Molecular Prevalence and Haematological Assessments of Avian Malaria in Wild Raptors of Thailand
, Ratiwan Sitdhibutr 2,3, Pornchai Pornpanom 4, Preeda Lertwatcharasarakul 5, Raveewan Ploypan 5, Aksarapak Kiewpong 6, Benya Chatkaewchai 7, Nithima To-adithep 6 and Chaiyan Kasorndorkbua 2,3,8,*
Dennis A. LaPointe
Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsThe manuscript is generally well-written and the few problem sentences can be revised easily. I do make a few comments where some method might need further clarification or feel a point needs elaboration. However, I do feel the Discussion needs some work. In short it needs a more central theme. More cohesion of the various scattered points. Please see the attached comments for a more detailed review.
Comments for author File: 
Comments.pdf
Overall, the quality of English if fine. The manuscript is well-written and mostly understandable. There are some confusing sentences that will need to be revised and some poor word choices that should be change. I have pointed these out in the comments to the authors and suggested possible changes.
Author Response
Abstract
A concise, well-written abstract. I made some suggested word changes that I think make the
methodology clearer. Since avian malaria does not always have a negative impact on infected individuals or populations, I added the word may on Line 23. Also, in Line 34 I felt the word vulnerable made better sense than susceptible since the authors consider the possibility of ecological factors as another explanation of observed differences.
Line 23 Avian malaria that causes the negative impact on…. change to Avian malaria that may cause a negative impact on…
Answer: Thank you. The sentence is revised.
Line 28 A total of 0.5–1 mL of each EDTA blood 28 sample was tested …… change Each EDTA blood sample (0.5–1 mL ) was tested ……..
Answer: Thank you. The sentence is revised.
Line 30 Amplicons of PCR positive samples were performed a….. change to Amplicons of PCR positive samples were analyzed for……
Answer: Thank you. The sentence is revised.
Line 32 , and it had a prevalence…. change to with a prevalence….
Answer: Thank you. The sentence is revised.
Line 34 more susceptible to…. change to more vulnerable to…..
Susceptible suggests an immunological difference in the hosts while vulnerable is a more general term that could include ecological factors like differences in vectors and local malarial prevalence.
Answer: Thank you. The sentence is revised.
Background
The Background is brief but adequately covers the who, what, where, and why of the study. Key supporting citations are present but I wonder why the earlier work by Pilai Poonswad is not included as a reference for perhaps the earliest survey for haematozoa in the birds of Thailand. (Manwell RD, McClure HE, Poonswad P, and Greiner E. 1979. Haematoza in the birds of eastern and southern Asia. J Parasitology 65(1): 7)
Answer: We are unable to access the book from Manwell et al (1978).
Line 47 In particular….. This is a complicated sentence as written. Consider revising the sentence to something like this. Of these non-breeding visitors, global populations of the Himalayan Vulture, Gyps himalayensis, and Cinereous Vulture, Aegypius monachus are threatened by the decline in natural food sources, poached for ornamental purposes, and poisoned by livestock treated with non-steroidal, anti-inflammatory drugs.
Answer: Thank you for your suggestion. The sentence is revised. “Particularly, Himalayan Vulture, Gyps himalayensis, and Cinereous Vulture, Aegypius monachus, (non-breeding visitors) that are threatened by several factors, such as the decline in natural food sources, poaching for ornamental purposes and toxic contamination in their prey (using ofnon-steroidal anti-inflammatory drugs in livestock). Thus, their global populations tend to be decreasing”
Line 52 In addition to anthropogenic threats, infectious diseases in tropical regions, such as haemosporidiosis, also negatively affect….. Delete the also.
Answer: We delete it.
Line 56 … both the prevalence and parasitemia levels are at small spatial scales… This sentence should be revised. As written, it is confusing because in the first part of the sentence avian malaria is described as globally widespread. Do the authors mean that reported studies of prevalence and parasitemias are still limited to small geographical areas?
Answer: We delete this sentence.
