Seroprevalence of viral and vector-borne bacterial pathogens in domestic dogs (Canis familiaris) in northern Botswana.

BACKGROUND: Domestic dogs (Canis familiaris) have the potential to act as disease reservoirs for wildlife and are important sentinels for common circulating pathogens. Therefore, the infectious disease seroprevalence among domestic dogs in northern Botswana may be indicative of pathogen exposure of various wildlife species. The objective of this study was to assess the seroprevalence of Ehrlichia spp., Borrelia burgdorferi, Anaplasma spp., Dirofilaria immitis, canine adenovirus, canine parvovirus, and canine distemper virus in domestic dogs as proxies of disease prevalence in the local wildlife in the Okavango Delta region of Botswana. Statistical analysis assessed crude and factor-specific seroprevalence proportions in relation to age, sex, and geographical location as predictors of seropositivity. Logistic regression was used to identify adjusted predictors of seropositivity for each of the pathogens of interest.
RESULTS: Samples from 233 dogs in a total of seven locations in Maun, Botswana, and surrounding villages were collected and serologically analyzed. No dogs were seropositive for B. burgdorferi, while low seroprevalence proportions were observed for Anaplasma spp. (2.2%) and D. immitis (0.9%). Higher seroprevalence proportions were observed for the tick-borne pathogen Ehrlichia spp. (21.0%), and 19.7% were seropositive for canine adenovirus (hepatitis). The highest seroprevalence proportions were for canine parvovirus (70.0%) and canine distemper virus (44.8%). The predictors of seropositivity revealed that adults were more likely to be seropositive for canine adenovirus, canine distemper virus, and canine parvovirus than juveniles, and location was a risk factor for canine adenovirus, canine distemper virus, canine parvovirus, and Ehrlichia spp.
CONCLUSIONS: Results indicate that increasing tick control and vaccination campaigns for domestic dogs may improve the health of domestic animals, and potentially wildlife and humans in the Okavango Delta since viral and vector-borne bacterial pathogens can be transmitted between them.

Introduction

Vaccination of domestic dogs has been reported as a method of wildlife conservation [1] with the implication that prevalence of transmissible diseases in the domestic canine population has the potential to affect disease burden in wildlife, including both wild felids and wild canids. African wild dogs (Lycaon pictus), a wild canid species in sub-Saharan Africa, are endangered according to the International Union for the Conservation of Nature Redlist [2], and the black-footed cat (Felis nigripes), cheetah (Acinonyx jubatus), and lion (Panthera leo) are all vulnerable species in Botswana [2] that can be negatively impacted by domestic dog viral and vector-borne bacterial pathogens [3, 4, 5]. Capturing a sufficient number of African wild dogs, black-footed cats, cheetah, or lion to perform seroprevalence surveys is not always feasible due to the risk associated with anesthesia necessary to collect blood samples from these animals. This is particularly concerning due to the low numbers of individuals as indicated by their conservation status. McRee, et al. [6] performed a prevalence evaluation of viral pathogens in domestic dogs in northwest Zimbabwe as a representation of wildlife viral disease prevalence in the region, particularly African wild dogs.

Ehrlichia spp., Anaplasma spp., and Borrelia burgdorferi (Lyme disease) are bacterial pathogens that are transmitted by tick bites [7, 8, 9]. Dirofilaria immitis, or heartworm disease, is a blood-borne parasite transmitted by mosquito bites [10], and canine distemper (CDV), parvovirus (CPV), and adenovirus (CAV) are viral diseases transmitted between individuals [11, 12, 13]. All of these common pathogens in dogs can cause significant morbidity and mortality. While the viral diseases, Lyme disease, and heartworm disease can be prevented either by vaccination or monthly heartworm preventative medication, many communities in southern Africa do not have the resources to pay for these medications for their animals. Thus, these preventable diseases may be widespread.

