High prevalence of extrapulmonary tuberculosis in dairy farms: Evidence for possible gastrointestinal transmission.

Bovine tuberculosis (bTB) caused by Mycobacterium bovis (M. bovis) represents one of major zoonotic diseases among cattle, it also affects the health of human, other domestic animals and wild life populations. Inhalation of infected aerosol droplets is considered as the most frequent route of the infection. This study aims to investigate the current forms of tuberculosis in cattle and identify the possible transmission modes in dairy farms of China. 13,345 cows from eight dairy farms in three provinces were comprehensively diagnosed by a multitude of assays, including SIT, CIT, IFN-γ assay and ELISA. It has been indicated that advanced infection of bTB was found in 752 (5.64%) cattle, suggesting a high prevalence of tuberculosis in these dairy farms. In the necropsy examination of 151 positive cattle, typical bTB lesions were observed in 131 cattle (86.75%), of which, notably, 90.84% lesions appeared in liver, spleen, mesenteric lymph nodes, mammary lymph nodes and other organs, taking up a large proportion among cattle with advanced bTB infection. 71.26% extrapulmonary tuberculosis (EPTB) was related to gastrointestinal system. M. bovis nucleic acid was further found in milk and feces samples and M. bovis was even isolated from milk samples. Phylogenetic analysis based on whole genome sequencing unraveled that six isolates were closely related to M. bovis AF2122/97 originated from UK, whereas four isolates shared close relation to M. bovis 30 from China, respectively. Our data demonstrate that the increase of EPTB transmitted by digestive tract is implicated in the current high prevalence rate of bTB in China, which also provides leads for bTB control in other countries with high prevalence of bTB in the future.

Introduction

Bovine tuberculosis (bTB) is a chronic bacterial disease caused by Mycobacterium bovis, which is harmful to humans, livestock and wildlife populations [1, 2]. Presently, bTB remains a major infectious disease in developing countries, and causes heavy burden in both public health and animal husbandry [2, 3]. bTB is mainly transmitted through the respiratory tract, which can result in typical symptoms, such as cough and dyspnea. Of note, the infection generally leads to the development of characteristic lesions, tuberculous nodular granuloma, and caseous necrosis or calcification in the lungs, pleura and pleural lymph [4]. In fact, digestive tract exposure contributes to another critical mode for tuberculosis transmission, as humans can become infected most commonly through consumption of unpasteurized milk products from infected cows [5]. Importantly, in the dairy farm, cattle can also become infected following ingestion of feed or water contaminated with nasal secretions, feces, urine, or unpasteurized milk from infected animals [4].

Current ante mortem diagnosis of bTB mainly relies on the single intradermal test (SIT) and IFN-γ assay, the comparative intradermal test (CIT) is also adopted alternatively to SIT. However, recent studies revealed that, in positive cattle by skin test, neither typical bTB-associated symptoms, such as cough and dyspnea, nor lung lesions upon necropsy were frequently found [6, 7]. This confusion has somehow even given rise to a loss of confidence among farmers and field veterinarians regarding the accuracy of delayed hypersensitivity test, and it also interfered the implementation of culling policy on the cattle suspected to bTB [6, 7]. Currently, in China, the postmortem examination of PPD positive cattle is often limited to lung and thorax in terms of bio-safety consideration. Additionally, in slaughterhouses, tissues and organs such as liver, spleen, intestine and stomach, are usually removed, the lesions in which are therefore frequently overlooked [8, 9]. It is believed that the route of infection is implicated to the nature, extent and distribution of tuberculous lesions [10]. For instance, oral transmission often leads to the development of primary foci in lymph tissues of the intestinal tract [11], during which, mesenteric lymph nodes become the most susceptible target organs [12]. However, there were still few details on the distribution of extrapulmonary tissues and organs of bTB. In this study, we aim to investigate the EPTB in cattle and seek to find clues for the possible transmission route in dairy farms.

Materials and methods

General information of dairy farms

Eight dairy farms from Xinjiang, Shandong and Guangxi provinces were chosen based on the epidemic state of bTB. These farms that raised Holstein cows adopted the intensive livestock production systems. The feeding of cows was based on total mixed ration (TMR) ad libitum. Previous bTB positive rates of these dairy farms were between 5%~15% as determined by twice-yearly SIT testing.

Skin test

The skin test was carried out in all animals after challenge. SIT was performed by inoculating 0.1 mL (3000 IU) of bovine-PPD (Prionics AG, Thermo Fisher Scientific, USA) in the mid-neck. CIT was conducted by inoculating 0.1 mL (3000 IU) of bovine-PPD and 0.1 mL (2500 IU) avian-PPD (Prionics AG, Thermo Fisher Scientific, USA) in the neck, and the distance between the two injection points was approximately 15 cm. The skin-fold thickness was measured before and at 72 h after injection. The results of skin thickness increase were interpreted according to the standards of official criteria (GB/T 18645–2002 and OIE Terrestrial Manual 2018) for both SIT and CIT. A cow was considered SIT positive, inconclusive or negative when the increase was 4 mm or greater, between 2 and 4 mm or less than 2 mm, respectively. For CIT, cattle were deemed positive, inconclusive or negative when the bovine injection site exceeded the avian site by greater than 4, 1~4 and 1 mm or less, respectively. CIT was conducted two months after SIT.

IFN-γ assay

Whole blood was collected from the jugular or caudal vein in tubes with lithium heparin. Stimulation of whole blood with bovine-PPD, avian-PPD or PBS (nil control) was carried out within 8 h after collection. The BOVIGAM® Mycobacterium bovis Gamma Interferon Test Kit for Cattle (Prionics AG, Thermo Fisher Scientific, USA) was used for the detection of the release of IFN-γ from sensitised lymphocytes according to the manufacturer’s protocol. The result interpretation was obtained based on the standard cut-off value of the kit.

ELISA

A commercial ELISA, the Mycobacterium bovis Antibody Test Kit (IDEXX, USAhttps://www.idexx.com/), was used to test all serum samples examined in this study. The assay and results interpretation were performed according to the manufacturer’s protocol. The results are expressed as a Sample to Positive (S/P) ratio, which is calculated for each sample according to the following equation: S/P  =  (Mean Sample OD450 − Mean Negative Control OD450) / (Mean Positive Control OD450 − Mean Negative Control OD450). The sample was positive with S/P ≥ 0.30.

Comprehensive diagnostic algorithm

Four immunological assays were employed to detect bTB in cattle from the eight dairy farms investigated, so as to reduce the false positive and false negative rates. Serological test (ELISA) detecting humoral immune responses can facilitate the detection if late stage diseased animals. In serial testing, positive results from two or more assays (including ELISA) were considered as advanced infection, and anatomical examination was performed in the local slaughterhouses named Kaerwan (Xinjiang), Musulin (Shandong), and Chengcheng (Guangxi). Different tissues and organs were collected for subsequent pathological and pathogenic examination. Milk and feces samples from cattle with advanced infection were detected by bacteria isolation and PCR assay (S1 Fig).