Line 79 Nevertheless, the abundance of vectors promoted by environmental factors can be considered a risk for the persistence of the diversity of parasitic species [16]. Furthermore may be a better word than Nevertheless for this sentence as it adds to the explanations for the diversity of malarial parasites. It is unclear if the authors mean the numbers of individuals of a vector species or the number of different vector species. Either way they are promoted (or increased) by environmental factors (which might be climatic or habitats).
Answer: We revise the sentence into “Additionally, the abundance of vectors promoted by environmental factors (temperature and water body) is considered a risk for the persistence of the diversity of parasitic species”
Line 83 In addition, genetic sequencing could reveal parasites at the lineages level, regarding to the MalAvi database [17]. Drop the regarding to the MalAvi database since the citation number refers to the MalAvi database as the source for lineage sequences.
Answer: Thank you. We delete it.
Methods
Lines 99 and 112 Why do the authors provide the results (Tables 1 and 2) in the methods section of the
manuscript. Shouldn’t these be cited in the Results section.
Answer: We explain about the sample population that are included in this study. The species of birds (Table 1) and localities (Table 2). This information is better to explain by the table and figure. Thus, we cite these table and figure in this section.
Line 97 A citation for the Bal-chatri trap would be appropriate. It is a more specific trap and probably
only familiar to people working with raptors.
Answer: We add the citation “https://doi.org.10.2307/4510726”
Table 1 Table one is clear and provides a valuable summary of specimens and results.
The first row of haematologic factors needs to be realigned with the overall values for diurnal raptors.
Answer: We would like to not provide the overall values for the diurnal raptors. The hematologic values can vary by species of birds.
Should the total number of Shikra be 6 or 7 since the parenthetical total number tested is 7?
Answer: Thank you very much. The total number of shikra is 6.
Figure 1 Clear figure, nicely labeled.
Answer: Thank you very much.
Line 111 …provinces; the raptors were obtained by either being caught in the wild or admitted to KURU……change to ….provinces. Raptors were caught in the wild or admitted to KURU.
Answer: The sentence is revised.
Table 2 Is a clear geographical summary of the diagnostic results.
Answer: Thank you very much.
Line 155 I am not certain what this sentence means. Can the authors elaborate or revise the sentence to
make the meaning clearer.
Answer: The sentence is revised to “The consensus length of our sequences generated by BT sequencing (Geumcheon-gu, Seoul, Korea) was 472 bp. Thus, we did not identify lineage following the MalAvi nomenclature”
Line 173 Were regions combined for the Fisher Exact Test? Are there enough degrees of freedom in each
cell for a valid test?
Answer: Each region contains the sample size that enough for calculation of Fisher exact. The region that contains small size are grouped with another region.
Results
The results section is clear and well-written
Answer: Thank you very much.
Figure 2 A clear and valuable phylogenic tree.
Answer: Thank you very much.
Line 232 We highlighted that there were the birds with sample size… change there to these
Answer: We change there to these.
Discussion
I feel the discussion may need some revision. Eliminating the first paragraph and changing the
paragraphs to focus on general ecological and epidemiological themes instead of individual species.
Answer: We revise the first paragraph.
Ecological patterns in malarial prevalence across species, parasite lineages and geography and how underlying epidemiological factors may contribute to these patterns. Given the small sample size the authors may want to avoid over speculation of the significance of these observations and offer general alternative explanations. Also, there is no need to reiterate the statistical values presented in the results section as in paragraph 2 and 3 of the Discussion.
Answer: Answer: We revise the 2nd and 3rd paragraphs.
What seems to be missing in both the Discussion and Conclusions is direct reference back to the conservation status of the two vulture species and the central question of whether or not avian malaria could be a contributing factor in their decline.
Answer: We add the conclusion “Additionally, other experiments may be needed for more understanding that whether the Plasmodium are the contributing factor in population decline of vulture and other raptors or not.:
As the introductory paragraph of the Background starts with the protected conservation status of raptors in Thailand and the global decline of the two vulture species, one would assume this theme would be revisited in the Discussion and Conclusion. The authors should consider making this the central theme to the paper and structure the discussion and concluding remarks to refocus on the potential impact of avian malaria to the conservation of raptors in general and the two vulture species specifically.
