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. 2023 Aug 11;18(8):e0289944. doi: 10.1371/journal.pone.0289944

Molecular detection of Coxiella burnetii in raw meat samples collected from different abattoirs in districts Kasur and Lahore of Punjab, Pakistan

Shahpal Shujat 1,2, Wasim Shehzad 1, Aftab Ahmad Anjum 3, Julia A Hertl 2, Muhammad Yasir Zahoor 1,*, Yrjö T Gröhn 2
Editor: Gizat Almaw4
PMCID: PMC10420375  PMID: 37566566

Abstract

Coxiella burnetii is the zoonotic pathogen that causes Q fever; it is widespread globally. Livestock animals are its main reservoir, and infected animals shed C. burnetii in their birth products, feces, vaginal mucus, urine, tissues, and food obtained from them, i.e., milk and meat. There were previously very few reports on the prevalence of C. burnetii in raw meat. This study aimed to determine the prevalence of C.burnetii and its molecular characterization in raw ruminant meat from the Kasur and Lahore districts in Punjab, Pakistan, as this has not been reported so far. In this study, 200 meat samples, 50 from each species of cattle, buffalo, goat, and sheep, were collected from the slaughterhouses in each district, Kasur and Lahore in 2021 and 2022. PCR was used for the detection of the IS1111 element of C. burnetii. The data were recorded and univariate analysis was performed to determine the frequency of C. burnetii DNA in raw meat samples obtained from different ruminant species using the SAS 9.4 statistical package. Of the total of 200 raw meat samples, C. burnetii DNA was present in 40 (20%) of them, tested by PCR using the IS1111 sequence. The prevalence of C.burnetii differed among the studied species of ruminants. When species were compared pairwise, the prevalence in cattle was statistically significantly lower than in sheep (P = 0.005). The sequence alignment based on origin implied that the strains are genetically diverse in different districts of Punjab, Pakistan. The findings demonstrated that the prevalence of C. burnetii, especially in raw meat samples, deserves more attention from the health care system and professionals from Punjab, Pakistan, i.e., abattoir workers and veterinarians.

Introduction

Coxiella burnetii is a gram-negative coccobacillus related to the Gamma subdivision of the Proteobacteria [1, 2]. It is an obligate intracellular bacterium replicating in eukaryotic cells and occurs in two forms [3]. The bacterium’s large-cell variant (LCV) resembles the intracellular replicative form. In contrast, the small-cell variant (SCV) is the non-replicating form of the bacterium that is released when the infected cells lyse and can resist long-term environmental stresses [1].

Coxiella burnetii is the zoonotic pathogen that causes Q fever worldwide [2]. It can infect different host species, including domestic, wild, and marine mammals, birds, reptiles, and arthropods. The main reservoirs of this infection are cattle, buffalo, goat and sheep, which are also a source of human infection [4]. It infects over 40 tick species, which is an important transmission vector in ruminants [5]. It replicates in ticks; thus sufficient amounts of this pathogen are eliminated in their faeces and deposited on the skin of animal hosts during feeding. Several studies have identified ticks as a potential risk for coxiellosis in livestock and other domesticanimals [6–10]. In humans, this infection can occur through inhalation of particles dispersed from environmental dust containing dried tick faeces [11–13] and direct contact with contagious wool, milk, meat, urine, semen and feces of animals [14]. In ruminants, infection can occur both asymptomatically and symptomatically. Clinical manifestation of this bacterium includes stillbirth, abortion, mastitis, endometritis, and other reproductive disorders in ruminants [15, 16]. Flu is a contagious respiratory illness caused by influenza viruses, fever, hepatitis, and endocarditis may also occur in humans [2, 17].

Coxiella burnetii is considered endemic and has a worldwide distribution, including Pakistan. It has gained international public health awareness with cases reported in humans throughout the globe, including 284 cases in Netherlands, 14 in Switzerland and 10 in France [16]. It has also been isolated from Australian abattoir workers [18]. Most cases remain undiagnosed due to a lack of proper diagnostic facilities in developing countries like Pakistan.

Molecular tests are commonly used to detect C. burnetii in samples of different origins, including blood, serum, milk, and meat. Several PCR assays have been used for the detection of C. burnetii. The IS1111 gene is a frequently used PCR target and is considered more sensitive than single-copy gene targets for detection [19].

