared to a standard curve. The serum titer of all samples was obtained after a direct reading on the standard curve and was expressed as Equivalent Units per milliliter (EU/ml), representing the quantitative determination.

Out of 110 samples from Kathmandu valley, 89.09% samples met or exceeded the required antibody titers level (≥ 0.5 EU/ml), another 9.09% did not reach the antibody titers level (0.125–0.5 EU/ml), and 1.81% samples had undetectable antibody titers (<0.125 EU/ml) according to PLATELIA™ RABIES II test (Fig 1). The r2 for both Plate 1 and 2 was 0.97 respectively. (S1 and S2 Files)

Fig 1

Quantity of anti-rabies antibodies in 110 dog serum samples determined by comparing the optical density of the sample to a standard curve (>4 EU/ml = high seroconversion level; 0.5–4 EU/ml = sufficient seroconversion level; 0.125–0.5 EU/ml = insufficient seroconversion level; <0.125EU/ml = undetectable seroconversion).

Many types of commercial rabies vaccine are available in the Nepalese market. Three of the most popular vaccines such as Defensor (Killed virus strain), Biocan R (Inactivated virus strain) and NeJa Rab (Inactivated cell culture rabies vaccine) were included in the research. The titer levels of the three different types of vaccine within a period of one year of vaccination are shown in Fig 2. The geometric mean and SD values for vaccine A, B and C were 1.96 and 1.30; 2.36 and 1.43; 2.74 and 1.26 respectively. However, there was no significant difference between these three vaccines.

Fig 2

Three commercially available vaccines and the antibody titer in dogs by months after vaccination.

* Titre: Rabies antibody Titre. Months: The months after which the samples were collected. A, B, C: Different types of Vaccines used.

The overall failure rate of the different vaccines varied from 3 to 17%. The vaccine B had high failure rate (17%). High vaccine failures were observed in dogs less than two years old (47%) followed by dogs over 7 years (33%). The highest rate of vaccine failures was observed in crossbreeds (55%), followed by exotic breeds (28%), and local breeds (17%). Medium size breeds (12.9%) showed higher failure rates compared to smaller breeds (9.09%) and larger breeds (8.1%).

Month wise comparison of antibody titer in dogs vaccinated with two different vaccines, A and C, is shown in Fig 3. Vaccine B was excluded from this comparison due to lack of data for all the months. The results confirmed the immunogenicity of both the vaccines. Both vaccines induced high seroconversion within a month of the booster vaccination.

Fig 3

Month by month comparison of median antibody titers with vaccines A and C.

Factors associated with rabies antibody titer level

Twenty eight different factors were analyzed univariately to identify associations between the factors and antibody titer level. Factors having P- values < 0.05 are shown in Table 2. Only one factor (how the dog is kept) was signficantly association with rabies antibody titer level.

Table 2

Factors associated with rabies antibody titer level.

Factors	Sub factors	T- (%)	T+ (%)	OR	95% CI	P- Value
Age	> 2 yrs	8 (7.27%)	89 (80.9%)	0.2	0.04–1.12	0.01
	2 yrs or less	4 (3.63%)	9 (8.18%)
Place where dog is kept	Indoor	0	26 (23.63%)	0	0–1.08	0.04
	Outdoor	12 (10.9%)	72 (65.45%)
How dog is kept	Free	6 (5.45%)	81 (73.63%)	0.21	0.05–0.90*	0.008
	Not Free	6 (5.45%)	17 (15.45%)
Provided Training	No	8 (7.27%)	87 (79.09%)	0.25	0.05–1.36	0.03
	Yes	4 (3.63%)	11 (10%)

* Has association significant

T- : Negative Treatment

T+ : Positive Treatment

OR : Odds Ratio

CI: Confidence Interval

Discussion

In this study, virus neutralizing antibody was detected in 110 pet dogs which had been vaccinated against rabies. It was found that out of the 110 vaccinated dogs, 89.09% had the necessary protective level of anti-rabies antibodies (≥0.5 IU/mL), which is in line with a study conducted by Berndtsson et al. [14] that reported 91.9% had a certified test result of ≥ 0.5 IU/mL. The current study found a higher percentage of dogs had a protective level of anti-rabies antibodies than that reported in a study by Singh et al. [15] which showed only 16% pet dogs in Chandigarh, India, had a protective level of anti-rabies antibodies. Millan et al. [16] found a seroprevalence of 20% in Uganda, while Kitala et al. [17] reported 21% in Kenya. In Nigeria, Mauti et al. [4] found that 43% of dogs had antibody titers exceeding the protective threshold.