Answer: Thank you for your suggestion.
Line 243-255 This is a confusing paragraph as written and tangential to the study as a whole. The specific concerns regarding the use of EDTA can be worked into the general discussion as a possible confounding factor for broader ecological interpretation of the results. A sentence in the conclusions summarizing the value and concerns of using EDTA in blood samples would be valuable. A simple solution, since raptors are generally large birds, is to take two blood samples one for standard haematology and one for molecular diagnostics.
Answer: We revise the first paragraph. Line 211-220.
Line 246 In an in vitro exflagellation of Haemoproteus tartakovskyi, blood containing gametocytes was mixed in 3.5% sodium citrate, and the blood was allowed to be exposed to air for 4 minutes [24]. This sentence highlights some of the problem with this paragraph. As written, it is unclear whether this sentence supports or disclaims the statement made before or how it relates to any of the sentences in this paragraph.
Answer: The sentence is revised.
How does EDTA interfere with gameotocyte development and why would this affect the ability to detect gametocytes in an infected individuals since the gametocytes would be present before exposure to EDTA.
Answer: The EDTA blood smear often show extracellular gametocytes.
I understand how EDTA is valuable for standard blood work but I do not know how it would affect the ability to detect haematozoan infection.
Answer: It not interfere the detection of haematozoan. However, it often shows extracellular gametocytes in smear. Normally, we describe haemosporidian parasite based on mature gametocytes within the red blood cells. For the extracellular gametocytes, we are not sure if the gametocytes are young or mature. We are talking for only parasite description not parasite detection.
Does it interfere with the PCR reaction because all that is discussed in this paragraph is blood smears and blood cell morphology.
Answer: We are discus only about EDTA blood smear.
This sentence – Line 252 Therefore, EDTA blood cannot used for parasite description but can be used for parasite detection and blood morphology observation. – suggests that EDTA may interfere with PCR and sequencing but it needs to be made clear.
Answer: Thank you very much for your suggestion. In this study, we have only the information about anticoagulant related to the parasite morphology. We have no information about EDTA and PCR results. Thus, we did not discuss more.
Line 257 End the sentence after …PCR analysis as PCR has been the standard diagnostic use in these types of studies for decades. see references by Bensch & Hellgren [17,19]
Answer: Thank you for your suggestion. The sentence is revised.
Line258-263 Remove the reiterated results. For example: The overall prevalence in DIRs was as low and
similar to the prevalence of DIR’s previously reported [23]. The prevalence in NORs in this study was similar to that in DIRs, however, much lower than reported previously (9.0% (95% CI: 4.65–13.32) [27].
Answer: Thank you for your suggestion. The sentence is revised.
Lines 260-267 I think its interesting to compare the current study with the previous studies in Thailand
but I think the authors should also look at studies of haematozoa prevalence in raptors from other parts
of the world to see if these patterns of infection are similar. Even going back to broader regional studies
like Manwell RD, McClure HE, Poonswad P, and Greiner E. (1979).
Answer: We are unable to access the book from Manwell et al (1978).
Line 263 Culicine vectors of avian malaria may be diurnal or nocturnal. For diurnal passerines the best vectors are likely nocturnal when passerines are sleeping and unlikely to display anti-mosquito behavior while for stealth predatory species like owls and herons remaining still might override. Taxonomic differences in haematozoan prevalence may be based on ecological aspects of epidemiology or more innate physiological aspects. I think the authors can elaborate here.
Answer: We add the sentence “Furthermore, the defensive behaviour of birds (foot stomping, head and wing movements and tail shaking) [27] might prevent mosquitoes bite, and this related with the prevalence in NORs and DIRs.” Line 228 – 230.
”
Line 267-269. This sentence just seems to be floating with no real connection to the rest of the paragraph.
This sentence may be better suited for another paragraph on rehabilitation and conservation effort of
species of concern.
