The authors have declared that no competing interests exist.
Current address: Department of Veterinary Biosciences, Ohio State University, Columbus, Ohio, United States of America
Current address: EVMS-Sentara Healthcare Analytics and Delivery Science Institute, Eastern Virginia Medical School, Norfolk, Virginia, United States of America
Current address: Universidade Estadual do Ceará, Fortaleza, CE, Brazil
Current address: Center of Innovation for Complex Chronic Healthcare, Edward Hines Jr. Veteran Affairs Hospital, Hines, Illinois, United States of America
Current address: Department of Pathology, Microbiology and Immunology, Vanderbilt University Medical Center, Nashville, Tennessee, United States of America
Dogs are the primary reservoir for human visceral leishmaniasis due to
We examined infectiousness of dogs vertically infected with
Together, our data implicate skin parasite burden and earlier clinical status as stronger indicators of outward transmission potential than blood parasite burden. Our studies of a population of dogs without vector transmission highlights the need to consider canine vertical transmission in surveillance and prevention strategies.
Sand flies transmit
Visceral leishmaniasis (VL) is caused by protozoan
Epidemiological modeling and experimental findings support that a fraction of
Importantly, sand flies are telmophages, feeding from blood pools formed by lacerating mammalian skin with a serrated proboscis [
When sand flies egest
To address these questions, we assayed skin parasite burden and performed xenodiagnosis on a cohort of dogs vertically infected with
All animal use involved in this work was approved by the University of Iowa Institutional Animal Care and Use Committee and was performed under the supervision of licensed and, where appropriate, board-certified veterinarians according to International AAALAC accreditation standards. Canine subjects in this study were donated to the University of Iowa after signed informed consent was obtained.
Canine subjects were obtained from a cohort of U.S. dogs where vertically transmitted
On the day of xenodiagnosis, a veterinarian conducted a physical exam on each dog for clinical signs of leishmaniosis including low body condition score, dermatitis, coat condition, lethargy, lymphdenomegaly, conjunctivitis, alopecia, cutaneous lesions, pale mucous membranes, or epistaxis. Whole blood and serum were collected. Whole blood was subjected to complete blood count (IDEXX Laboratories Inc.) and DNA isolation using the QIAamp Blood DNA Mini Kit according to manufacturer instructions (Qiagen). Real Time-quantitative PCR (RT-qPCR) for
Dogs were sedated with dexmedetomidine (Zoetis Inc.). Heartrate and respiratory rate were monitored throughout procedure. Dogs were placed within an enclosed mesh chamber, and two feeding cups per dog were placed on the axillary region and inner pinna for 30 minutes [
Following feeding, sand flies were incubated for 48 hours at 26°C in a humidified chamber with access to sucrose solution. Due to institutional requirements, 48hrs was the maximum allowable incubation time post-feeding on infected dogs to minimize risk of infection to staff. After incubation, individual engorged blood-fed female sand flies were separated into Eppendorf tubes containing 100 uL of DNA lysis buffer (Gentra Puregene Tissue Kit, Qiagen) and stored at -20°C.
Following xenodiagnosis and humane euthanasia per AVMA guidelines, a full necropsy was performed by a board-certified veterinary pathologist. Canine spleen samples were obtained, flash frozen on dry ice, and stored at -80°C until DNA isolation. Skin biopsies were taken from axillary and pinnal sand fly feeding sites as well as contralateral, unfed sites from each study animal using 6 mm punch biopsies (Integra) and placed into zinc formalin for histology or frozen in saline at -80°C for nucleic acid isolation.
For preparations of standard curves, low passage
Sand fly and canine sample DNA were isolated with the Gentra Puregene Tissue Kit (Qiagen). For
Normality of data was assessed using the D’Agostino-Pearson test. For comparisons between tissues obtained from the same subject, Wilcoxon matched-pairs signed rank test was performed. For comparisons between subjects, Kruskal-Wallis ANOVA was performed. When appropriate, Dunn’s post-test was used for multiple comparisons. Spearman correlation was computed for correlation analyses. For all analyses, significance is: *p≤0.05; **p<0.01; ***p<0.001; ****p<0.0001.
