Case Report of a Fatal Babesia vulpes Infection in a Splenectomised Dog

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors The manuscript is well structured and clear. The manuscript addresses an interesting and current topic; the follow-up given to the patient was very meticulous with good technical support using sensitive and specific diagnostic tests. Line 67 Mycoplasma haematoparvum change to italics.

Author Response

Thank you for the kind evaluation of our manuscript. Regarding the change of the name Candidatus Mycoplasma haematoparvum to italics: as this species has only the Candidatus status it should not be italic as stated by the International Code of Nomenclature of Prokaryotes.

Author Response File: Author Response.docx

Reviewer 2 Report

Comments and Suggestions for Authors

Concerns/Suggestions:

 On line 22 in the Abstract the authors state that treatment “led to a decrease in cycle threshold in real-time PCR and an absence of B. vulpes in the blood smear”.   Based on the context it seems likely that what the authors intended to say is that there was an increase in Ct value, indicating less parasites in the blood.

In the Materials and Methods it appears that the primers and probes used to amplify the cytochrome c oxidase gene of B. vulpes and the 16S rRNA gene of Mycoplasma have not been previously reported in the literature.  If this is the case, the sequences of the primers and probes should be reported here.

Minor concerns/Typos:

Lines 45-46  Suggest either moving the symptoms of fever, anorexia and apathy to before the phrase “haemoglobinuria due to haemolysis” or adding a phrase such as “haemoglobinuria due to haemolysis as well as fever, anorexia and apathy.

Line 64 –  It is not clear what is meant by the dog showing intensified breathing.  Does this mean tachypnea or labored breathing?

Line 99 -   Suggest breaking up the sentence.…markedly improved. Erythrocyte counts and rt-PCR Ct values increased, pointing towards decreasing organism numbers, although the thrombocyte count was markedly elevated.

Line 79 -101 – Where possible suggest adding dates to correlate with figure 1 to clarify the time points and responses to treatment.

Line 129 - Legend of figure 3 – anlanesne?

Line 145 - Delete “as well” at the end of the sentence.

Lines 156-157  Reorder the sentence.   Suggest  …but only rarely do cases in dogs occur outside of north-western Spain.

Lines 165-169 For better clarity consider breaking up the sentence starting with “The fact that…

Lines 169-173 Similarly, for better clarity consider breaking up the sentence starting with “However, the vector competence of this tick vector has to be confirmed…”

Lines 189 - 190 Delete “(corresponding to a decrease of organism numbers)” as the reader should understand Ct values and this statement occurs late in the text after Ct values have been presented without explanation.

Line 190 Delete the word “additionally” as it is redundant.

Lines 191-193 Rewrite the sentence starting with “Damage to the liver…” Suggest -  Damage to the liver and kidneys may be a sequela of haemolysis and could have been exacerbated by drug treatment, which may have been too aggressive.

Line 192  The word sequel should be sequela.

Author Response

Concerns/Suggestions: On line 22 in the Abstract the authors state that treatment “led to a decrease in cycle threshold in real-time PCR and an absence of B. vulpes in the blood smear”.   Based on the context it seems likely that what the authors intended to say is that there was an increase in Ct value, indicating less parasites in the blood.

This is correct, we have corrected the error: The dog was treated with a combination of atovaquone (20 mg/kg BW, BID) and azithromycin (10 mg/kg BW, SID), which led to an increase in cycle threshold in real-time PCR and absence of B. vulpes in the blood smear.

In the Materials and Methods it appears that the primers and probes used to amplify the cytochrome c oxidase gene of B. vulpes and the 16S rRNA gene of Mycoplasma have not been previously reported in the literature.  If this is the case, the sequences of the primers and probes should be reported here.

            Unfortunately, the primers and probes cannot be specified, due to proprietary rights regulations of IDEXX: The sample was therefore reanalysed with reported primers and the resulting sequence was uploaded to GenBank. If of interest, reference GenBank accession numbers with the targeted fragment of the gene can be included: Babesia vulpes: target gene cytochrome c oxidase subunit 1 (KX169167), Mycoplasma haemocanis: target gene 16S rRNA (MN294708), Candidatus Mycoplasma haematoparvum: target gene 16S rRNA (GQ129114).