Anatomical and pathological examination

Anatomical examination was systematically conducted to 151 cattle. Numerous organs and tissues were thoroughly inspected and collected for examination of bTB-compatible lesions, including lung, liver, spleen, kidney, intestine, thoracic cavity and pleura, abdominal cavity and peritoneum, hilar lymph nodes, mammary lymph nodes, mesenteric lymph nodes, inguinal lymph nodes. Tissues were fixed in 10% formalin neutral solution. Conventional paraffin sectioning and H&E staining were conducted for histopathological examination. Furthermore, paraffin sections were stained with Ziehl-Neelsen acid-fast staining for detection of tubercle bacillus.

Bacteria isolation

M. bovis was isolated and cultured at the Biosafety Level-3 Laboratory for Zoonoses, China Animal Health and Epidemiology Center (CAHEC) (Qingdao, Shandong, China). Briefly, approximately 2 g of tissue were collected, homogenized in 15 mL of 0.85% physiological saline and vortexed at high speed for 1 minute. 15 mL of homogenate was transferred to a centrifuge tube, after which 15 mL of hexadecylpyridinium chloride monohydrate (HPC) 1.5% (w/v) was added and mixed at room temperature for 30 minutes. The mixture was centrifuged at 3,000 × g for 20 minutes at 22°C, and the pellets were resuspended in 10 mL of 0.85% physiological saline as inoculum. Milk samples were similarly treated. 20 mL of milk were centrifuged at 3,000 × g for 10 minutes, and pellets were decontaminated in 20 mL of HPC 0.75% (w/v) for 20 minutes. After centrifugation and suspension, 100 μL of tissue or milk samples were inoculated on to Lowenstein-Jensen medium supplemented with pyruvate (BD, USA) or Lowenstein-Jensen medium (BD, USA), at 37°C for 4~10 weeks. The growth of bacteria was monitored weekly.

DNA extraction and purification

DNAs from all positive cultures, milk, and feces were extracted by QIAamp® DNA Mini Kit (QIAGEN, Germany) and QIAamp® DNA Stool Kit (QIAGEN, Germany). Purified DNA concentrations were determined using a NanoPhotometer® NP80 (Implen, Germany).

Detection of M. bovis by quantitative PCR (qPCR)

The qPCR assay targeting IS1561 of Mycobacterium tuberculosis complex (MTBC) species was performed. The amplicon was then further analyzed using the RD4 based qPCR assay to confirm the presence of M. bovis [13]. Taqman Universal PCR Master Mix was used according to the manufacturer’s directions with a final primer concentration of 0.4 μM each and 0.2 μM probe. The qPCR was conducted with Applied Biosystems™ QuantStudio 3 Real-Time PCR Systems (Thermo Fisher Scientific, USA). Initial denaturation was made at 95°C for 15 min, followed by 45 cycles with denaturation at 95°C for 15 s, annealing and elongation at 60°C for 1 min.

Whole genome sequencing (WGS)

Ten bacterium strains were isolated from eight dairy farms, while six strains from lungs (2016–4, 2017–12, 2017–14, 2017–15, 2017–18) and four strains from extrapulmonary tissues (2016–1, 2016–2, 2016–6, 2017–13, 2017–17). WGS was performed on the BGISEQ-500 platform (BGI, Shenzhen, China). Reads were generated and assembled into contigs, while the sequence of M. bovis AF2122/97 (GenBank: NC_002945.4) was set as reference.

We introduced Prokka to fully annotate draft bacteria genomes. All annotated assemblies in GFF3 format were used as input files to conduct a core-pan analysis. SNP sites were extracted from a core gene alignment file generated by Roary. Missing and incomplete data were excluded from analysis. A matrix dataset containing the orthologous SNPs was generated, and the filtered dataset was applied to conduct evolutionary analyses using MEGA version 5. A neighbor-joining tree was constructed using the Jukes–Cantor model and the percentage bootstrap confidence levels of internal branches were calculated from 1,000 resamplings of the original data. The M. tuberculosis H37Rv strain (GenBank: NC_000962.3) was selected as the outgroup. Six M. bovis strains from NCBI were selected as reference. Specifically, M. bovis AF2122/97 (GenBank: NC_002945.4) was isolated from the lung and bronchomediastinal lymph nodes of a cow in the UK, M. bovis 1595 (GenBank: NZ_CP012095.1) was isolated from the larynopharyngeal lymph node of a cow in South Korea, M. bovis 30 (GenBank: CP010332.1) was isolated from the lymph node of a cow in China, M. bovis AN5 (GenBank: NZ_AWPL00000000.1) was obtained from Brazil and used for PPD production, M. bovis SP38 (GenBank: NZ_CP015773.2) was isolated from the lymph node of a cow in Brazil, and M. bovis BCG str. Tokyo 172 (GenBank: NC_012207.1) was vaccine strain.

Ethical statement

bTB is a notifiable disease and there are control and surveillance campaigns in China. Official diagnostic methods for bTB are immunological tests, culture, PCR and histopathology. Skin tests and tissue collection were included as part of routine surveillance strategy of bTB. Sample collection and subsequent detection were conducted under the condition of permission of farmer owners. In this study, no animal experiment was involved. All datasets were in complete agreement with national and OIE regulations.

Results

High prevalence of tuberculosis in dairy farms

The bTB infection in dairy farms from multiple provinces of China was investigated. In this study, the positive rates from a total of 13,345 cattle from eight dairy farms were determined by using SIT, CIT, IFN-γ assay and ELISA. Our result showed that the positive rates from different dairy farms varied, ranging from 6.15% to 31.38% (SIT), 2.75% to 24.13% (CIT), 3.00% to 24.44% (IFN-γ assay), and 1.60% to 13.94% (ELISA), respectively (Table 1). The average positive rates among 13,345 cattle were 14.79%, 12.25%, 12.28%, 5.55%, by using immunological detection methods. Collectively, 752 (5.64%) cattle were further diagnosed with advanced infection, confirmed by both IFN-γ assay and ELISA, suggesting a high prevalence of tuberculosis in these dairy farms.

Table 1

Results of immunological detection.