Coxiella burnetii is a neglected pathogen in Pakistan, although it substantially affects public health. Farm management and public awareness are required to control this infection. Moreover, the infection remains largely undetected, mainly due to limited diagnostic facilities, misdiagnosis for other diseases with similar symptoms (e.g., Brucellosis) and insufficient training of healthcare workers and clinical physicians in handling this contagious disease in developing countries like Pakistan. Notably, there have been only about six previous publications on human and animal Q fever from Pakistan in the international databank [20–25]. Information regarding C. burnetii’s manifestation in raw meat obtained from small and large ruminants for human consumption has not been collected so far. The objective of the current study was to estimate the prevalence of C. burnetii in raw meat samples collected from ruminants intended for human consumption in districts Kasur and Lahore.

Methodology

Study area and sampling

The Advanced Studies and Research Board at the University of Veterinary and Animal Sciences in Lahore, Pakistan approved this study in its 50th meeting held on 8 -02- 2019. The sampling was conducted between 2021 and 2022 and skeltal muscle meat samples were collected from the slaughterhouses of Districts Kasur and Lahore. This study included 200 skeltal muscle meat samples, 50 from each species of cattle, buffalo, goat, and sheep, from the slaughterhouses in each district, Kasur and Lahore.

Sample processing and DNA isolation

Meat samples were stored at -20°C and further processed for DNA extraction using a manual method [26]. 0.15 g of meat samples were minced and washed with distilled water and 70% ethanol and placed in a microcentrifuge tube. Then, 800 μl of Digestion buffer and 20 μl of Proteinase K, and 30 μl of 10% Sodium Dodiecyl Sulphate, remained pellet were added, and incubated at 56°C overnight. After overnight incubation, 500 μl Phenol Chloroform Isoamylalcohol was added and vortexed until the solution turned milky. Then it was centrifuged at 13500 RPM and 4°C as mentioned above. Three layers were formed, and the upper transparent layer containing DNA was placed into a separate microcentrifuge tube. Two parts of isopropanol and 200 μl of chilled absolute ethanol were added in 1 part aqueous transparent layer and incubated for 20 min at -20°C. It was then centrifuged under the same conditions, the supernatant was discarded, and the remained pellet. The taken pellet was washed using 200 μl of 70% ethanol and centrifuged under the same conditions. Then the supernatant was discarded, leaving the pellet for overnight drying to evaporate ethanol that act as a PCR inhibitor. The dried pellet was resuspended in 20 μl of distilled water in the water bath and heat shocked at 70°C for 40 minutes. DNA quality and quantity were assessed using a spectrophotometer.

Molecular assay and sequence analysis

Coxiella burnetii was detected using multiple copy gene amplification assay targeting the transposase gene, i.e., IS1111, and particular primers were used for this assay. The set of primers used for PCR amplification assay was sequenced as 5’-GTCTTAAGGTGGGCTGCGTG-3’ and 5`-CCCCGAATCTCATTGATCAGC-3` for forward and reverse primer [27]. The diagnostic assay was validated using Vircell Amplirun® Coxiella DNA Control. Each PCR reaction test contained 12.5 μl of 2X master mix, 1.25 μl of 10μM forward and reverse primer, and 1 μl of 50–100 ng DNA in a final volume of up to 25 μl by adding nuclease-free water. PCR reaction was performed using 96 well Applied Biosystems by Thermo Fisher Scientific thermal cycler. The reaction conditions were as follows: for initial denaturation at 95°C for 5 min, denaturation at 94°C for 30 sec, annealing at 60°C for 30 sec, extension at 72°C for 1 min, repeating steps 1 to 3 for 30 cycles, and final extension at 72°C for 10 min. PCR products were analyzed on 2% agarose gel, and specific product was identified, i.e., 294 bp was observed during analysis. The obtained positive samples were sequenced commercially by Macrogen, Korea. Phylogenetic analysis was conducted on the sequences using the MEGA version 6.0 bioinformatics tool. Alignment and phylogenetic tree construction of 12 sequences, including two query sequences, were performed using the MEGA tool by the maximum likelihood method [28]. The nucleotide substitution model was verified and adjusted according to the data type and tamura nei model was selected for the analyzed data. Bootstrap value was adjusted as 100 number of replications.

Data analysis

The data were recorded in a Microsoft Excel spreadsheet and univariate analysis was performed to determine the prevalence of C. burnetii in raw meat samples obtained from different ruminant sources using the SAS 9.4 statistical package. Chi-square tests were performed in PROC FREQ, and logistic regression models (PROC LOGISTIC) were fitted, with occurence of C. burnetii DNA as the outcome.