Botswana is a land-locked country in southern Africa and is home to the Okavango Delta, a diverse wetland habitat. Not only is the Okavango Delta home to countless species, it is the center for tourism in the country, which has become the second most important industry in Botswana after diamond mining [14]. The Okavango Delta of Botswana is rich with wildlife which have the chance to interact with domestic animal populations. This potentially results in cross-species transmission of infections between domestic and wild animals implying that infectious disease exposure in domestic animals might mirror those of wildlife. As disease prevalence of common infectious diseases in wild carnivores is unknown in the Okavango Delta, this presents the opportunity to use domestic dogs as sentinels for infectious disease exposure in wildlife. Therefore, the objective of this study was to assess the seroprevalence of common infectious diseases (Ehrlichia spp., Borrelia burgdorferi, Anaplasma spp., Dirofilaria immitis, CDV, CAV, and CPV) in domestic dogs in Maun, Botswana, an area adjacent to the Okavango Delta, as a proxy for seroprevalence that would be expected in the wild canid and felid populations.

Materials and methods

Animals

The University of Tennessee Knoxville Institutional Animal Care and Used Committee approved this research proposal on March 27, 2015; #2333–0315. All blood collections were done under the direct supervision of the veterinary members of the Maun Animal Welfare Society (MAWS). The majority (n = 128/233) of blood samples from domestic dogs were collected using a convenience sampling strategy at MAWS in Maun, Botswana, from dogs who were presented for castration and vaccination. The remaining blood samples (n = 105/233) were collected in surrounding villages. The uncastrated domestic dogs in this area were highly unlikely to have been vaccinated and be seropositive to the viral diseases due to vaccination cross-reaction because MAWS is the main veterinary clinic in the area for the low income population, and they will not vaccinate animals unless they are also castrated at the time of vaccination. Therefore, animals that were reproductively intact are likely to be unvaccinated. Blood samples were collected from a peripheral vein, transported on ice, and stored at -20°C until testing.

Sample analyses

Seroprevalence for CAV, CPV, and CDV were assessed using Biogal Titer Check (Biogal Galed Laboratories, Kibbutz Galed, Israel) following manufacturer’s instructions. The Biogal Titer Check results were reported with a change of color and ‘negative‘, ‘positive’, ‘highly positive’, or ‘inconclusive’ as the range of possible results. The vector-borne diseases, Ehrlichia spp., B. burgdorferi, Anaplasma spp., and D. immitis, were assessed with IDEXX 4DX SNAP ELISA (IDEXX, Westbrook, Maine, USA) following manufacturer’s instructions. The IDEXX SNAP ELISA results were reported as either ‘positive’ or ‘negative’ by a color change.

Statistical analysis

Crude and factor-specific seroprevalence proportions of Ehrlichia spp., B. burgdorferi, Anaplasma spp., D. immitis, CDV, CAV and CPV, as well as their 95% exact confidence intervals were computed. The factors considered were age, sex, and location. Associations between seroprevalence and each of the above factors were assessed using the Chi-square or Fishers Exact tests as appropriate. Significance was set at α = 0.05 for all statistical tests. Logistic regression was used to identify adjusted predictors of seropositivity for each of the pathogens of interest.

Results

Animal demographics

A total of 233 dogs were tested (Table 1). Female dogs made up the majority (54.5%) of the sampled dogs. Dogs were sampled during the months of June and July 2015 with the majority (55.4%) of the dogs being sampled in July. The age group distribution was 70% adults and 30% juveniles. Samples were collected at seven locations in Maun, Botswana, and surrounding villages: MAWS (n = 128), Khumaga (n = 13), Boro (n = 18), Mathapane (n = 16), Shorobe (n = 18), Sexaxa (n = 8), and Etsha (n = 32).

Table 1

Characteristics of dogs included in a seroprevalence assessment of prior exposure to common pathogens in Botswana, 2015.