The influencing factors associated with antibody titers level and their P-value are presented in Table 2. Of the 110 dogs, 48 dogs had been vaccinated with vaccine A, 31 with vaccine B, and 31 with vaccine C. All three vaccines A, B and C induced a high seroconversion within a month of the booster vaccination. This is similar to the findings of Minke et al. [18] who reported a peak antibody response in pets between 3 to 6 weeks after vaccination. Poor response of vaccine B (17% failure rate) may be due to the age of the vaccinated pet dogs (as 3/ 31 dogs were below the age of two years and 2/31 dogs were above seven years). Also the formulation and concentration of vaccine B was different in comparison to the other two vaccines. However, there was no significant relationship between the type of vaccine used and the antibody titer level obtained. One of the vaccines included in the study was commercially produced in Nepal. All three commercially available vaccines had the same potency to generate a sufficient protective level of anti-rabies antibody (≥ 0.5 IU/mL), indicating that the Nepalese anti-rabies vaccine was not significantly different from the imported vaccines in terms of generating immunogenicity in dogs.

Higher numbers of vaccine failures in the dogs below two years of age may be due to poor immune response in younger dogs, because their immune system is less well developed compared to older dogs. A similar decline in immune response in older humans has been documented [19], which may explain the higher failure rates in older dogs. Life expectancy is another complicating factor, i.e., "old" and "young" varies according to the breed as well. In the univariate analysis, however, no significant relationship between the age of the dog and the titer level was found.

Vaccination failure rates were higher in cross-breeds than in exotic and local breeds. Univariate analysis did not find any significant differences between breed and titer, similar to the findings of Jakel et al. [20] and Berndtsson et al. [14]. Higher failure rates were observed in medium size dogs compared to small and large dogs. Kennedy et al. [21] reported that most failures occurred in larger breeds, while some smaller breeds also showed higher failure rates. But in this study, there was no relationship between the size of the dog and the antibody titer level. The higher failure rates in cross-breeds may be due to the genetic diversity resulting from cross breeding in different countries.

In Kathmandu valley, there is a general practice to give core vaccine to pet dogs which includes anti- rabies vaccine along with canine distemper combined vaccine DHPPiL (which includes vaccines for canine distemper, hepatitis, parvo virus, para influenza and leptospirosis), while corona virus vaccination is optional. However, there was no difference in antibody titer level or vaccine performance with two or more vaccines given at the same time (anti-rabies and corona, anti-rabies and canine distemper combined vaccine DHPPiL or anti-rabies, corona and canine distemper combined vaccine DHPPiL together). As yet, no studies on variation in canine parvovirus, has been published from Nepal. Mansfield et al. [22] and Jakel et al. [20] found differences in antibody response related to gender, but this study found no such differences, which was similar to the findings of Berndtsson et al. [14].

Vaccine failure was observed in twelve dogs in this study. Ten of those dogs had an insufficient titer level, while two dogs had an undetectable titer level. Those results could be due to influencing factors like age, breeds, type of vaccines, poor administration, out of date and poor storage etc. Vaccines produced by different manufacturers have significantly different failure rates, and significantly different median titers of response. This is presumably due to their formulation and the production differences between vaccines together with the concentration and integrity of antigen content and the adjuvant used [21].

The twelve dogs with low antibody titer were revaccinated and titer tested again. It was found that most people preferred to keep their dogs tied up outside the house, allowed to roam free, or caged outside the house. Dogs that were not kept at home had a greater chance to interact with stray dogs and wildlife that could be risk of infection for other animals. Dogs which stayed outdoors were comparatively less well cared for than the dogs which stayed inside and had frequent contact with their owners. Due to some health conditions, prior to the vaccination or after the vaccination, the immune system in some dogs might have been compromised which led to a delayed immune response. Among the five classes of immunoglobulins, IgG has the predominant role in protecting against infections. Some animals have normal levels of immunoglobulins but does not produce sufficient, specific IgG antibodies, while others lack the ability to produce protective IgG antibodies against specific diseases due to abnormalities in their genetic makeup [23].

Implications of the research

Based on the results from the research, all the vaccines showed similar potency and efficacy. However, in the market, there is a tendency to charge extra for some vaccines, claiming that the vaccine has higher potency in comparison to the other commonly available vaccines. It can be said that the consumers are free to choose any of the three available vaccines without any risk. The antibody titer level after one year of vaccination is sufficient to fight against rabies. Therefore, it can be safely said that anti-rabies vaccination should be done yearly.

Conclusions

Although only one factor examined was found to have a significant association with the rabies antibody titer, it is important to realize that vaccination is one of the most important measures for the prevention and control of rabies. By breaking the disease transmission cycle, rabies can be controlled in the dog population and simultaneously, reduce the human rabies burden as well. Based on the results of this study, yearly booster vaccination of dogs is recommended. Titer testing post vaccination would aid in evaluating the efficacy of the vaccines. Further study is necessary to be conducted in the stray and community dog population.