Results

A total of 200 meat samples was assessed for C. burnetii DNA; it was found in 40 (20%) samples tested by PCR using the IS1111 sequence. It was further distributed in four species of ruminants as 8% in buffalo, 12% in cattle, 22% in goats, and 38% in sheep (Table 1). Coxiella burnetii DNA was thus found more frequently in the raw meat samples obtained from small ruminants (goats and sheep) compared to large ruminants (cattle and buffalo). Coxiella burnetii DNA was observed in 30% of mutton (sheep and goats) and 10% of beef (buffalo and cattle) samples. The C. burnetii prevalence differed significantly by species. There was no statistically significant difference between the two districts, however.

Table 1. Detection of Coxiella burnetii in meat samples collected from different species in Districts Lahore and Kasur, Pakistan, by PCR, 2021–2022.

Sample type No. of examined samples Number of positive samples Percentage P Value
Cattle 50 6 12 Species
Buffalo 50 4 8 0.0008
Goat 50 11 22
Sheep 50 19 38
Lahore 100 20 20 Districts
Kasur 100 20 20 1.0000
Total 200 40 20
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Chi-square tests showed that animal species, meat type, and animal age were associated with the occurence of C. burnetii DNA. Furthermore, logistic regression showed that meat samples from sheep were 7 times more likely to test positive than were samples from buffalo, 4.5 times more likely to test positive than were those from cattle and 2.2 times were more likely to test positive than from goat samples. When the 4 animal species were grouped into meat type (beef (buffalo, cattle) and mutton (sheep, goats)), mutton was 3.9 times more likely to test positive than was beef. Meat from 6-month-old animals was 3.9 times more likely to test positive than was meat from 1-year-old animals, and was 3.6 times more likely to test positive than was meat from 2-year-old animals (Table 2).

Table 2. Detection of Coxiella burnetii in meat samples collected from different age groups in Districts Lahore and Kasur.

Animal Age Prob SE Asymp.LCL Asymp.UCL
0.5y 0.364 0.0649 0.2481 0.497
1 y 0.129 0.0348 0.0748 0.214
1.5y 0.174 0.0790 0.0668 0.382
2y+ 0.333 0.2722 0.0434 0.846
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When the sequences were assessed on the basis of their origin, the results showed that in the current study, the sequences obtained from district Lahore were clustered with previously reported sequences from district Sahiwal because of their close genetic similarity. In comparison, the sequences obtained from district Kasur were clustered separately from those obtained from district Attock (Fig 1). Thus, the results showed that the strains in different districts of Punjab, Pakistan are genetically diverse (Fig 2).

Fig 1. The phylogenetic relationship of C. burnetii IS1111 gene sequence recovered from meat samples of district Lahore and Kasur, Pakistan, 2021–2022.

Fig 1

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Labelled sequences are query sequences.

Fig 2. The map showing all districts of Punjab, Pakistan.

Fig 2

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Discussion

The current study was designed to estimate the prevalence of C. burnetii in raw meat intended for human consumption. This study was the first to determine whether C. burnetii DNA occurs in raw meat from livestock animals. It included beef and mutton samples from cattle, buffalo, goats, and sheep from districts Lahore and Kasur, Pakistan. In previously reported studies C. burnetii DNA was detected in food obtained from livestock animals, including milk and its products [29, 30]. The past studies suggested the risks for Coxiella burnetii through consumption of unpasteurized milk and its products are not negligible [31]. There were previously no reports of C. burnetii DNA occurrence in raw meat for human consumption. Past studies’ limitations encouraged the current experiment to detect C. burnetii DNA in raw meat of livestock origin for human consumption.

A One Health approach was adopted in the current study to investigate raw meat samples from small and large ruminants. This study determined the prevalence of C. burnetii DNA to be 20% in raw meat samples collected from small ruminants. A previous study estimated the prevalence of C. burnetii in the blood samples of ruminants from district Kasur; it was estimated as 36.8% in small ruminants for the district Kasur [32]. For district Lahore, C. burnetii was reported in 4.8% of environmental samples [24]. Coxiella burnetii thus occurred more frequently in the blood of small ruminants than in raw meat.