Answer: The sentence is revised.
Line 279 While latitudinal differences in prevalence may be due to climatic and geographical factors influence on vector abundance and diversity, I think the small sample size for the Southern region may skew any true difference in prevalence. It is also interesting to note that two of the three positive birds are migratory vultures and may have acquired the infections in their breeding range.
Answer: We change to not statistically compare the prevalence among the region.
Line 285 The phylogenetic analyses revealed that they were identical to each other. By they I assume the authors mean the haematozoan sequences that were detected. Please revise this sentence because as written it sounds like the birds have identical lineages.
Answer: We revise the sentence “The phylogenetic analyses revealed that Plasmodium infected in both Himalayan Vulture and Eagle-owl were identical to each other.”
Line 291 Is there any reasons why the vulture could not have acquired the parasite on its summer range. This seems equally as likely as acquiring it in captivity in Thailand. In the next paragraph, regarding the cinereous vulture, it is suggested that the P. elongatum - like parasite might have been acquire on the vulture summer, breeding grounds. Any of these scenarios seem likely and should probably be discuss as a group and not species by species. Of the four species found to be infected 3 are migratory or potential migratory so an origin for their infection could be outside of Thailand. Only the owl Ketupa sumatrana is a resident species so its infection had to be acquired locally.
Answer: We delete the sentence.
Line 307 This sentence suggests that malaria is probably not a threat to these declining vulture species. I feel this is an opportunity to re-address the significance of malaria to the conservation of raptors and vultures specifically.
Answer: We discus that immune mechanism works against the severity of chronic infection. The immune may depend on individual health. Susceptible vulture may be dead quickly.
Conclusions
The conclusions might benefit from a brief summary of result highlights that might include 1) a statement
about the concerns of using EDTA in this type of study 2) the observation that primarily migratory and
potentially migratory species were infected, 3) that infected birds were asymptomatic and 4) that lineages
included unique sequences, lineages formerly reported in Thailand as well as lineages with a broader
distribution across Asia.
Line 341-344 This study provides valuable insights into the prevalence and genetic diversity of Plasmodium spp. in raptors in Thailand and offers crucial information for effective disease management strategies for endangered species. I would agree with this statement but I wish the authors would have stated in the Discussion section how this information might be used in the management of raptors in Thailand. A couple of concerns are made about migratory species bring potentially new parasites to Thailand as well as chicken and fighting cockerals serving as a reservoir of P. gallinaceum that may be transmitted to raptors. But these concerns may all be mote if there is no evidence of pathology in infected raptors.
Answer: The conclusion is revised.
Reviewer 2 Report
Comments and Suggestions for AuthorsI believe that the work is of utmost importance for a better understanding of haemoparasites found in raptors, especially in the studied region
Comments for author File: Comments.pdf
Author Response
Line 3: This is related to a disease right? If you did not correlate the findings with the disease, may be more adequate to refer to the parasites
Answer: This article works on the Plasmodium. This protozoan parasite causes the malaria. Thus, we would like to report in the topic of “Molecular Prevalence and Haematological Assessments of Avian Malaria”.
Line 47: “Sedentary” is it the right term?
Answer: The word is changed into “resident”.
Line 63: “age” is it the right term?
Answer: We would like to explain that any age of birds (chicks, young or adults) is not only one factor that relates to the severity of infection.
Round 2
Reviewer 1 Report
Comments and Suggestions for AuthorsMuch improved. I only have a few suggested changes in word or sentence structure that I feel will improve the manuscript.
Simple Abstract Line 21 Avian malaria caused by Plasmodium. It is considered.... change to Avian malaria, caused by Plasmodium spp., is considered....
Abstract Line 45 more vulnerable... change to are more vulnerable....
Background Line 89 Does water body refer to precipitation or larval vector habitat. Please use a more specific word.
Methods Line 110 What percentage of the 109 birds sampled were wild captured vs. birds at KURU? Can the authors report this number as infirmed birds may have a greater likelihood of being infected.
Discussion 1st paragraph is greatly improved.