Variable	Number	Percent	95% Exact Binomial Confidence Interval
Sex
Female	127	54.5	47.9, 61.0
Male	106	45.5	3.90, 52.1
Age Category
Adult	163	69.7	63.6, 75.8
Juvenile	70	30.0	24.2, 36.3
Location
Boro	18	7.7	4.6, 11.9
Etsha	32	13.7	9.6, 18.8
Khumaga	13	5.6	3.0, 9.4
MAWS	128	54.9	48.3, 61.4
Mathapane	16	6.9	4.0, 10.9
Sexaxa	8	3.4	1.5, 6.7
Shorobe	18	7.7	4.6, 11.9
Month
June 2015	104	44.6	38.1, 51.0
July 2015	129	55.4	48.7, 61.8

Crude seroprevalence

Of 232 individuals, 0% were seropositive for B. burgdorferi, 2.2% were seropositive for Anaplasma spp., and 0.9% were seropositive for D. immitis (Table 2). Out of the 233 animals, 21.0% were seropositive for Ehrlichia spp., 19.7% were seropositive for CAV, 70% were seropositive for CPV, and 46.8% were seropositive for CDV.

Table 2

Crude seroprevalence of selected pathogens among domestic dogs in Botswana, 2015.

Pathogen	n	Number of seropositive samples	Percentage of seropositive samples	95% Confidence Interval
Anaplasma spp.	232a	5	2.2	0.7, 5.0
B.burgdorferi	232 a	0	0	0, 1.6
D.immitis	232 a	2	0.9	0.1, 3.1
CDV	233	109	46.8	40.2, 53.4
Ehrlichia spp.	233	49	21.0	16.0, 26.8
CAV	233	46	19.7	14.8, 24.4
CPV	233	163	70.0	63.6, 75.8

a One record had missing information for results of Anaplasma, B.burgdorferi and D.immitis

Predictors of seropositivity

Based on the results of the logistic model, there were no statistically significant predictors (sex, age, month, or location) (P>0.05) for seropositivity of either Anaplasma spp. or D. immitus (Table 3).

Table 3

Factor-specific seroprevalence of Anaplasma spp. and D.immitis among dogs in Botswana, 2015.

Pathogen	n	Number of seropositive samples	Percentage of seropositive samples	P-value
Anaplasma spp.	232	5	2.2
Sex
Female	126	4	3.2	0.379
Male	106	1	0.9
Age Category
Adult	162	5	3.1	0.326
Juvenile	70	0	0
Month
June	104	1	1.0	0.383
July	128	4	3.1
Location
Boro	18	0	0	0.938
Etsha	32	0	0
Khumaga	13	0	0
MAWS	127	5	3.9
Mathapane	16	0	0
Sexaxa	8	0	0
Shorobe	18	0	0
D.immitis	232	2	0.9
Sex
Female	126	2	1.6	0.502
Male	106	0	0
Age Category
Adult	162	2	1.2	1.000
Juvenile	70	0	0
Month
June	104	1	1.0	1.000
July	128	1	0.8
Location
Boro	18	0	0	0.242
Etsha	32	0	0
Khumaga	13	0	0
MAWS	127	1	0.8
Mathapane	16	0	0
Sexaxa	8	1	12.5
Shorobe	18	0	0

Although age of the dog and location had significant unadjusted associations with the odds of CAV seropositivity, only age (OR = 4.4; p<0.0003) was significant in the final analysis; implying that the odds of CAV seropositivity was 4.4 times higher in adults dogs than in juveniles (Table 4). Similarly, only age had a significant association in the final model for CPV with adults having 4.4 times higher odds of CPV seropositivity (OR 4.4; p<0.0001) than juveniles (Table 4).

Table 4

Factor-specific seroprevalence of CAV and CPV among dogs in Botswana, 2015.

Pathogen	n	Number of seropositive samples	Percentage of seropositive samples	P-value
CAV	233	46	19.7
Sex
Female	127	20	15.6	0.101
Male	106	26	24.5
Age Category
Adult	163	41	25.2	0.001
Juvenile	70	5	7.1
Month
June	104	26	25.0	0.097
July	129	20	15.5
Location
Boro	18	6	33.3	0.043
Etsha	32	5	15.6
Khumaga	13	2	15.4
MAWS	128	22	17.2
Mathapane	16	8	50
Sexaxa	8	0	0
Shorobe	18	3	16.7
CPV	233	163	70.0
Sex
Female	127	92	72.4	0.392
Male	106	71	67.0
Age Category
Adult	163	130	79.8	<0.001
Juvenile	70	33	47.1
Month
June	104	78	75.0	0.152
July	129	85	65.9
Location
Boro	18	15	83.3	0.025
Etsha	32	16	50
Khumaga	13	6	46.2
MAWS	128	92	71.9
Mathapane	16	12	75
Sexaxa	8	6	75
Shorobe	18	16	88.9