Farm	No. cattle	No. bTB positive cattle (positive rate/%)				No. cattle with advanced infection (positive rate/%)	No. necropsy
		SIT	CIT	IFN-γ assay	ELISA
A	2,000	123(6.15)	55(2.75)	60(3.00)	32(1.60)	56(2.80)	23
B	1,690	280(16.57)	245(14.50)	226(13.37)	65(3.85)	58(3.43)	38
C	1,600	502(31.38)	386(24.13)	391(24.44)	223(13.94)	241(15.06)	10
D	1,300	95(7.31)	85(6.54)	83(6.38)	71(5.46)	68(5.23)	25
E	2,300	359(15.61)	322(14.00)	396(17.22)	111(4.83)	123(5.35)	17
F	1,755	412(23.48)	378(21.54)	352(20.06)	89(5.07)	76(4.33)	8
G	1,200	79(6.58)	69(5.75)	45(3.75)	44(3.67)	38(3.17)	25
H	1,500	124(8.27)	95(6.33)	86(5.73)	106(7.07)	92(6.13)	5
Total	13,345	1,974(14.79)	1,635(12.25)	1,639(12.28)	741(5.55)	752(5.64)	151

A large proportion of EPTB among cattle with advanced bTB infection

One hundred and fifty one animals from all eight dairy farms were selected and received further anatomical examination. In fact, typical bTB lesions were observed in 131 (86.75%) cattle, among which, 119 cattle (90.84%) were found with lesions regarding EPTB (Fig 1A), including extensive cavitary lesions in liver, tuberculous granuloma in spleen, granulomas with caseous necrosis and mineralization in mammary lymph node, gastric lymph node, mesenteric lymph node, intestinal lymph node, hilar lymph node and lung, miliary generalised tuberculosis on the thoracic cavity pleura (tuberculous “pearls”) (Fig 2). H&E staining result further validated typical tuberculous granulomas with evident necrosis and mineralization, numerous epithelioid cells and Langhans’ giant cells in the intermediate layer of the granuloma, a large number of lymphocytes in the external layer of the granuloma, as well as caseous necrosis of granulomata (Fig 3). There were bTB lesions associated with gastrointestinal system, which accounted for 71.26% of EPTB (Fig 1B and 1C).

Fig 1

Lesion distribution.

(A) Proportion of extrapulmonary tuberculosis and pulmonary tuberculosis in slaughtered cattle. (B) Proportion of lesion sites in extrapulmonary tuberculosis. (C) Proportion of lesions associated with gastrointestinal system and other system.

Fig 2

Lesions observed by necropsy.

(A) Extensive cavitary lesions in liver. (B) Granulomas with caseous necrosis in mammary lymph node. (C) Granulomas with caseous necrosis in gastric lymph node. (D) Granulomas with caseous necrosis and mineralization in mesenteric lymph node. (E) Tuberculous granuloma in spleen. (F) Granulomas with caseous necrosis and mineralization in intestinal lymph node. (G) Granulomas with caseous necrosis and mineralization in hilar lymph node. (H) Big granulomas with caseous necrosis and mineralization in lung. (I) Miliary generalised tuberculosis on the thoracic cavity pleura (tuberculous “pearls”).

Fig 3

H&E staining of sections of hilar lymph nodes.

(A) Typical tuberculous granulomas consisted of three parts, as shown at the box: the center of the granuloma displays evident necrosis and mineralization (*); numerous epithelioid cells and Langhans’ giant cells are present in the intermediate layer of the granuloma (†); A large number of lymphocytes are present in the external layer of the granuloma (‡). Magnification ×200. Box (*), Box (†), and Box (‡) indicate areas enlarged in panel B, C, and D, respectively. (B) Amorphous pink caseous material composed of the necrotic elements of the granuloma as well as the infectious organisms. Magnification ×400. (C) Epithelioid cells aggregation and Langhans’ giant cells could be seen subsequently. Magnification ×400. (D) Redundant lymphocytes infiltrated. Magnification ×400.

M. bovis isolation and identification

Sections of all paraffin-embedded tissues isolated from 151 cattle were treated with Ziehl-Neelsen acid-fast staining, and red-stained rod-shaped bacteria were observed from samples of 26 cattle (S2 Fig). The isolation of MTBC was then performed from 289 diverse kinds of tissues of 151 cattle. We successfully isolated the bacteria from 113 tissue samples of 91 cattle, accounting for 60.26% (91/151) of the total cattle examined. Bacterial colonies exhibited pellet, tubercle, or cauliflower shapes with a beige and creamy-white color on solid medium (S3 Fig). These bacteria were then identified as M. bovis by qPCR (S4 Fig).

As shown in the phylogenetic tree, 17 MTBC strains from different species clustered into two major clades, all 10 isolates in this study belonged to the M. bovis clade (Fig 4). Notably, 6 isolates were closely related to M. bovis AF2122/97 originated from UK, whereas 4 isolates shared close relation to M. bovis 30 from China, indicating the complicated epidemic status of M. bovis in dairy farms of China.

Fig 4

Phylogenetic trees of ten isolates.

The optimal tree with the sum of branch length = 1.71277593 was shown in Fig 4. The evolutionary distances were calculated using the Maximum Composite Likelihood method and were in the units of the number of base substitutions per site. This analysis involved 17 nucleotide sequences. All ambiguous positions from each sequence pair were removed (pairwise deletion option). There were a total of 3,914 positions in the final dataset. Evolutionary analyses were conducted in MEGA 5.

Evidence for possible gastrointestinal transmission

Field investigations at these dairy farms identified that the colostrum and regular milk for calves feed were not fully sterilized through pasteurization. In addition to the large proportion of bTB lesions sites associated with gastrointestinal system among EPTB, we further investigated the possible routes of tuberculosis transmission in these dairy farms. 54 milk samples, including 46 from positive cattle and 8 from calving room, were collected. qPCR result revealed M. bovis nucleic acid positive in all 8 samples from calving room and 39 samples from positive cattle. 12 M. bovis isolates were further cultured from 54 milk samples. We also tested the 54 feces samples. Similar to milk samples, M. bovis nucleic acid was detected in all 8 samples from fecal pool and 34 feces samples from bTB-positive cattle. However, no M. bovis was successfully isolated from these 54 feces samples (Table 2). These results unraveled the possibility of gastrointestinal transmission mode of EPTB in dairy farms.

Table 2

The results of tissue, milk and feces samples by culture and qPCR.