In the current study, the estimated prevalence of C. burnetii in raw meat samples collected from sheep was 38%, while in districts Kasur and Lahore it was estimated to be 36% and 40%, respectively. In another study, the prevalence of C. burnetii in sheep blood was reported to be 46.9% in the Kasur district [23]. The prevalence of C. burnetii in sheep carcasses was reported to be 6.7% in Iran, while in other countries, there were no reports on sheep carcasses [24]. According to the findings, it is inferred that C. burnetii is common in sheep. Like goat meat, there is a substantial interest in consuming sheep meat and its products in Pakistan and other parts of the world. Therefore, paying attention to the food-borne pathogens in such communities is essential, and veterinary organizations must prioritize control and prevention strategies in livestock. The healthcare system should also provide training for at-risk people.

The current study found C. burnetii DNA in 22% of raw meat samples collected from goats. The prevalence of C. burnetii in districts Lahore and Kasur was 20% and 24%, respectively. There were no previous studies available for estimating C. burnetii DNA in meat samples obtained from goats. In contrast, C. burnetii prevalence was reported previously in cattle and sheep meat from Iran [33]. Goat meat is consumed in many countries. Therefore, serious attention must be paid to C. burnetii in goat meat.

Coxiella burnetii DNA was detected in 12% of cattle meat samples in this study. Its prevalence was estimated as 16% and 8% in districts Lahore and Kasur, respectively. In comparison, C. burnetii was present in 8% of overall buffaloes. Its prevalence for district Kasur was estimated as 8% and for district Lahore it was estimated as 8%. Previously, C. burnetii prevalence was reported in Iran in 5.7% of the samples collected from cattle carcasses [24]. Therefore, meat samples can be considered a source of C. burnetii in livestock animals. The limitations to the current study includes viability count and the remaining districts of the Punjab, Pakistan. In future studies, considerations must include the other districts of Punjab and risk evaluation in the human population for the infection, especially in professionals, i.e., abattoir workers and veterinarians. Based on our results, there was molecular evidence of C. burnetii in meat samples collected from livestock animals of the districts of Kasur and Lahore. These findings imply that C. burnetii prevalence, especially in meat samples, could pose a severe risk of Q fever to abattoir workers and consumers in Punjab, Pakistan.

Conclusion

Molecular evidence of C. burnetii was observed in meat samples of cattle, buffalo, goats, and sheep collected from the slaughterhouses in two districts of Punjab, Pakistan. These findings emphasized that the prevalence of C. burnetii, especially in raw meat samples, deserves more attention from the health care system and meat industry in Kasur and Lahore of Punjab, Pakistan. Future studies must include other districts and risk evaluation in the human population for the infection, especially in professionals, i.e., abattoir workers and veterinarians.

Supporting information

S1 Table. Univariable analysis of Coxiella burnetii in raw meat samples collected from slaughter houses in districts Kasur and Lahore in Punjab province, Pakistan, 2021–2022.

(DOCX)

Click here for additional data file. (16KB, docx)

Acknowledgments

The authors are grateful to HEC, Assistant Director Livestock District Kasur Dr. Musarat, Veterinary assistant in Livestock Department, and Dr. Saqib, Veterinary Assistant, for assisting in sample collection.

Data Availability

All relevant data are within the manuscript and its Supporting information files.

Funding Statement

This study was funded by International Research Support Initiative Program, Higher Education Comission, Pakistan fellowship. Grant:I -8/HEC/HRD/2021/11529 awarded to student. The funder has no role in study design, data collection and analysis, decision to publish, or preparation of manuscript.

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Decision Letter 0

Gizat Almaw

13 Mar 2023

PONE-D-23-03478Molecular detection of Coxiella burnetii in raw meat samples collected from different abattoirs in districts Kasur and Lahore of Punjab, PakistanPLOS ONE

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

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

Reviewer #1: Partly

Reviewer #2: Partly

**********

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

Reviewer #1: I Don't Know

Reviewer #2: Yes

**********

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

Reviewer #1: Yes

Reviewer #2: Yes

**********

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Reviewer #1: Yes

Reviewer #2: Yes

**********

5. 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: Overall, the present article is pertinent since it explored the frequency of C. burnetii and its molecular characterization in raw meat from ruminant sources in Pakistan. However, some items need attention and adjustment:

-Please, all paper needs a complete revision of references.

For example, references 1 and 4 are de same (duplicated)

Reference 26 is incomplete.