(XLSX)

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Optical densities values for ELISA plate 1 and 2.

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Antibody titer for ELISA plate 1 and 2.

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Questionnaire used for data collection.

(DOCX)

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Consent form for blood collection.

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Consent form translated to local language (Nepali).

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Animal ethical clearance letter.

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Human ethical clearance letter.

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Certificate of approval for use of laboratory animals at laboratory animal center.

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27 Jan 2020

PONE-D-19-32427

Factors Associated with Rabies Antibody Titer Levels 1 of Vaccinated Dogs in Kathmandu Valley, Nepal

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Reviewer #1: Overall, the manuscript is good and worth publishing. However, it needs refining and edition to make the information clear. One of the important point to change would be to align the aim of the study and the title of the study.

Reviewer #2: This is a very well written, and interesting article, which I thoroughly enjoyed reading. I have made a few minor comments which are mainly grammatical, but also have a few questions regarding the study and interpretation. Most of the comments are minor. I anticipate being asked to review this again, so if it is a minor grammatical change, please don’t feel that you need to write a big long rebuttal.

Line 21- I am not a fan of using first person words like our in scientific writing. Maybe change this to this study identified….. ?

Line 27- you can remove the word out, as separated is sufficient

Lines 35-37- you talk about seroconversion levels. I do wonder if antibody titre would be better as undetectable seroconversion levels suggests that the animal hasn’t seroconverted?

Line 42- you talk about the Nepalese vaccine, and it almost sounds as if you expect it not to be as good as international vaccines. Is there a reason for that?

Line 50- a high level of rabies antibody titre sounds strange- please reword

Line 55- comma after preventable, and after neglected will help flow

Line 58- delete out

Line 61- a semi colon after India may aid flow

Line 66- a comma after There may aid flow

Line 74- you talk about spill over from stray and community dogs- is there any spill over from wildlife?

Line 78- you talk about high level vaccination coverage- is this in Nepal or elsewhere? It would be nice to clarify that to make it clearer

Line 98- as part of the questionnaire, you ask about recipient of vaccination. In the Uk and US you get a confirmation of vaccination. Is that available in Nepal? And if not, how do you know that they were telling the truth?

Line 103- How was the blood separated out?

Line 109- you mention the specificity and sensitivity of the assay- is this done as part of the study or is it what the manufacturer has recommended? Please state that in the manuscript

Line 110- sample dilution- how was this done? In what? And to what concentration?

Line 112- 3 needs to be in full, and it would be three washing steps (rather than washings)

Line 114- 5 needs to be in full, and it would read better as five washing steps

Line 115- comma after well

Line17- what is 1N- should this be 1M for molarity?

Line 149- required may be better than requested

Line 149-151- again seroconversion may be better written as antibody titres?

Line 154- It would be very good to name the vaccines in here as the company, and the people would benefit from knowing from this relative lack of efficacy.

You also talk about three different types of vaccine- are they different makes, killed, modified live? A bit more detail in here would be useful

Line 155-156- this reads a bit unclearly, but I cannot work out a better way to put it

Line 159- you discuss vaccine failures, but I think to call it a failure is difficult due to so many potential reasons why they may not work. Poor administration, out of date, poor storage etc. Maybe tone this down, or discuss reasons why they may have failed

Line 161- 7 in words

Line 162-163- it is reported that black and tan dogs respond poorly, particularly to parvovirus vaccines. Is there any evidence of this here?

Line 170- you analyse 28 different factors but don’t mention which ones. It would be good to have some idea of what you analysed

Line 174- again, We and our would be better replaced with non first person words

Line 179- can remove the P. from P. Singh

Line 179 … showed only 16% of household ….

Lines 177-183- you discuss different studies which have different antibody titres. How many of these are vaccine induced? Or doesn’t it say?

Line 186- 3 in words

Line 189- as stated above, I feel calling it a vaccine failure is a bit misleading, and maybe need quantifying.

Line 193-196- a bit more detail on the vaccines and how they differ maybe useful here- strains, administration, DOI, boosters, preparation etc

Line 197- 2 should be in words

Line 197- How much of the vaccine failures could be associated with MDA? Could this be a cause of poor vaccine response in dogs under 2 years?

Line 202- again, replace we

Line 208- But in this study, no relationship ….

Line 212- again, replace we

Line 213- I have not encountered a coronavirus vaccine before? Is this common in Nepal? Is parvovirus not commonly vaccinated against?