Based on the results of the logistic model, CDV seropositivity was significantly associated with age (p<0.0001), month (p = 0.0002), and geographical location of sampling (p = 0.0437) (Table 5). Although both age of the dog and geographical location had significant unadjusted association with the odds of Ehrlichia spp. seropositivity (Table 5), when both were offered to the model in a multivariable analysis, neither was significant. Therefore, there was no final multivariable model for Ehrlichia spp.

Table 5

Factor-specific seroprevalence of CDV and Ehrlichia spp. among dogs in Botswana, 2015.

Pathogen	n	Number of seropositive samples	Percentage of seropositive samples	P-value
CDV	233	109	46.8
Sex
Female	127	62	48.8	0.512
Male	106	47	44.3
Age Category
Adult	163	99	60.7	<0.001
Juvenile	70	10	14.3
Month
June	104	71	68.3	<0.001
July	129	38	29.5
Location
Boro	18	14	77.8	<0.001
Etsha	32	6	18.8
Khumaga	13	2	15.4
MAWS	128	51	39.8
Mathapane	16	13	81.3
Sexaxa	8	6	75.0
Shorobe	18	17	94.4
Ehrlichia spp.	233	49	21.0
Sex
Female	126	28	22.1	0.748
Male	106	21	19.8
Age Category
Adult	163	40	24.5	0.054
Juvenile	70	9	12.9
Month
June	104	22	21.2	1.000
July	129	27	20.9
Location
Boro	18	3	16.7	<0.001
Etsha	32	0	0
Khumaga	13	0	0
MAWS	128	40	31.3
Mathapane	16	3	18.8
Sexaxa	8	2	25
Shorobe	18	1	7.6

Controlling for the other two factors in the model, the odds of CDV seropositivity is 12 times higher among adult dogs than the juveniles (Table 6). Similarly, the odds of the dogs having seropositive results for CDV were 7.8 times higher in June than in July (Table 6). With respect to geographical location, only Khumaga (p = 0.0014) and Shorobe (p = 0.0481) had significantly different odds of canine seropositivity from MAWS, with the odds of the dogs in Shorobe being 5.8 times higher than those of the reference group (MAWS) (Table 6). By contrast, the dogs in Khumaga had significantly lower odds (OR = 0.072) of testing seropositive to CDV than dogs in MAWS. The odds of being seropositive for CDV among dogs from the other locations were not significantly different from that of MAWS. Based on the Hosmer-Lemeshow Goodness-of-fit test, there is no evidence that the canine distemper model did not fit the data well (p = 0.4813).

Table 6

Results of multivariable logistic regression showing predictors of CDV sero-positivity among domestic dogs in Botswana, 2019.

Predictor	Odds Ratio	95% Confidence Interval	P-value
Age
Adult	12.4	4.8, 32.1	<0.0001
Juvenile	Referent	Referent
Month
June	7.8	2.6, 23.1	0.0002
July	Referent	Referent
Location
Boro	0.7	0.2, 3.3	0.8022
Etsha	0.7	0.2, 1.9	0.7233
Khumaga	0.07	0.01, 0.5	0.0014
Mathapane	1.2	0.2, 6.5	0.5889
Sexaxa	1.2	0.2, 9.6	0.6601
Shorobe	5.8	0.6, 57.8	0.0481
MAWS	Referent	Referent