Line 39: About following affirmation and references: "It infects over 40 tick species, which is an important transmission vector in ruminants (5). It replicates in ticks; thus sufficient 41 amounts of this pathogen are eliminated in their faeces and deposited on the skin of animal hosts during feeding" It is recommended to review and address this issue more assertively and support it with recent references. The role of ticks as vectors of this bacterium is still controversial...

Line 71. The following phrase, "PCR was used to detect the IS1111 element of the C. burnetii genome" could be deleted from here.

Line 73: Is there any reference supporting the DNA extraction protocol from the meat sample? Please include it.

Line 87: To explain the PCR condition, please include the following:

-The final concentrations of the primers

-Reference of the type of taq used

- How were possible PCR amplification inhibitors in the sample evaluated?

Line 98 For the Phylogenetic analysis, please clarify with more detail. It needs to be improved:

-Was the Nucleotide substitution model (model test) verified and adjusted for the data before the ML method?

-Did Bootstrap? Please present it.

-Outgroup? Please include and present it.

Line 118: It is unclear in the methodology; how was it possible to verify the age of the animals from which the meat sample came?

In the discussion, the authors should address the issue of whether or not good meat cooking could inactivate this bacterium. Furthermore, how much could molecular detection be expected to ensure viable transmission of this species?

Reviewer #2: The manuscript entitled “Molecular detection of Coxiella burnetii in raw meat samples collected from different abattoirs in districts Kasur and Lahore of Punjab, Pakistan” investigates 200 meat specimens collected in slaughter houses in two districts of Punjab, Pakistan. Samples obtained from 4 species (goat, sheep,cattle and buffalo) were tested for the presence of specific C.burnetii sequence by PCR, also phylogenetic analysis was performed. The manuscript is easy to follow and provides valuable information on presence of C.burnetii DNA in raw meat samples – a subject that has been poorly known so far.

Major concerns:

1. The data about tested meat samples are too general. There is no information whether skeletal muscles or offal samples were collected. It might be an important issue, taking into consideration that one of the studies showed significant difference between prevalence of pathogen DNA in skeletal muscle meat and offal. Moreover there is lack of information if the same type of meat was collected from all animal species.

2. The data about age of tested animals are not present in the manuscript, but they are included as one of variables in chi square analysis.

3. Lack of essential information in phylogenetic tree i.e. scale bar and bootstap values of the nodes makes the figure non-informative. Once completed, the data should be analysed and discussed.

4. The discussion is a little bit one-sided, possible limitation of the research are not adressed. Moreover, the conclusion that „C. burnetii prevalence, especially in meat samples, pose a severe risk of Q fever” seems to be far-reaching. The presence of C. burnetii DNA was confirmed but the viability of bacteria in specimens was not evaluated and the risk level is unknown, so far. In adition, it would be advisable to refer to findings about milk and dairy products and importance of transmission via alimentary route.

Minor comments:

line 17: I suggest “in raw ruminant meat”

line 22: Please replace “ruminant sources” with “ruminant species”

line 26: I suggest “strains are genetically diverse (…)” rather than “strains are not genetically identical”

line 38: reservoirs of

line 46: Flu is a contagious respiratory illness caused by influenza viruses. Please correct

49: Delete dot after word France

line 52: Please correct as follows: to detect Coxiella burnetii in samples

lines 62-63: Please precise the aim of this study (add regions where prevalance was estimated)

line 63: Please correct “used for human consumption” to “intended for human consumption”

Methodology

It would be interested for readers to have more information about tested meat. Could you add information about type of tested meat? Were it skeletal muscle meat samples or offal? Did you collect the same type of meat from all animals? Is it possible to present some details i.e. types of offal or meat cuts that were incuded in the study?

line 68: between 2021 and 2022/in 2021 and 2022- please correct

line 69: I suggest: This study included 200 meat samples(…)

line 73: Please delete word temperature, it is unnecessary

line 75 and 76: Please add full names of the reagents SDS and PCI

line 77: Then it was centrifuged under the same conditions as mentioned above – Please complete the information about centrifugation conditions as none were mentioned in this section.

line 81: I suggest “remained pellet“

line 83: “(…) evaporate ethanol and act as a PCR inhibitor”- it is uncelar, please rephrase

line 83: I suggest: “pellet was resuspended”

Section molecular assay and sequence analysis

Please add information about controls that were used in DNA extraction and PCR.

line 87: Assay (not assays), please correct

lines 93-94: I suggest rephrasing the sentence e.g. The reaction conditions were as follows (…)

line 94: delete final

line 97: by Macrogen

line 100: Please add information about bootstap value.

lines 115-116: What about sheep vs. goat meat?

lines 118-120: The results of chi square analysis on animals age as one of variables were described, but the data about age of tested animals are not present in the manuscript. Please complete this information!

line 124: the sequences were clustered because their genetic similarity not geographical proximity, please rephrase to clarify.

lines 122-126: A map showing all district of the Punjab province would make easier for the readers to follow the analysis presented in this paragraph.

line 126: I suggest: genetically diverse.