Minor concerns/Typos:

Lines 45-46  Suggest either moving the symptoms of fever, anorexia and apathy to before the phrase “haemoglobinuria due to haemolysis” or adding a phrase such as “haemoglobinuria due to haemolysis as well as fever, anorexia and apathy.

            Has been changed accordingly:

Clinically affected dogs show fever, anorexia, apathy, severe anaemia and haemoglobinuria due to haemolysis [16,26,27].

Line 64 –  It is not clear what is meant by the dog showing intensified breathing.  Does this mean tachypnea or labored breathing?

            The sentence has been changed:

In August 2021, inappetence was noticed and the following day the dog was apathetic and showed tachypnoea.

Line 99 -   Suggest breaking up the sentence.…markedly improved. Erythrocyte counts and rt-PCR Ct values increased, pointing towards decreasing organism numbers, although the thrombocyte count was markedly elevated.

            Has been changed accordingly:

After two weeks, the dog’s condition markedly improved. Erythrocyte counts and Ct values in rt-PCR increased, pointing toward decreasing organism numbers, although the thrombocyte count was markedly elevated.

Line 79 -101 – Where possible suggest adding dates to correlate with figure 1 to clarify the time points and responses to treatment.

            The correlating dates have been mentioned throughout the case history:

Line 65-66: (Fig. 1: 2021-09-09)

Line 77: (Fig. 1: 2021-10-16)

Line 80: (Fig. 1: 2021-12-30)

Line 89: (Tab. 1: 2022-01-06)

Line 91-92: (Fig. 1, Tab. 1: 2022-01-06)

Line 100: (Fig. 1, Tab. 1: 2022-01-22)

Line 103: (Fig.1, Tab. 1: 2022-02-12)

Line 129 - Legend of figure 3 – anlanesne?

            Mistakes in the figure legend have been corrected:

Figure 3. Gel electrophoresis of PCR-RFLP 18S rRNA gene fragments. Lane 1 presents a 100 bp molecular weight marker, lanes 2 to 4, and lanes 5 to 7 present PCR-RFLP products digested with endonucleases HinfI and TaqI respectively. Lanes 2, 3, 5, and 6 present samples of the patient from two different time points, and lanes 4 and 7 present a sample with B. canis as positive controls.

Line 145 - Delete “as well” at the end of the sentence.

            Has been changed accordingly:

Apart from the residence in another country, the origin and travel history rely solely on the information provided by the owner, so an autochthonous infection in Germany cannot be considered as confirmed [38].

Lines 156-157  Reorder the sentence.   Suggest  …but only rarely do cases in dogs occur outside of north-western Spain.

            The sentence has been changed:

It is not clear why B. vulpes is found in high prevalence in foxes in many European countries, but cases rarely occur in dogs outside north-western Spain.

Lines 165-169 For better clarity consider breaking up the sentence starting with “The fact that…

            The sentence has been divided:

Ixodes hexagonus is found on nearly 40% of the dogs there and that B. vulpes is the predominant Babesia species in dogs in north-western Spain. In other regions, lower infestation rates with I. hexagonus and rare autochthonous cases of B. vulpes infection in dogs are reported. These observations support the hypothesis that I. hexagonus could be the vector [40].

Lines 169-173 Similarly, for better clarity consider breaking up the sentence starting with “However, the vector competence of this tick vector has to be confirmed…”

            The sentence has been divided:

However, the vector competence of this tick vector has to be confirmed in further studies. Which other factors contribute to the high infestation rates with I. hexagonus and infection rate with B. vulpes of dogs in Spain in contrast to other regions (including habitat, climate, wildlife reservoir, lifestyle of dogs among others) should also be further investigated.

Lines 189 - 190 Delete “(corresponding to a decrease of organism numbers)” as the reader should understand Ct values and this statement occurs late in the text after Ct values have been presented without explanation.

            Has been changed accordingly:

Even though the laboratory findings were suggestive of an improvement of the infection, seen by the increase of the Ct value and the absence of organisms in blood smear, the dog had to be euthanized because clinical signs deteriorated.