In
Calculated
Real Time-qPCR for
The spleen is a major target organ of
To investigate skin parasite burden, we assayed between two and four unique skin biopsy sites per dog. The parasite quantification shown in
Parasitemia has long been considered a focal determinant of
Spearman’s correlation (A) and paired Wilcoxon test (B) between skin parasite load and blood parasite load from the same animals (n = 16). Spearman’s correlation (C) and paired Wilcoxon test (D) between skin parasite load and splenic parasite load from the same animal (n = 14). r, Spearman correlation coefficient.
Although it was demonstrated that vertically infected dogs could transmit parasites to sand flies [
(A) Frequency of sand flies containing a blood meal and (B) frequency of blood meal containing sand flies containing >1 calculated parasite after xenodiagnosis on dogs at indicated LeishVet clinical stages. (C-F) Calculated number of parasites per individual sand fly after xenodiagnosis on dogs with indicated LeishVet clinical stage (C), number of VL clinical signs (D), age range (E), or DPP serological value (F). Kruskal Wallis ANOVA with Dunn’s post-test. **p<0.01; ***p<0.001; ****p<0.0001.
Although flies successfully fed on all dogs at a similar rate, we measured variable parasite equivalents in each sand fly after feeding. (
Clinicopathological abnormalities such as signs of non-regenerative anemia, hypergammaglobulinemia, hypoalbuminemia, and increased creatinine are used to inform LeishVet stage categorization. As the sample size in LeishVet stage 3 and 4 was relatively low, we also stratified tissue parasite burden and xenodiagnosis results by whether a dog has progressed to present clinicopathological alterations (
The age range of dogs with the highest average parasite uptake per sand fly in this cohort was 3–4 years old (
Parasitemia is known to correlate with
Correlation between calculated parasite uptake by individual sand flies after xenodiagnosis and paired (A) average skin parasite burden (n = 16), (B) blood parasite burden (n = 16), (C) or splenic parasite burden (n = 14). Skin parasite burden was averaged between the two biopsies collected from ipsilateral feeding sites. Spearman correlation p-value and correlation coefficient (r) are shown.
This study is the first investigation into parasite dermotropism and its relationship to xenodiagnosis in a vertically infected canine VL cohort. Our data supports significant parasite burdens in sand fly accessible tissues, such as blood and particularly skin, as the driving factor determining infectiousness of
Dermal parasite load also differentiates human hosts contributing to the infectious sand fly pool. In people with post-Kala Azar dermal leishmaniasis (PKDL) due to
The mechanisms
The proficient
We saw that by grouping dogs presenting complete blood count or serum chemistry alterations had significantly higher tissue parasite burdens, which lead to increased parasite uptake by xenodiagnosis, compared to dogs with no clinicopathological abnormalities. However, using the higher resolution LeishVet scale to stage disease revealed an interesting dynamic. Mild to moderately diseased dogs (LeishVet levels 2–3) were the most infectious to
It is interesting to consider why parasites from dogs with severe VL disease may not be as transmissible. Here,
Diagnosing VL early in the infectious course in dogs is difficult due to the non-specific nature of initial clinical signs and clinicopathological results. Interestingly we observed several dogs with undetectable parasitemia already had detectable parasites in the skin by qPCR. This could implicate skin biopsy as a more sensitive diagnostic in these instances. Importantly, some sand flies fed on these dogs were able to successfully take up parasites, demonstrating that these early-stage dogs are also infectious to sand flies as seen in our experiments, despite having a low blood burden.