Line 265 This is the evidence..... change to This is evidence......
Line 269 as low as previously ... change to as low as prevalences previously reported.
Line 275 daytimes.... change to daytime.....
Line 277 prevent mosquitoes bite... change to that prevent mosquito bites might be related to prevalence differences in DIRs and NORs.
Line 299 Since the environment in Southern Thailand may promote the abundance of parasites and vector, and Plasmodium is not described for species, thus, future studies should investigate Haemosporidian parasites in Southern Thailand. This area may allow to collect the samples that are positive for parasites. Change to Since the environment in Southern Thailand may promote the abundance of parasites and vectors, future studies should investigate Haemosporidian parasites in Southern Thailand. The last sentence can be dropped. It is implied.
Line 320 Considering that it might be the undescribed... change to It might be an undescribed....
Line 388 Regarding the locality, southern Thailand, which may contribute to the distribution of avian malaria among raptors, as was revealed by the phylogenetic lineages in this study. Change to a more direct sentence. For example. Southern Thailand, with its humid rainforest ecological niche, had the highest prevalence and diversity of Plasmodium spp.
Line 342 Additionally, other experiments may be needed for more understanding that whether the Plasmodium are the contributing factor in population decline of vulture and other raptors or not. Change to Additionally, experiments may be needed to understand whether or not Plasmodium is a contributing factor in the decline of vultures and raptors in general.
Comments on the Quality of English LanguageMuch improved. Still a few awkward sentences and word choices.
Author Response
Simple Abstract Line 21 Avian malaria caused by Plasmodium. It is considered.... change to Avian malaria, caused by Plasmodium spp., is considered....
Answer: The sentence is revised.
Abstract Line 45 more vulnerable... change to are more vulnerable....
Answer: The sentence is revised.
Background Line 89 Does water body refer to precipitation or larval vector habitat. Please use a more specific word.
Answer: Thank you for suggestion. The “water body” is changed into larval vector habitat.
Methods Line 110 What percentage of the 109 birds sampled were wild captured vs. birds at KURU? Can the authors report this number as infirmed birds may have a greater likelihood of being infected.
Answer: The sentence “Of these 109 birds, 56 birds were admitted to the KURU (KU samples) and the other 53 birds were captured from the wild (R samples)” is added. Line 115-117.
Discussion 1st paragraph is greatly improved.
Answer: Thank you very much.
Line 265 This is the evidence..... change to This is evidence......
Answer: The sentence is revised.
Line 269 as low as previously ... change to as low as prevalences previously reported.
Answer: The sentence is revised.
Line 275 daytimes.... change to daytime.....
Answer: We made the change
Line 277 prevent mosquitoes bite... change to that prevent mosquito bites might be related to prevalence differences in DIRs and NORs.
Answer: The sentence is revised.
Line 299 Since the environment in Southern Thailand may promote the abundance of parasites and vector, and Plasmodium is not described for species, thus, future studies should investigate Haemosporidian parasites in Southern Thailand. This area may allow to collect the samples that are positive for parasites. Change to Since the environment in Southern Thailand may promote the abundance of parasites and vectors, future studies should investigate Haemosporidian parasites in Southern Thailand. The last sentence can be dropped. It is implied.
Answer: The sentence is revised.
Line 320 Considering that it might be the undescribed... change to It might be an undescribed....
Answer: The sentence is revised.
Line 388 Regarding the locality, southern Thailand, which may contribute to the distribution of avian malaria among raptors, as was revealed by the phylogenetic lineages in this study. Change to a more direct sentence. For example. Southern Thailand, with its humid rainforest ecological niche, had the highest prevalence and diversity of Plasmodium spp.
Answer: The sentence is revised.
Line 342 Additionally, other experiments may be needed for more understanding that whether the Plasmodium are the contributing factor in population decline of vulture and other raptors or not. Change to Additionally, experiments may be needed to understand whether or not Plasmodium is a contributing factor in the decline of vultures and raptors in general.
Answer: The sentence is revised.