Line 216 this reads a bit unclearly, consider rewording.

Line 218- 2 needs to be in words

Line 219- titre testing. Is there commonly done post vaccination in Nepal? Maybe this could be a suggestion for the conclusions?

Line 220- again replace we

Line 222- outdoor dogs would also have chance to interact with wildlife. Is this considered a risk for infection for these animals?

Line 227-230- you discuss a low IgG response in some animals. How many animals is this issue thought to affect? Could it be a cause of your poor vaccine response?

Line 237- decreased may sound better than less?

Line 239- exposure instead of exposer

Line 248- again, replace we

Line 249- this seems a strange comment considering that you have said that vaccine B gives a poor response?

Line 251- does anti rabies vaccination need capitals?

Line 252- the alteration from bi-yearly to yearly vaccination is an interesting idea. How does this work with costs? And regulatory bodies?

Line 253- how can yearly vaccination be as economically viable as bi-yearly? You need 2 vaccines instead of one which will cost more?

Line 257- again, replace we

Line 258- comma after simultaneously

Line 260 again replace we

Line 261- you say here that you collected samples from clinics, but the methods implies more that you go out to houses and test. Could you make this clearer in the methods?

Could you remove some of the figure legends, as they are in 3 or 4 times?

Figure 1-= a map of where these areas are in Nepal may be useful as my Nepalese geography is terrible. Is there any differences between the areas?

Figure 2- it would be good to label which bit means vaccines, and what the titre is, because these values are low and you mention over 120 in the text which doesn’t link to the figure

Table 1- your second line adds up to 89%- where is the other 11% . Seroconverted could be one word too

Table 2- a bit more detail in the table legend as to what all the abbreviations means may be helpful too

**********

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Reviewer #2: No

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Submitted filename: Comments.docx

Click here for additional data file.

17 Mar 2020

Comments to authors

Lines 17 and 19: In line 17, you have mentioned rabies impacts heavily on rural but in line 19, there is mention of dogs being the principal vector for rabies transmission in the urban areas. There is a disconnect in the flow of information. Could you please make it clearer. Also, how rabies impacts heavily in the rural and what is the principal vector for rabies in the rural area? Same goes for lines 45 and 46 under author summary.

Response: We appreciate your comments. To make the sentences more coherence and follow information, we have modified the sentences in lines 17, 19, 45 and 46 which are highlighted by red color for your kind information.

Line 21: Here, you have mentioned that the study aimed to measure the efficacy of vaccination …….. but the title of this article is “Factors Associated with Rabies Antibody Titer Levels of Vaccinated Dogs…….”. to me, the aim of the study and title do not match. In the aim, it seems that to evaluate vaccine efficacy was your primary investigation while the factors to be secondary. However, study title indicates factors for vaccine efficacy to be your primary investigation.

Response: Thank you so much for your comments. We have changed title and aim of study as per your suggestions.

Lines 55 and 56: Please include references.

Response: As per your suggestions, we have incorporated reference in lines 55 and 56 that is highlighted by red color.

Line 68 and 69: Please clarify and elaborate as to what is meant by “true status”.

Response: Thank you for your comment “ true status” meant rabies burden in Nepal which has been corrected in the lines 68 and 69.

Lines 74, 75 and 76: High rabies cases in humans in Nepal is due to more stray and community dogs or due to more rabies in the pets? Please clarify.

What is the overall prevalence of rabies or prevalence in pet, stray and community dogs? If you have this data then the burden of human rabies due to dogs can be explained.

Response: Thank you for your comments. Host wise prevalence study also suggests that 61.7% rabies cases were detected in stray and community dogs (Pant et al, 2013). Therefore, high human rabies cases are due to stray and community dogs in Nepal. We have added one supporting reference in line 74, 75 and 76.

Lines 78, 79 and 80: could you please rephrase the sentence to make the point clear.

Response: As per your suggestions, we rephrased in lines 78, 79 and 80 for your kind information that is indicated by red color.

Lines 90 and 91: Please clarify your aim of the study and the title of the study. Your title says factors associated …… but your says different.

Response: Thank you very much for your kind comments. We have corrected it as per your previous suggestions mentioned in line 21.

Lines 92 and 93: Are the pet dogs vaccinated as a campaign program or as and when requested by the owner?

Response: Thank you very much for your comments. In Nepal, the pet dogs are vaccinated both in the campaign program as well as requested of dog owners.

Line 218 and 219: Here, it is mentioned that the insufficient titres could be due to recent vaccination. But I understand from the inclusion criteria that the blood collection was done one month after the last booster. It would be clear if you had data on the number of dogs that were vaccinated recently. In addition, you could also look for information on the non-respondents to vaccination as some animals innately do not respond irrespective of time since vaccination and type of vaccines.