Sample type	Sample source (eight farms)	Number	Culture (+)	qPCR (+)	Ct value
Tissue	Positive cattle	151	91	91	17.56–30.11
Milk	Positive cattle	46	10	39	30.04–35.59
	Calving room	8	2	8	31.79–35.13
Total		54	12	47
Feces	Positive cattle	46	0	34	34.85–36.80
	Fecal pool	8	0	8	34.60–36.07
Total		54	0	42

Discussion

Bacteriological and histopathological examination of tissues are recognized as the most accurate and reliable methods for tuberculosis detection in cattle [14]. Given that isolation of M. bovis preferably requires 10~12 weeks, macroscopic lesions in carcasses at slaughterhouse are of great necessity for rapid detection and prompt actions for disease intervention [15]. So far, tuberculin test has been the standard method for detection of bTB worldwide [16]. For instance, the SIT has been extensively employed in the Irish bTB eradication program and has proven to be a very safe means to test and screen the Irish cattle population [17]. In several dairy farms of China, a high prevalence of bTB is reported by using the same method [18]. The complexity of bTB pathogenesis influences the conclusive presence of infection based on a single detection test, while the specificity and sensitivity may vary among the different immunological methods depending on the stages of infection. The combination of pathological and etiological methods is typically used to confirm M. bovis infection. In this scenario, we performed a comprehensive diagnosis by employing four immunological assays, SIT, CIT, IFN-γ assay and ELISA to reduce the false positive and false negative rates, in addition to pathological changes. By screening 13,345 cattle from 8 dairy farms, we found high prevalence of tuberculosis (2.8~15.06% of advanced infection) in these dairy farms. We further isolated and identified the pathogen and the result of WGS-SNP analysis of ten isolates confirmed they belonged to different origins of M. bovis clade.

The previous study on emergence of virulent isolates of M. bovis in the Nile Delta present that positive CIT results were identified in 3% of the animals spread among 40% of the examined herds. Post-mortem examination of slaughtered cattle then revealed the presence of both pulmonary and/or digestive forms of tuberculosis in > 50% of the examined animals [19]. Another finding regarding badgers naturally infected with M. bovis exhibited that most transmission occurred by the respiratory route, due to predominance of lesions in the respiratory tract [20]. The evidence on naturally infected dromedary reported that a total of 18 (19.56%) camels out of 92 examined revealed two different TB-lesion patterns, pulmonary (n  =  15) and disseminated (n  =  3) forms, suggesting that the pulmonary form of the TB was more common in camels [21]. Intriguingly, based on experimental infection studies with M. bovis that the transmission route affects the distribution of tuberculose focus and tissue tropism [10], our postmortem finding revealed that the majority of bTB occurred was EPTB (90.84%), as lesions in the lung were only detected among 9.16% of infected cattle in this study, suggesting EPTB as the major manifestation of infection in these dairy farms. This result was in accordance with previous finding in Ethiopia that the majority of bTB lesions located in the mesenteric lymph nodes, and the frequency and severity of the lesions were higher in the mesenteric lymph nodes than the thoracic lymph nodes [22]. They suggested that shedding of M. bovis in the feces and ingestion of the bacilli from contaminated pasturage and/or water may be the main route of transmission in pasture cattle, as lesions were primarily observed in mesenteric lymph nodes. Similarly, our data suggests that the gastrointestinal tract lesions in EPTB were initial foci, for M. bovis was found in colostrum and regular milk and feces.

In developed countries, effective control and eradication strategies include sterilization of milk by strict pasteurization procedures. In this scenario, lesions are mainly found in the lungs and lymph nodes in most of sporadic cases, suggesting the primary transmission route via respiratory tract [23]. It is therefore believed that milk, urine and feces play a minor role in transmission of bTB [24]. However, there are few cases reported regarding gastrointestinal tract infection with mesenteric lymph node lesions and intestinal wall tuberculosis nodules, the cause of which is related to contaminated pasture and drinking water by wildlife [25]. In these reports gastrointestinal tract lesions were far less common than respiratory tract lesions [26]. Nevertheless, our studies indicate that EPTB was mainly found among PPD-positive cattle in dairy farms, especially tuberculosis in digestive tract. We speculate that calves were mainly infected by drinking colostrum or regular milk that were not completely sterilized through pasteurization, or by drinking bacteria-contaminated forage or water in the dairy farms. It is proposed that the gastrointestinal transmission mode may lead to high prevalence of bTB in herds, on account of inadequate pasteurization and wildlife reservoirs in many developing countries [27]. Our preliminary data indicate the presence of EPTB in cattle farms throughout multiple provinces of China, owing to transmission via digestive tract. However, the limitation in this study still exists that the high prevalence rate of bTB in cattle farms caused by gastrointestinal transmission requires to be further investigated throughout China.

Conclusions

In conclusion, our data demonstrate that the bovine EPTB is the major manifestation of bTB infection in dairy farms, linking the evidence of oral transmission route. It attaches the attention on pasteurization programs in M. bovis–prevalent areas to restrict possible transmission of bTB through the consumption of dairy products.

Comprehensive diagnosis of bovine tuberculosis in dairy farms.

SIT, single intradermal test; CIT, comparative intradermal test; IFN-γ, gamma-interferon; ELISA, enzyme-linked immunosorbent assay; PCR, polymerase chain reaction.

(TIF)

Click here for additional data file.

Ziehl-Neelsen acid-fast staining of mesenteric lymph node.

Sections were stained by Ziehl-Neelsen acid-fast staining and images were captured and shown at ×400. Acid fast bacilli were stained with red and were observed under microscope.

(TIF)

Click here for additional data file.

Isolation and culture of M. bovis.

(A) Creamy-white pellet colony. (B) Beige granular colony. (C) Nodular colony. (D) Cauliflower like colony.

(TIF)

Click here for additional data file.

The results of qPCR.

(TIF)

Click here for additional data file.

18 Aug 2020

PONE-D-20-23226

High prevalence of extrapulmonary tuberculosis in dairy farms: evidence for possible gastrointestinal transmission

PLOS ONE

Dear Dr. Zeng,

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

Please submit your revised manuscript. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.

A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.

An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

We look forward to receiving your revised manuscript.

Kind regards,

Frederick Quinn

Academic Editor

PLOS ONE

Journal Requirements:

When submitting your revision, we need you to address these additional requirements.

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at

https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and

https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

2. In your Methods section, please provide the name of the slaughterhouse where the animals were sacrificed.

3. Thank you for including your ethics statement:  "All ante-mortem testing and post-mortem samples were obtained in compliance with national standards of the People's Republic of China (PRC): Diagnostic techniques for tuberculosis of animal (GB/T 18645-2002), and Bovine tuberculosis diagnosis-Assay on IFN-γ in vitro (GB/T 32945-2016); in accordance with national guidelines for the prevention and treatment of bovine tuberculosis (2017-2020) (Ministry of Agriculture and Rural Affairs of PRC); in accordance with legislation: Animal Epidemic Prevention Law of PRC (1997, order No.87 of president of PRC).".

Please amend your current ethics statement to confirm that your named ethics committee specifically approved this study.

For additional information about PLOS ONE submissions requirements for ethics oversight of animal work, please refer to http://journals.plos.org/plosone/s/submission-guidelines#loc-animal-research

Once you have amended this/these statement(s) in the Methods section of the manuscript, please add the same text to the “Ethics Statement” field of the submission form (via “Edit Submission”).