Although we quantified parasite uptake after xenodiagnosis with a high degree of sensitivity utilizing qPCR, a limitation of this study is that parasite viability in the sand fly midgut was not verified by microscopy, and therefore we cannot assume all parasites survived ingestion. Due to institutional requirements, this study was not able to capture whether all infected sand flies would go on to develop high levels of infection and virulent metacyclic promastigotes. Longer incubation times are needed for parasites to undergo logarithmic replication and metacyclogenesis in the sand fly. We expect, with longer incubation times, the correlation between skin parasite load and sand fly infection level would be even stronger. Additionally, Serafim
It is evident high skin parasite burden significantly improves the probability of transmission to sand flies in dogs with CanL and PKDL patients, however whether skin parasite accumulation occurs to the same extent during active human VL is not well described. Dermotropism of
Xenodiagnosis is the most accurate way to ascertain reservoir infectivity to vectors, however it is not trivial to perform. More easily measured correlates of xenodiagnosis need to be identified for practical application. In agreement with studies in vector-infected dog cohorts, our data supports skin parasite load as a surrogate marker of dogs capable of
Overview of the age in years, sex, and LeishVet status of xenodiagnosis cohort.
(DOCX)
Overview of serum chemistry and blood count findings for dogs in each LeishVet clinical grouping. Bolded values indicate the mean is outside of the normal reference range.
(DOCX)
Overview of dogs, sand flies, and sample handling workflow (left). Description of feeding and skin biopsy sites, ipsilateral pertains to side of sand fly feeding and contralateral indicates same location on opposite side where no sand fly feeding occurred.
(TIFF)
(A-B) Calculated
(TIFF)
(A) Standard curve derived from
(TIFF)
Dogs within normal limits (WNL) were compared against dogs with abnormal complete blood count or serum chemistry clinicopathological findings (Abnormal). Calculated parasite burden in blood (A, n = 16), spleen (B, n = 14), average of 2–4 skin biopsies (C, n = 16). (D) Frequency of female sand flies containing a blood meal after feeding at each site. (E) Frequency of female sand flies containing >1 parasite equivalent 48hrs post-feeding. (F) The calculated parasite burden within sand flies 48hrs post-feeding. (A-F) Mann-Whitney test. *p<0.05; **p<0.01; ****p<0.0001.
(TIFF)
We would like to thank the participating animal caretakers who donated subjects to the University of Iowa for this work. We also acknowledge the University of Iowa Comparative Pathology Core technicians for their valuable necropsy assistance. Figure artwork was created using
Dear Dr. Petersen,
Thank you very much for submitting your manuscript "Leishmania infantum xenodiagnosis from vertically infected dogs reveals significant skin tropism" for consideration at PLOS Neglected Tropical Diseases. As with all papers reviewed by the journal, your manuscript was reviewed by members of the editorial board and by several independent reviewers. In light of the reviews (below this email), we would like to invite the resubmission of a significantly-revised version that takes into account the reviewers' comments.
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Mitali Chatterjee
Associate Editor
PLOS Neglected Tropical Diseases
Epco Hasker
Deputy Editor
PLOS Neglected Tropical Diseases
***********************
Reviewer's Responses to Questions
As you describe the new analyses required for acceptance, please consider the following:
-Are the objectives of the study clearly articulated with a clear testable hypothesis stated?
-Is the study design appropriate to address the stated objectives?
-Is the population clearly described and appropriate for the hypothesis being tested?
-Is the sample size sufficient to ensure adequate power to address the hypothesis being tested?
-Were correct statistical analysis used to support conclusions?
-Are there concerns about ethical or regulatory requirements being met?
Reviewer #1: The objectives of the study are articulated with a clear testable hypothesis stated.
The study design is appropriate to address the stated objectives.
Statistical analysis used to support conclusions were correct.
There is no concern about meeting ethical or regulatory requirements.
Reviewer #2: (No Response)
Reviewer #3: 1. Line 91-92 : VL is also caused by L. donovani, and author should make it clear by revising the sentence.