Discussion

Domestic animals can serve as disease reservoirs for wildlife. Wild animal populations, including various canid and felid species, have the potential to be infected by CDV, CPV, CAV, D. immitis, B. burgdorferi, Ehrlichia spp. and Anaplasma spp., which are pathogens carried by the domestic animal population. Free-ranging cheetah (Acinonyx jubatus) in Namibia have been reported to have antibodies to CPV (though this test cross-reacts with feline panleukopenia virus) and CDV [15]. A captive breeding group of African wild dogs in Tanzania showed 94% mortality after infection with CDV [16], and another group of African wild dogs in Kenya had increasing disease-related mortality rates (from 21% to 50%) and CDV antibodies (from 1–4% to 76%) over a three-year period [3]. In Chobe National Park, Botswana, in 1996, a pack of twelve African wild dogs was reduced to two animals following an outbreak of CDV [17]. While the prevalence in the current study was low (0.9%), D. immitis can infect wildlife, with reports of D. immitis in a captive lion in Spain [18] and a captive black-footed cat in Florida [19]. Butler, et al. [20] indicated that domestic dogs in northwest Zimbabwe are a source of disease transmission for leopards (Panthera pardus), lions, and spotted hyenas (Crocuta crocuta), as these predator species feed on domestic dogs as prey. A survey of domestic dogs and African wild dogs in Kenya from 2001 to 2009, showed 16% of African wild dogs and 48% of domestic dogs had been exposed to CDV, 25% of African wild dogs and 64% of domestic dogs had been exposed to CPV, and 80% of African wild dogs and 86% of domestic dogs had been exposed to Ehrlichia canis [21]. Similar to the Kenya evaluation, the results of the present study revealed 46.8% CDV seroposititivity and 70.0% CPV seropositity in domestic dogs. However, it was found that lower seropositivity rates for Ehrlichia spp. (21.0%) were present as compared to the Kenya study.

When comparing regional infectious disease prevalence differences, similar seroprevalence for viral diseases in domestic dogs were determined in Botswana as was reported in a similar study performed in Zimbabwe in 2012 [6]. A study of domestic dogs in northwest Zimbabwe reported that 34% had antibodies to CDV, 84% had antibodies to CPV, and 13% had antibodies for CAV [6]. These results are similar to those of the present study of 46.8% for CDV, 70.0% for CPV, and 19.7% for CAV. Another seroprevalence study evaluating domestic dogs in northeast Namibia in 1993 and 1994 found similar exposure to CDV (44.3%), but lower prevalence of CPV (47.1%) and higher prevalence of CAV (64.3%) [22]. Both Zimbabwe and Namibia border Botswana on its eastern and western edges, respectively. Viral diseases spread from animal to animal so localized differences in exposure are expected, but vectored pathogens depend on prevalence of the pathogen, prevalence of the vector, and on contact between the vector and the susceptible animal host. Williams, et al. [23] assessed prevalence of several hemoparasites, including Ehrlichia spp., in domestic dogs, lions, spotted hyena, and African wild dogs in 2009 to 2011 in Zambia, another country in southern Africa that borders Botswana, and samples were evaluated by polymerase chain reaction which only reveals active infections rather than current and past exposure. No carnivores had positive results for E. canis or E. ewingii [23], which does not rule out presence of Ehrlichia in the study area.

As ticks are the vectors for B. burgdorferi, Ehrlichia spp. and Anaplasma spp, tick prevalence is a crucial factor in the spread of these pathogens. While no recent studies have reported tick prevalence in northern Botswana, Eygelaar, et al. [24] reported that African buffalo (Syncerus caffer) in the Okavango Delta had a lower prevalence of tick-borne diseases than African buffalo in Chobe National Park, a region in northeast Botswana that is closer to the study region assessed by McRee, et al. [6]. Perhaps the same cause is responsible for the reduced tick-vectored diseases in African buffalo and domestic dogs in the Okavango Delta compared to northwest Zimbabwe. Eygelaar, et al. [24] hypothesized that veterinary fences in the Okavango Delta prevented direct contact between the African buffalo and cattle, which were not present in Chobe National Park. Perhaps these same fences reduce tick spread from wildlife to domestic dogs and vice versa, which limits the spread of the vector-borne diseases in the Okavango Delta which were not present in northwest Zimbabwe. More research must be performed to determine if the differences in Ehrlichia spp. seroprevalence is due to a reduction in total tick numbers or another cause, such as physical barriers. The high viral disease seroprevalence, similar to those in northwest Zimbabwe, is likely due to lack of vaccination. While strong efforts are being actively put forth by local not-for-profit organizations, the number of unvaccinated dogs remains much greater than the number of vaccinated dogs.