Line 190 Delete the word “additionally” as it is redundant.

            Has been changed accordingly (see above).

Lines 191-193 Rewrite the sentence starting with “Damage to the liver…” Suggest -  Damage to the liver and kidneys may be a sequela of haemolysis and could have been exacerbated by drug treatment, which may have been too aggressive.

            The sentence has been changed:

Damage to the liver and kidneys might have been too severe at that stage. This damage may be a sequela of haemolysis and could have been exacerbated by drug treatment [50,51].

Line 192  The word sequel should be sequela.

            Has been changed accordingly (see above).

Author Response File: Author Response.docx

Reviewer 3 Report

Comments and Suggestions for Authors

Reviewer’s comment to Unterköfler et al.

This case report described a seplenectomized dog with Babesia vulpes infection. B. vulpes is a minor etiologic agent for canine babesiosis and is rarely reported from Germany. In this case report, the dog was imported from Spain and received splenectomy and immune-suppressing drug. Though it looks the B. vulpes infection was controlled by chemotherapy, the general condition of the dog was worsened, and finally euthanized. This report should be of interest to small animal clinicians and the manuscript is well written. I have a few minor suggestions for the manuscript.

Minor comments

1. Page 2, Line 85-87: At what time point did the blood donor dog from Spain confirm for Babesia negative? Is there any possibility that the donor dog is the source of B. vulpes?

2. Table 1: The authors showed rt-PCR data, but there was no parasitemia data. If they can count from the blood smear, it will be appreciated to show the data.

3. Page 5 Line 121-122, and Figure 3: It is unnecessary to italicize the restriction enzyme name (please see PMID: 12654995).

4. Figure 3: B. canis should be italicized.

5. Page 8 Line 213: It may be better to write the product name and company name of Taq polymerase.

Author Response

This case report described a seplenectomized dog with Babesia vulpes infection. B. vulpes is a minor etiologic agent for canine babesiosis and is rarely reported from Germany. In this case report, the dog was imported from Spain and received splenectomy and immune-suppressing drug. Though it looks the B. vulpes infection was controlled by chemotherapy, the general condition of the dog was worsened, and finally euthanized. This report should be of interest to small animal clinicians and the manuscript is well written. I have a few minor suggestions for the manuscript.

Minor comments

1. Page 2, Line 85-87: At what time point did the blood donor dog from Spain confirm for Babesianegative? Is there any possibility that the donor dog is the source of B. vulpes?

The dog was tested retrospectively 3 weeks after application of the blood donation. We consider the possibility that this dog was the source of infection therefore very low. We added the information in the text:

Two blood transfusions (250 ml) were administered, one from a dog that had never travelled outside Germany and one from a dog from Spain, which tested negative for Babesia spp. and B. vulpes by rt-PCR retrospectively 3 weeks after application.

2. Table 1: The authors showed rt-PCR data, but there was no parasitemia data. If they can count from the blood smear, it will be appreciated to show the data.

Thank you for the suggestion; unfortunately, parasite count was not performed and only photographs but not the original blood smears are available.

3. Page 5 Line 121-122, and Figure 3: It is unnecessary to italicize the restriction enzyme name (please see PMID: 12654995).

Has been changed throughout the manuscript:

Line 122: 111 bp (HinfI), and 452 bp, 192 bp, 146 bp (TaqI) length

Figure 3. HinfI and TaqI

Line 213: Taq polymerase

Line 219: HinfI and TaqI

4. Figure 3: B. canis should be italicized.

            Has been changed accordingly:

Figure 3. Gel electrophoresis of PCR-RFLP 18S rRNA gene fragments. Lane 1 presents a 100 bp molecular weight marker, lanes 2 to 4, and lanes 5 to 7 present PCR-RFLP products digested with endonucleases HinfI and TaqI respectively. Lanes 2, 3, 5, and 6 present samples of the patient from two different time points, and lanes 4 and 7 present a sample with B. canis as positive controls.

5. Page 8 Line 213: It may be better to write the product name and company name of Taq polymerase.

                Has been added:

Taq polymerase (5 u/µl, GoTaq® G2 DNA Polymerase, Promega, Madison, USA)

Author Response File: Author Response.docx