2. Reference 4 is about transplacental transmission in dog but not in humans. Please add appropriate reference.
3. Line 133: How did author confirm the infection?
4. Line: What is the meaning of reader value 10? Is it optical density or percent positives, and how this threshold value change with sample to sample?
5. Line 157 – 160: I guess then approx. 40-50 flies (including males) were exposed at a time during xenodiagnosis, if yes then how did this number come? This is too high number in feeding cups. Please support it with appropriate reference.
6. Line 164: Again, no reference for 30 min exposure to justify best exposure time in dogs.
7. Line 165-166: Why fed flies were incubated for 48 hrs and how this time period was established. As with many other xenodiagnosis studies, researchers use up to 72 hrs flies for outcomes. Please discuss it in detail as this is one of the important factors that can affect the outcomes.
8. Line 165-168: Is there any reason not to perform microscopy for providing direct clinal evidence?
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-Does the analysis presented match the analysis plan?
-Are the results clearly and completely presented?
-Are the figures (Tables, Images) of sufficient quality for clarity?
Reviewer #1: The analysis match the analysis plan.
The results are clearly and completely presented.
Tables and figures in the manuscript and in supporting information are of sufficient quality for clarity.
Reviewer #2: (No Response)
Reviewer #3: 9. Figure 1 do not include negative control (blood, spleen and skin) from healthy dogs and it is difficult to predict threshold without proper control. Author should make it clear in the figure legend. In addition, Figure-1 (A and B) shows the data only 14 dogs instead of 16. I would like to suggest to make schematic flow chart (as supplementary figure) showing all details e.g. recruitment, inclusion and exclusion criteria, samples collection, processing, xenodiagnosis etc. with time periods. This chart is essential to make the study process clear.
10. Figure 1A: how this 2.5 ug DNA was calculated? I guess it should be parasites genome/ml of blood.
11. Figure 1C: Since 2 -4 skin biopsy per dog was sampled as mentioned in line: 230 therefore, please explain the figure about other biopsies data? If it is Figure S2A, then please use the colour dots representing each sample in each category.
12. Line 281- 283: This is what something strange and may be due to false positivity of DNA? Singh et al 2020 (PLoS NTD) has also discussed about this issue with PCR and thus gold standard microscopy xenodiagnosis is needed to confirm such findings. I would like to suggest to perform one set experiment in all these four categories of dog with microscopy xenodiagnosis not only in support of findings but also for direct clinical evidence with live parasite.
13. I don’t understand the need of Figure 3D. Please move it to supplementary or explain the meaning of clinical sign in context to LeishVet Guidelines stages.
14. Fig 3C-F: Please define LOD and how it was calculated?
15. Figure 3A is confusing with regard biopsy number per dogs? Is it cumulative parasite burden or one biopsy only? Please make it clear.
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-Are the conclusions supported by the data presented?
-Are the limitations of analysis clearly described?
-Do the authors discuss how these data can be helpful to advance our understanding of the topic under study?
-Is public health relevance addressed?
Reviewer #1: The conclusions are supported by the data presented.
The limitations of analysis are clearly described.
The authors discuss in great detail the usefulness of the data to advance our understanding of the topic under study.
Yes, the public health relevance is addressed.
Reviewer #2: (No Response)
Reviewer #3: 16. Line 358-359: I do not agree with authors as data presented by authors are limited by qPCR. Please discuss it as one of the limitations of study and further studies with microscopy xenodiagnosis are needed to support the findings.
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Use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity. If the only modifications needed are minor and/or editorial, you may wish to recommend “Minor Revision” or “Accept”.
Reviewer #1: The subject of this manuscript is within the field of interest for PNTD. It is well written and concise and therefore is suitable for publication in PNTD as Research Article, following some minor revision indicated below.