Table 1: Please redesign the table and add information about age of animals (see comment for lines 118-120)

Figure 1: Phylogenetic tree has not got a scale bar and bootstap values of the nodes, please add them. Moreover, the tree would be more informative if you add the data about host orgin of the sequences and year of isolation.

line 142: I suggest: for human consumption

line 147: - the sentence is misleading. It was mentioned that in other study the prevalence of C.burnetii DNA was determined for blood samples collected from ruminants in Lahore and Kasur. Nextly you referred to publication that describes prevalence only in Kasur. The publication 19 ( also do not analyse the prevalence of C.burnetii DNA in animals from Lahore. Please correct the sentence or add appropriate publication.

line 152: Please correct the reference number 23 as the publication describes first isolation of pathogen from cheese in Brasil…

lines 154-156: It would be worthwhile to familiarize readers with culinary habits in Pakistan. What is the most consumed meat type in Pakistan? Do Pakistanis often consume raw meat and thus what might be the zoonotic risk?

lines 165-166: What was the prevalence for buffalo samples in each district (Kasur and Lahore)?

line 167: Please correct the reference number

References need to be adjusted to the requirements of the journal.

**********

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Reviewer #1: No

Reviewer #2: No

**********

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PLoS One. 2023 Aug 11;18(8):e0289944. doi: 10.1371/journal.pone.0289944.r002

Author response to Decision Letter 0

1 Jun 2023

Thanks for the detailed review of the manuscript. All the suggestions are incorporated. The followings are the answer to the comments:

Reviewer 1

1. Overall, the present article is pertinent since it explored the frequency of C. burnetii and its molecular characterization in raw meat from ruminant sources in Pakistan. However, some items need attention and adjustment:

-Please, all paper needs a complete revision of references.

For example, references 1 and 4 are de same (duplicated)

Reference 26 is incomplete.

Ans. The article has been reviewed and all the references are checked and adjusted accordingly.

2. Line 39: About following affirmation and references: "It infects over 40 tick species, which is an important transmission vector in ruminants (5). It replicates in ticks; thus sufficient 41 amounts of this pathogen are eliminated in their faeces and deposited on the skin of animal hosts during feeding" It is recommended to review and address this issue more assertively and support it with recent references. The role of ticks as vectors of this bacterium is still controversial...

Ans. Reviewed and provided with recent references.

3. Line 71. The following phrase, "PCR was used to detect the IS1111 element of the C. burnetii genome" could be deleted from here.

Ans. The phrase is deleted.

4. Line 73: Is there any reference supporting the DNA extraction protocol from the meat sample? Please include it.

Ans. Reference is incorporated.

5. Line 87: To explain the PCR condition, please include the following:

A. The final concentrations of the primers

Ans. The working concentrations of primers used were 10 µM. It is incorporated in the manuscript.

B. Reference of the type of taq used

Ans. The taq was not used separately; it was already added in the master mix of Macrogen Company.

6. How were possible PCR amplification inhibitors in the sample evaluated?

Ans. The samples were analyzed for determining contamination using UV spectrophotometer and concentration of DNA was also evaluated and DNA was diluted in working concentration before PCR.

7. Line 98 for the Phylogenetic analysis, please clarify with more detail. It needs to be improved :

A. Was the Nucleotide substitution model (model test) verified and adjusted for the data before the ML method? Data type, data alignment base tamura was suitable

Ans. Nucleotide substitution model was verified and adjusted according to the data set and Tamura Nei model was suitable for the analyzed data.

B. Did Bootstrap? Please present it.

Ans. Bootstrap value was adjusted as 100 numbers of replications.

C. Outgroup? Please include and present it.

Ans . The outgroup from obtained results were from the sequences obtained from France and India.