Response: As per your suggestions, we have incorporated influencing factors for insufficient production of antibody titer level. The factor like age, breeds, type of vaccines could be influencing factor. We have also inserted supporting information and reference.

Lines 228-230: Please add references.

Response: As per your suggestions, we have inserted reference number 23 in line 228-230 for your kind information.

Lines 231-244: The paragraph is informative but does not quite connect with the present study.

Response: We appreciate your comments and we also realized that the paragraph is more informative but irrelevant in this study so, we removed it from this manuscript.

Reviewer #1: Overall, the manuscript is good and worth publishing. However, it needs refining and edition to make the information clear. One of the important point to change would be to align the aim of the study and the title of the study.

Response: Thank you very much for your comments. We have corrected all comments assigned by reviewer 1.

Reviewer #2: This is a very well written, and interesting article, which I thoroughly enjoyed reading. I have made a few minor comments which are mainly grammatical, but also have a few questions regarding the study and interpretation. Most of the comments are minor. I anticipate being asked to review this again, so if it is a minor grammatical change, please don’t feel that you need to write a big long rebuttal.

Line 21- I am not a fan of using first person words like our in scientific writing. Maybe change this to this study identified….. ?

Response: Thank you very much for your kind comments. We have changed first personal pronoun “our” into this study for your kind information.

Line 27- you can remove the word out, as separated is sufficient

Response: As per your suggestions, we removed “out” from the line 27 that is highlighted by red color.

Lines 35-37- you talk about seroconversion levels. I do wonder if antibody titre would be better as undetectable seroconversion levels suggests that the animal hasn’t seroconverted?

Response: Thank you very much for your kind comments. We followed WHO guidelines. According to WHO, the recommended level of protection is (≥ 0.5 IU/ml) but we used (PlateliaTM Rabies II kit ad usum Veterinarium, Biorad, China. This test had 98.6% specificity and 88.8% sensitivity. For detecting antibody titer level there are other gold standard methods like rapid fluorescent focus inhibition test (RFFIT) it can detect seroconversion level of rabies in dogs.

Line 42- you talk about the Nepalese vaccine, and it almost sounds as if you expect it not to be as good as international vaccines. Is there a reason for that?

Response: In Nepal, cell culture rabies vaccine was produced by rabies vaccine production laboratory (RVPL) and animal trials were done in 2002. Since then, vaccine efficacy has not been conducted till date. This is our first comparative efficacy trials of rabies vaccination in Nepal. This study will generate base line data for the control and eliminated rabies in Nepal.

Line 50- a high level of rabies antibody titre sounds strange- please reword

Response: We have changed high level of rabies antibody titer in line 50 that is highlighted by red color.

Line 55- comma after preventable, and after neglected will help flow

Response: We did it.

Line 58- delete out

Response: Thank you sir, we deleted out from manuscript.

Line 61- a semi colon after India may aid flow

Response: We have put a semi colon after India in text that is highlighted by red color.

Line 66- a comma after There may aid flow

Response: We did it for your kind information.

Line 74- you talk about spill over from stray and community dogs- is there any spill over from wildlife?

Response: Thank you for your comments. Sylvatic rabies cycle exists in Nepal but human rabies cases spilled from wild life animal has not been reported yet.

Line 78- you talk about high level vaccination coverage- is this in Nepal or elsewhere? It would be nice to clarify that to make it clearer

Response: Thank you very much for your comments. It is WHO recommendation and we have modified these sentences as per reviewers 1 comments and inserted reference also for your kind information.

Line 98- as part of the questionnaire, you ask about recipient of vaccination. In the Uk and US you get a confirmation of vaccination. Is that available in Nepal? And if not, how do you know that they were telling the truth?

Response: Thank for your comments. Dog owners are provided receipt of vaccination cards of their dogs in Nepal. Before blood sample collection, we had checked vaccination cards in details.

Line 103- How was the blood separated out?

Response: We have inserted blood separation method in methodology parts.

Line 109- you mention the specificity and sensitivity of the assay- is this done as part of the study or is it what the manufacturer has recommended? Please state that in the manuscript

Response: The manufacturers company has mentioned specificity and sensitivity in the kits 98.6% specificity and 88.8% sensitivity. We have also mentioned its sensitivity and specificity in manuscript as per you suggestions.

Line 110- sample dilution- how was this done? In what? And to what concentration?

Response: Thank you very much. The sample dilution was done 1/10 ratio (10 μl of sample in 990 μl of dilution solution).

Line 112- 3 needs to be in full, and it would be three washing steps (rather than washings)

Response: We have corrected it.