1. There is no doubt that the skin parasite load seems a better predictor than peripheral blood in canine leishmaniasis and probably in PKDL patients. However, in the case of human VL caused by L. infantum this is not so clear. Although the skin seems to play some role in immunosupressed L. donovani-infected patients it must also be taken into account that venous blood also is an important source of parasites for sand flies in the case of HIV / L. infantum coinfection. Furthermore, in the case of the human visceral leishmaniasis caused by Leishmania infantum the proportion of patients harboring parasites in their healthy skin is relatively low and such parasitism, although unusual, may be a source of infection for phlebotomine sand flies (Moura CRLP Costa CHN, Moura RD, Braga ARF, Silva VC, Costa DL. Cutaneous parasitism in patients with American visceral leishmaniasis in an endemic área. Revista da Sociedade Brasileira de Medicina Tropical. 2020; 53:e20190446.
2. Lines 330-339. I think that in the discussion addressed in this paragraph it would be very enriching also discuss the work of Mondal et al, 2018 (Mondal D, Bern C, Ghosh D, Rashid M, Molina R, Chowdhury R, Nath R, Ghosh P, Chapman LAC, Alim A, Bilbe G, Alvar J. Quantifying the infectiousness of post-kala-azar dermal leishmaniasis toward sand flies. Clinical Infectious Diseases. 2018; ciy891.
3. Why the study was not able to capture whether all infected sand flies would go on to develop high levels of infection and virulent metacyclic promastigotes?.
Reviewer #2: (No Response)
Reviewer #3: N/A
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Use this section to provide overall comments, discuss strengths/weaknesses of the study, novelty, significance, general execution and scholarship. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. If requesting major revision, please articulate the new experiments that are needed.
Reviewer #1: This is an excellent work well-designed and conducted. The authors show in this study that canine vertical transmission of leishmaniasis should be taken into consideration in surveillance and prevention strategies. Furthermore the authors corroborate that parasite burden of dog skin can be a strong indicator of outward transmission potential.
With this paper one more piece is provided to the puzzle of the canine leishmaniasis epidemiology.
Reviewer #2: COMMENTS TO AUTHORS
There are some issues that the authors must consider. The manuscript at this moment should not be recommended or needs major revisions before to evaluate its publication.
Major comments
I suggest review the experimental design used to classify CanL disease. The authors described that CanL classification used in the study was according to Leishvet guideline. However, the stages defined in this study as "subclinical (stage 1), mild (stage 2), moderate (stage 3), or severe disease (stage 4)" are not according to the LeishVet reference. The Leishvet guideline establishes CanL disease as "stage I (mild disease), stage II (moderate disease), stage III (severe disease) and stage IV (very severe disease)". If the author adapted the CanL classification, the used criteria should be described. Since small sample was evaluated in this study, I suggest the authors consider the reclassification of dogs according to "no clinicopathological alterations versus with clinicopathological alterations".
The authors describe that skin parasite burden is more important than dog´s clinical status. I suggest to reword the sentences with these statements, since LeishVet stage 3 and 4 had few dogs, 2 and 1, respectively. Their emphasized does not appear appropriate.
In Material and methods section should provide more information about dogs sampling. What is their origin? How was performed the follow up of these dogs since birth? It is not clear if after the birth, the dogs of this study have never been exposed to areas with potential risk for L. infantum infection transmitted by the vector.
In figure 1, is not clear how statistical analysis was performed. How was performed each group comparison? For example, parasite burden in blood was significantly higher compared with what? Line 215-216 in the statement L. infantum parasite burden in (A) blood (p=0.004; n=16), (B) spleen (p=0.153; 216 n=14), or (C) skin (p=0.072; n=16), these P value refers to comparison between blood and spleen or skin..., please clarify. I suggest include in figure 1 another graph to show parasite burden per tissue without CanL stages.
Minor comments
I suggest using the term "canine leishmaniasis (CanL)” when authors refer the dog disease.
Line 97. References (21, 22) are not correctly numbered in the order that they appear in the text.