4. We note that you have included the phrase “data not shown” in your manuscript. Unfortunately, this does not meet our data sharing requirements. PLOS does not permit references to inaccessible data. We require that authors provide all relevant data within the paper, Supporting Information files, or in an acceptable, public repository. Please add a citation to support this phrase or upload the data that corresponds with these findings to a stable repository (such as Figshare or Dryad) and provide and URLs, DOIs, or accession numbers that may be used to access these data. Or, if the data are not a core part of the research being presented in your study, we ask that you remove the phrase that refers to these data.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

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

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

Reviewer #1: Partly

Reviewer #2: Yes

Reviewer #3: No

**********

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

Reviewer #1: No

Reviewer #2: Yes

Reviewer #3: Yes

**********

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

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

Reviewer #1: Yes

Reviewer #2: No

Reviewer #3: Yes

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

**********

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: The current paper investigates extra-pulmonary tuberculosis in dairy farms. 8 farms have been included in the study with a total of 13345 cattle in 3 different provinces in China. bTB diagnostic was performed using SIT,  CIT, IFN-γ assay, PPD eye drop reaction and ELISA. In addition 151 positive cattle were examined by necropsy and revealed 71.26% of the lesions localized in the liver, spleen, mesenteric lymph nodes, mammary lymph nodes and other organs and were classified as extra-pulmonary TB, while only 9.16% of the animals had lesions in the lungs.

The study discusses the possibility of transmission of bTB through consumption of infected colostrum, regular milk, forage and water rather than aerosol transmission, since a small proportion of the animals had lesions in their lungs.

Overall, the manuscript present interesting data but needs to be improved by a in-depth data analysis and more exhaustive discussion, suggestions are included below, in addition to some questions:

Please explain from an immunological point of view how did you chose the different tests to design the early infection versus the advanced infection.

I suggest you add the methods of each test briefly and then reference the instructions given by the vendor when appropriate. The same for the qPCR, it will be good to add a brief description of the experiment

In the methods section, I suggest you add a description of the livestock production systems (intensive or extensive) of the farms included in the study, in addition to a brief description of the animal housing conditions and the breeds of the cattle screened. Those are all elements that can influence the transmission and persistence of bTB in livestock.

Table 1 gives a great summary of the results of bTB diagnosis, however, it will be interesting to perform a multivariate analysis in order to detect any significant statistical difference between the different methods used.

In addition, it will be useful to calculate the apparent and true prevalence considering the sensitivity and specificity of each test used (or the tests for which sensitivity and specificity have been previously determined)

I suggest to add some information to the introduction part, some questions to answer in the introduction:

- is the comparative tuberculin skin test using avian PPD in addition to bovine PPD used in China

- Are there any available molecular data of bTB in cattle in China (pulmonary and extra pulmonary)

- In Slaughterhouses, usually many organs in addition to the lungs can be condemned (collected as samples if a study is undertaken), does anything similar exist in China, how is the slaughterhouse surveillance undertaken

- What are the last official prevalence data of bTB in cattle in China?

Line 50-54: add references

Line 239: you mention bTB nucleic acid, do you mean M. bovis or mycobacteria in general? please specify. Bovine tuberculosis is mostly caused by M. bovis, but other mycobacteria have been shown to cause the disease, so it's better to refer to the pathogen instead of the disease when talking about DNA. In addition, if you mean mycobacteria here, then please mention if you are talking about tuberculous or non tuberculous mycobacteria?

Table 2: I suggest you add brief details of the qPCR you used here (as mentioned above)

In the following study, performed in Ethiopia, the researchers found that the majority of bTB lesions were in the mesenteric lymph nodes, I would suggest to include this reference and also perform a deeper bibliographic search to supplement the discussion

1. Ameni G, Aseffa A, Engers H, Young D, Gordon S, Hewinson G, et al. High Prevalence and Increased Severity of Pathology of Bovine Tuberculosis in Holsteins Compared to Zebu Breeds under Field Cattle Husbandry in Central Ethiopia. Clin Vaccine Immunol. 2007 Oct 1;14(10):1356–61.

Line 208: Sections of all paraffin-embedded tissues isolated from ....

Line 259-260: I suggest you reword this sentence :

In several dairy farms of China, a high prevalence of bTB is reported by using the same method

Line 267: by screening 13,385.....

I suggest to english edit the manuscript by an english native speaker or a professional language editing service.

Reviewer #2: Several paragraphs need to be reviewed with regard to their style in English.

The authors should specify the conditions under which in vivo testing was conducted. Especially, if the animals have been subjected to parallel or serial testing. This is critical for result comparisons. According to International standards (OIE standard), two in vivo tuberculin tests for screening of bTB should be conducted 6 weeks apart at least . In addition the Eye drop reaction used by the authors is not considered as a reference test to screen for BTB. Published as such, would be misleading, inciting others researchers to use the eye drop reaction and to classify the reactors accordingly, as being in advanced stage of disease. Furthermore, the pathological post mortem findings in 9,16% of infected animals do not precise if the gastrointestinal associated lesions were primary or secondary foci. bTB is known to spread from primary foci (i.e. pulmonary...) to various tissues and organs via different routes. These informations should be provided to acertain data validity.

On the other hand, the provided ethical statement refers to technical standards of the Chinese competent authority and not to a university ethic committee.

Reviewer #3: In general terms, the manuscript shows a significant amount of information and reflects the great workload invested and the important investigation tools deployed.

The English style needs an improvement and homogenization. Reading by a native English speaker is recommended.

Description of the used screening tests did not mention their conditions of application and chronology (serial or parallel testing) on which the results interpretation is depending.

Objectives and purposes of using such a large number of screening tests have not been clearly defined, especially since one of the tests is not recognized as a reference test by international standards (PPD eye drop reaction). Furthermore, the authors did not specify how they selected animals with advanced infection based on test results.

Comparison of test results and the subsequent discussion will certainly bring an added value to this work, especially in terms of practical test use and related cost effectiveness.

**********

6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

29 Oct 2020

Dear Prof. Quinn and Reviewers,

Thanks very much for your letter and for the reviewers’ comments concerning our manuscript entitled “High prevalence of extrapulmonary tuberculosis in dairy farms: evidence for possible gastrointestinal transmission” (ID: PONE-D-20-23226). I really appreciate all your comments and suggestions. We made revision as required with changes marked in red. We also made point-to-point response as highlighted in blue.

Replies to the editor:

Journal Requirements:

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

Response: The manuscript has revised according to the PLOS ONE style templates, including those for file naming.

2. In your Methods section, please provide the name of the slaughterhouse where the animals were sacrificed.

Response: Thanks. The names of the slaughterhouse where the animals were sacrificed were provided in Methods section (Lines 125-126, page 6).