Factor-specific seroprevalence indicated significant associations between seropositivity to these common canine infectious diseases and age, month, and location. Adults are more likely to be seropositive for CAV, CDV, and CPV, which is likely due to having more time to be exposed to the viruses than juveniles. There was a significant association between seropositivity for CDV and month with June, having higher risk than July. Lastly, geographical location is a risk factor for CAV, CDV, and CPV because viral pathogens are transmitted either by direct contact or contact with bodily fluids. Therefore, geographical locations with high rates of these pathogens allow easy transmission to naïve individuals. Location was also a risk factor for E. canis perhaps because of tick concentrations in certain locations or due to an increase in the pathogen in the dogs of certain locations that perpetuates the elevated infection rate (ticks can spread the pathogen transstadially, but not transovarially [7]).

While the viral pathogens evaluated in this study cannot infect humans, some of the vectored pathogens can affect humans. In addition to affecting domestic and wild animal populations, E. canis and E. ewingii have been reported in humans [25, 26]. Borrelia burgdorferi, the causative organism for Lyme disease, and A. phagocytophilum [27, 28] are also zoonotic. The ‘One Health’ paradigm, a collaborative approach to animal, human, and environmental health that recognizes their interconnectivity, is particularly important, since reducing disease risk in domestic animals will reduce disease risk in wildlife and human populations. By increasing vaccination and reducing tick burden in domestic dogs, human health and environmental health, in the case of wildlife, are improved [29, 30].

One of the limitations of this study was that serology detects exposure to the pathogen, but it does not determine the rate of active infections. Thus, seroprevalence indicates that pathogen exposure has occurred, but current risk of infection is unknown. This aspect is important for human and wildlife health because new infections may increase disease burden in these populations.

Information regarding disease prevalence is necessary to determine domestic animal, wildlife, and human disease risk. This study reveals the need for local tick surveys to determine the cause of tick-borne pathogen prevalence differences between Botswana and surrounding countries. In conclusion, further disease testing and vaccination of both domestic dogs and wildlife would benefit domestic animals, wildlife, and humans in the Okavango Delta region of Botswana.

Data analyzed including ELISA results for 233 blood samples collected from dogs in northern Botswana.

(XLS)

Click here for additional data file.

30 Aug 2019

PONE-D-19-20072

Seroprevalence of viral and vector-borne bacterial pathogens in domestic dogs (Canis familiaris) in northern Botswana

PLOS ONE

Dear Melissa Kennedy

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Reviewer #1: Dear Editor

I have read through the MS entitled ’Seroprevalence of viral and vector-borne bacterial pathogens in domestic dogs (Canis familiaris) in northern Botswana’ sent to me for review. The MS is technically sound and the information contributes to the understanding of disease dynamics in domestic dog population in northern Botswana and by implication the disease condition in the wild felid and canid populations. However, there are some aspect of the study that needs to be addressed in order to enhance the clarity of the paper and improve on its quality in general. My comments to the authors is attached

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Joshua Kamani

Reviewer #2: Ms. No. PONE-D-19-20072

Seroprevalence of viral and vector-borne bacterial pathogens in domestic dogs (Canis

familiaris) in northern Botswana

Plos one

________________________________________________________

This study is a survey on the main viral, bacterial and parasitic pathogens in domestic dogs living in an area that has been little investigated such as the Botswana.

It is interesting to use domestic dogs as sentinels for infectious disease exposure in wildlife.

However, this study's objectives are not clearly stated and the methods are incomplete and, at times, difficult to follow.

The epidemiologic study was completely descriptive.

In addition, sampling methods are unclear.

It would be optimal to better specify the age of the dogs examined and how age was determined.

The withdrawal period is so short that I don't think it can really influence the positivity differences. I would eliminate it.