Line 114- 5 needs to be in full, and it would read better as five washing steps

Response: We have corrected it.

Line 115- comma after well

Response: We have corrected it.

Line17- what is 1N- should this be 1M for molarity?

Response: 1 N mean Normal solution of sulphuric acid (H2SO4) which was provide in test kit.

Line 149- required may be better than requested

Response: We have changed it.

Line 149-151- again seroconversion may be better written as antibody titres?

Response: We have corrected it.

Line 154- It would be very good to name the vaccines in here as the company, and the people would benefit from knowing from this relative lack of efficacy.

You also talk about three different types of vaccine- are they different makes, killed, modified live? A bit more detail in here would be useful

Response: We have mentioned vaccine details with company name in manuscript.

Line 155-156- this reads a bit unclearly, but I cannot work out a better way to put it

Response: We have changed it as per your suggestions.

Line 159- you discuss vaccine failures, but I think to call it a failure is difficult due to so many potential reasons why they may not work. Poor administration, out of date, poor storage etc. Maybe tone this down, or discuss reasons why they may have failed

Response: Thank you very much for your comments. We have discussed the influencing factors associated with vaccine failures in manuscript.

Line 161- 7 in words

Response: We have corrected it.

Line 162-163- it is reported that black and tan dogs respond poorly, particularly to parvovirus vaccines. Is there any evidence of this here?

Response: This is not any evidence of poor vaccine efficacy of parvovirus reported in Nepal.

Line 170- you analyse 28 different factors but don’t mention which ones. It would be good to have some idea of what you analysed

Response: Thank you for the comments. We have analyzed 28 different factors which are described in the methodology parts. Only four influencing factors were associated with antibody titer and these are mentioned in the analysis table.

Line 174- again, We and our would be better replaced with non first person words

Response: We have corrected it.

Line 179- can remove the P. from P. Singh

Response: We have corrected it.

Line 179 … showed only 16% of household ….

Response: We have corrected it.

Lines 177-183- you discuss different studies which have different antibody titres. How many of these are vaccine induced? Or doesn’t it say?

Response: Thank you for your comments. Details vaccination status and administrated doses has not clearly mentioned in that reference paper.

Line 186- 3 in words

Response: We have corrected it.

Line 189- as stated above, I feel calling it a vaccine failure is a bit misleading, and maybe need quantifying.

Response: Thank you. We have corrected it Sir.

Line 193-196- a bit more detail on the vaccines and how they differ maybe useful here- strains, administration, DOI, boosters, preparation etc

Response: Three of the most popular vaccines such as Defensor (Killed Virus), Biocan R (Inactivated Strain) and NeJa Rab (Inactivated Cell culture rabies Vaccine) were included in the research. All of the vaccines are administered subcutaneously that have 1 ml capacity vial. Yearly booster of the vaccine is given.

Line 197- 2 should be in words

Response: We have corrected it.

Line 197- How much of the vaccine failures could be associated with MDA? Could this be a cause of poor vaccine response in dogs under 2 years?

Response: Only one dog included in the study was four months of age, the vaccine failure of which might be associated with MDA.

Line 202- again, replace we

Response: We have corrected it.

Line 208- But in this study, no relationship ….

Response: We have corrected it.

Line 212- again, replace we

Response: We have corrected it.

Line 213- I have not encountered a coronavirus vaccine before? Is this common in Nepal? Is parvovirus not commonly vaccinated against?

Response: The core vaccination of pets in Kathmandu valley comprises of Anti Rabies Vaccine and Canine Distemper combined vaccine (DHPPiL) which includes Canine Distemper, Canine Hepatitis, Parvo Virus Vaccine, Para Influenza Vaccine and Leptospira Vaccine. Parvo virus vaccination is included in the core vaccination. Corona Virus vaccination is an optional vaccination.

Line 216 this reads a bit unclearly, consider rewording.

Response: We have corrected it.

Line 218- 2 needs to be in words

Response: We have corrected it.

Line 219- titre testing. Is there commonly done post vaccination in Nepal? Maybe this could be a suggestion for the conclusions?

Response: Titer testing is not commonly done after post vaccination in Nepal. We have recommended it in our conclusion part.

Line 220- again replace we

Response: We have corrected it.

Line 222- outdoor dogs would also have chance to interact with wildlife. Is this considered a risk for infection for these animals?

Response: Dogs that were not kept inside the home had a greater chance to interact with stray dogs and wild life that could be risk of infection for other animals as well as humans.

Line 227-230- you discuss a low IgG response in some animals. How many animals is this issue thought to affect? Could it be a cause of your poor vaccine response?

Response: Poor vaccine response had reported in twelve dogs in this study it could be cause due to low IgG titer level.