3. Thank you for including your ethics statement: "All ante-mortem testing and post-mortem samples were obtained in compliance with national standards of the People's Republic of China (PRC): Diagnostic techniques for tuberculosis of animal (GB/T 18645-2002), and Bovine tuberculosis diagnosis-Assay on IFN-γ in vitro (GB/T 32945-2016); in accordance with national guidelines for the prevention and treatment of bovine tuberculosis (2017-2020) (Ministry of Agriculture and Rural Affairs of PRC); in accordance with legislation: Animal Epidemic Prevention Law of PRC (1997, order No.87 of president of PRC).".

Please amend your current ethics statement to confirm that your named ethics committee specifically approved this study.

For additional information about PLOS ONE submissions requirements for ethics oversight of animal work, please refer to http://journals.plos.org/plosone/s/submission-guidelines#loc-animal-research

Once you have amended this/these statement(s) in the Methods section of the manuscript, please add the same text to the “Ethics Statement” field of the submission form (via “Edit Submission”).

Response: We are aware of the reviewer’s concern. In this epidemiological investigation, we detected different types of samples by using multiple methods, on the basis of the national standards, national guidelines and the legislation.

We have made correction and interpretation accordingly. (Lines 194-200, page 9-10).

“Ethical statement: Bovine tuberculosis (bTB) is a notifiable disease and there are control and surveillance campaigns in China. Official diagnostic methods for bTB are immunological tests, culture, PCR and histopathology. In this study, no animal experiment was involved. All datasets were in complete agreement with national and OIE regulations.”

4. We note that you have included the phrase “data not shown” in your manuscript. Unfortunately, this does not meet our data sharing requirements. PLOS does not permit references to inaccessible data. We require that authors provide all relevant data within the paper, Supporting Information files, or in an acceptable, public repository. Please add a citation to support this phrase or upload the data that corresponds with these findings to a stable repository (such as Figshare or Dryad) and provide and URLs, DOIs, or accession numbers that may be used to access these data. Or, if the data are not a core part of the research being presented in your study, we ask that you remove the phrase that refers to these data.

Response: Thanks. We added that as S4 Fig.

Special thanks to you for your good comments.

Replies to the reviewers’ comments:

Reviewer #1:

1. Please explain from an immunological point of view how did you chose the different tests to design the early infection versus the advanced infection.

Response: We accept the reviewer’s critique and added the explanation in an immunological point of view (Lines 121-128, page 6).

2. I suggest you add the methods of each test briefly and then reference the instructions given by the vendor when appropriate. The same for the qPCR, it will be good to add a brief description of the experiment.

Response: As suggested, the methods of each test were briefly added in “Materials and Methods” section (Lines 89-117, page 5-6).

We added the part of “DNA extraction and purification” in “Materials and Methods” section (Lines 158-162, page 8).

We also added a brief description of the experiment to the qPCR (Lines 164-172, page 8).

3. In the methods section, I suggest you add a description of the livestock production systems (intensive or extensive) of the farms included in the study, in addition to a brief description of the animal housing conditions and the breeds of the cattle screened. Those are all elements that can influence the transmission and persistence of bTB in livestock.

Response: We added a description of the livestock production systems of the farms, the animal housing conditions and the breeds of the cattle screened (Lines 85-87, page 4-5).

4. Table 1 gives a great summary of the results of bTB diagnosis, however, it will be interesting to perform a multivariate analysis in order to detect any significant statistical difference between the different methods used.

Response: That’s a good point. Basically, bovine tuberculosis (bTB) is a chronic bacterial disease, the diagnosis of which is implicated to multiple methods during the whole course of infection. For instance, currently, three assays (SIT, CIT, and IFN-γ assay) that depended on the pro-inflammatory cell-mediated immune (CMI) response are generally performed for detection of cattle with early infection, while the ELISA that depended on humoral responses is somehow performed for detection of cattle with advanced infection. That is, at different periods of bTB infection, there are appropriate methods. We believe it is interesting to further focus on the limitations of these diverse methods at different periods of infection based on a large number of samples.

5. In addition, it will be useful to calculate the apparent and true prevalence considering the sensitivity and specificity of each test used (or the tests for which sensitivity and specificity have been previously determined).

Response: Thanks. As similar to Q4 that required the comparison among the different methods, at different periods of bTB infection, there are appropriate methods. Our further study may systematically focus on the true prevalence considering the sensitivity and specificity of each test based on true positive and negative samples.

6. I suggest to add some information to the introduction part, some questions to answer in the introduction:

- is the comparative tuberculin skin test using avian PPD in addition to bovine PPD used in China

- Are there any available molecular data of bTB in cattle in China (pulmonary and extra pulmonary)

- In Slaughterhouses, usually many organs in addition to the lungs can be condemned (collected as samples if a study is undertaken), does anything similar exist in China, how is the slaughterhouse surveillance undertaken

- What are the last official prevalence data of bTB in cattle in China?

Response: We added the information to the introduction part according to the Reviewer’s suggestion, and all the questions above have answered in the introduction (Lines 46-47, 56-58, 64-72, 76-78, page 3-4).

7. Line 50-54: add references.

Response: Thanks. The references have been added (Lines 53-55, page 3).

8. Line 239: you mention bTB nucleic acid, do you mean M. bovis or mycobacteria in general? please specify. Bovine tuberculosis is mostly caused by M. bovis, but other mycobacteria have been shown to cause the disease, so it's better to refer to the pathogen instead of the disease when talking about DNA. In addition, if you mean mycobacteria here, then please mention if you are talking about tuberculous or non tuberculous mycobacteria?

Response: Thanks. We modified the expression in these sentences according to the previous comment (Lines 292-297, page 14).

9. Table 2: I suggest you add brief details of the qPCR you used here (as mentioned above).

Response: Thanks. We added brief details of the qPCR in “Materials and Methods” section (Lines 164-172, page 8).

10. In the following study, performed in Ethiopia, the researchers found that the majority of bTB lesions were in the mesenteric lymph nodes, I would suggest to include this reference and also perform a deeper bibliographic search to supplement the discussion.

1. Ameni G, Aseffa A, Engers H, Young D, Gordon S, Hewinson G, et al. High Prevalence and Increased Severity of Pathology of Bovine Tuberculosis in Holsteins Compared to Zebu Breeds under Field Cattle Husbandry in Central Ethiopia. Clin Vaccine Immunol. 2007 Oct 1;14(10):1356–61.

Response: Thank you for the suggestion. We added this reference and the information required as explained above (Lines 345-353, page 16-17).

11. Line 208: Sections of all paraffin-embedded tissues isolated from ....

Response: We modified the sentence according to the previous comment (Line 262, page 13).

12. Line 259-260: I suggest you reword this sentence:

In several dairy farms of China, a high prevalence of bTB is reported by using the same method.

Response: We reworded the sentence according to the previous comment (Lines 316-317, page 15).

13. Line 267: by screening 13,385.....

Response: We modified the sentence according to the previous comment (Line 324, page 16).