Line 127 change ‘confirms’ to ‘suggests’
Line 287 change ‘subclinical disease’ to subclinical infection
In table S1, I suggest to provide age class frequency (young, adult or elderly). Exclude ‘mean (SD)’. Also, I suggest include information on breed.
In table S2, I suggest to include the reference source which was take out the value normal range of the laboratory data.
Lines 172-173 the statement “Skin biopsies were taken from axillary and pinnal sand fly feeding sites as well as contralateral”, I suggest to provide more information on number fragment tissues were obtained from each skin region.
Line 292-293. On the statement “the age range of dogs with the highest average parasite uptake per sand fly in this cohort was 3-4 years old”, what is hypothesis for this finding?
Line 305-306. In the statement “Parasitemia is known to correlate with Leishmania parasite transmission to sand flies”, provide reference.
Line 360-362. The statement “Moderately diseased dogs (LeishVet levels 2-3) were the most infectious to Lu. longipalpis sand flies compared to dogs with mild (LeishVet level 1) or severe disease (LeishVet level 4)” is confused. It is not accordign to CanL classification used by authors (line 151-152).
Line 394-396 statement “Our data supports skin parasite load as a surrogate marker of dogs with L. infantum transmission potential from both horizontally and vertically infected dogs”, I suggest exxclude “horizontally”, since this study not compared parasite skin load from horizontally infected dogs.
Reviewer #3: The study by Scorza et al have conducted xenodiagnosis on cohort of dogs to examined infectiousness of vertically infected dogs with
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Reviewer #2: No
Reviewer #3: No
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Dear Dr. Petersen,
We are pleased to inform you that your manuscript 'Leishmania infantum xenodiagnosis from vertically infected dogs reveals significant skin tropism' has been provisionally accepted for publication in PLOS Neglected Tropical Diseases.
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Thank you again for supporting Open Access publishing; we are looking forward to publishing your work in PLOS Neglected Tropical Diseases.
Best regards,
Mitali Chatterjee
Associate Editor
PLOS Neglected Tropical Diseases
Epco Hasker
Deputy Editor
PLOS Neglected Tropical Diseases
***********************************************************
Reviewer's Responses to Questions
As you describe the new analyses required for acceptance, please consider the following:
-Are the objectives of the study clearly articulated with a clear testable hypothesis stated?
-Is the study design appropriate to address the stated objectives?
-Is the population clearly described and appropriate for the hypothesis being tested?
-Is the sample size sufficient to ensure adequate power to address the hypothesis being tested?
-Were correct statistical analysis used to support conclusions?
-Are there concerns about ethical or regulatory requirements being met?
Reviewer #3: Yes
**********
-Does the analysis presented match the analysis plan?
-Are the results clearly and completely presented?
-Are the figures (Tables, Images) of sufficient quality for clarity?
Reviewer #3: Yes
**********
-Are the conclusions supported by the data presented?
-Are the limitations of analysis clearly described?
-Do the authors discuss how these data can be helpful to advance our understanding of the topic under study?
-Is public health relevance addressed?
Reviewer #3: Yes
**********
Use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity. If the only modifications needed are minor and/or editorial, you may wish to recommend “Minor Revision” or “Accept”.
Reviewer #3: Authors have substantially modified the draft and now it is more clear.
**********
Use this section to provide overall comments, discuss strengths/weaknesses of the study, novelty, significance, general execution and scholarship. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. If requesting major revision, please articulate the new experiments that are needed.
Reviewer #3: I would like to suggest to add Flow diagram of experimental design to make the process including exclusion and inclusion criteria clear!
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If you choose “no”, your identity will remain anonymous but your review may still be made public.
Reviewer #3: No
Dear Dr. Petersen,
We are delighted to inform you that your manuscript, "Leishmania infantum xenodiagnosis from vertically infected dogs reveals significant skin tropism," has been formally accepted for publication in PLOS Neglected Tropical Diseases.
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