The discussion should be re-evaluated with greater attention and the hypotheses on the role of ticks in the transmission of some pathogens rewritten, please.

I would reduce the tables to only one.

Tables 1 and 2 show 232 dogs analyzed while 3 and 4 show 233 dogs, please check the tables.

References

Check the bibliographic references

Insert in italic the different pathogens

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Reviewer #1: Yes: Joshua Kamani

Reviewer #2: No

[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.]

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Submitted filename: Review Result for PONE.pdf

Click here for additional data file.

15 Oct 2019

Reviewers’ comments:

Laboratory methods – given that the only techniques used involved ELISA kits per manufacturers’ recommendation, none were deposited.

The style requirements were reviewed.

Owners’ verbal consent for participation, the locale of participants, and the volume of blood collected were addressed in the text.

Reviewer #1:

The short title was modified per the reviewer’s recommendations.

The italics for “spp.” designations were removed.

“total of” was removed from the sentence in line 37.

The issue of tick infestation and control were addressed in the text

Lines 60-61 and line 64 were modified per the reviewer’s recommendations.

The tick vector for each organism was included.

Rephrasing of lines 87-89 was made.

As stated in the manuscript, rabies was not assessed because of unknown vaccination status for rabies, and the lack of a point-of-care assay for this assessment.

The various aspects of demographic data were addressed in the text.

The use of whole blood was addressed

The various properties of the kits were addressed.

Color change evaluation was addressed.

Tables were condensed.

Lines 202-206 were modified per the reviewer’s recommendation.

The reference corrections were made.

Reviewer #2:

The hypothesis, objectives and methods were clarified.

Demographic data was addressed.

The discussion section was adjusted.

Tables were reduced:

Reviewer Comment

Line 134: is it 232 or 233?

Response

The total sample size was 233. However, there was one missing data point for test results of the following pathogens: Anaplasma spp., B.burgdorferi and D.immitis. Due to that missing data, these pathogens had 232 instead of 233. The rest of the pathogens did not have missing data and therefore had a total of 233. These explanations have been added as footnotes on tables 2, 3a and 3b

Reviewer Comment

Table 2: I am not sure the authors explained why there are two populations; 232 233 being tested for different pathogens.

Response

There is only one population of dogs. However, as explained above there was there was one missing data point for test results for Anaplasma spp., B.burgdorferi and D.immitis. Therefore these pathogens had data for only 232 dogs instead of 233 that the rest of the pathogen had.

Reviewer Comment

Table 3: The table can be presented in a better format without having to repeat the variables in columns 1 and 2 for the two pathogens. Why are some variables in column 1 in italics? (see throughout the tables)

Response

We thank the reviewer for their suggestion on improving the presentation of our results. As per the suggestion, tables 3, 4 and 5 have now been combined into Table 3. However, since the new Table 3 cannot fit on one page, it has been split into Table 3a and Table 3b. This change has allowed us to stop repeating information from column 1. However, we have retained column 2 because the data changes between pathogens depending on whether there was missing data or not.

Reviewer Comment

Tables 4 and 5 should be merged similar to comments made for table 3 to avoid duplicating the variables in columns 1 and 2.

Response:

As stated above tables 3, 4 and 5 have not been combined into Table 3. Since the Table 3 cannot fit on one page, it has been split into Table 3a and Table 3b. This change has allowed us to stop repeating information from column 1. However, we have retained column 2 because the data changes between pathogens depending on whether there was missing data or not.

Reviewer Comment

Line 240-41: What could influence the significant association of CDV with June than July? The study duration of 2 months to me is too short to make any meaningful deduction other than chance findings.

Response:

We thank the reviewer for noting this association and seeking an explanation. Unfortunately, this being a sero-survey, the scope of the study was limited and so we were not able to investigate this association further. However, due to the very strong association (OR = 7.8; p=0.002), it is unlikely to be due to chance. Suffice it to say that future studies will need to investigate this association further. We have added this comment in the manuscript.

Reference corrections were made.