14. I suggest to English edit the manuscript by an English native speaker or a professional language editing service.

Response: Thanks. We improved the language.

Special thanks to you for your good comments.

Reviewer #2:

1. Several paragraphs need to be reviewed with regard to their style in English.

Response: Thanks. We modified that.

2. The authors should specify the conditions under which in vivo testing was conducted. Especially, if the animals have been subjected to parallel or serial testing. This is critical for result comparisons. According to International standards (OIE standard), two in vivo tuberculin tests for screening of bTB should be conducted 6 weeks apart at least.

Response: We specified the conditions under which in vivo testing was conducted in “Materials and Methods” section (Line 101, page 5).

3. In addition the Eye drop reaction used by the authors is not considered as a reference test to screen for BTB. Published as such, would be misleading, inciting others researchers to use the eye drop reaction and to classify the reactors accordingly, as being in advanced stage of disease.

Response: Considering the Reviewer’s comments, we deleted the PPD eye drop reaction method and the related data which had no effect on the test results. We have corrected the Table 1 and S1 Fig.

4. Furthermore, the pathological post mortem findings in 9.16% of infected animals do not precise if the gastrointestinal associated lesions were primary or secondary foci. bTB is known to spread from primary foci (i.e. pulmonary...) to various tissues and organs via different routes. These informations should be provided to a certain data validity.

Response: We provided the information to a certain data validity according to the Reviewer’s comment (Lines 343-351, page 16-17).

5. On the other hand, the provided ethical statement refers to technical standards of the Chinese competent authority and not to a university ethic committee.

Response: We are aware of the reviewer’s concern. In this epidemiological investigation, we detected different types of samples by using multiple methods, on the basis of the national standards, national guidelines and the legislation.

We have made correction and interpretation accordingly. (Lines 196-200, page 9-10).

“Ethical statement: Bovine tuberculosis (bTB) is a notifiable disease and there are control and surveillance campaigns in China. Official diagnostic methods for bTB are immunological tests, culture, PCR and histopathology. In this study, no animal experiment was involved. All datasets were in complete agreement with national and OIE regulations.”

Special thanks to you for your good comments.

Reviewer #3:

1. The English style needs an improvement and homogenization. Reading by a native English speaker is recommended.

Response: Thanks. We improved the language.

2. Description of the used screening tests did not mention their conditions of application and chronology (serial or parallel testing) on which the results interpretation is depending.

Response: We added the conditions of application and chronology of screening tests in “Materials and Methods” section (Lines 89-128, page 5-6).

3. Objectives and purposes of using such a large number of screening tests have not been clearly defined, especially since one of the tests is not recognized as a reference test by international standards (PPD eye drop reaction). Furthermore, the authors did not specify how they selected animals with advanced infection based on test results.

Response: Considering the Reviewer’s comments, we deleted the PPD eye drop reaction and the related data which had no effect on the test results. We have defined the objectives and purposes of using a large number of screening tests (Lines 119-121, page 6).

4. Comparison of test results and the subsequent discussion will certainly bring an added value to this work, especially in terms of practical test use and related cost effectiveness.

Response: We thank the comments.

Special thanks to you for your good comments.

Other changes:

1. Line 24-25: “8” was corrected as “eight”, “3” was corrected as “three”.

2. Line 30: “12” was corrected as “twelve”, “%” was corrected as “percent”.

3. Line 33: “10” was corrected as “ten”.

4. Line 35: “bTB” was corrected as “M. bovis”.

5. Line 59: “PPD” was corrected as “purified protein derivative (PPD)”.

6. Line 119: “Five” was corrected as “Four”, “8” was corrected as “eight”.

7. Line 174-175: “8” was corrected as “eight”, “6” was corrected as “six”, “4” was corrected as “four”.

8. Line 207: “8” was corrected as “eight”.

9. Line 238: “%” was corrected as “percent”.

10. Line 266: “mycobacterium tuberculosis complex” was corrected as “MTBC”.

11. Line 272-275: “7” was corrected as “seven”, “17” was corrected as “seventeen”, “10” was corrected as “ten”, “6” was corrected as “six”, “4” was corrected as “four”.

12. Line 279: “10” was corrected as “ten”.

13. Line 284: “17” was corrected as “seventeen”.

14. Line 293: “8” was corrected as “eight”.

15. Line 295: “12” was corrected as “Twelve”.

16. Line 297: “8” was corrected as “eight”.

17. Line 306: Table 2, “8” was corrected as “eight”.

18. Line 324: “4” was corrected as “four”.

19. Line 326: “8” was corrected as “eight”.

20. Line 329: “10” was corrected as “ten”.

Response: Thanks. We made revision.

Submitted filename: Response to Editor.docx

Click here for additional data file.

6 Jan 2021

PONE-D-20-23226R1

High prevalence of extrapulmonary tuberculosis in dairy farms: evidence for possible gastrointestinal transmission

PLOS ONE

Dear Dr. Zeng,

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

Please submit your revised manuscript. If you will need significantly more time to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.

A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.

An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

We look forward to receiving your revised manuscript.

Kind regards,

Frederick Quinn

Academic Editor

PLOS ONE

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: (No Response)

**********

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

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

Reviewer #1: Yes

**********

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

Reviewer #1: Yes

**********

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

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

Reviewer #1: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: No

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: Thank you for submitting a revised version and answering all the comments, the manuscript has improved so much, however, I still think it needs professional proofreading and english editing

One more question: did you collect the cervical lymph nodes and lung associated lymph nodes? if not, can you please explain why ? I'm asking this question because very often TB disease in cattle is contained in the lymph nodes and does not progress to other organs and tissues, so it is important to examine those specific lymph nodes for gross visible lesions (in addition to the other lymph nodes you mentioned you have examined)

Below few typos and suggestions

Line 29: were tested

Line 30: the results....had advanced infection of bTB

Line 34: replace of note by in fact

Line 38-39: the phylogenetic results... (reword this sentence please)

Line 60 The Single Intradermal Test (SIT) and IFN-γ assay are the current standard diagnostic

diagnosis for bTB in some cattle farms with good economic conditions: reword

Line 69: what do you mean by harmless treatment

Line 72: is frequently overlooked

Line 82: ...and extrapulmonary bTB....

Line 126 to 129: Reword this sentence

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

18 Jan 2021

Dear Prof. Quinn and Reviewers,

Thanks very much for your letter and for the reviewers’ comments concerning our manuscript entitled “High prevalence of extrapulmonary tuberculosis in dairy farms: evidence for possible gastrointestinal transmission” (ID: PONE-D-20-23226R1). I really appreciate for all your comments and suggestions. We made revision as required with changes marked in red. We also made point-to-point response as highlighted in blue.