Line 237- decreased may sound better than less?

Response: We have removed this paragraph according to previous reviewer comments.

Line 239- exposure instead of exposer

Response: According to first reviewers suggestion, we removed this paragraph from 231-244.

Line 248- again, replace we

Response: We have corrected it.

Line 249- this seems a strange comment considering that you have said that vaccine B gives a poor response?

Response: We have corrected it.

Line 251- does anti rabies vaccination need capitals?

Response: Generally free rabies vaccination program conducted by government, private clinics, kennel club and other NGOs do not charge for this vaccine in the world rabies day (September 28 of each year). Other than that, private clinic and government hospital charge for anti rabies vaccination in regular vaccination schedule.

Line 252- the alteration from bi-yearly to yearly vaccination is an interesting idea. How does this work with costs? And regulatory bodies?

Response: Usually a yearly booster vaccine is given. At places, local technicians tend to give a booster vaccination every six months. The cost for the pet owners is more as they pay double the money for the same vaccine. We do not have a regulatory body that monitors this malpractice.

Line 253- how can yearly vaccination be as economically viable as bi-yearly? You need 2 vaccines instead of one which will cost more?

Response: Yearly vaccination is more cost effective than bi-yearly. The cost of single dose vaccine is less by half than two doses of the same vaccine.

Line 257- again, replace we

Response: We have corrected it.

Line 258- comma after simultaneously

Response: We did it.

Line 260 again replace we

Response: We have corrected it.

Line 261- you say here that you collected samples from clinics, but the methods implies more that you go out to houses and test. Could you make this clearer in the methods?

Response: We corrected this sentences linking with methodology part.

Could you remove some of the figure legends, as they are in 3 or 4 times?

Response: We have corrected it.

Figure 1-= a map of where these areas are in Nepal may be useful as my Nepalese geography is terrible. Is there any differences between the areas?

Response: We have included an area map as per your suggestions.

Figure 2- it would be good to label which bit means vaccines, and what the titre is, because these values are low and you mention over 120 in the text which doesn’t link to the figure

Table 1- your second line adds up to 89%- where is the other 11% . Seroconverted could be one word too

Response: We have added as per your suggestions.

Table 2- a bit more detail in the table legend as to what all the abbreviations means may be helpful too

Response: We have added as per your suggestions.

Submitted filename: Response of reviewers comments 20200312.docx

Click here for additional data file.

25 Mar 2020

PONE-D-19-32427R1

Detection of virus-neutralizing antibodies and associated factors against rabies in the vaccinated household dogs of Kathmandu Valley, Nepal

PLOS ONE

Dear Dr Shah

Thank you for submitting your manuscript to PLOS ONE. A few very minor changes have been recommended, These are mainly just grammatical points, which will expedite publication, and will only take around 10 minutes to complete. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

==============================

Many thanks for resubmitting your manuscript to PLOS One

The expert reviewers are happy. I have reviewed the manuscript and highlighted a few minor grammatical issues

If you could modify these, I can recommend it for publication without need for another review

This will hopefully expedite publication at the editorial office

Please do not write a detailed response to reviewers, just a single line saying that the changes have all been made will suffice.

I wish you good luck with your modifications

Hope you are keeping safe in these difficult times

Thanks

Simon

==============================

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Academic Editor

PLOS ONE

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

**********

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The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

**********

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Reviewer #1: Yes

Reviewer #2: Yes

**********

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The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

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6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: (No Response)

Reviewer #2: I have reviewed your manuscript to ensure that it is ready to go for publication. I have made a few minor suggestions for grammatical changes which will increase the flow of the reading. It may look like there is a lot, but it will probably only take you 10 minutes maximum.

This will hopefully increase the speed of publication when it gets to the editorial office.

Line 22- …the aim of detecting virus-neutralising…..

Line 36- …(<125 EU/ml) suggesting that the ….

Line 42- …. manufactured brand of vaccine, which is available in Nepal, is potent …

Line 49- …efficacy of vaccination programmes, and to determine whether ….

Line 57- ….is a vaccine preventable, viral zoonosis ….

Line 58- …all human deaths from rabies are caused ….

Line 59- …59,000 people due from this disease …

Line 60- …of which 30-50% are below ….

Line 61- …60% of the total number of cases ….

Line 70- … the year, and dogs are ….

Line 73 -…involves domesticated dogs, and the other is ….

Line 76- Large numbers of human rabies cases …..

Line 82- For the dog rabies mass vaccination program, achieving a ….

Line 89-… vaccines been used, or to determine if the ….

Line 98- …were taken from the Nepal Veterinary ….