8. Line 118: It is unclear in the methodology; how was it possible to verify the age of the animals from which the meat sample came? (Changed to Line 126)

Ans. It was verified by adult teeth count method

9. In the discussion, the authors should address the issue of whether or not good meat cooking could inactivate this bacterium. Furthermore, how much could molecular detection be expected to ensure viable transmission of this species?

Ans. The data regarding inactivating pathogen while cooking meat has not been reported so far. The PCR method is fast, sensitive and specific means to detect disease causing pathogen. However, it cannot distinguish viable bacterial cells from dead cells or from free DNA or RNA in sample. In order to detect viable cells specific assays are developed that measure the production of species specific pre-RNA in samples. But the current study was designed on bacterial diagnosis not viable transmission. In this research we focused on the transmission of pathogen in butchers, veterinarians and meat handlers in kitchen rather than cooked meat.

Reviewer 2

Major concerns

1. The data about tested meat samples are too general. There is no information whether skeletal muscles or offal samples were collected. It might be an important issue, taking into consideration that one of the studies showed significant difference between prevalence of pathogen DNA in skeletal muscle meat and offal. Moreover there is lack of information if the same type of meat was collected from all animal species.

Ans. The skeletal muscle meat samples were collected for the study. The same type of meat was collected from the studied animal species. It is incorporated in the manuscript methodology section.

2. The data about age of tested animals are not present in the manuscript, but they are included as one of variables in chi square analysis.

Ans. The data regarding age of the animals tested is incorporated in the manuscript.

3. Lack of essential information in phylogenetic tree i.e. scale bar and bootstap values of the nodes makes the figure non-informative. Once completed, the data should be analysed and discussed

Ans. The scale bar and the bootsrap values of the nodes are incorporated in the manuscript.

4. The discussion is a little bit one-sided, possible limitation of the research is not adressed. Moreover, the conclusion that „C. burnetii prevalence, especially in meat samples, pose a severe risk of Q fever” seems to be far-reaching. The presence of C. burnetii DNA was confirmed but the viability of bacteria in specimens was not evaluated and the risk level is unknown, so far. In adition, it would be advisable to refer to findings about milk and dairy products and importance of transmission via alimentary route.

Ans. The limitations of the study are incorporated in the discussion section. This study has no evidence in the previous records. Literature for meat samples does not show any viability rather calculated for future studies. The findings about milk and its products consumption risks are incorporated. The current study was focused on detection of pathogen but not distinguished between viable bacterial cells from dead cells.

Minorconcerns

1.line 17: I suggest “in raw ruminant meat”

Ans. Followed.

2. .line 22: Please replace “ruminant sources” with “ruminant species

Ans. Replaced.

3. line 26: I suggest “strains are genetically diverse (…)” rather than “strains are not genetically identical”

Ans.Followed.

4. line 38: reservoirs of

Ans.Corrected

5. line 46: Flu is a contagious respiratory illness caused by influenza viruses. Please correct(Line 46 to 48)

Ans. Corrections are incorporated.

6. 49: Delete dot after word France

Ans.Deleted.

7. line 52: Please correct as follows: to detect Coxiella burnetii in samples

Ans.Corrected

8. lines 62-63: Please precise the aim of this study (add regions where prevalance was estimated)

Ans. The aim of the study is précised.

9. line 63: Please correct “used for human consumption” to “intended for human consumption”

Ans.Corrected

10.Methodology

It would be interested for readers to have more information about tested meat. Could you add information about type of tested meat? Were it skeletal muscle meat samples or offal? Did you collect the same type of meat from all animals? Is it possible to present some details i.e. types of offal or meat cuts that were included in the study?

Ans. The details regarding meat type are incorporated.

11. line 68: between 2021 and 2022/in 2021 and 2022- please correct

Ans.Corrected.

12. line 69: I suggest: This study included 200 meat samples(…)

Ans.Followed.

13. line 73: Please delete word temperature, it is unnecessary

Ans.Deleted.

14. line 75 and 76: Please add full names of the reagents SDS and PCI

Ans. Abbreviations are replaced with full names.

15. line 77: Then it was centrifuged under the same conditions as mentioned above – Please complete the information about centrifugation conditions as none were mentioned in this section.

Ans. The details are incorporated.

16. line 81: I suggest “remained pellet“

Ans.Followed.

17. line 83: “(…) evaporate ethanol and act as a PCR inhibitor”- it is uncelar, please rephrase

Ans. Rephrased.

18. line 83: I suggest: “pellet was resuspended”

Ans. Corrected.