Replies to the reviewers’ comments:

Reviewer #1:

1. Did you collect the cervical lymph nodes and lung associated lymph nodes? if not, can you please explain why? I'm asking this question because very often TB disease in cattle is contained in the lymph nodes and does not progress to other organs and tissues, so it is important to examine those specific lymph nodes for gross visible lesions (in addition to the other lymph nodes you mentioned you have examined).

Response: That’s a good point. We examined lymph nodes and numerous organs and tissues in order to identify gross visible lesions, and we then collected all the organs and tissues with lesions, including the cervical lymph nodes and lung associated lymph nodes, such as submandibular lymph nodes and hilar lymph nodes.

2. Below few typos and suggestions.

Line 29: were tested

Line 30: the results....had advanced infection of bTB

Line 34: replace of note by in fact

Line 38-39: the phylogenetic results... (reword this sentence please)

Line 60 The Single Intradermal Test (SIT) and IFN-γ assay are the current standard diagnostic

diagnosis for bTB in some cattle farms with good economic conditions: reword

Line 69: what do you mean by harmless treatment

Line 72: is frequently overlooked

Line 82: ...and extrapulmonary bTB....

Line 126 to 129: Reword this sentence

Response: Thank you for the suggestions. We made modification according to the previous comments. We double checked the spelling and improved the language.

Submitted filename: Response to Reviewers.docx

Click here for additional data file.

3 Feb 2021

PONE-D-20-23226R2

High prevalence of extrapulmonary tuberculosis in dairy farms: evidence for possible gastrointestinal transmission

PLOS ONE

Dear Dr. Zeng,

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

Please submit your revised manuscript. If you will need significantly more time to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.

A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.

An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

We look forward to receiving your revised manuscript.

Kind regards,

Frederick Quinn

Academic Editor

PLOS ONE

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

**********

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

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

Reviewer #1: Yes

**********

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

Reviewer #1: N/A

**********

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

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

Reviewer #1: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: No

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: Thank you for addressing all the comments, the manuscript has improved a lot, however, I still have few suggestions and questions listed below. In addition, I highly suggest detailed english editing of the manuscript, by an english native speaker or a professional editing service.

Line 30_31:

"The result indicated that 752 (5.64%) had advanced infection of bTB"

Line 39: delete "positive"

Line 41_42:

"Our data demonstrate that the increase of EPTB transmitted by digestive tract is implicated in the current high prevalence rate of bTB in China"

Line 50 : delete "concerne"

Line 61: "the comparative intradermal test (CIT) is also used often."

Line 62 to 64: here you mentioned studies showing that PPD positive cattle don't present typical Tb associated symptoms, and they don't possess lung lesions upon necropsy. Can you please reference those studies.

Line 65-66: "as well as interfered **with** the implementation of culling policy on the cattle suspected to be infected with bTB."

Line 132-133: I suggest to rewrite this part of the sentence as follow:

", were slaughtered and anatomical examination was performed in the local slaughterhouses named Kaerwan (Xinjiang), Musulin (Shandong), and Chengcheng (Guangxi)."

Line 139 anatomical examination was or anatomical examinations were

please modify accordingly

Line 150: "approximately"

Line 300-302: "Similar to milk samples, M. bovis nucleic acid was detected in all eight samples from fecal pool and from 34 positive cattle"

In the last sentence, which specific samples from 34 positive cattle are you talking about here? are they also fecal samples, or milk? please specify

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

4 Mar 2021

Dear Prof. Quinn and Reviewers,

Thanks very much for your letter and for the reviewers’ comments concerning our manuscript entitled “High prevalence of extrapulmonary tuberculosis in dairy farms: evidence for possible gastrointestinal transmission” (ID: PONE-D-20-23226R2). We really appreciate all your comments and suggestions. We revised our manuscript, and we have sent the revised manuscript, and a version containing all the changes to be visible. We also made point-to-point response as highlighted in blue.

Replies to the reviewers’ comments:

Reviewer #1:

1. Below few typos and suggestions.

Line 30_31: "The result indicated that 752 (5.64%) had advanced infection of bTB"

Line 39: delete "positive"

Line 41_42: "Our data demonstrate that the increase of EPTB transmitted by digestive tract is implicated in the current high prevalence rate of bTB in China"

Line 50: delete "concerne"

Line 61: "the comparative intradermal test (CIT) is also used often."

Line 65-66: "as well as interfered **with** the implementation of culling policy on the cattle suspected to be infected with bTB."

Line 132-133: I suggest to rewrite this part of the sentence as follow:

", were slaughtered and anatomical examination was performed in the local slaughterhouses named Kaerwan (Xinjiang), Musulin (Shandong), and Chengcheng (Guangxi)."

Line 139: anatomical examination was or anatomical examinations were

please modify accordingly

Line 150: "approximately"

Response: Thank you for the suggestions. We modified and reworded the sentences according to the previous comment.

2. Line 62 to 64: here you mentioned studies showing that PPD positive cattle don't present typical Tb associated symptoms, and they don't possess lung lesions upon necropsy. Can you please reference those studies.

Response: Thanks. We had listed the references behind the sentence.

3. Line 300-302: "Similar to milk samples, M. bovis nucleic acid was detected in all eight samples from fecal pool and from 34 positive cattle"

In the last sentence, which specific samples from 34 positive cattle are you talking about here? are they also fecal samples, or milk? please specify

Response: Thank you for the suggestion. We modified the sentences according to the previous comment. In the last sentence, we had specified the 34 feces samples from bTB-positive cattle.

4. In addition, I highly suggest detailed english editing of the manuscript, by an english native speaker or a professional editing service.

Response: Thank you for the suggestion. We modified other spelling and improved the language. English expression has been carefully improved throughout the manuscript. And we made a marked-up copy of manuscript that highlights changes to the original version named 'Revised Manuscript with Track Changes'.

Special thanks to you for your good comments.

Thanks. We made revision.

Submitted filename: Response to Reviewers.docx

Click here for additional data file.

17 Mar 2021

High prevalence of extrapulmonary tuberculosis in dairy farms: evidence for possible gastrointestinal transmission

PONE-D-20-23226R3

Dear Dr. Zeng,

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.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Frederick Quinn

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

**********

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

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

Reviewer #1: Yes

**********

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

Reviewer #1: Yes

**********

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

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

Reviewer #1: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: Following few suggestions and typos:

Line 72: become the most.... (delete as)

Line 80-81: reword this sentence please

Line 114: ...immune responses can facilitate the detection if late stage....

Line 173-178: cattle is the plural form please replace by the singular form, which is the age or sex-specific terms of the animal (e.g: cow, bull....)

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

22 Mar 2021

PONE-D-20-23226R3

High prevalence of extrapulmonary tuberculosis in dairy farms: evidence for possible gastrointestinal transmission

Dear Dr. Zeng:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

If we can help with anything else, please email us at plosone@plos.org.

Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Frederick Quinn

Academic Editor

PLOS ONE