Line 102- agriculture doesn’t need to be capitalised

Lines 98-104- this doesn’t need to be in bold

Line 107- ….districts of Kathmandu valley, shown in map….

Line 108- Inclusion criteria were a dog ages above 4 months ….

Line 120- Biorad, China) was used, following the ….

Line 131- adding to each well, a peroxidase substrate …..

Line 135- ….quantification standards (S1 to S6) , obtained by serial ….

Line 137- …titers in quantification tests, obtained after a ….

Line 143- ….regarding each pet dog (age, sex ….

Line 162- …optical density compared to a standard curve ….

Line 173- please put 3 in words

Line 175. However, there was no significant ….

Line 178- …less than two years old (47%) …

Line 193- …antibody was detected in 110 pet dogs….

Line 194- …89.09% had the necessary ….

Line 195- …..(>0.5 IU/ml), which is in line ….

Line 207- …may be due to the age of the vaccinated ….

Line 217- Higher numbers of vaccine failures ….

Line 218- …in younger dogs, because their ….

Line 219- ….has been documented [19], which may ….

Line 222- …analysis, however, no significant relationship….

Line 223- dog and their titer level was found

Line 228- double space between higher and failure (please remove 1)

Line 232- double space between give and core (please remove 1)

Line 232- …which includes the anti-rabies. …

Line 233- Canine distemper doesn’t need capitals

Line 234- diseases from canine distemper to leptospirosis don’t need capitals

It may also be worth mentioning that there is, as yet, no studies on variation in canine parvovirus published from Nepal. This is one of the diseases which I work on so it maybe worth a chat in the future to get some samples and do this study?

Line 235- coronavirus doesn’t need a capital

Line 235- However, there was no….

Line 236 and 237- Anti-rabies, corona, canine and distemper, don’t need capitals

Line 242- ….insufficient titer level, while two dogs ….

Line 251- wildlife is one word

Line 253- Due to some health conditions, prior to the vaccination….

Line 257- …does not produce sufficient, specific IgG….

Line 262- However, in the market, there is a tendency ….

Line 266- …safely said that anti rabies vaccination ….

Line 273- Titre testing post vaccination …..

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: Yes: Simon Clegg

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files to be viewed.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at figures@plos.org. Please note that Supporting Information files do not need this step.

2 Apr 2020

Comments to authors

Reviewer #1: (No Response)

Reviewer #2: I have reviewed your manuscript to ensure that it is ready to go for publication. I have made a few minor suggestions for grammatical changes which will increase the flow of the reading. It may look like there is a lot, but it will probably only take you 10 minutes maximum.

This will hopefully increase the speed of publication when it gets to the editorial office.

Response: Thank you very much sir for your kind valuable suggestions and comments for grammatical changes to enhance our revised manuscript flow of the reading. As per your kind suggestions, we have modified and made all single inline grammatical changes in revised manuscript with red color according to assigned by reviewer 2 for your kind information

Submitted filename: Response to reviewer comments 20200331.docx

Click here for additional data file.

6 Apr 2020

Detection of virus-neutralising antibodies and associated factors against rabies in the vaccinated household dogs of Kathmandu Valley, Nepal

PONE-D-19-32427R2

Dear Dr. Shah

We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements.

Within one week, you will receive an e-mail containing information on the amendments required prior to publication. When all required modifications have been addressed, you will receive a formal acceptance letter and your manuscript will proceed to our production department and be scheduled for publication.

Shortly after the formal acceptance letter is sent, an invoice for payment will follow. To ensure an efficient production and billing process, please log into Editorial Manager at https://www.editorialmanager.com/pone/, click the "Update My Information" link at the top of the page, and update your user information. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, you must inform our press team as soon as possible and no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

With kind regards,

Simon Russell Clegg, PhD

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Many thanks for submitting your manuscript to PLOS One

I have reviewed your manuscript. Many thanks for making the suggested changes.

I have recommended your manuscript for publication, so you should hear from the editorial office soon.

There are two minor changes which need to be made, and if these could be made during editing for publication, I would be very grateful

Line 59- change due to die

Line 88- change been to being

I wish you all the best for your future research. I also recommend looking into a study on canine parvovirus in Nepal, and if I can be of any assistance with this then please let me know as its one of my research interests

Please stay safe and well during the current difficult times

Thanks

Simon

10 Apr 2020

PONE-D-19-32427R2

Detection of virus-neutralising antibodies and associated factors against rabies in the vaccinated household dogs of Kathmandu Valley, Nepal

Dear Dr. Shah:

I am pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please notify them about your upcoming paper at this point, to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

For any other questions or concerns, please email plosone@plos.org.

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With kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Simon Russell Clegg

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PLOS ONE