19. Section molecular assay and sequence analysis

Please add information about controls that were used in DNA extraction and PCR.

line 87: Assay (not assays), please correct

Ans. Corrected.DNA control details are mentioned in the manuscript.

20. lines 93-94: I suggest rephrasing the sentence e.g. The reaction conditions were as follows (…)

Ans. Rephrased.

21. line 94: delete final

Ans. Deleted.

22. line 97: by Macrogen

Ans. Corrected

23. line 100: Please add information about bootstap value.

Ans. Added

24. lines 115-116: What about sheep vs. goat meat?

Ans. Incorporated in the manuscript.

25. lines 118-120: The results of chi square analysis on animals age as one of variables were described, but the data about age of tested animals are not present in the manuscript. Please complete this information!

Ans. The information regarding the age of the studied animals is incorporated in the manuscript.

26. line 124: the sequences were clustered because their genetic similarity not geographical proximity, please rephrase to clarify.

Ans. Rephrased.

27. lines 122-126: A map showing all district of the Punjab province would make easier for the readers to follow the analysis presented in this paragraph.

Ans. Incorporated.

28. line 126: I suggest: genetically diverse.

Ans.Corrected

29. Table 1: Please redesign the table and add information about age of animals (see comment for lines 118-120)

Ans. The table is designed for studied animal age.

30. Figure 1: Phylogenetic tree has not got a scale bar and bootstap values of the nodes, please add them. Moreover, the tree would be more informative if you add the data about host orgin of the sequences and year of isolation.

Ans. Scale bar and bootstrap values are incorporated.

31. line 142: I suggest: for human consumption

Ans.Followed

32. line 147: -the sentence is misleading. It was mentioned that in other study the prevalence of C.burnetii DNA was determined for blood samples collected from ruminants in Lahore and Kasur. Nextly you referred to publication that describes prevalence only in Kasur. The publication 19 (also do not analyse the prevalence of C.burnetii DNA in animals from Lahore. Please correct the sentence or add appropriate publication.

Ans. Corrected.

33. line 152: Please correct the reference number 23 as the publication describes first isolation of pathogen from cheese in Brasil…

Ans. Added

34. lines 154-156: It would be worthwhile to familiarize readers with culinary habits in Pakistan. What is the most consumed meat type in Pakistan? Do Pakistanis often consume raw meat and thus what might be the zoonotic risk?

Ans. The skeletal muscle meat type is consumed more often and there is no report on raw meat consumption so it is not possible to explain the zoonotic risk.

35. lines 165-166: What was the prevalence for buffalo samples in each district (Kasur and Lahore)?

Ans. Prevalence is added in the manuscript.

36. line 167: Please correct the reference number

References need to be adjusted to the requirements of the journal.

Ans. Corrected. I followed Vancouver style with little variation from the accepted paper.

Attachment

Submitted filename: Answer to the comments.docx

Click here for additional data file. (30.4KB, docx)

Decision Letter 1

Gizat Almaw

23 Jun 2023

PONE-D-23-03478R1Molecular detection of Coxiella burnetii in raw meat samples collected from different abattoirs in districts Kasur and Lahore of Punjab, PakistanPLOS ONE

Dear Dr. Zahoor,

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.

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PLoS One. 2023 Aug 11;18(8):e0289944. doi: 10.1371/journal.pone.0289944.r004

Author response to Decision Letter 1

11 Jul 2023

Thanks for the review of the manuscript. The suggestions of academic editor and reviewers have been incorporated and article has been adjusted to the journal requirements accordingly i.e., Style and format are adjusted, and references are reviewed, and incomplete references are completed.

Attachment

Submitted filename: Answer to the comments.docx

Click here for additional data file. (30.4KB, docx)

Decision Letter 2

Gizat Almaw

31 Jul 2023

Molecular detection of Coxiella burnetii in raw meat samples collected from different abattoirs in districts Kasur and Lahore of Punjab, Pakistan

PONE-D-23-03478R2

Dear Dr. Zahoor,

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

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Gizat Almaw

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Associated Data

    This section collects any data citations, data availability statements, or supplementary materials included in this article.

    Supplementary Materials

    S1 Table. Univariable analysis of Coxiella burnetii in raw meat samples collected from slaughter houses in districts Kasur and Lahore in Punjab province, Pakistan, 2021–2022.

    (DOCX)

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    Data Availability Statement

    All relevant data are within the manuscript and its Supporting information files.

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