19 Nov 2019

PONE-D-19-20072R1

Seroprevalence of viral and vector-borne bacterial pathogens in domestic dogs (Canis familiaris) in northern Botswana

PLOS ONE

Dear Melissa Kennedy

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process. Please note that the modifications here are very minor.

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

Many thanks for resubmitting your manuscript to PLOS One

It has again been reviewed by two expert reviewers, and they have come back with some very minor typographical and grammatical comments

If you can address these comments, then I can recommend the article for publication

Please do not feel that you need to write a full rebuttal to reviewers comments. Merely a line saying that all comments were addressed, and a comment on which ones were not and why.

Wishing you the best of luck with your minor revisions

Thanks

Simon

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

We would appreciate receiving your revised manuscript by Jan 03 2020 11:59PM. When you are ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

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Simon Russell Clegg, PhD

Academic Editor

PLOS ONE

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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: (No Response)

Reviewer #3: (No Response)

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

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: (No Response)

Reviewer #3: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: (No Response)

Reviewer #3: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

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: (No Response)

Reviewer #3: 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: (No Response)

Reviewer #3: Yes

**********

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 #3: I found this manuscript very interesting, and current, particularly with the sad plight of the African wild dog. It is an interesting way of looking at potential disease risk to these animals.

As this is a second submission, I have made mainly minor comments to the manuscript as I think it is well written and interesting.

Line 36- numbers under 10 should be written in full

Line 63- et al., should be in italics throughout as it is Latin

Line 70- would serious illness and/or death maybe be better as morbidity and mortality?

Line 80- would cross species transmission sound better?

Line 105- following manufacturers instructions may sound better, or as per manufacturers instructions

Line 108- you can shorten B. burgdorferi and D. immitis as you have already mentioned them in full. The only time it needs to be in full after the first time is at the start of a sentence. Please modify throughout

Line 114- comma after CPV

Line 125- you mention age distribution, but there is 0.3% missing. Where does that animal fit?

Tables may look better center aligned

Table 2. Why is there a difference in number tested for Anaplasma, Borrelia and Dinofilaria compared to the others? I don’t remember reading that in the text anywhere

Line 147- in the final what? It didn’t make sense to me

Line 153- comma after (Table 5)

Line 161- comma after MAWS

Table 3, 4 and 5- Is it possible to fill in some of the gaps (even a hyphen may help), or merge cells?

Line 187-190- this didn’t quite read correctly, but I cant work out how to reword it. Maybe have a look at it and see if you could modify it?

Line 196- wild doesn’t need capitalising

Line 197- comma after 2009

Line 199 – I think this is the first mention of E. canis so it needs to be in full

Line 201- Avoid using us, we, our etc in scientific writing

Line 203- change was to were

Line 218- E. ewingii needs to be in full as it’s the first mention of it

Line 219- Ehrlichia needs capitalising

Line 223- had a lower prevalence

Line 241- comma after month

Line 250- B. burgdorferi needs to be in full as it’s the start of a sentence

Reference 10- names don’t need capitalising

**********

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: None

Reviewer #3: No

[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.

Submitted filename: Cor PONE-D-19-20072 R1.pdf

Click here for additional data file.

Submitted filename: Cor PONE Rev 1.docx

Click here for additional data file.

25 Nov 2019

Minor revisions were addressed.

Submitted filename: Response to Reviewers (minor revisions).doc

Click here for additional data file.

23 Dec 2019

Seroprevalence of viral and vector-borne bacterial pathogens in domestic dogs (Canis familiaris) in northern Botswana

PONE-D-19-20072R2

Dear Dr.Kennedy

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 revised manuscript to PLOS One

As you have addressed all the comments (thank you) I have recommended the manuscript for publication

I wish you all the best for your future research

Many thanks

Simon

Reviewers' comments:

30 Dec 2019

PONE-D-19-20072R2

Seroprevalence of viral and vector-borne bacterial pathogens in domestic dogs (Canis familiaris) in northern Botswana

Dear Dr. Kennedy:

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.

Thank you for submitting your work to PLOS ONE.

With kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Simon Russell Clegg

Academic Editor

PLOS ONE